CN117957316A

CN117957316A - Fucosyltransferase for converting lactose-N-disaccharide

Info

Publication number: CN117957316A
Application number: CN202280049885.7A
Authority: CN
Inventors: S·埃萨尔特; J·博普雷兹; G·彼得斯; K·梵德瓦勒; A·瓦考特兰
Original assignee: Inbiose NV
Current assignee: Inbiose NV
Priority date: 2021-07-16
Filing date: 2022-07-15
Publication date: 2024-04-30

Abstract

The invention belongs to the technical field of synthetic biology and metabolic engineering. More specifically, the invention is in the technical field of metabolically engineered cells and the use of said cells in culture, preferably in fermentation. Cells and methods for producing compounds are described. The cells express an alpha-1, 2-fucosyltransferase having a galactoside alpha-1, 2-fucosyltransferase activity on galactose residues of Gal-b1,3-GlcNAc (LNB, lactose-N-disaccharide). Furthermore, the invention provides for purifying the compounds from the culture.

Description

Fucosyltransferase for converting lactose-N-disaccharide

Technical Field

The invention belongs to the technical fields of synthetic biology and metabolic engineering. More specifically, the invention is in the technical field of metabolically engineered cells and the use of said cells in culture, preferably in fermentation. Cells and methods for producing compounds are described. The cells express an alpha-1, 2-fucosyltransferase having galactoside alpha-1, 2-fucosyltransferase activity on galactose residues of Gal-b1,3-GlcNAc (LNB, lactose-N-disaccharide). Furthermore, the invention provides for purifying the compounds from the culture.

Background

Oligosaccharides often exist in glycoconjugated forms with proteins and lipids and are involved in a number of important phenomena such as differentiation, development and biological recognition processes associated with fertilization, embryogenesis, inflammation, transfer and development and progression of host pathogen adhesion. Oligosaccharides may also exist in body fluids and human milk as unconjugated glycans, where they also regulate important developmental and immune processes (Bode, early hum dev.1-4 (2015); reily et al, nat rev. Neprol.15, 346-366 (2019); varki, glycobiology 27,3-49 (2017)). Fucose-alpha 1, 2-galactose-beta 1, 3-N-acetylglucosamine (Fuc-a 1,2-Gal-b1, 3-GlcNAc) or 2 '-fucosyllacto-N-disaccharide (2' FLNB), also known as H1 type antigen (H1), is a structure in type I Lewis antigens reported to be involved in inflammatory reactions, rotavirus infections, and cancer pathogenesis, etc. (Blanas et al, 2018, front.Oncol., https:// doi.org/10.3389/fonc.2018.00039). Fuc-a1,2-Gal-b1,3-GlcNAc groups are also present in lactose-N-fucopentaose I (LNFP-I, fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc) and lactose-N-difucose-hexaose I (LNDFH I, fuc-a-1,2-Gal-b1,3- [ Fuc-a1,4] -GlcNAc-b1,3-Gal-b 1.4-Glc), which are abundant oligosaccharides present in human milk. Human Milk Oligosaccharides (HMOs) have a variety of functions including prebiotic, immune, intestinal, and cognitive benefits (Reverri et al, nutrients (10), 1346 (2018)). LNDFH I has been reported to have immunomodulatory capacity to participate in viral infection (Triantis et al, front. LNFP-I is an important immunomodulator that prevents infants in lactation from suffering severe infectious diarrhea by inhibiting adhesion of pathogenic bacteria and viruses such as E.coli (EPEC, UPEC). LNFP-I is also involved in the binding of pathogen toxins, growth inhibition of group B streptococci and selective stimulation of bifidobacterium populations (Derya et al, J.Biotechnol.318,31-38 (2020); gotoh et al, sci.Rep.8,13958 (2018); lin et al, J.biol.chem.292,11243-11249 (2017); sotgiu et al, int.J.biomed.Sci.2 (2), 114-120 (2006)).

There is great scientific and commercial interest in these structures or compounds, but the availability is limited because the production relies on chemical or chemoenzymatic synthesis or purification from natural sources (e.g. animal milk). Chemical synthesis methods are laborious and time-consuming and difficult to scale up due to the large number of steps involved. Enzymatic methods offer advantages over chemical synthesis, but the stereospecificity and regioselectivity of the desired enzyme remain a significant challenge.

It is an object of the present invention to provide means and methods by which these structures can be produced from cells, and preferably in an efficient, time and cost effective manner, and produce large amounts of the desired compounds. This and other objects are achieved according to the present invention by providing a cell and a method for producing a compound comprising a structure of formula I, II or III according to the present invention, wherein the cell is genetically modified to produce the compound.

Disclosure of Invention

Surprisingly, it has now been found that compounds comprising the structure of formula I, II or III can be produced by a single cell:

Wherein:

R ¹ is a monosaccharide, disaccharide, oligosaccharide, protein, glycoprotein, peptide, glycopeptide, lipid or glycolipid; r ² is a monosaccharide, disaccharide or oligosaccharide.

The invention provides cells and methods for producing the compounds comprising the structures of formulas I, II or III. The method comprises the following steps: providing a cell expressing an α -1, 2-fucosyltransferase having a galactosidase α -1, 2-fucosyltransferase activity on galactose residues of Gal-b1,3-GlcNAc (LNB, lactose-N-disaccharide), the cell being capable of producing the compound comprising the structure of formula I, II or III, and culturing and/or incubating the cell under conditions that allow production of the compound comprising the structure of formula I, II or III. The invention also provides methods for isolating and purifying the compounds comprising the structures of formulas I, II or III. Furthermore, the invention provides cells genetically engineered to produce compounds comprising the structure of formula I, II or III.

Definition of the definition

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

The various aspects and embodiments of the various aspects of the invention disclosed herein are to be understood not only in the order and context specifically described in the present specification, but also in any order and any combination thereof. Where the context requires, all words 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. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Nucleic acid and peptide synthesis uses standard techniques. Typically, the purification step is performed according to manufacturer specifications.

In the specification, embodiments 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 embodiments have been set forth only for the purposes of example and that it should not be taken as limiting the invention. It will be apparent to one skilled in the art that changes, other embodiments, modifications, details and applications that are consistent with the text and spirit disclosed herein and within the scope of the invention are to be interpreted in accordance with the principles of patent law, including the doctrine of equivalents. In the appended claims, the reference numerals used to designate claim steps are provided for the convenience of description only and do not imply any particular order of performing the steps.

In this document and in its claims, the verbs "comprise/include," "have" and "contain" and their conjugations are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. The verb "to consist essentially of … …" means that there may be more ingredients than specifically identified, but that the additional ingredients do not alter the unique features of the present invention. Throughout the application and claims, the verbs "comprise/include," "have" and "contain" and their variants may preferably be replaced by "consisting of … …" (and their variants) or "consisting essentially of … …" (and their variants) and vice versa, unless otherwise specifically indicated. Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there be only one element. Thus, the indefinite article "a" or "an" generally means "at least one".

Throughout this application, unless explicitly stated otherwise, the terms "a" and "an" are preferably replaced by "at least two" s, more preferably by "at least three" s, even more preferably by "at least four" s, even more preferably by "at least five" s.

Throughout this application, the features "synthesized", "synthesized" and "produced" are used interchangeably with the features "produced", "generated" and "produced", respectively, unless explicitly stated otherwise.

Each of the embodiments 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.

According to the present invention, the term "polynucleotide" generally means any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to: single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions or single-and double-stranded, double-and triple-stranded regions, single-and double-stranded RNA, and RNA that is a mixture of single-and double-stranded regions, hybrid molecules (which comprise DNA and RNA that may be single-or more generally double-or triple-stranded regions, or a mixture of single-and double-stranded regions). In addition, "polynucleotide" as used herein refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The chains in such regions may be from the same molecule or from different molecules. The region may include all of one or more molecules, but more typically only regions involving some molecules. One of the molecules of the triple helical region is often an oligonucleotide. The term "polynucleotide" as used herein also includes DNA or RNA as described above, which contains one or more modified bases. Thus, DNA or RNA having a backbone modified for stability or other reasons is a "polynucleotide" as described herein. In addition, DNA or RNA comprising rare bases (such as inosine) or modified bases (such as tritylated bases) should be understood to be encompassed by the term "polynucleotide". It will be appreciated that numerous modifications have been made to DNA and RNA for a number of purposes known to those skilled in the art. The term "polynucleotide" as used herein includes 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 includes short polynucleotides, often referred to as oligonucleotides.

"Polypeptide" means any peptide or protein comprising two or more amino acids linked to each other by peptide bonds or modified peptide bonds. "Polypeptides" means short chains, commonly referred to as peptides, oligopeptides and oligomers, and longer chains, commonly referred to as proteins. The polypeptide may contain amino acids other than those encoded by the 20 genes. "Polypeptides" include those modified by natural processes such as processing and other post-translational modifications, but also include those modified by chemical modification techniques. Such modifications are well described in basic textbooks and in more detailed monographs and in numerous research literature, and are well known to the skilled artisan. The same type of modification may be present at several sites in a given polypeptide to the same or different extents. 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 flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, 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 glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, 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 cyclic polypeptides may be produced by posttranslational natural processes, and may also be produced by entirely synthetic methods.

The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides comprising sequences encoding polypeptides of the invention. The term also encompasses polynucleotides that include a single contiguous region or a discontinuous region encoding a polypeptide (e.g., interrupted by an integrated phage or insert sequence or edit) and may also include additional regions of coding and/or non-coding sequences.

"Isolated" means altered from its natural state "by hand", i.e., if it is naturally occurring, it has been 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 separated from coexisting materials in its natural state is "isolated," as that term is used herein. Similarly, as the term is used herein, "synthetic" sequence refers to any sequence that has been synthetically produced and is not isolated directly from natural sources. As the term is used herein, "synthesized" refers to any synthetically produced sequence and is not isolated directly from natural sources.

Throughout this application, the features "synthetic," "synthetic," and "co-produced" are used interchangeably with the features "produced," "produced," and "manufactured," respectively, unless explicitly stated otherwise.

The terms "recombinant" or "transgenic" or "metabolically engineered" or "genetically modified" as used herein with respect to a cell or host cell are used interchangeably and refer to the cell replicating a heterologous nucleic acid, or expressing a peptide or protein encoded by a heterologous nucleic acid (i.e., "sequence foreign to the cell" or "sequence foreign to the location or environment in the cell"). Such cells are described as transformed with at least one heterologous or exogenous gene, or as transformed by the introduction of at least one heterologous or exogenous gene. The metabolically engineered or recombinant or transgenic cell may contain genes not found in the native (non-recombinant) form of the cell. Recombinant cells may also contain genes found in the native form of the cell, wherein the genes are modified and reintroduced into the cell by artificial means. The term also encompasses cells containing nucleic acid endogenous to the cell, which nucleic acid has been modified or whose expression or activity has been modified, but which nucleic acid has not been removed from the cell; such modifications include those obtained by gene replacement, promoter replacement, site-specific mutation, and related techniques. Thus, a "recombinant polypeptide" is a polypeptide that has been produced by a recombinant cell. As used herein, a "heterologous sequence" or "heterologous nucleic acid" is a sequence or nucleic acid that originates from a foreign source (e.g., from a different species) of a particular cell, or if from the same source, is modified from its original form or position in the genome. Thus, a heterologous nucleic acid operably linked to a promoter is from a source that is different from the source of the promoter, or if from the same source, is modified from its original form or position in the genome. Heterologous sequences can be stably introduced, for example, by transfection, transformation, conjugation, or transduction into the genome of a host microbial cell, wherein the technique that can be applied will depend on the cell and the sequence to be introduced. Various techniques are known to those skilled in the art and are disclosed, for example, in Sambrook et al, molecular Cloning: A Laboratory Manual, version 2, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (1989). The term "mutated" cell or microorganism as used in the context of the present invention means a genetically modified cell or microorganism.

In the context of the present invention, the term "endogenous" means 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 is unchanged compared to the natural control mechanism acting on its expression. The term "exogenous" refers to any polynucleotide, polypeptide, or protein sequence that originates outside of the cell under study and is not a natural part of the cell, or that is not present at its natural location in the chromosome or plasmid of the cell.

The term "heterologous" when used with reference to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme, refers to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme derived from a source other than the species of the host organism. Conversely, a "homologous" polynucleotide, gene, nucleic acid, polypeptide, or enzyme is used herein to refer to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme derived from a host organism species. When referring to a gene regulatory sequence or a helper nucleic acid sequence for maintaining or manipulating a gene sequence (e.g., a promoter, 5 'untranslated region, 3' untranslated region, polyadenylation addition sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genomic homologous region, recombination site, etc.), by "heterologous" is meant that the regulatory sequence or helper sequence is not naturally associated with the gene in which the regulatory or helper nucleic acid sequence is juxtaposed in a construct, genome, chromosome, or episome. Thus, a promoter operably linked to a gene that is not operably linked in its natural state (i.e., in the genome of a non-genetically engineered organism) is referred to herein as a "heterologous promoter," even though the promoter may be derived from the same species (or in some cases, the same organism) as the gene to which it is linked.

The term "altered activity" of a protein or enzyme is related to a change in the activity of the protein or enzyme compared to the wild type (i.e., the natural activity of the protein or enzyme). The altered activity may be an elimination, impairment, reduction or delay of the activity 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 activity of the protein or enzyme compared to the wild-type activity of the protein or enzyme. Altered activity of a protein or enzyme is obtained by altered expression of the protein or enzyme, or by expression of a modified (i.e., mutated) form of the protein or enzyme. The altered activity of the enzyme further relates to an alteration of the apparent Mie constant Km and/or apparent maximum speed (Vmax) of the enzyme.

The term "altered expression" of a gene relates to a change in expression compared to the wild-type expression of said gene at any stage of the production process of the desired di-and/or oligosaccharides. The altered expression is lower or higher expression compared to the wild type, wherein the term "higher expression" is also defined as "overexpression" of the gene in the case of an endogenous gene or "expression" in the case of a heterologous gene not present in the wild type strain. Reduced expression was obtained as follows: these techniques are used to alter genes in a manner that renders the genes less capable (i.e., statistically significantly "less capable" compared to the functional wild-type gene) or completely incapable (such as the knocked-out gene) of producing a functional end product by common techniques well known to the skilled artisan, such as the use of siRNA, crispR, crispRi, riboswitches, recombinant engineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutant genes, knockdown genes, transposon mutagenesis, … …. The term "riboswitch" as used herein is defined as a portion of messenger RNA that folds into a complex structure that blocks expression by interfering with translation. Binding of effector molecules induces conformational changes, allowing expression to be regulated post-transcriptionally. In addition to altering the gene of interest in such a way that reduced expression is obtained as described above, reduced expression may also be obtained by altering transcription units, promoters, untranslated regions, ribosome binding sites, SD (Shine Dalgarno) sequences or transcription terminators. Reduced expression or reduced expression may be obtained, for example, as follows: one or more base pairs in the promoter sequence are mutated or the promoter sequence is completely changed to a constitutive promoter having a lower expression strength than the wild type or an inducible promoter resulting in regulated expression or a repressible promoter resulting in regulated expression.

Overexpression or expression is obtained as follows: the genes are part of an "expression cassette" which relates to any sequence in which a promoter sequence, an untranslated region sequence (containing a ribosome binding sequence, SD (Shine Dalgarno) sequence or Kozak sequence), a coding sequence and optionally a transcription terminator are present, and which leads to the expression of a functionally active protein, by means of common techniques well known to the skilled person, such as the use of artificial transcription factors, the de novo design of promoter sequences, ribosome engineering, the introduction or re-introduction of expression modules at the euchromatin, the use of high copy number plasmids. The expression is constitutive or regulatable.

The term "constitutive expression" is defined as expression that is not regulated under certain growth conditions by transcription factors other than subunits of the RNA polymerase (e.g., bacterial sigma factors such as s ⁷⁰、s⁵⁴ or related s-factors and the yeast mitochondrial RNA polymerase specific factor MTF1 that is co-bound to the 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. The RNA polymerase is the catalytic mechanism for the synthesis of RNA from a DNA template. RNA polymerase binds to a specific DNA sequence to initiate transcription, for example by sigma factor in a prokaryotic host or by MTF1 in yeast. Constitutive expression will provide a constant level of expression without induction or inhibition.

The term "regulated expression" is defined as a facultative or regulated or regulatable expression of a gene that is expressed only under certain natural conditions of the host (e.g., the joining phase of budding yeast, resting phase of bacteria), as a response to an inducer or repressor such as, but not limited to, glucose, allo-lactose, galactose, glycerol, arabinose, rhamnose, fucose, IPTG, methanol, ethanol, acetate, formate, aluminum, copper, zinc, nitrogen, phosphate, xylene, carbon or nitrogen depletion or a substrate or produced product or chemical inhibition, as a response to an environmental change (e.g., anaerobic or aerobic growth, oxidative stress, pH change, temperature change such as, for example, heat or cold shock, osmotic pressure, light conditions, starvation), or depending on the developmental stage or cell cycle location of the host cell, including, but not limited to apoptosis and autophagy. Regulated expression allows control of when the gene is expressed. The term "natural inducer-induced expression" is defined as the facultative or regulated expression of a gene that is expressed only under the specific natural conditions of the host (e.g., in a parturition or mammalian organism), as a response to environmental changes (e.g., including but not limited to hormones, heat, cold, pH changes, light, oxidative or osmotic stress/signaling), or depending on the developmental stage or location of the cell cycle of the host cell, including but not limited to apoptosis and autophagy. The term "inducible expression after chemical treatment" is defined as the facultative or regulated expression of a gene that is expressed only when treated with a chemical inducer or repressor, where the inducer and repressor include, but are not limited to, alcohols (e.g., ethanol, methanol), carbohydrates (e.g., glucose, galactose, glycerol, lactose, arabinose, rhamnose, fucose, isolactose), metal ions (e.g., aluminum, copper, zinc), nitrogen, phosphate, IPTG, acetate, formate, xylene.

The term "control sequence" refers to a sequence recognized by a cellular transcription and translation system, thereby allowing transcription and translation of a polynucleotide sequence into a polypeptide. Thus, such DNA sequences are necessary for expression of the 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, SD (Shine Dalgarno) sequences, kozak sequences, transcription terminator sequences. Suitable control sequences for prokaryotes include, for example, promoters, optional operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers. The DNA of the pre-sequence or secretory leader may be operably linked to the DNA of the polypeptide provided that it is expressed as a pre-protein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence provided that it affects transcription of the sequence; or the ribosome binding site is operably linked to a coding sequence provided that it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence provided that it is positioned so as to facilitate translation. The control sequences may also be controlled by an inducible promoter or by a genetic loop (which induces or inhibits transcription of the polynucleotide or translation into a polypeptide) with an external chemical such as, but not limited to, IPTG, arabinose, lactose, iso-lactose, rhamnose or fucose.

Typically, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in the reading phase. Enhancers need not be contiguous.

The term "wild-type" refers to a well-known genetic or phenotypic condition that occurs in nature.

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

As used herein, the term "mammary gland cell" generally refers to a mammary gland epithelial cell, a mammary gland 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 that has a phenotype/genotype similar (or substantially similar) to a native breast cell but is derived from a non-breast cell source. Such breast-like cells may be engineered to remove at least one undesired 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. Further 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 cells having a phenotype similar (or substantially similar) to that of natural breast epithelial cells. Cells having a phenotype similar (or substantially similar) to a native mammary gland cell or mammary gland epithelial cell or exhibiting at least one characteristic may comprise cells that naturally exhibit or have been engineered to be capable of expressing at least one milk component (e.g., derived from a mammary gland cell line or a non-mammary gland cell line).

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 can be 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 "can … < verb >" and "can … < verb >" are preferably replaced with the active language of the verb, and vice versa. For example, the expression "capable of expressing" is preferably replaced with "expressing" and vice versa, i.e. the expression is preferably replaced with "capable of expressing".

As the term is used herein, a "variant" is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. In the context of the present invention, variants of an α -1, 2-fucosyltransferase (i.e., reference polypeptide) as disclosed herein are polypeptides that differ from the reference polypeptide but retain their enzymatic activity as described herein, e.g., the galactosyl α -1, 2-fucosyltransferase activity for galactose residues of Gal-b1,3-GlcNAc (LNB, lactose-N-disaccharide), or the galactosyl α -1, 2-fucosyltransferase activity for galactose residues of Gal-b-1,3-GlcNAc and additional galactosyl a-1, 2-fucosyltransferase activity for galactose residues of the non-reducing end of LNT, depending on the identity of the α -1, 2-fucosyltransferase described herein. Typical variants of a polynucleotide differ in nucleotide sequence from another reference polynucleotide. Variations in the nucleotide sequence of the variants may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. As discussed below, nucleotide changes may result in 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. Typically, the differences are limited, so the sequences of the reference polypeptides and variants are very similar overall, and are identical in many regions. Variants and reference polypeptides may differ 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 those encoded by the genetic code. The variant of the polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or it may be an unknown naturally occurring variant. Non-naturally occurring variants of polynucleotides and polypeptides may be produced by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those of skill in the art. In the context of the present invention, a "variant" of a reference polypeptide is preferably a polypeptide having an amino acid sequence with at least 80% sequence identity to the full-length sequence of the reference polypeptide.

The term "derivative" of a polypeptide as used herein is a polypeptide that: it may contain deletions, additions or substitutions of amino acid residues within the amino acid sequence of the polypeptide, but which result in a silent change, thereby producing a functionally equivalent polypeptide (i.e., retaining the enzymatic activity of the polypeptides described herein). 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 glutamine; positively charged (basic) amino acids include arginine, lysine and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In the context of the present invention, a derivative polypeptide as used herein refers to a polypeptide that is capable of exhibiting in vitro and/or in vivo activity substantially similar to the original polypeptide, as judged by any of a number 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 as substitutions or additions. In the context of the present invention, a "derivative" of a reference polypeptide is preferably a polypeptide having an amino acid sequence with at least 80% sequence identity to the full-length sequence of the reference polypeptide.

In some embodiments, the invention contemplates the creation of functional variants by modifying the structure of the enzymes used in the invention. Variants may be produced by amino acid substitutions, deletions, additions, or combinations thereof. For example, it is reasonably expected that the isolation of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, asparagine with glutamine, lysine with arginine, cysteine with methionine or similar substitutions 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 those that occur within the family of related amino acids in their side chains. By assessing the response of a variant polypeptide in a cell in a similar manner to the wild-type polypeptide, it can be readily determined whether a change in the amino acid sequence of the polypeptide of the invention results in a functional homolog.

By "fragment" in the context of a polynucleotide is meant a clone or any portion of a polynucleotide molecule, particularly a portion of a polynucleotide that retains the functional characteristics available for a full length polynucleotide molecule. Useful fragments include oligonucleotides and polynucleotides, which can be used in hybridization or amplification techniques or in the regulation of replication, transcription or translation. By "polynucleotide fragment" is meant any subsequence of a polynucleotide SEQ ID NO (or Genbank No.), typically comprising or consisting of at least about 9, 10, 11, 12 consecutive nucleotides from said polynucleotide SEQ ID NO (or Genbank No.), for example at least about 30 nucleotides or at least about 50 nucleotides of any polynucleotide sequence provided herein. Exemplary fragments may additionally or alternatively include fragments comprising, consisting essentially of, or consisting of regions encoding conserved family domains of polypeptides. Exemplary fragments may additionally or alternatively include conserved domain-containing fragments of polypeptides. Thus, a fragment of a polynucleotide SEQ ID NO (or Genbank No.) preferably refers to a nucleotide sequence comprising or consisting of said 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 functional characteristics (e.g. activity) of the available full length polynucleotide molecule are retained, which can be assessed by the skilled person by routine experimentation. Alternatively, a fragment of a polynucleotide SEQ ID NO (or Genbank No.), preferably refers to a nucleotide sequence comprising or consisting of a quantity of consecutive nucleotides from said polynucleotide SEQ ID NO (or Genbank No.), wherein said 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 80.0%, more preferably at least 87.0%, even more preferably at least 90.0%, even more preferably at least 95.0%, most preferably at least 97.0% of the full length of said polynucleotide SEQ ID NO (or Genbank No.), and retains the functional characteristics (e.g. activity) of a useful full length polynucleotide molecule. Thus, a fragment of a polynucleotide SEQ ID NO (or Genbank No.), preferably refers to a nucleotide sequence comprising or consisting of said polynucleotide SEQ ID NO (or Genbank No.), wherein an amount of consecutive nucleotides is deleted, and wherein said 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 15.0%, even more preferably not more than 10.0%, most preferably not more than 2.5% of the full length of said polynucleotide SEQ ID NO (or Genbank No.), wherein said fragment retains the functional characteristics (e.g. activity) available for a full length polynucleotide molecule, which can be assessed by conventional techniques.

By "fragment" in reference to a polypeptide is meant a subsequence of the polypeptide that performs at least one biological function of the intact polypeptide in substantially the same manner or to a similar extent as the intact polypeptide. "subsequence of a 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 site, an activation domain, or a protein-protein interaction domain, and may initiate transcription. Fragments may vary in size 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 refers to a polypeptide sequence comprising or consisting of said 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 at least one biological function of the complete polypeptide, such as the complete polypeptide, is performed in substantially the same manner (preferably similar or greater), which can be routinely assessed by the skilled person. Alternatively, a fragment of a polypeptide SEQ ID NO (or UniProt ID or Genbank No.) preferably refers to a polypeptide sequence comprising or consisting of a quantity of consecutive amino acid residues from said polypeptide SEQ ID NO (or UniProt ID or Genbank No.), wherein said 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 80.0%, more preferably at least 87.0%, even more preferably at least 90.0%, even more preferably at least 95.0%, most preferably at least 97.0% of the full length of said polypeptide SEQ ID NO (or UniProt ID or Genbank No.), and which performs at least one biological function of the complete polypeptide in substantially the same way (preferably similar or greater) as the complete polypeptide, as can be routinely assessed by the skilled person. Thus, a fragment of a polypeptide SEQ ID NO (or Unit Prot ID or Genbank NO.) preferably refers to a polypeptide sequence comprising or consisting of (or Unit Prot ID or Genbank NO.) a certain amount of consecutive amino acid residues is absent and said 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 15.0%, even more preferably not more than 10.0%, even more preferably not more than 5.0%, most preferably not more than 2.5% of the full length of said polypeptide SEQ ID NO (or Unit Prot ID or Genbank NO.), and this may be assessed in a manner similar manner to the full length polypeptide (or by a conventional biological assay), e.g. by a human, as a human, or by evaluating the full-length polypeptide, e.g. by a human, or by a human, or by evaluating the full-length-like means. Throughout the application and claims, the terms "at least one biological function", "at least one property" and "at least one activity" preferably refer to an alpha-1, 2-fucosyltransferase activity as described herein.

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

A "functional fragment" of a polypeptide has at least one property or activity of the polypeptide from which it is derived, preferably to a similar or greater extent. For example, a functional fragment may comprise a functional domain or a conserved domain of a polypeptide. It is understood that a polypeptide or fragment thereof may have conservative amino acid substitutions that have substantially no effect on the activity of the polypeptide. Conservative substitutions are substitutions of one hydrophobic amino acid for another, or one polar amino acid for another, or one acidic amino acid for another, or one basic amino acid for another, etc. Preferably, conservative substitutions are combinations contemplated, such as glycine for alanine and vice versa; methionine replaces valine, isoleucine and leucine and vice versa; glutamic acid replaces aspartic acid, and vice versa; glutamine for asparagine and vice versa; threonine replaces serine and vice versa; arginine replaces lysine and vice versa; methionine replaces cysteine and vice versa; and tryptophan replaces phenylalanine and tyrosine, and vice versa.

Homologous sequences as used herein describe those nucleotide sequences that have sequence similarity and encode polypeptides having at least one functional characteristic (e.g., biochemical activity). More specifically, the term "functional homolog" as used herein describes those polypeptides (Altenhoff et al, PLoS comp. Biol.8 (2012) e 1002514) that have sequence similarity (in other words, homology) and at the same time at least one functional similarity (e.g., biochemical activity). In the context of the present invention, a functional homolog of a reference polypeptide is preferably a polypeptide having an amino acid sequence that has at least 80% sequence identity to the full-length sequence of the reference polypeptide.

Functional homologs are sometimes referred to as orthologs, where "ortholog" refers to a homologous gene or protein that is functionally equivalent to a reference gene or protein in another species. Orthologous sequences are homologous sequences in different species that originate from a vertical descent of a single sequence of the last common ancestor, where the sequence and its main function are conserved. Homologous sequences are sequences inherited in two species from a common ancestor. When used in reference to an amino acid or nucleotide/nucleic acid sequence from a given species, the term "ortholog" refers to the same amino acid or nucleotide/nucleic acid sequence from a different species. It will be appreciated that two sequences are orthologs of each other when they are derived from a common ancestral sequence by linear descent and/or are closely related in terms of their sequence and biological function. Orthologs often have a high degree of sequence identity, but may (and will not always) share 100% sequence identity. Paralogs are homologous sequences that result from sequence repeat events. Paralogous sequences generally belong to the same species, but this is not required. Paralogs can be divided into internal paralogs (pair of paralogs occurring after a speciation event) and external paralogs (pair of paralogs occurring before a speciation event). An inter-species paralog is a pair of paralogs that exist between two organisms due to repetition prior to speciation. Intraspecies and extraspecies paralogs are paralog pairs that exist in the same organism, but whose repeat events occur after speciation. Paralogs generally have the same or similar functions.

Functional homologs will generally produce similar, but not necessarily identical, features. Functionally homologous polypeptides produce the same characteristics, wherein the quantitative measurement produced by one ortholog is at least 10% of the other homolog; more generally, at least 20%, about 30% to about 40% of the results produced by the original molecule; for example, about 50% to about 60%; about 70% to about 80%; or about 90% and about 95%; about 98% to about 100%, or greater than 100%. Thus, in case the molecule has enzymatic activity, the functional homologue will have the above percentage of enzymatic activity compared to the original enzyme. Where the molecule is a DNA binding molecule (e.g., a polypeptide), the homolog will have the above-described percentage of binding affinity as measured by the weight of the bound molecule as compared to the original molecule.

The functional homolog and the reference polypeptide may be naturally occurring polypeptides, and the sequence similarity may be due to convergent or divergent evolutionary events.

Functional homologs can be identified by analyzing nucleotide and polypeptide sequence alignments. For example, querying a database of nucleotide or polypeptide sequences can identify homologs of a polypeptide of interest (e.g., a biomass-modulating polypeptide, a glycosyltransferase, a protein involved in nucleotide-activated sugar synthesis, or a membrane transporter). Sequence analysis may involve BLAST, reciprocal BLAST or PSI-BLAST analysis using the amino acid sequences of biomass-modulating polypeptides, glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis, or membrane transporters, respectively, as non-redundant databases of reference sequences. In some cases, the amino acid sequence is deduced from the nucleotide sequence. In general, those polypeptides having greater than 40% sequence identity in the database are candidates for further evaluation of suitability as biomass-modulating polypeptides, glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis, or membrane transporters, respectively. Amino acid sequence similarity allows conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or one polar residue for another or one acidic amino acid for another or one basic amino acid for another, and the like. Preferably, conservative substitutions refer to a desired combination, such as substitution of glycine for alanine, and vice versa; substitution of methionine for valine, isoleucine and leucine, and vice versa; substitution of glutamic acid for aspartic acid and vice versa; substitution of glutamine for asparagine and vice versa; replacement of serine with threonine, and vice versa; substitution of arginine for lysine and vice versa; substitution of methionine for cysteine and vice versa; and replacement of phenylalanine and tyrosine with tryptophan, and vice versa. If desired, such candidates may be manually inspected, reducing the number of candidates that need further evaluation. Manual examination may be performed by selecting those candidates that appear to have domains present in the productivity-modulating polypeptide (e.g., conserved functional domains).

The domain may be characterized, for example, by: pfam (El-Gebali et al, nucleic Acids Res.47 (2019) D427-D432), IPR (InterPro domain) (Mitchell et al, nucleic Acids Res.47 (2019) D351-D360), protein fingerprint domain (PRINTS) (Attwood et al, nucleic Acids Res.31 (2003) 400-402), SUBFAM domain (Gough et al, J.mol. Biol.313 (2001) 903-919), TIGRFAM domain (Selengut et al, nucleic Acids Res.35 (2007) D260-D264), conserved Domain Database (CDD) nomenclature (https:// www.ncbi.nlm.nih.gov/CDD) (Lu et al, nucleic Acids Res.48 (2020) D-268), HR domain (PT386/www.pantherdb.org) (Mi et al, nucleic Acids Res.41-377.41D-377); thomas et al Genome Research 13 (2003) 2129-2141) or PATRIC identifier or PATRIC DB general family domain (https:// www.patricbrc.org /) (Davis et al Nucleic Acids Res.48 (D1) (2020) D606-D612). It will be appreciated by those skilled in the art that for the databases used herein, including Pfam 32.0 (release at month 9 of 2018), CDD v3.17 (release at month 3 of 2019), egnogdb 4.5.1 (release at month 9 of 2016), interPro 75.0 (release at month 4 of 2019), TCDB (release at month 17 of 2019) and PATRIC 3.6.9 (release at month 17 of 2019), the contents of each database are fixed in each release and will not change. 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 and the specific content annotated at these specific release dates are available and known to those skilled in the art.

Protein or polypeptide sequence information and functional information may be provided by comprehensive resources of protein sequences and annotation data such as, for example, universal Protein Resource (UniProt) (www.uniprot.org) (Nucleic Acids res.2021,49 (D1), D480-D489). UniProt contains a specialized and well-planned protein database called UniProt knowledge base (UniProt Knowledgebase, uniProtKB), as well as UniProt reference clusters (UniProt Reference Clusters, uniRef) and UniProt files (UniProt Archive, uniParc). The UniProt identifier (UniProt ID) is unique for each protein present in the database. The UniProt ID as used herein is the UniProt ID in the 2021 month 5 day UniProt database version 2021_02. Proteins without UniProt ID are indicated herein using the corresponding GenBank accession number (Genbank NO.), which is found in the NIH genetic sequence database (https:// www.ncbi.nlm.nih.gov/GenBank /) (Nucleic Acids Res.2013,41 (D1), D36-D42) version 5 of year 5 of 2021.

In the case of two or more nucleic acid or polypeptide sequences, the term "identical" or "percent identity" or "% identity" means that two or more sequences or subsequences that are the same or have a specified percentage of the same amino acid residues or nucleotides, when aligned and aligned for maximum correspondence, are the same, as measured using a sequence alignment algorithm or by visual inspection. For alignment, one sequence serves as a reference sequence against which test sequences are aligned. When using the sequence alignment algorithm, the test sequence and reference sequence are entered into a computer, subsequence coordinates are designated (if necessary), and sequence algorithm program parameters are designated. The sequence alignment algorithm then 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 overall over the full length sequence of the reference sequence, resulting in an overall percent identity score. Alternatively, the percent identity may be calculated over a portion 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 overall identity score between the test sequence and the reference sequence.

The percent 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.

BLAST (search tool based on local alignment algorithm) alignment is an algorithm provided by the National Center for Biotechnology Information (NCBI) for comparing sequences using default parameters. The program compares the nucleotide or protein sequence to a sequence database and calculates statistical significance. PSI-BLAST (position-specific iterative basic local alignment algorithm search tool) uses protein-protein BLAST (BLASTP) to derive a position-specific scoring matrix (PSSM) or pattern from a multiple sequence alignment of sequences detected above a given scoring threshold. The BLAST method can be used for pairwise or multiplex sequence alignment. The pairwise sequence alignment is used to identify similar regions that may indicate functional, structural and/or evolutionary relationships between two biological sequences (proteins or nucleic acids). The web interface of BLAST is located: https:// blast.ncbi.nlm.nih.gov/blast.cgi.

Clustal Omega (Clustal W) is a multiple sequence alignment program that uses seed guide trees (seeded guide trees) and HMM profile-profile technology (HMM profile-profile technique) to generate alignments between three or more sequences. It produces biologically interesting multiple sequence alignments of different sequences. The Clustal W web interface is located at https:// www.ebi.ac.uk/tools/msa/clustalo/. The default parameters for the multiple sequence alignment and calculation of percent protein sequence identity using the Clustal W method are: enabling a de-alignment of the input sequence: FALSE; enabling mbed-like clustering guide-tree: TRUE; cluster iterations similar to mbed are enabled: TRUE; (combined guide tree/HMM) iteration number: default value (0); maximum number of guide tree iterations: default value [ -1]; maximum HMM iteration number: default value [ -1]; a command: and (5) comparison.

MatGAT (matrix Global alignment tool) is a computer application that can generate a similarity/identity matrix for DNA or protein sequences without pre-aligning the data. The program performs a series of pairwise alignments using Myers and Miller global alignment algorithms, calculates similarity and identity, and then places the results into a distance matrix. The user may specify which type of alignment matrix (e.g., BLOSUM50, BLOSUM62, and PAM 250) to use in the protein sequence examination.

EMBOSS Needle (https:// galaxy-iuc. Gitub. Io/EMBOSS-5.0-docs/Needle. Html) when considering the entire length of two sequences, a Needleman-Wunsch global alignment algorithm was used to find the best alignment (including gaps) of the two sequences. By exploring all possible routes and selecting the best route, the best route is ensured by a dynamic planning method. The Needleman-Wunsch algorithm is 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 the two sequences). Gap opening penalty (default value of 10.0) is the score subtracted when the gap is created. Default values assume you are using the EBLOSUM62 matrix for protein sequencing. Gap extension (default value of 0.5) penalties will be added to the standard gap penalties for each base or residue in the gap. This is how the long gaps are penalized.

As used herein, a polypeptide having an amino acid sequence (or polypeptide sequence) that has at least 80% sequence identity to the full length sequence of a reference polypeptide sequence is understood to have 80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、91.50％、92.00％、92.50％、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 identities to the full length of the amino acid sequence of the reference polypeptide sequence. Throughout this application, unless explicitly stated otherwise, polypeptides comprising, having, or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of a reference polypeptide (typically represented by SEQ ID NO, uniProt ID, or Genbank NO.), preferably having at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, or 99.0%, more preferably having at least 85.0%, even more preferably having at least 90.0%, most preferably having at least 95.0% sequence identity to the full-length reference sequence. In addition, unless explicitly stated otherwise, a polynucleotide sequence comprising/having/consisting of a nucleotide sequence having at least 80.0% sequence identity to the full length nucleotide sequence of a reference polynucleotide sequence (typically represented by SEQ ID NO or Genbank No.), preferably having at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, more preferably having at least 85.0%, even more preferably having at least 90.0%, most preferably having at least 95.0% sequence identity to the full length reference sequence.

For the purposes of the present invention, the percent identity was determined using MatGAT2.01 (Campanella et al 2003,BMC Bioinformatics 4:29). Proteins used the following default parameters: (1) vacancy cost present (Gap cost Existence): 12, and expansion: 2; the matrix used in (2) is BLOSUM65. In a preferred embodiment, sequence identity is calculated based on the full length sequence of a given SEQ ID NO (i.e., the reference sequence) or a portion thereof. Part of this preferably means at least 50%, 60%, 70%, 80%, 90% or 95% of the complete reference sequence.

The terms "mannose-6-phosphate isomerase", "phosphomannose isomerase", "mannose phosphate isomerase", "phosphohexose isomerase", "phosphomannose isomerise", "phosphomannose-isomerase", "phosphohexose mutase", "D-mannose-6-phosphate ketol isomerase" and "manA" are used interchangeably and refer to enzymes that catalyse the reversible conversion of D-fructose 6-phosphate to D-mannose 6-phosphate.

The terms "phosphomannose mutase," "mannose phosphomutase," "phosphomannose-mutase," "D-mannose 1, 6-phosphomutase," and "manB" are used interchangeably to refer to an enzyme that catalyzes the reversible conversion of D-mannose 6-phosphate to D-mannose 1-phosphate.

The terms "mannose-1-phosphate guanylate transferase", "GTP-mannose-1-phosphate guanylate transferase", "PIM-GMP (phosphomannose isomerase-guanosine 5 '-diphosphate-D-mannose pyrophosphorylase)", "GDP-mannose pyrophosphorylase", "guanosine 5' -diphosphate-D-mannose pyrophosphorylase", "guanosine diphosphate pyrophosphorylase", "guanosine triphosphate mannose 1-phosphate guanylate transferase", "mannose 1-phosphate guanylate transferase (guanosine triphosphate)", and "manC" are used interchangeably to refer to enzymes that convert D-mannose-1-phosphate sugars to GDP-mannose and diphosphate using GTP.

The terms "GDP-mannose 4, 6-dehydratase", "guanosine 5' -biphosphate-D-mannose oxidoreductase", "guanosine diphosphate mannose 4, 6-dehydratase", "GDP-D-mannose 4, 6-dehydratase", "GDP-mannose 4, 6-water lyase (GDP-4-dehydro-6-deoxy-D-mannose formation)" and "gmd" are used interchangeably and refer to enzymes forming the first step of GDP-rhamnose and GDP-fucose biosynthesis.

The terms "GDP-L-fucose synthase", "GDP-4-keto-6-deoxy-D-mannose-3, 5-epimerase-4-reductase", "GDP-L-fucose: NADP+4-oxidoreductase (3, 5-epimer) "and" fcl "are used interchangeably and refer to the enzymes forming the second step of GDP-fucose biosynthesis.

The terms "L-fucose kinase/GDP-fucose pyrophosphorylase", "L-fucose kinase/L-fucose-1-P guanylate transferase", "GDP-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", "glutamine- - -fructose-6-phosphate aminotransferase (isomerisation)", "hexose phosphate aminotransferase", "glucosamine-6-phosphate isomerase (formation of glutamine)", "glutamine-fructose-6-phosphate aminotransferase (isomerisation)", "D-fructose-6-phosphate amidotransferase", "glucosamine phosphate isomerase", "glucosamine 6-phosphate synthase", "GlcN6P synthase", "GFA" and "glmS" are used interchangeably and refer to enzymes that catalyse the conversion of D-fructose-6-phosphate to D-glucosamine-6-phosphate using L-glutamine.

The terms "glucosamine-6-P deaminase", "glucosamine-6-phosphate deaminase", "GlcN6P deaminase", "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" and "glmM" are used interchangeably and refer to an enzyme that catalyzes the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate. Phosphoglucomutase 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-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 the 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 the enzyme include UDP-N-acyl glucosamine 2-epimerase, UDP-GlcNAc-2-epimerase and UDP-N-acetyl-D-glucosamine 2-epimerase.

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

Bifunctional UDP-GlcNAc 2-epimerase/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 an acetyl group from acetyl-CoA to D-glucosamine-6-phosphate, thereby producing free CoA and N-acetyl-D-glucosamine 6-phosphate. Alternative names include aminodeoxyglucose phosphoacetyl transferase, D-glucosamine-6-P N-acetyl transferase, glucosamine 6-phosphoacetyl transferase, glucosamine 6-phosphate N-acetyl transferase, glucosamine 6-phosphoacetyl transferase, N-acetylglucosamine-6-phosphate synthase, phosphoglucamine acetylase, phosphoglucamine N-acetylase (phosphoglucosamine N-acetylase), phosphoglucamine acetyl transferase, GNA and GNA1.

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

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

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

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

The terms "N-acetylglucosamine 1-phosphate uridylyltransferase", "UDP-N-acetylglucosamine pyrophosphorylase", "uridine diphosphate acetylglucosamine pyrophosphorylase", "UTP: 2-acetamido-2-deoxy-alpha-D-glucose-1-phosphate uridylyltransferase", "UDP-GlcNAc pyrophosphorylase", "GlmU uridine acyl transferase", "acetylglucosamine 1-phosphate uridylyltransferase", "UDP-acetylglucosamine pyrophosphorylase", "uridine diphosphate-N-acetylglucosamine pyrophosphorylase" and "acetylglucosamine 1-phosphate uridylyltransferase" are used interchangeably and refer to enzymes that catalyze the conversion of N-acetylglucosamine 1-phosphate (GlcNAc-1-P) to UDP-N-acetylglucosamine (UDP-NAc) by the transfer of uridine 5-monophosphate (from uridine 5-triphosphate (UTP)).

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

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

The terms "NeunAc synthase", "N-acetylneuraminic acid synthase", "sialic acid synthase", "NeuAc synthase", "NeuB", "NeuB1", "NeunAc synthase", "NANA condensed enzyme", "N-acetylneuraminic acid lyase synthase", "N-acetylneuraminic acid condensed enzyme" are used interchangeably herein, and refer to an enzyme capable of synthesizing sialic acid from N-acetylmannosamine (ManNAc) using phosphoenolpyruvate (PEP) in a reaction.

The terms "N-acetylneuraminic acid lyase", "Neu5Ac lyase", "N-acetylneuraminic acid pyruvate-lyase", "N-acetylneuraminic acid aldolase", "NALase", "sialyltransferase", "sialylaldehyde aldolase", "sialyltransferase" and "nanA" are used interchangeably and refer to enzymes that degrade N-acetylneuraminic acid into N-acetylmannosamine (ManNAc) and pyruvate.

The terms "N-acyl neuraminic acid-9-phosphate synthase", "NANA synthase", "NANAS", "NANS", "NmeNANAS", "N-acetylneuraminic acid pyruvate-lyase (pyruvate phosphorylation)" are used interchangeably herein and refer to an enzyme capable of synthesizing N-acyl neuraminic acid-9-phosphate from N-acetylmannosamine-6-phosphate (ManNAc-6-phosphate) using phosphoenolpyruvate (PEP) in a 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.

The terms "CMP-sialic acid synthase", "N-acyl neuraminic acid cytidylyltransferase", "CMP-sialic acid synthase", "CMP-NeuAc synthase", "NeuA" and "CMP-N-acetylneuraminic acid synthase" are used interchangeably herein, and refer to an enzyme capable of synthesizing CMP-N-acetylneuraminic acid from N-acetylneuraminic acid using CTP in a reaction.

The terms "galactose-1-epimerase", "aldose 1-epimerase", "mutarotase", "aldose mutarotase", "galactose 1-epimerase" and "D-galactose 1-epimerase" are used interchangeably and refer to enzymes that catalyze the conversion of β -D-galactose to α -D-galactose.

The terms "galactokinase", "galactokinase (phosphorylating)" and "ATP: D-galactose-1-phosphotransferase" are used interchangeably to refer to enzymes that catalyze the conversion of alpha-D-galactose to alpha-D-galactose 1-phosphate using ATP.

The terms glucokinase and "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", "Gal-1-P uridylyltransferase", "UDP-glucose-hexose-1-phosphate uridylyltransferase", "hexose-1-phosphate uridylyltransferase", "uridylyltransferase"; "hexose 1-phosphate uridyltransferase", "UDP-glucose: α -D-galactose-1-phosphate uridyltransferase "," 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-epimerase", "UDP-galactose 4-epimerase", "uridine diphosphate glucose epimerase", "galactose Walder converting enzyme (galactowaldenase)", "UDPG-4-epimerase", "uridine diphosphate galactose 4-epimerase", "UDP-glucose epimerase", "4-epimerase", "uridine diphosphate glucose 4-epimerase", "uridine diphosphate 4-epimerase" and "UDP-D-galactose 4-epimerase" are used interchangeably and refer to enzymes that catalyze the conversion of UDP-D-glucose into UDP-galactose.

The terms "glucose-1-phosphate uridylyltransferase", "UTP-glucose-1-phosphate uridylyltransferase", "UDP-glucose pyrophosphorylase", "UDPG phosphorylase", "UDPG pyrophosphorylase", "uridine 5' -diphosphate glucose pyrophosphorylase", "uridine diphosphate-D-glucose pyrophosphorylase", "uridine diphosphate glucose pyrophosphorylase" and "galU" are used interchangeably and refer to enzymes that catalyze the conversion of D-glucose-1-phosphate to UDP-glucose using UTP.

The terms "phosphoglucomutase (α -D-glucose-1, 6-biphosphoric acid dependency)", "phosphoglucomutase (ambiguous)" and "phosphoglucomutase (ambiguous)" are used interchangeably, which refer to enzymes catalyzing the conversion of D-glucose 1-phosphate to D-glucose 6-phosphate.

The terms "UDP-N-acetylglucosamine 4-epimerase", "UDP-acetylglucosamine epimerase", "uridine diphosphate N-acetylglucosamine 4-epimerase", "uridine 5' -diphosphate N-acetylglucosamine 4-epimerase" and "UDP-N-acetyl-D-glucosamine 4-epimerase" are used interchangeably and refer to enzymes that catalyze the epimerization of UDP-N-acetylglucosamine (UDP-GlcNAc) into UDP-N-acetylgalactosamine (UDP-GalNAc).

The terms "N-acetylgalactosamine kinase", "GALK", "GK2", "GalNAc kinase", "N-acetylgalactosamine (GalNAc) -1-phosphokinase" and "ATP: N-acetyl-D-galactosamine 1-phosphotransferase" are used interchangeably and refer to enzymes that catalyze the synthesis of N-acetylgalactosamine 1-phosphate (GalNAc-1-P) from N-acetylgalactosamine (GalNAc) using ATP.

The terms "UDP-N-acetylgalactosamine pyrophosphorylase" and "UDP-GalNAc pyrophosphorylase" are used interchangeably to 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", "ManNAc 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 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, thereby forming a glycosidic bond. Glycosyltransferases have been described as classified into different sequence-based families using nucleotide diphosphate-sugar, nucleotide monophosphate-sugar and sugar phosphate 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, the glycosyltransferase may be selected from the list including, but not limited to: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosyltransferase, N-acetylmannosyltransferase, xylosyltransferase, glucuronyltransferase, galacturonate transferase, glucosaminyltransferase, N-glycolylneuraminidase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-arabinoxylan-Zhuo Tangan aminotransferase, UDP-N-acetylglucosaminenolpyruvyltransferase and fucosyltransferase.

Fucosyltransferases are glycosyltransferases that transfer a fucose residue (Fuc) from a GDP-fucose (GDP-Fuc) donor to an 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 the Fuc residue from GDP-Fuc to a receptor via an alpha-glycosidic bond. Fucosyltransferases may be present in, but are not limited to, the GT10, GT11, GT23, GT65, GT68 and FT74 CAZy families.

The terms "alpha-1, 2-fucosyltransferase", "alpha 1, 2-fucosyltransferase", "2-FT" or "2FT" as used herein are used interchangeably and refer to glycosyltransferases that catalyze the transfer of fucose from donor GDP-L-fucose to an acceptor molecule in alpha-1, 2-linkages.

The expression "a-1, 2-fucosyltransferase having a galactoside α -1, 2-fucosyltransferase activity on galactose residues of Gal-b1,3-GlcNAc (LNB, lactose-N-disaccharide)" refers to an α -1, 2-fucosyltransferase which catalyzes the transfer of fucose from donor GDP-L-fucose to galactose residues of Gal-b1,3-GlcNAc in α -1, 2-linkage to produce Fuc-a1,2-Gal-b1,3-GlcNAc (2 '-fucosyl lactose-N-disaccharide, 2' FLNB).

In the present invention, a polypeptide sequence segment is used to refer to a fragment of an alpha-1, 2-fucosyltransferase as used herein, which is common to those alpha-1, 2-fucosyltransferases. Such polypeptide fragments are written in the form of an amino acid sequence of one letter code. If the amino acid at a particular position in such a polypeptide fragment can be several amino acids, that particular position will have the amino acid code X. The letter "X" refers to any possible amino acid unless otherwise indicated herein. The term "X (not M)" refers to any possible amino acid other than methionine (Met, M). The term "X (not F)" refers to any possible amino acid other than phenylalanine (Phe, F). The term "X (not N)" refers to any possible amino acid other than asparagine (Asn, N). The term [ ILMV ] in the sequence refers to isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M) or valine (Val, V) as possible amino acids for that particular position. The term "X (not E, S)" refers to any possible amino acid other than glutamic acid (Glu, E) and serine (Ser, S). The term "X (not E)" refers to any possible amino acid other than glutamic acid (Glu, E). The term "X (not F, S)" refers to any possible amino acid other than phenylalanine (Phe, F) and serine (Ser, S). The term "X (not Y)" refers to any possible amino acid other than tyrosine (Tyr, Y). The term "X (not H, S, Y)" refers to any possible amino acid except histidine (His, H), serine (Ser, S) and tyrosine (Tyr, Y). The term [ DE ] in the sequence refers to aspartic acid (Asp, D) or glutamic acid (Glu, E) as possible amino acids in this particular position. The term [ FWY ] in the sequence refers to phenylalanine (Phe, F), tryptophan (W) or tyrosine (Y) as possible amino acids in this particular position. The term "X (not D, E)" refers to any possible amino acid other than aspartic acid (Asp, D) and glutamic acid (Glu, E). The term "(Xn)" with n from 10 to 40 refers to a polypeptide fragment of 10 to 40 amino acid residues X, where X refers to any possible amino acid.

Sialyltransferases are glycosyltransferases that transfer sialic acid (e.g., neu5Ac or Neu5 Gc) from a donor (e.g., CMP-Neu5Ac or CMP-Neu5 Gc) to an acceptor. Sialyltransferases include alpha-2, 3-sialyltransferases, alpha-2, 6-sialyltransferases, and alpha-2, 8-sialyltransferases, which catalyze the transfer of sialic acid to a receptor via an alpha-glycosidic bond. Sialyltransferases may be present in, but are not limited to, the GT29, GT42, GT80 and GT97 CAZy families. Galactosyltransferases are glycosyltransferases that transfer a galactosyl group (Gal) from a UDP-galactose (UDP-Gal) donor to an acceptor. Galactosyltransferases include beta-1, 3-galactosyltransferases, N-acetylglucosamine beta-1, 3-galactosyltransferases, beta-1, 4-galactosyltransferases, N-acetylglucosamine beta-1, 4-galactosyltransferases, alpha-1, 3-galactosyltransferases, and alpha-1, 4-galactosyltransferases, which transfer Gal residues from UDP-Gal to a receptor via an alpha-or beta-glycosidic linkage. Galactosyltransferases may be present in, but are not limited to, the GT2, GT6, GT8, GT25 and GT92 CAZy families. Glucosyltransferases are glycosyltransferases that transfer a glucosyl group (Glc) from a UDP-glucose (UDP-Glc) donor to an 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 the receptor via alpha-or beta-glycosidic linkages. Glucosyltransferases may be present in, but are not limited to, the GT1, GT4 and GT25 CAZy families. Mannosyltransferases are glycosyltransferases that transfer mannose groups (Man) from a GDP-mannose (GDP-Man) donor to an acceptor. Mannosyltransferases include alpha-1, 2-mannosyltransferases, alpha-1, 3-mannosyltransferases and alpha-1, 6-mannosyltransferases, which transfer the Man residues from GDP-Man to the receptor via an alpha-glycosidic bond. Mannosyltransferases may be present in, but are not limited to, the GT22, GT39, GT62 and GT69 CAZy families. N-acetylglucosamine transferase is a glycosyltransferase that transfers an N-acetylglucosamine group (GlcNAc) from a UDP-N-acetylglucosamine (UDP-GlcNAc) donor to an acceptor. N-acetylglucosaminyl transferases may be present in, but are not limited to, the GT2 and GT4 CAZy families. The galactoside β -1, 3-N-acetylglucosaminyl transferase is part of an N-acetylglucosaminyl transferase and transfers GlcNAc from a UDP-GlcNAc donor to terminal galactose units present in the acceptor via β -1, 3-linkages. Beta-1, 6-N-acetylglucosaminyl transferases are N-acetylglucosaminyl transferases that transfer GlcNAc from UDP-GlcNAc donors to acceptors via beta-1, 6-linkages. N-acetylgalactosamine transferase is a glycosyltransferase transferring N-acetylgalactosamine groups (GalNAc) from UDP-N-acetylgalactosamine (UDP-GalNAc) donor to acceptor. N-acetylgalactosamine transferase may be present in the GT7, GT12 and GT27 CAZy families, but is not limited thereto. The α -1, 3-N-acetylgalactosamine transferase is part of an N-acetylgalactosamine transferase and transfers GalNAc from a UDP-GalNAc donor to an acceptor via an α -1, 3-linkage. N-acetylmannosamine transferase is a glycosyltransferase that transfers an N-acetylmannosamine group (ManNAc) from a UDP-N-acetylmannosamine (UDP-ManNAc) donor to an acceptor. Xylosyltransferases are glycosyltransferases that transfer xylose residues (Xyl) from a UDP-xylose (UDP-Xyl) donor to an acceptor. The xylosyltransferases may be present in, but are not limited to, the GT14, GT61 and GT77 CAZy families. Glucuronyl transferase is a glycosyltransferase that transfers glucuronic acid from a UDP-glucuronate donor to an acceptor through an alpha-or beta-glycosidic linkage. Glucuronyltransferases may be present in, but are not limited to, the GT4, GT43 and GT93 CAZy families. Galacturonic acid transferase is a glycosyltransferase that transfers galacturonic acid from a UDP-galacturonic acid donor to an acceptor. N-glycolyl neuraminic acid transferase is a glycosyltransferase that transfers an N-glycolyl neuraminic acid group (Neu 5 Gc) from a CMP-Neu5Gc donor to an acceptor. Rhamnosyl transferase is a glycosyltransferase that transfers a rhamnose residue from a GDP-rhamnose donor to an acceptor. Rhamnosyltransferases may be present in, but are not limited to, the GT1, GT2 and GT102 CAZy families. N-acetylrhamnosyl transferase is a glycosyltransferase that transfers an N-acetylrhamnosylamine residue from a UDP-N-acetyl-L-rhamnosylamine donor to an acceptor. UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-Zhuo Tangan aminotransferase is a glycosyltransferase that uses UDP-2-acetamido-2, 6-dideoxy-L-arabino-4-hexulose, which is a sialic acid-like sugar for modifying flagellin, in the biosynthesis of pseudo-amino acids (pseudaminic acid). UDP-N-acetylglucosamine enolpyruvyl transferase (murA) is a glycosyltransferase that transfers an enolpyruvyl group from phosphoenolpyruvic acid (PEP) to UDP-N-acetylglucosamine (UDPAG) to form UDP-N-acetylglucosamine enolpyruvic acid. Fucosyl amine transferases are glycosyltransferases that transfer an N-acetylfucosyl amine residue from a dTDP-N-acetylfucosyl amine or UDP-N-acetylfucosyl amine donor to an acceptor.

The terms "activated monosaccharide", "nucleotide-activating sugar", "nucleotide-sugar", "activated sugar", "nucleoside" or "nucleotide donor" are used interchangeably herein and refer to an activated form of a monosaccharide. Examples of activated monosaccharides include UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2, 6-dideoxy-L-arabino-4-hexulose, UDP-2-acetamido-2, 6-dideoxy-L-lyxol-4-hexulose, UDP-N-acetyl-L-rhamnose amine (UDP-L-RhaNAc or UDP-2-acetamido-L-mannose), dTDP-N-acetylfucose amine, UDP-N-acetylfucose amine (UDP-L-FucNAc or UDP-2-acetamido-2, 6-dideoxy-L-acetylgalactosamine), UDP-N-acetylfucose amine (UDP-L-FucNAc or UDP-2-acetamido-6-diacetyl-L-5-hexuloe), UDP-4-hexuloe, UDP-N-acetamido-N-acetylgalactosamine (UDP-L-RhaNAc or UDP-2-acetamido-N-acetylmanno-6-acetylglucosamine), UDP-N-acetyl-L-quiniosamine (UDP-L-QuiNAc or UDP-2-acetamido-2, 6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac)、CMP-Neu4Ac、CMP-Neu5Ac9N3、CMP-Neu4,5Ac2、CMP-Neu5,7Ac2、CMP-Neu5,9Ac2、CMP-Neu5,7(8,9)Ac2、CMP-N- hydroxyacetyl neuraminic acid (CMP-Neu 5 Gc), GDP-rhamnose or UDP-xylose. Nucleotide-sugars act as glycosyl donors in glycosylation reactions. These reactions are catalyzed by glycosyltransferases.

The term "monosaccharide" as used herein means a sugar that is not broken down by hydrolysis into simpler sugars, which are classified as aldoses or ketoses, and which contain one or more hydroxyl groups per molecule. Monosaccharides are sugars that contain only one simple sugar. Examples of monosaccharides include hexose, D-glucopyranose, D-furanose, D-galactopyranose, L-galactopyranose, D-mannopyranose, D-allose, L-altrose, D-gulose, L-iodopyranose, D-talose, D-ribose furanose, D-ribose, D-arabinose, L-arabinose, D-xylose, D-lyxose, D-erythrose, D-threose, D-erythrose, D-arabinose heptose, L-glycero-D-mannopyranose (LDmanHep), D-glycero-D-mannopyranose (DDmanHep), 6-deoxy-L-altrose, 6-deoxy-D-glucopyranose, 6-deoxy-D-galactopyranose, 6-deoxy-L-galactopyranose, 6-deoxy-D-mannopyranose, 6-deoxy-L-mannopyranose, 6-deoxy-D-glucopyranose, 2-deoxy-D-arabino-hexose, 2-deoxy-D-erythro-pentose, 2, 6-dideoxy-D-arabino-glucopyranose, 3, 6-dideoxy-D-arabinopyranose, 3, 6-dideoxy-L-arabinopyranose, 3, 6-dideoxy-D-xylopyranose, 3, 6-dideoxy-D-ribopyranose, 2, 6-dideoxy-D-ribopyranose, 3, 6-dideoxy-L-xylopyranose, 2-amino-2-deoxy-D-glucopyranose, 2-amino-2-deoxy-D-galactopyranose, 2-amino-2-deoxy-D-glucopyranose, and 2-amino-2-deoxy-D-pyranoid allose, 2-amino-2-deoxy-L-pyranoid allose, 2-amino-2-deoxy-D-pyranoid gulose, 2-amino-2-deoxy-L-iodopyranose, 2-amino-2-deoxy-D-pyranoid talose, 2-acetamido-2-deoxy-D-pyranoid glucose, 2-acetamido-2-deoxy-D-pyranoid galactose, 2-acetamido-2-deoxy-D-pyranoid mannose, 2-acetamido-2-deoxy-D-pyranoid allose, and, 2-acetamido-2-deoxy-L-pyranoid altrose, 2-acetamido-2-deoxy-D-pyranoid gulose, 2-acetamido-2-deoxy-L-iodopyranose, 2-acetamido-2-deoxy-D-pyranoid talose, 2-acetamido-2, 6-dideoxy-D-pyranoid galactose, 2-acetamido-2, 6-dideoxy-L-pyranoid mannose, 2-acetamido-2, 6-dideoxy-D-pyranoid glucose 2-acetamido-2, 6-dideoxy-L-pyran type altrose, 2-acetamido-2, 6-dideoxy-D-pyran type talose, D-glucopyranose aldehyde acid, D-galactopyranose aldehyde acid, D-mannopyranose aldehyde acid, D-allopyranose aldehyde acid, L-altranose aldehyde acid, D-gulranose aldehyde acid, L-idopyranose aldehyde acid, D-talose aldehyde acid, sialic acid, 5-amino-3, 5-dideoxy-D-glycero-D-galacto-non-2-You Luosuo nicotinic acid, 5-acetamido-3, 5-dideoxy-D-galacto-2-You Luosuo-nicotinic acid, 5-glycolyl-amido-3, 5-dideoxy-D-glycero-D-galacto-2-You Luosuo-nicotinic acid, erythritol, arabitol, xylitol, ribitol, glucitol, galactitol, mannitol, D-core-hexo-2-one pyranose, D-arabino-hexo-2-one furanose (D-furanose), D-arabino-hexo-2-one pyranose, L-xylo-hexo-2-one pyranose, D-lyxo-2-one pyranose, D-threo-pent-2-one pyranose, D-arabino-Zhuo Shi-hept-2-one pyranose, 3-C- (hydroxymethyl) -D-furanose, 2,4, 6-trideoxy-2, 4-diamino-D-deoxy-glucopyranose, 6-core-hexo-2-one furanose (D-furanose), D-arabino-hexo-2-one pyranose, L-xylo-hexo-2-one pyranose, D-lyxo-2-one pyranose, D-xylo-hept-2-one pyranose, D-arabino-erythro-2-furanose, 2, 4-trideoxy-D-deoxy-2, 4-diamino-2-D-2-ribofuranose, 6-oxo-2-oxo-glucose, R-2-methyl-2-oxo-2-one glucose 2-acetamido-3-O- [ (R) -carboxyethyl ] -2-deoxy-D-pyrano-glucose, 2-glycolyl-amido-3-O- [ (R) -1-carboxyethyl ] -2-deoxy-D-pyrano-glucose, 3-deoxy-D-lyxohept-2-one pyranose acid (ulopyranosaric acid), 3-deoxy-D-mann-oct-2-one pyranose acid, 3-deoxy-D-glycero-D-galacto-non-2-You Luo pyrano-type Song-acid, 5, 7-diamino-3, 5,7, 9-tetradeoxy-L-glycero-L-manno-non-2-You Luo pyrano-type Song-acid 5, 7-diamino-3, 5,7, 9-tetradeoxy-L-glycero-L-Zhuo Shi-non-2-You Luo pyranoid type of Song Nic acid, 5, 7-diamino-3, 5,7, 9-tetradeoxy-D-glycero-D-galacto-non-2-You Luo pyranoid type of Song Nic acid, 5, 7-diamino-3, 5,7, 9-tetradeoxy-D-glycero-D-Tacrow-non-2-You Luo pyranoid type of Song Nic acid, 2-acetamido-2, 6-dideoxy-L-arabino-4-hexulose, 2-acetamido-2, 6-dideoxy-L-lyxol-4-hexulose, N-acetyl-L-rhamnose amine, N-acetyl-D-fucose amine, N-acetyl-L-neotame amine, N-acetyl muramic acid, N-acetyl-L-quinitol amine, glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc), glucosamine (Glcn), mannose (Man), xylose (Xyl), N-acetylmannosamine (ManNAc), N-glycolyl neuraminic acid, N-acetylgalactosamine (GalNAc), galactosamine (Galn), fucose (Fuc), rhamnose (Rha), glucuronic acid, gluconic acid, fructose (Fru) and polyols.

The term polyol refers to an alcohol containing multiple hydroxyl groups. For example glycerol, sorbitol or mannitol.

The terms "sialic acid", "N-acetylneuraminic acid" are used interchangeably to refer to acidic sugars having a nine carbon backbone, including but not limited to ：Neu4Ac;Neu5Ac;Neu4,5Ac2;Neu5,7Ac2;Neu5,8Ac2;Neu5,9Ac2;Neu4,5,9Ac3;Neu5,7,9Ac3;Neu5,8,9Ac3;Neu4,5,7,9Ac4;Neu5,7,8,9Ac4,Neu4,5,7,8,9Ac5 and Neu5Gc.

Neu4Ac is also known as 4-O-acetyl-5-amino-3, 5-dideoxy-D-glycero-D-galacto-non-2-You Luo pyran type Song Nic acid or 4-O-acetylneuraminic acid and has the molecular formula C11H19NO9.Neu5Ac is also known as 5-acetamido-3, 5-dideoxy-D-glycero-D-galacto-non-2-You Luo-pyran type of sorbic acid, D-glycero-5-acetamido-3, 5-dideoxy-D-galacto-non-2-You Luo-pyran type of sorbic acid, 5- (acetamido) -3, 5-dideoxy-D-glycero-D-galacto-2-non You Luo-pyran type of sorbic acid, 5- (acetamido) -3, 5-dideoxy-D-glycero-D-galacto-2-non You Luosuo-nicotinic acid, 5- (acetamido) -3, 5-dideoxy-D-glycero-D-galacto-non-2-You Luosuo-nicotinic acid, or 5- (acetamido) -3, 5-dideoxy-D-glycero-D-galacto-non-2-You Luo pyran type of sorbic acid, of formula C11H19NO9.Neu4,5Ac2 is also known as N-acetyl-4-O-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid ester, 4-acetic acid 5-acetamido-3, 5-dideoxy-D-glycero-D-galacto-nonene You Luosuo-nicacid ester, 4-acetic acid 5- (acetamido) -3, 5-dideoxy-D-glycero-D-galacto-2-nonene You Luosuo-nicacid ester, 4-acetic acid 5-acetamido-3, 5-dideoxy-D-glycero-D-galacto-nonene You Luosuo-nicacid, or 4-acetic acid 5- (acetamido) -3, 5-dideoxy-D-glycero-D-galacto-2-nonene You Luosuo-nicacid, having the molecular formula C13H21NO10.Neu5,7Ac2 is also known as 7-O-acetyl-N-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, 7-O-acetyl-N-acetylneuraminic acid ester, 7-acetic acid 5-acetamido-3, 5-dideoxy-D-glycero-D-galacto-nonene You Luosuo-nicacid ester, 7-acetic acid 5- (acetamido) -3, 5-dideoxy-D-glycero-D-galacto-2-nonene You Luosuo-nicacid ester, 7-acetic acid 5-acetamido-3, 5-dideoxy-D-glycero-D-galacto-nonene You Luosuo-nicacid or 7-acetic acid 5- (acetamido) -3, 5-dideoxy-D-glycero-D-galacto-2-nonene You Luosuo-nicacid, and has the molecular formula C13H21NO10.Neu5,8Ac2 is also known as 5-n-acetyl-8-o-acetylneuraminic acid and has the molecular formula C13H21NO10.Neu5,9Ac2 is also known as N-acetyl-9-O-acetylneuraminic acid, 9-anana, 9-O-acetylsialic acid, 9-O-acetyl-N-acetylneuraminic acid, 5-N-acetyl-9-O-acetylneuraminic acid, N, 9-O-diacetylneuraminic acid or N, 9-O-diacetylneuraminic acid, of formula C13H21NO10.Neu4,5,9Ac3 is also known as 5-N-acetyl-4, 9-di-O-acetylneuraminic acid. Neu5,7,9Ac3 is also known as 5-N-acetyl-7, 9-di-O-acetylneuraminic acid. Neu5,8,9Ac3 is also known as 5-N-acetyl-8, 9-di-O-acetylneuraminic acid. Neu4,5,7,9ac4 is also known as 5-N-acetyl-4, 7, 9-tri-O-acetylneuraminic acid. Neu5,7,8,9Ac4 is also known as 5-N-acetyl-7, 8, 9-tri-O-acetylneuraminic acid. Neu4,5,7,8,9ac5 is also known as 5-N-acetyl-4, 7,8, 9-tetra-O-acetylneuraminic acid. Neu5Gc is also known as N-glycolyl-neuraminic acid, N-glycolyl neuraminic acid ester, N-glycolyl-neuraminic acid, N-glycolyl neuraminic acid, 3, 5-dideoxy-5- ((glycolyl) amino) -D-glycero-D-galacto-2-none You Luosuo-nicotinic acid, 3, 5-dideoxy-5- (glycolylamino) -D-glycero-D-galacto-2-nones You Luo pyran type of sorbic acid, 3, 5-dideoxy-5- (acetylamino) -D-glycero-D-galacto-non-2-You Luo pyran type of sorbic acid, 3, 5-dideoxy-5- [ (glycolyl) amino ] -D-glycero-D-galacto-nono-2-You Luo pyran type of sorbic acid, D-glycero-5-glycolylamino-3, 5-dideoxy-D-galacto-2-nono-You Luo-pyran type of C acid, of formula C11H.

The term "disaccharide" as used herein means a sugar polymer containing two simple sugars (i.e. monosaccharides). Such disaccharides contain monosaccharides preferably selected from the list of monosaccharides used herein. Examples of disaccharides include lactose (Gal-b 1, 4-Glc), lactose-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 pyranoid fucosyl- (1-4) -N-hydroxyacetylneuraminic acid (Fuc- (1-4) -Neu5 Gc).

As the term is used herein and as is commonly understood in the art, "oligosaccharides" means sugar polymers containing small amounts, typically 3 to 20 simple sugars (i.e., monosaccharides). Preferably, the oligosaccharides described herein contain monosaccharides selected from the list used herein. The oligosaccharides used in the present invention may be of a linear structure or may include a branched chain. The bond between two sugar units (e.g., glycosidic bond, galactosidic bond, glucosidic bond, etc.) may be represented as, for example, 1,4, 1- >4, or (1-4), which are used interchangeably herein. For example, the terms "Gal-b1,4-Glc", "b-Gal- (1- > 4) -Glc", "Galβ1-4-Glc" and "Gal-b (1-4) -Glc" have the same meaning, i.e. the β -glycosidic bond connects carbon-1 of galactose (Gal) with carbon-4 of glucose (Glc). Each monosaccharide may be in a cyclic form (e.g., pyranose or furanose form). The linkage 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-and beta-glycosidic linkages or may contain only alpha-glycosidic linkages or only beta-glycosidic linkages. The term "polysaccharide" means a compound consisting of a large number of typically more than 20 glycosidically linked monosaccharides.

Examples of oligosaccharides include, but are not limited to, lewis-type antigenic oligosaccharides, mammalian (including human) milk oligosaccharides, O-antigens, E.coli co-antigens (ECA), glycan chains present in Lipopolysaccharide (LPS), oligosaccharide repeat units present in capsular polysaccharides, peptidoglycans (PG), amino sugars and antigens of the human ABO blood group system.

"Mammalian milk oligosaccharides" as used herein means oligosaccharides such as, but not limited to, 3-fucosyllactose, 2' -fucosyllactose, 6-fucosyllactose, 2', 3-difucosyllactose, 2', 2-Difucosyllactose, 3, 4-Difucosyllactose, 6' -sialyllactose, 3, 6-polysialiyllactose, 6' -disialyllactose, 8, 3-disialyllactose, 3, 6-disialyllactose-N-tetraose, lacto-di-fuctetraose, lacto-N-tetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI sialyllactose-N-tetraose c, sialyllactose-N-tetraose b, sialyllactose-N-tetraose a, lacto-N-difucose hexaose I, lacto-N-difucose hexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, monofucosyl Shan Tuoye-acid lacto-N-tetraose c, monofucosyl para-lacto-N-hexaose, monofucosyl lacto-N-hexaose III, isomerised fucosylated lacto-N-hexaose I, sialyllactose-N-hexasaccharide, sialyllactose-N-neohexasaccharide II, difucosyl-p-lacto-N-hexasaccharide, difucosyl lacto-N-hexasaccharide a, difucosyl lacto-N-hexasaccharide c, galactosylated chitosan, fucosylated oligosaccharide, neutral oligosaccharide and/or sialylated oligosaccharide.

As used herein and as generally understood in the art, a "fucosylated oligosaccharide" is an oligosaccharide bearing a fucose residue. Examples include 2 '-fucosyllactose (2' FL), 3-fucosyllactose (3 FL), 4-fucosyllactose (4 FL), 6-fucosyllactose (6 FL), dif-fucosyllactose (diFL), lacto-di-fucose (LDFT), lacto-N-fucose I (LNF I), lacto-N-fucose II (LNF II), lacto-N-fucose III (LNF III), lacto-N-fucose V (LNF V), lacto-N-fucose VI (LNF VI), lacto-N-neofucose I, lacto-N-dif-fucose I (LDFH I), lacto-N-dif-fucohexaose II (ldii), mono-fucose-N-hexasaccharide III (MFLNH III), dif-fucose-N-hexasaccharide (DFLNHa), dif-fucose-N-neohexasaccharide.

"Sialylated oligosaccharide" as used herein is understood to mean an oligosaccharide containing charged sialic acid, i.e. an oligosaccharide having sialic acid residues. It has acidic properties. Some examples are 3-SL (3 '-sialyllactose or 3' SL or Neu5Ac-a2,3-Gal-b1, 4-Glc), 3 '-sialyllactoamine, 6-SL (6' -sialyllactose or 6'SL or Neu5Ac-a2,6-Gal-b1, 4-Glc), 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), 6 '-sialyllactosamine, oligosaccharides comprising 6' -sialyllactose, SGG hexoses (neu5ac alpha-2, 3gal beta-1, 3galnac beta-1, 3gal alpha-1, 4gal beta-1, 4gal), sialylated tetrasaccharides (neu5ac alpha-2, 3gal beta-1, 4glcnac beta-14 GlcNAc), pentose LSTD (neu5ac alpha-2, 3gal beta-1, 4glcnac beta-1, 3gal beta-1, 4glc), sialylated lactose N-trisaccharides, sialylated lactose N-tetrasaccharides, sialyllacto-N-neotetrasaccharides, monosialyllacto-N-hexasaccharides, disialyllacto-N-hexasaccharides I, monosialyllacto-N-neohexasaccharides II, disialyllactose-N-neohexose, disialyllactose-N-tetrasaccharide, disialyllactose-N-hexasaccharide II, sialyllactose-N-tetrasaccharide a, disialyllactose-N-hexasaccharide I, sialyllactose-N-tetrasaccharide b, 3' -sialyllactose-3-fucosyllactose, disialomo-fucosyllactose-N-neohexasaccharide, monosialyllactose-Shan Tuoye-yl lactose-N-octasaccharide (sialyl Lea), sialyllactose-N-hexasaccharide II, disialyllactose-N-fucopyranose II, monosialyllactose-N-tetrasaccharide 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, neu5acα -2,3galβ -4 Glc), and oligosaccharides 、GD3Neu5Acα-2,8Neu5Acα-2,3Galβ-1,4GlcGT3(Neu5Acα-2,8Neu5Acα-2,8Neu5Acα-2,3Galβ-1,4Glc);GM2GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc、GM1Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc、GD1aNeu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc、GT1aNeu5Acα-2,8Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc、GD2 GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc、GT2GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc、GD1b、Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc、GT1bNeu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc、GQ1bNeu5Acα-2,8Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc、GT1cGalβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc、GQ1cNeu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc、GP1cNeu5Acα-2,8Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc、GD1aNeu5Acα-2,3Galβ-1,3(Neu5Acα-2,6)GalNAcβ-1,4Galβ-1,4Glc、 fucosyl-GM 1 fucα -1,2galβ -1,3galnacβ -1,4 (neu5acα -2, 3) galβ -1,4Glc comprising the GM3 motif; they can be extended to the production of the corresponding gangliosides by reacting the oligosaccharide moiety with or synthesizing the oligosaccharide on a ceramide.

As used herein and as generally understood in the art, a "neutral oligosaccharide" is an oligosaccharide that does 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), lacto-N-trisaccharide II, lacto-N-tetrasaccharide, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucosohose I, lacto-N-difucosohose II, 6' -galacto-lactose, 3' -galactolactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-neohexaose, difucosyl-lacto-N-hexaose and difucose-N-neohexaose.

Mammalian milk oligosaccharides or MMOs comprise oligosaccharides present in milk seen at any stage during lactation, including colostrum from humans (human milk oligosaccharides or MMOs) and mammals including, but not limited to: cattle (Bos Taurus), sheep (Ovis aries), goats (Capra aegagrus hircus)), bactrian camels (Camelus bactrianus), horses (European wild horses (Equus ferus caballus)), pigs (Sus scropha), dogs (Canis lupus familiaris)), patina brown bear (ezo brown bears) (Japanese brown bear (Ursus arctos yesoensis)), polar bear (fur seal (Ursus maritimus)), japanese black bear (Ursus thibetanus japonicus), striped skulls (MEPHITIS MEPHITIS), crown seal (Cystophora cristata), asian elephant (Elephas maximus), african elephant (Loxodonta africana), giant formica (Myrmecophaga tridactyla), bottle nasal dolphins (Tursiops truncates), northern whales (Balaenoptera acutorostrata)), eugene kangaroos (Macropus eugenii), red kangaroos (Macropus rufus), brush tail negative rats (Trichosurus Vulpecula)), kola (Phascolarctos cinereus), eastern ferrets (Dasyurus viverrinus), duckbill (Ornithorhynchus anatinus). Human milk oligosaccharides, also known as human breast milk oligosaccharides, have the same chemical composition as human milk oligosaccharides found in human milk, but are produced by biotechnology (e.g., using cell-free systems or cells and organisms comprising bacterial, fungal, yeast, plant, animal or protozoan cells, preferably genetically engineered cells and organisms). Human breast milk oligosaccharides are sold under the name HiMO.

The term "Lewis-type antigen" as used herein comprises the following oligosaccharides: an H1 antigen which is Fucα1-2Galβ1-3GlcNAc or abbreviated as 2' FLNB; lewis, which is the trisaccharide Galβ1-3[ Fucα1-4] GlcNAc or abbreviated as 4-FLNB; lewis b, which is the tetrasaccharide Fucα1-2Galβ1-3[ Fucα1-4] GlcNAc or abbreviated DiF-LNB; sialyl Lewis, which is 5-acetylneuraminic acid- (2-3) -galactosyl- (1-3) - (pyranyl fucosyl- (1-4)) -N-acetylglucosamine or abbreviated as Neu5 Ac. Alpha.2-3 Gal. Beta.1-3 [ Fucα1-4] GlcNAc; an H2 antigen which is fucα1-2galβ1-4GlcNAc or otherwise known as 2 'fucosyl-N-acetyl-lactosamine, abbreviated as 2' flicnac; lewis x, which is the trisaccharide Galβ1-4[ Fucα1-3] GlcNAc, or alternatively referred to as 3-fucosyl-N-acetyl-lactosamine, abbreviated as 3-FLacNAc; lewis, which is the tetrasaccharide Fucα1-2Galβ1-4[ Fucα1-3] GlcNAc; and sialyl Lewis x, which is 5-acetylneuraminic acid- (2-3) -galactosyl- (1-4) - (pyranyl fucosyl- (1-3)) -N-acetylglucosamine or Neu5 Ac. Alpha.2-3 Gal. Beta.1-4 [ Fuc. Alpha.1-3 ] GlcNAc.

The term "O-antigen" as used herein refers to the recurring glycan component of the surface Lipopolysaccharide (LPS) of gram-negative bacteria. The term "lipopolysaccharide" or "LPS" refers to glycolipids found in the outer membrane of gram-negative bacteria, which consist of lipid A, core oligosaccharides and O-antigen. The term "enterobacteriaceae common antigen" or "ECA" refers to a specific carbohydrate antigen constructed from repeating units of three amino sugars, i.e., N-acetylglucosamine, N-acetyl-d-mannosamine, and 4-acetamido-4, 6-dideoxy-d-galactose, which is shared by all members of the enterobacteriaceae family and is located in the outer leaflet and periplasm of the outer membrane. The term "capsular polysaccharide" means a long chain polysaccharide with oligosaccharide repeating structures that is present in the bacterial capsule, which is a polysaccharide layer located outside the cell envelope. The term "peptidoglycan" or "murein" refers to an essential structural element in the cell wall of most bacteria, consisting of a sugar and an amino acid, wherein the sugar component consists of alternating residues of β -1,4 linked GlcNAc and N-acetyl murein. The term "amino-sugar" as used herein means a sugar molecule in which a hydroxyl group has been replaced by an amine group. Antigens of the human ABO blood group system as used herein are oligosaccharides. 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-, the B determinants Gal- α1,3 (Fuc- α1, 2) -Gal-and the H determinants Fuc- α1,2-Gal-, present on a disaccharide core structure comprising Gal- β1,3-GlcNAc, gal- β1,4-GlcNAc, gal- β1,3-GalNAc and Gal- β1,4-Glc.

The terms "LNT II", "LNT-II", "LN3", "lactose-N-trisaccharide II", "lactose-N-trisaccharide" or "GlcNAcβ1-3Galβ1-4Glc" as used in the present invention are used interchangeably.

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

The terms "LNnT", "lactose-N-neotetraose", "neolnt" or "galβ1-4glcnacβ1-3galβ1-4Glc" as used in the present invention are used interchangeably.

The terms "LSTa", "LS-tetrasaccharide a", "sialyl-lactose-N-tetrasaccharide a", "sialyl lactose-N-tetrasaccharide a" or "Neu5Ac-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", "sialyl-lactose-N-tetrasaccharide b", "sialyl lactose-N-tetrasaccharide b" or "Gal-b1,3- (Neu 5Ac-a2, 6) -GlcNAc-b1,3-Gal-b1,4-Glc" as used in the present invention are used interchangeably.

The terms "LSTc", "LS-tetrasaccharide c", "sialyl-lactose-N-tetrasaccharide c", "sialyl lactose-N-neotetrasaccharide c" or "Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc" as used in the present invention are used interchangeably.

The terms "LSTd", "LS-tetrasaccharide d", "sialyl-lactose-N-tetrasaccharide d", "sialyl lactose-N-neotetrasaccharide d" or "Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc" as used in the present invention are used interchangeably.

The terms "DSLNnT" and "disialo-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 "disialo-N-tetrasaccharide" 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", "lactose-N-fucopentaose I", "LNFP I", "LNF IOH type I determinant", "LNF I", "LNF1" and "blood group H antigen pentasaccharide 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" are used interchangeably and refer to GalNAc-a1,3- (Fuc-a 1, 2) -Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc.

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

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

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

The terms "LNnFP I" and "lactose-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", "lactose-N-difucose I", "LNDFH-I", "LDFH I", "Le ^b -lactose" and "Lewis-b hexose" 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", "lactose-N-disaccharide hexasaccharide 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", "lactose-N-neo-difucose" and "Lewis x hexose" are used interchangeably to refer to Gal-b1,4- (Fuc-a 1, 3) -GlcNAc-b1,3-Gal-b1,4- (Fuc-a 1, 3) -Glc.

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

The terms "Fuc-a1,2-Gal-b1,3-GlcNAc", "2-fucosyllacto-N-disaccharide", "2FLNB", "2-FLNB" and "2' FLNB" are used interchangeably and refer to trisaccharides in which fucose residues are linked to galactose residues of lactose-N-disaccharide (LNB, gal-b1, 3-GlcNAc) by an a-1, 2 bond.

The term "glycopeptide" as used herein refers to a peptide containing one or more sugar groups, which are monosaccharides, disaccharides, oligosaccharides, polysaccharides and/or glycans, covalently attached to the side chains of the amino acid residues of the peptide. Glycopeptides include natural glycopeptide antibiotics, such as glycosylated non-ribosomal peptides produced by a variety of soil actinomycetes that target gram-positive bacteria by binding to the acyl-D-alanyl-D-alanine (D-Ala-D-Ala) termini of peptidoglycans grown on the outer surface of the cytoplasmic membrane, as well as synthetic glycopeptide antibiotics. The common core of natural glycopeptides consists of a cyclic peptide consisting of 7 amino acids, to which 2 saccharides are bound. Examples of glycopeptides include vancomycin, teicoplanin, oritavancin, chloromycins, telavancin, and dalbavancin.

The terms "glycoprotein" and "glycopolypeptide" are used interchangeably to refer to polypeptides containing one or more sugar groups, which are monosaccharides, disaccharides, oligosaccharides, polysaccharides and/or glycans, covalently attached to the side chains of amino acid residues of the polypeptide.

As used herein, the term "glycolipid" refers to any glycolipid generally known in the art. Glycolipids (GL) can be subdivided into Simple Glycolipids (SGL) and Complex Glycolipids (CGL). Simple glycolipids, sometimes referred to as glycolipids, are two-component (glycosyl and lipid moieties) glycolipids in which the glycosyl and lipid moieties are directly linked to each other. Examples of SGLs include glycosylated fatty acids, fatty alcohols, carotenoids, agastache-alkanes, sterols or Zhong Kangsuan. Bacterial produced SGLs can be divided into rhamnolipids, glycolipids, trehalose lipids, other glycosylated (trehalose-free) mycoates, trehalose-containing oligosaccharide lipids, glycosylated fatty alcohols, glycosylated macrolides and macrolactams, glycosylated macrodiols (glycosylated macrolides), saccharide carotenes and saccharide terpenes, and glycosylated agastaches/sterols. However, complex Glycolipids (CGLs) are more structurally heterogeneous in that they contain other residues in addition to the glycosyl and lipid moieties, such as glycerol (glycoglycerolipid), peptides (glycopeptidic lipid), acylated sphingosines (glycosphingolipid) or other residues (lipopolysaccharide, phenolic glycolipid, nucleoside lipid).

The term "membrane transporter" as used herein refers to a protein that is part of or interacts with a cell membrane and controls the flow of molecules and information through the cell. Thus, membrane proteins are involved in transport, whether into or out of cells.

Such membrane transporters may be transporters, P-P bond hydrolysis-driven transporters, β -barrel porins, auxiliary transporters, putative transporters, and phosphotransporter-driven group translocations, which are defined by the transporter class database operated and managed by the Saier Lab bioinformatics group, available through www.tcdb.org, and provide functional and phylogenetic classification of membrane transporters. The transporter class database details the class system of IUBMB approved synthetic membrane transporters, known as the Transporter Class (TC) system. The TCDB category search described herein is defined based on tcdb.org published on month 17 of 2019.

The transporter is a generic name for a one-way transporter, a symporter and an antiporter that utilizes a vector-mediated process (Saier et al, nucleic Acids Res.44 (2016) D372-D379). They belong to the class of electrochemical potential driven transporters and are also referred to as second carrier-type promoters. Membrane transporters are included in this classification in the following cases: when they utilize a carrier-mediated process to catalyze unidirectional transport, where the single species is transported by promoted diffusion or in a membrane potential dependent process (if the solute is charged); when they utilize a carrier-mediated process to catalyze counter-transport, where two or more species are transported in opposite directions during the close coupling process, are not coupled to direct forms of energy other than chemical osmotic energy; and/or when they utilize a carrier-mediated process to catalyze co-transport, where two or more species are transported together in the same direction during close coupling, without coupling to direct forms of energy other than the chemical osmotic energy of the secondary carrier (Forrest et al, biochem. Biophys. Acta 1807 (2011) 167-188). These systems are generally stereospecific. Solute: solute transport in reverse is a characteristic feature of secondary carriers. Dynamic association of transporter and enzyme produces functional membrane transport metabolites (metabolons) that direct substrates normally obtained from extracellular compartments into their cellular metabolism (morae and REITHMEIER, biochem. Biophys. Acta 1818 (2012), 2687-2706). Solutes transported by the transport system include, but are not limited to, cations, organic anions, inorganic anions, nucleosides, amino acids, polyols, phosphorylated glycolytic intermediates, osmotic agents, iron conjugates.

If the membrane transporter hydrolyzes the diphosphate bond of inorganic pyrophosphate, ATP or other nucleoside triphosphates to drive the active uptake and/or extrusion of one or more solutes, the transporter is included in the class of P-P bond hydrolytically driven transporters (Saier et al, nucleic Acids Res.44 (2016) D372-D379). The membrane transporter may or may not be transiently phosphorylated, but the substrate is not. Substrates transported by P-P bond hydrolysis driven transporter classes include, but are not limited to, cations, heavy metals, beta-glucans, UDP-glucose, lipopolysaccharides, teichoic acids.

Beta-barrel porin membrane transporters form transmembrane pores, generally allowing solutes to pass through the membrane independent of energy. The transmembrane portion of these proteins consists solely of the beta-strands forming the beta-barrel (Saier et al, nucleic Acids Res.44 (2016) D372-D379). These porin proteins are present in the outer membrane of gram-negative bacteria, mitochondria, plastids and possibly acid-resistant gram-positive bacteria. Solutes transported by these 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. These membrane transporters always function with one or more established transport systems, such as, but not limited to, outer Membrane Factor (OMF), polysaccharide (PST) transporter, ATP-binding cassette (ABC) type transporter. They may provide functions related to energy coupled transport, structural in complex formation, biological or stability functions or functions in regulation (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, and 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 established, or will be eliminated from the transporter classification system if proposed transport functions prove incorrect. These families include one or more members that have proposed a transport function, but evidence of this function is not convincing (Saier et al, nucleic Acids Res.44 (2016) D372-D379). Examples of putative transporters categorized as this group under the TCDB system published 6.17.2019 include, but are not limited to, copper transporters.

Acid transport driven group translocators are also known as bacterial phosphoenolpyruvate: PEP-dependent phosphotransferase driven group transporter of the sugar phosphotransferase system (PTS). The reaction product from extracellular sugar is cytoplasmic sugar phosphate. The enzyme components that catalyze the phosphorylation of sugars are superimposed on the transport process in a tightly coupled process. PTS systems are involved in many different aspects including regulation and chemotaxis, biofilm formation and pathogenesis the membrane transporter family classified as acid transport driven group translocators under TCDB system published at month 6, 17 of (Lengeler,J.Mol.Microbiol.Biotechnol.25(2015)79-93;Saier,J.Mol.Microbiol.Biotechnol.25(2015)73-78).2019 includes PTS systems associated with glucose-glucoside, fructose-mannitol, lactose-N, N' -diacetyldisaccharide β -glucoside, glucitol, galactitol, mannose-fructose-sorbose and ascorbate transport.

The Major Facilitator Superfamily (MFS) is the superfamily of membrane transporters that catalyze unidirectional transport, solutes: cations (h+, but little na+) co-transport and/or solutes: h+ or solute: solute transport in reverse. Most are 400-600 aminoacyl residues in length and have 12, 14 or occasionally 24 transmembrane α -helices (transmembrane α -HELICAL SPANNERS, TMS) according to the definition of the transport protein classification database operated by Saier laboratory bioinformatics group (www.tcdb.org).

As used herein, "SET" or "sugar efflux transporter" refers to a membrane protein of the SET family, which is a protein having the InterPRO domain IPR004750 and/or a protein belonging to the egnogv 4.5 family ENOG410XTE 9. Default values may be used to identify the InterPro domain using https:// www.ebi.ac.uk/InterPro/online tools or independent versions of InterPro scan (https:// www.ebi.ac.uk/InterPro/download. Html). The direct homology family in eggNOGv4.5 can be identified using either an online version or an independent version eggNOG-mapperv1 (http:// eggnogdb. Embl. De/#/app/home).

The term "iron conjugate" as used herein refers to the secondary metabolites of various microorganisms that are primarily iron ion specific chelators. These molecules are classified into catecholates, hydroxamates, carboxylates and mixtures. Iron conjugates are typically synthesized by the non-ribosomal peptide synthase (NRPS) dependent pathway or the NRPS independent pathway (NIS). The most important precursor in the NRPS-dependent iron conjugate 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-dihydroxybenzoic acid-2, 3-dehydrogenase. The iron conjugate may also be formed from salicylic acid, which is formed from isochorismate by an isochorismate pyruvate lyase. When ornithine is used as a precursor for iron conjugates, biosynthesis depends on the hydroxylation of ornithine catalyzed by L-ornithine N5 monooxygenase. In the NIS pathway, an important step in iron conjugate biosynthesis is N (6) -hydroxylysine synthase.

Transport proteins are required to export the iron conjugate outside the cell. To date, four superfamilies of membrane proteins have been identified in this process: major Facilitator Superfamily (MFS); a multidrug/oligosaccharyl lipid/polysaccharide invertase superfamily (MOP); resistance, nodulation and cell division superfamily (RND); and the ABC superfamily. Typically, genes involved in the export of iron conjugates are clustered together with the iron conjugate biosynthesis genes. The term "iron conjugate exporter" as used herein refers to a transporter required to transport an iron conjugate out of a cell.

The ATP-binding cassette (ABC) superfamily contains both uptake and efflux transport systems, which are typically loosely aggregated together. ATP hydrolysis without protein phosphorylation provides energy for transport. There are tens of families in the ABC superfamily, which are generally associated with substrate specificity. Members were classified according to class 3.A.1 defined by the transporter classification database operated by Saier laboratory bioinformatics group, which is available through www.tcdb.org and provides a functional and phylogenetic classification of membrane transporters.

It will be appreciated by those skilled in the art that for the databases used herein, including egnogdb 4.5.1 (release 9 in 2016) and InterPro 75.0 (release 4 in 2019), the contents of each database are fixed in each version and will not change. When the contents of a particular database change, the particular database receives a new release version with a release date. All release versions of each database and their corresponding release dates and the specific content annotated at these specific release dates are available and known to those skilled in the art.

As used herein, a "fucosylation pathway" is a biochemical pathway comprising at least one enzyme and its respective gene selected from the list comprising: mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanyl transferase, GDP-mannose 4, 6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-1-phosphate guanyl transferase, in combination with fucosyl transferase, to produce alpha 1,2; α1,3; alpha 1,4 and/or alpha 1,6 fucosylated oligosaccharides.

A "sialylation pathway" is a biochemical pathway comprising at least one enzyme selected from the list comprising: n-acyl glucosamine 2-epimerase, UDP-N-acetyl glucosamine 2-epimerase, N-acetyl mannosamine 6-phosphate 2-epimerase, UDP-GlcNAc 2-epimerase/kinase hydrolysis, N-acyl neuraminic acid-9-phosphate synthase, phosphatase, N-acetyl neuraminic acid synthase, N-acyl neuraminic acid cytidylyltransferase and sialic acid transporter, in combination with sialyltransferase, to produce α2,3; α2,6 and/or α2,8 sialylated oligosaccharides.

As used herein, a "galactosylation pathway" is a biochemical pathway comprising at least one enzyme and its respective gene selected from the list comprising: galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, in combination with galactosyltransferase, produce a galactosylated compound comprising a mono-, di-or oligosaccharide having alpha or beta-bound galactose on any one or more of the hydroxyl groups in the 2, 3, 4 and 6 positions of said mono-, di-or oligosaccharide.

As used herein, the "N-acetylglucosaminylation pathway" is a biochemical pathway comprising at least one enzyme and its respective gene selected from the list comprising: L-glutamine-D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, in combination with a glucosyltransferase, produces a GlcNAc-modified compound comprising a monosaccharide, disaccharide or oligosaccharide having an alpha or beta bound N-acetylglucosamine (GlcNAc) at any one or more of the hydroxyl groups at positions 3,4 and 6 of the monosaccharide, disaccharide or oligosaccharide.

As used herein, the "N-acetylgalactosamine amination pathway" is a biochemical pathway comprising at least one enzyme and its respective gene selected from the list comprising: L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucomutase, 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, in combination with glycosyltransferase, produce GalNAc modified compounds comprising mono-, di-or oligosaccharides having alpha or beta bound N-acetylgalactosamine on said mono-, di-or oligosaccharides.

As used herein, a "mannosylation pathway" is a biochemical pathway comprising at least one enzyme and its respective gene selected from the list comprising: mannose-6-phosphate isomerase, phosphomannose mutase, and/or mannose-1-phosphate guanylate transferase, in combination with a glycosyltransferase, produce a mannosylated compound comprising a monosaccharide, disaccharide, or oligosaccharide having an alpha or beta-bound mannose on the monosaccharide, disaccharide, or oligosaccharide.

As used herein, the "N-acetylmannosylation pathway" is a biochemical pathway comprising at least one enzyme and its respective gene selected from the list comprising: L-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate N-acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, UDP-GlcNAc 2-epimerase and/or ManNAc kinase, in combination with a glycosyltransferase, produces a ManNAc-modified compound comprising a monosaccharide, disaccharide or oligosaccharide having alpha or beta bound N-acetylmannosamine on the monosaccharide, disaccharide or oligosaccharide.

The term "efflux" refers to an activity that directs transport of solutes across the cytoplasmic membrane and/or cell wall. The transport may be achieved by introducing and/or increasing the expression of the membrane transporter described in the present invention. The term "enhanced efflux" refers to an improvement in the trafficking activity of solutes across the cytoplasmic membrane and/or cell wall. By introducing and/or increasing the expression of the membrane transporter proteins described in the present invention, transport of solutes across the cytoplasmic membrane and/or cell wall can be enhanced. "expression" of a membrane transporter is defined as "over-expression" (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) of the gene encoding the membrane transporter.

The terms "acetyl-CoA synthase", "acs", "acetyl-CoA synthase", "AcCoA synthase", "acetate-CoA ligase", "acyl-activating enzyme" and "yfaC" are used interchangeably and refer to an enzyme that catalyzes the conversion of acetate to acetyl-CoA (AcCoA) in an ATP-dependent reaction.

The terms "pyruvate dehydrogenase", "pyruvate oxidase", "POX", "poxB" and "pyruvate: ubiquinone-8 oxidoreductase" are used interchangeably and refer to enzymes that catalyze the oxidative decarboxylation of pyruvate to produce acetic acid and CO 2.

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

The term "Cell Production Index (CPI)" as used herein means 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. For cells, sugars, nucleic acids, polypeptides, peptides, glycoproteins, glycopeptides, lipids, and glycolipids, the term "purified" refers to a substance that is substantially or essentially free of components found in the natural state of the substance that are normally associated with the substance. Typically, the purified sugar, oligosaccharide, peptide, glycopeptide, protein, glycoprotein, lipid, glycolipid 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 on silver stained gel or other method of determining purity. Purity or homogeneity may be indicated by a variety of methods well known in the art, such as polyacrylamide gel electrophoresis of protein or nucleic acid samples, followed by visualization after staining. For some purposes, high resolution will be required and HPLC or similar purification methods will be used. For di-and 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 "culture" refers to the medium in which the cells are cultured or fermented, the cells themselves, and the Fuc-a1,2-Gal-b1,3-GlcNAc- (Rn) produced by the cells throughout the culture broth, i.e., both inside (intracellular) and outside (extracellular) the cells.

The term "precursor" as used herein refers to a substance that is taken up and/or synthesized by a cell for the specific production of Fuc-a1,2-Gal-b1,3-GlcNAc- (Rn) as described herein. In this sense, a precursor may be a receptor as defined herein, but may also be another substance, metabolite, which is first modified intracellularly as part of the biochemical synthetic pathway of Fuc-a1,2-Gal-b1,3-GlcNAc (Rn). Examples of such precursors include the receptors and/or glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, dihydroxyacetone, glucosamine, N-acetylglucosamine, mannosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, phosphorylated sugars, such as, but not limited to, glucose-1-phosphate, galactose-1-phosphate, glucose-6-phosphate, fructose-1, 6-biphosphoric acid, mannose-6-phosphate, mannose-1-phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylmannosamine-1-phosphate, N-acetylneuraminic acid-9-phosphate and/or nucleotide activated sugars as defined herein (e.g., UDP-glucose, UDP-galactose, UDP-N-acetylneuraminic acid, GDP-D-glucose, GDP-D-glucose-alpha-glucose.

Optionally, the cell is transformed to comprise and express at least one nucleic acid sequence encoding a protein selected from the group consisting of lactose transporter, fucose transporter, transporter of a nucleotide-activated sugar, wherein internalization of the transporter is used to produce Fuc-a1,2-Gal-b1,3-GlcNAc- (Rn) of the invention by a precursor added in a culture medium.

The term "receptor" as used herein refers to a monosaccharide, disaccharide or oligosaccharide, protein, glycoprotein, peptide, glycopeptide, lipid or glycolipid that can be modified by a glycosyltransferase. Examples of such receptors include: glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose-N-disaccharide (LNB), lactose-N-trisaccharide, lactose-N-tetrasaccharide (LNT), lactose-N-neotetraose (LNnT), N-acetyllactosamine (LacNAc), lactose-N-pentose (LNP), lactose-N-neopentose, para-lactose-N-pentose, para-lactose-N-neopentose, lactose-N-neopentose I, lactose-N-hexaose (LNH)), lactose-N-neohexaose (LNnH), para-lactose-N-neohexaose (pLNnH), for lactose-N-hexose (pLNH), lactose-N-heptasaccharide, lactose-N-neoheptasaccharide, lactose-N-heptasaccharide, lactose-N-octasaccharide (LNO), lactose-N-neooctasaccharide, isolactose-N-octasaccharide, lactose-N-octasaccharide, isolactose-N-neooctasaccharide, neolactose-N-neooctasaccharide, lactose-N-neooctasaccharide, isolactose-N-nonose, neolactose-N-nonose, lactose-N-decose, isolactose-N-decose, neolactose-N-decose, lactose-N-neodecose, galactosyllactose and oligosaccharides containing 1 or more N-acetyllactosamine units and/or 1 or more lactose-N-disaccharide units or intermediates in the synthesis of: oligosaccharides, fucosylated and sialylated forms thereof, peptides, polypeptides, lipids, sphingolipids, cerebrosides, ceramide lipids, phosphatidylinositol lipids in glycosylated forms of peptides, polypeptides, lipids, sphingolipids, cerebrosides, ceramide lipids, phosphatidylinositol lipids.

Detailed Description

According to a first aspect, the present invention provides a method of producing a compound comprising a structure of formula I, II or III:

Wherein:

r ¹ is a monosaccharide, disaccharide, oligosaccharide, protein, glycoprotein, peptide, glycopeptide, lipid or glycolipid; and, when present, R ² is a monosaccharide, disaccharide, or oligosaccharide. The method comprises the following steps:

i. As described herein, there is provided a cell, preferably a single cell, expressing an alpha-1, 2-fucosyltransferase having galactosidase alpha-1, 2-fucosyltransferase activity on galactose residues of Gal-b1,3-GlcNAc (LNB, lactose-N-disaccharide), and

Culturing and/or incubating the cells under conditions allowing expression of the alpha-1, 2-fucosyltransferase and production of the compound comprising the structure of formula I, II or III,

Preferably, the compound comprising the structure of formula I, II or III is isolated from the culture.

In a second aspect, the invention provides a genetically engineered cell for use in the production of a compound comprising a structure of formula I, II or III as described herein. In the context of the present invention, the compound comprising the structure of formula I, II or III as described herein is preferably not present in the wild-type progenitor cells of the cells.

A genetically engineered cell, preferably a single cell, is provided that expresses an alpha-1, 2-fucosyltransferase having galactosyl alpha-1, 2-fucosyltransferase activity on galactose residues of Gal-b1,3-GlcNAc (LNB, lactose-N-disaccharide).

According to the invention, the methods for producing the compounds comprising the structures of formulae I, II or III may utilize non-genetically engineered cells, or may utilize genetically engineered cells as disclosed herein.

In the context of the present invention, it will be appreciated that the compound comprising the structure of formula I, II or III is preferably produced intracellularly. Those of skill in the art will further appreciate that a portion or substantially all of the resulting compound comprising the structure of formula I, II or III remains intracellular and/or is excreted outside the cell passively or by active transport.

Throughout this application, unless explicitly stated otherwise, "genetically modified cell" or "genetically engineered cell" preferably refers to a genetically modified cell or a genetically engineered cell, respectively, used to produce the compounds of the invention comprising the structure of formulae I, II or III.

In the context of the present invention, the term "compound comprising a structure of formula I, II or III" refers to the trisaccharides Fuc-a1,2-Gal-b1,3-GlcNAc as well as to the following compounds: wherein the trisaccharide Fuc-a1,2-Gal-b1,3-GlcNAc has an N-acetylglucosamine (GlcNAc) residue: (a) To one R group, wherein the R is a monosaccharide, disaccharide, oligosaccharide, protein, glycoprotein, peptide, glycopeptide, lipid, or glycolipid, or (b) to two R groups, wherein one of the two R groups is a monosaccharide, disaccharide, oligosaccharide, protein, glycoprotein, peptide, glycopeptide, lipid, or glycolipid, and the other of the two R groups is a monosaccharide, disaccharide, or oligosaccharide. Preferably, each of the two R groups is a monosaccharide, disaccharide or oligosaccharide.

In a preferred embodiment of the method and/or cell of the invention, the compound comprising the structure of formula I, II or III is an oligosaccharide, preferably the oligosaccharide is a Mammalian Milk Oligosaccharide (MMO) as defined herein, more preferably the oligosaccharide is a Human Milk Oligosaccharide (HMO).

In another preferred embodiment of the method and/or cell of the invention, the compound comprising the structure of formula I, II or III is a charged oligosaccharide. In a more preferred embodiment of the method and/or cell of the invention, the compound comprising the structure of formula I, II or III is a sialylated oligosaccharide. In an alternative preferred embodiment of the method and/or cell of the invention, the compound comprising the structure of formula I, II or III is a neutral oligosaccharide.

In another preferred embodiment of the method and/or cell of the invention, the compound comprising the structure of formula I, II or III is the trisaccharide Fuc-a1,2-Gal-b1,3-GlcNAc, also known as 2 '-fucosyllacto-N-disaccharide or 2' flnb.

In another preferred embodiment of the method and/or cell of the invention, the compound comprising the structure of formula I, II or III is a compound comprising one R group selected from the group consisting of a monosaccharide, disaccharide, oligosaccharide, protein, glycoprotein, peptide, glycopeptide, lipid or glycolipid, wherein the GlcNAc residue of Fuc-a1,2-Gal-b1,3-GlcNAc is linked to the R group via an α -or β -glycosidic bond.

In another preferred embodiment of the method and/or cell of the invention, the compound comprising the structure of formula I, II or III is a compound comprising two R groups, wherein one of the two R groups is a monosaccharide, disaccharide, oligosaccharide, protein, glycoprotein, peptide, glycopeptide, lipid or glycolipid and the other of the two R groups is a monosaccharide, disaccharide or oligosaccharide, wherein the GlcNAc residue of Fuc-a1,2-Gal-b1,3-GlcNAc is bound to each of the R groups via a glycosidic bond. Here, the GlcNAc residue may be bound to a first R group via an α -glycosidic bond and to a second R group via a β -glycosidic bond. Alternatively, the GlcNAc residue may be bound to two R groups via a β -glycosidic bond.

In another preferred embodiment of the method and/or cell of the invention, the compound comprising the structure of formula I, II or III is an oligosaccharide comprising two R groups, wherein each of the R groups is a monosaccharide, disaccharide or oligosaccharide, and wherein the GlcNAc residue of Fuc-a1,2-Gal-b1,3-GlcNAc is bound to each of the R groups via a glycosidic bond. Here, the GlcNAc residue may be bound to a first R group via an α -glycosidic bond and to a second R group via a β -glycosidic bond. Alternatively, the GlcNAc residue may be bound to two R groups via a β -glycosidic bond.

In another preferred embodiment of the method and/or cell of the invention, the compound comprising the structure of formula I, II or III is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R comprising one R group, wherein the N-acetylglucosamine (GlcNAc) residue of the trisaccharide Fuc-a1,2-Gal-b1,3-GlcNAc is bound to said R group via a β -1,3 glycosidic bond, wherein said R group is selected from the list comprising monosaccharides, disaccharides or oligosaccharides. In a more preferred embodiment, the compound comprising the structure of formula I, II or III is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R comprising one R group selected from the list comprising monosaccharides, disaccharides or oligosaccharides. In an even more preferred embodiment, the compound comprising the structure of formula I, II or III is lactose-N-fucose I (LNFP-I, fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc), wherein the trisaccharide Fuc-a1,2-Gal-b1,3-GlcNAc is linked to the galactose residue (Gal-b 1, 4-Glc) of lactose through a GlcNAc residue in its β -1,3 linkage.

In another preferred embodiment of the method and/or cell of the invention, the compound comprising the structure of formula I, II or III is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,6-R comprising one R group, wherein the GlcNAc residue of the trisaccharide Fuc-a1,2-Gal-b1,3-GlcNAc is bound to said R group via a β -1,6 glycosidic bond, wherein said R group is selected from the list comprising mono-, di-or oligosaccharides.

In another preferred embodiment of the method and/or cell of the present invention, the compound comprising the structure of formula I, II or III is LNDFH I (Fuc-a 1,2-Gal-b1,3- [ Fuc-a1,4] -GlcNAc-b1,3-Gal-b1, 4-Glc).

In another preferred embodiment of the method and/or cell of the invention, the compound comprising the structure of formula I, II or III is an oligosaccharide selected from the list:

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal；Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,6-[Gal-b1,4-GlcNAc-b1,3]-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,3-GalNAc；Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[NeuAc-a2,6]-GalNAc；

NeuAc-a2,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,6-[Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3]-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[Fuca1,2]-Gal-b1,3-[Fuc-a1,2-Gal-b1,4-GlcNAc-b1,6]-GalNAc；

Fuc-a1,3-[Gal-b1,4]-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,6-[Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3]-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[NeuAc-a2,3-Gal-b1,4-GlcNAc-b1,6]-Gal-b1,3-GalNAc；

Fuca1,2-Gal-b1,4-[Fuca1,3]-GlcNAc-b1,6-[Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3]-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[Fuca1,2-Gal-b1,4-GlcNAc-b1,6]-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,3-[Fuc-a1,2-[GalNAc-a1,3]-Gal-b1,4-GlcNAc-b1,6]-GalNAc；

Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,6-[Fuc-a1,2-Gal1,3-GlcNAc-b1,3]-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[Fuc-a1,3-[Gal-b1,4]-GlcNAc-b1,6]-Gal-b1,4-Glc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[Fuc-a1,2-Gal-b1,4-GlcNAc-b1,6]-Gal-b1,4-GlcNAc-b1,3-[NeuAc-a2,6]-Gal-b1,3-[Fuc-a1,2-Gal-b1,4-GlcNAc-b1,6]-GalNAc;

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,3-[Gal-b1,4-GlcNAc-b1,6]-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[Gal-b1,4-[HSO3(-6)]GlcNAc-b1,6]-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3]-GalNAc；

Fuc-a1,4-[HSO3(-3)Gal-b1,3]-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,6-[Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3]-GalNAc;

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[GlcNAc-b1,6]-Gal-b1,4-GlcNAc-b1,3-[NeuAc-a2,6]-Gal-b1,3-[Fuc-a1,2-Gal-b1,4-GlcNAc-b1,6]-GalNAc;

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[NeuAc-a2,3-Gal-b1,4-GlcNAc-b1,6]-GalNAc；

Fuc-a1,3-[HSO3(-3)Gal-b1,4]-GlcNAc-b1,6-[Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3]-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,6-[NeuAc-a2,3-Gal-b1,3]-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[Fuc-a1,2-Gal-b1,4-GlcNAc-b1,6]-Gal-b1,3-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[Gal-b1,4-GlcNAc-b1,6]-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[HSO3(-3)Gal-b1,4-GlcNAc-b1,6]-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,6-[Fuc-a1,2-Gal-b1,3]-GalNAc；

Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[Gal-b1,4-GlcNAc-b1,6]-Gal-b1,3-[Gal-b1,4-GlcNAc-b1,6]-GalNAc;Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-Gal-b1,3-GalNAc;

Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,6-[NeuAc-a2,3-Gal-b1,3]-GalNAc;

Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-Gal-b1,3-[Gal-b1,4-GlcNAc-b1,6]-GalNAc；

Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,6-[Gal-b1,3]-GalNAc；

Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-[Fuc-a1,2]-Gal-b1,4-GlcNAc-b1,6-[GlcNAc-b1,3]-GalNAc；

Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,6-[Fuc-a1,2-Gal-b1,3]-GalNAc；

Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-[Gal-b1,4-GlcNAc-b1,6]-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,6-[NeuAc-a2,3-Gal-b1,3]-GalNAc;

Fuc-a1,2-Gal-b1,3-[NeuAc-a2,6]-GlcNAc-b1,3-Gal-b1,3-Glc；

Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,6-[GlcNAc-b1,3]-GalNAc;Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-[Fuc-a1,3-[Gal-b1,4]-GlcNAc-b1,6]-Gal-b1,4-GlcNAc-b1,6-[NeuAc-a2,3-Gal-b1,3]-GalNAc And

Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,6-[Gal-b1,4-GlcNAc-b1,3]-GalNAc。

Within the scope of the present invention, "permissible conditions to produce a compound comprising a structure of formula I, II or III" are understood to mean conditions associated with physical or chemical parameters, including, but not limited to, temperature, pH, pressure, osmotic pressure, and product/precursor/acceptor concentrations.

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

In a preferred embodiment of the method, the allowing conditions comprise using a medium comprising at least one precursor and/or receptor as defined herein to produce said compound comprising the structure of formula I, II or III. In alternative and/or other preferred embodiments of the method, the allowing conditions include adding at least one precursor and/or acceptor feed to the medium to produce the compound comprising the structure of formula I, II or III.

According to a preferred embodiment of the invention, the cell is modified with one or more expression modules. 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, an untranslated 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 multiple recombinant genes, or may be organized in an operon structure to integrally express two or more recombinant genes. The polynucleotides may be produced by recombinant DNA techniques using techniques well known in the art. Methods for constructing expression modules well known to those skilled 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 the following documents: sambrook et al (2001) Molecular Cloning: a laboratory manual, 3 rd edition, cold Spring Harbor Laboratory Press, CSH, new York or Current Protocolin Molecular Biology, john Wiley and Sons, N.Y. (1989 and yearly updates).

The expression of each of the expression modules may be constitutive or produced by natural or chemical inducers. Constitutive expression, as used herein, is understood to be the expression of a gene transcribed continuously in an organism. Expression by a natural inducer is understood to be the facultative or regulated expression of a gene expressed only under certain natural conditions of the host (e.g., an organism in childbirth or lactation), as a response to environmental changes (e.g., including but not limited to hormones, heat, cold, pH changes, light, oxidation, or osmotic/signaling), or depending on the developmental stage or location of the host cell, including but not limited to apoptosis and autophagy. Expression by a chemical inducer is understood to be the facultative or regulated expression of a gene expressed only after induction of an external chemical agent (e.g. IPTG, arabinose, lactose, iso-lactose, rhamnose or fucose), either by an inducible promoter or by inducing or inhibiting the transcription or translation of the polynucleotide into a genetic loop of a polypeptide.

The expression module may be integrated into the genome of the cell or may be presented to the cell on a vector. The vector may be in the form of a plasmid, cosmid, phage, liposome or virus, which will be stably transformed/transfected into the metabolically engineered cell. Such vectors include, inter alia, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacterial phages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacterial phage genetic elements, such as cosmids and phagemids. These vectors may contain selectable markers such as, but not limited to, antibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNA sense/antisense markers. The expression system construct may contain control regions that regulate and produce expression. In general, any system or vector suitable for maintaining, propagating or expressing a polynucleotide and/or expressing a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well known and conventional techniques such as those described, for example, by Sambrook et al, supra. For recombinant production, the cells may be genetically engineered to incorporate the expression systems of the invention or portions or polynucleotides thereof. Polynucleotides can be introduced into cells by methods described in many standard laboratory manuals, such as Davis et al, basic Methods in Molecular Biology, (1986) and Sambrook et al, 1989 (supra).

As used herein, an expression module comprises a polynucleotide for expressing at least one recombinant gene. The recombinant gene is involved in the expression of a polypeptide that plays a role in the production of a compound comprising the structure of formula I, II or III; or the recombinant gene is associated with other pathways in the host cell not involved in the production of a compound comprising the structure of formula I, II or III. The recombinant gene encodes an endogenous protein having altered expression or activity, preferably the endogenous protein is overexpressed; or the recombinant gene encodes a heterologous protein, which is introduced and expressed, preferably overexpressed, heterologous in the modified cell. Endogenous proteins may have altered expression in cells that also express heterologous proteins.

In a preferred embodiment of the method and/or the cell according to the invention, the cell is capable of producing a compound comprising a structure of formula IV, V or VI:

Wherein:

R ¹ is a monosaccharide, disaccharide, oligosaccharide, protein, glycoprotein, peptide, glycopeptide, lipid or glycolipid; and, when present, R ² is a monosaccharide, disaccharide, or oligosaccharide.

In a more preferred embodiment of the method and/or the cell according to the invention, the cell is capable of producing Gal-b1,3-GlcNAc or lactose-N-disaccharide (LNB). The production of LNB in cells can be achieved by expression and/or overexpression of the N-acetylglucosamine beta-1, 3-galactosyltransferase gene, which transfers galactose (Gal) residues from UDP-Gal to N-acetylglucosamine (GlcNAc) moiety in beta-1, 3-linkages. The GlcNAc and UDP-Gal required in the reaction may be fed into the culture and/or may be produced by cellular metabolism and/or may be provided by enzymes expressed in the cells.

In alternative and/or additional preferred embodiments of the method and/or the cell according to the invention, the cell is capable of producing GlcNAc-b1,3-Gal-b1,4-Glc or lactose-N-trisaccharide (LN 3). LN3 production in cells can be obtained by over-expression of the galactoside beta-1, 3-N-acetylglucosaminyl transferase gene, which transfers the GlcNAc residue from UDP-GlcNAc to lactose to form LN3. The UDP-GlcNAc and lactose required in the reaction may be fed into the culture and/or may be produced by cellular metabolism and/or may be provided by enzymes expressed in the cells.

In alternative and/or additional preferred embodiments of the method and/or the cell according to the invention, the cell is capable of producing Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc or lactose-N-tetraose (LNT). LNT production in cells can be obtained by over-expressing a galactoside beta-1, 3-N-acetylglucosaminyl transferase gene and an N-acetylglucosaminyl beta-1, 3-galactosyltransferase gene, which transfer GlcNAc residues from UDP-GlcNAc to lactose to form LN3, and Gal residues from UDP-Gal to LN3, respectively, to form LNT. The UDP-GlcNAc, UDP-Gal and lactose required in the reaction may be fed into the culture and/or may be produced by cellular metabolism and/or may be provided by enzymes expressed in the cells.

The GlcNAc producing cells may express a phosphatase, e.g., selected from the list comprising any one or more of: coli genes including aphA、Cof、HisB、OtsB、SurE、Yaed、YcjU、YedP、YfbT、YidA、YigB、YihX、YniC、YqaB、YrbL、AppA、Gph、SerB、YbhA、YbiV、YbjL、Yfb、YieH、YjgL、YjjG、YrfG and YbiU, or PsMupP from pseudomonas putida (Pseudomonas putida), DOG1 from saccharomyces cerevisiae and AraL from bacillus subtilis, as described in WO18122225, which phosphorylate GlcNAc-6-phosphate (GlcNAc-6P) to GlcNAc. Preferably, the cells are modified to produce GlcNAc. More preferably, the cells are modified to enhance the production of GlcNAc. The modification may be any one or more selected from the group consisting of: knocking out N-acetylglucosamine-6-phosphate deacetylase, knocking out glucosamine-6-phosphate deaminase, and overexpressing any one or more genes comprising L-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine 6-phosphate N-acetyltransferase, and phosphatase, as described in WO 18122225.

Cells producing UDP-Gal can express, for example, an enzyme that converts UDP-glucose to UDP-Gal. For example, this enzyme may be the UDP-glucose 4-epimerase GalE, which is known, for example, from several species including Chile, E.coli and brown rat. Preferably, the cells are modified to produce UDP-Gal. More preferably, the cells are modified to enhance UDP-Gal production. The modification may be any one or more selected from the group consisting of: knocking out the coding gene of the bifunctional 5' -nucleotidase/UDP-glycosylase, knocking out the coding gene of galactose-1-phosphate uridylyltransferase and overexpressing the coding gene of UDP-glucose 4-epimerase.

The UDP-GlcNAc-producing cell may express, for example, an enzyme that converts GlcNAc to be added to the cell into UDP-GlcNAc. These 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 human and E.coli. Preferably, the cells are modified to produce UDP-GlcNAc. More preferably, the cells are modified to enhance UDP-GlcNAc production. The modification may be any one or more selected from the group consisting of: knock-out of N-acetylglucosamine-6-phosphate deacetylase, overexpression of L-glutamine-D-fructose-6-phosphate aminotransferase, overexpression of phosphoglucomutase, and overexpression of N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase.

Lactose-producing cells may express a beta-1, 4-galactosyltransferase that transfers a Gal residue from UDP-Gal to glucose in a beta-1, 4-linkage, wherein the glucose may be fed into the culture and/or may be produced by metabolism of the cell and/or may be provided by an enzyme expressed in the cell, such as UDP-glucose 4-epimerase. Preferably, the cells in which lactose is used for LN3, LNT and/or derivatives thereof do not have an active galactosidase, e.g. lacZ, which degrades lactose into glucose and galactose.

In a preferred embodiment of the method and/or the cell according to the invention, the cell resists lactose killing phenomena when grown in an environment where lactose is combined with one or more other carbon sources. The term "lactose killing" refers to the inhibition of cell growth in a medium in which lactose is present with another carbon source. 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 includes expression and/or overexpression of exogenous and/or endogenous lactose transporter genes by heterologous promoters, the expression or oversurface does not result in a lactose killing phenotype, and/or modification of codon usage of lactose transporter proteins to produce altered expression of the lactose transporter proteins, the altered expression does not result in a lactose killing phenotype. The contents of WO 2016/075243 in this regard are incorporated by reference. In the context of the present invention lactose is preferably taken up by the cells disclosed herein, wherein the lactose is further glycosylated by the glycosyltransferases disclosed herein to synthesize MMO, preferably HMO.

GDP-fucose may be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such GDP-fucose producing cells may express, for example, enzymes that convert fucose to be added to the cells into GDP-fucose. Such enzymes may be, for example, bifunctional fucose kinase/fucose-1-phosphate guanyl transferase, such as Fkp from Bacteroides fragilis, or a combination of a single fucose kinase with a single fucose-1-phosphate guanyl transferase, which are known, for example, from several species including homo sapiens, wild boars and brown rats.

Preferably, the cells are modified to produce GDP-fucose. More preferably, the cells are modified to enhance the production of GDP-fucose. The modification may be any one or more selected from the group consisting of: knock-out UDP-glucose: undecenyl phosphoglucose-1-phosphotransferase coding gene, over-expression of GDP-L-fucose synthase coding gene, over-expression of GDP-mannose 4, 6-dehydratase coding gene, over-expression of mannose-1-phosphogguanylate transferase coding gene, over-expression of phosphomannose mutase coding gene and over-expression of mannose-6-phosphate isomerase coding gene.

In the context of the present invention, the cells express an alpha-1, 2-fucosyltransferase which has the galactosyl alpha-1, 2-fucosyltransferase activity on the galactose residues of Gal-b1,3-GlcNAc (LNB, lactose-N-disaccharide). More specifically, the α -1, 2-fucosyltransferase transfers a fucose residue from GDP-fucose to a galactose residue in the disaccharide Gal-b1,3-GlcNAc (LNB) of the compound comprising the structure of formula I, II or III.

According to embodiments of the methods and/or cells of the invention, the alpha-1, 2-fucosyltransferase is a polypeptide belonging to the family of gt11 fucosyltransferases and comprises the motif X (not M) X (not F) XXXGNX (not N) [ ILMV ] X (not E, S) X (not E) XXXX (not F, S) X (not Y) XXXXX (not H, S, Y) shown in SEQ ID NO 38, wherein X may be any amino acid residue. In an alternative embodiment, the alpha-1, 2-fucosyltransferase is a polypeptide that belongs to the family of gt74 fucosyltransferases.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase comprises any one of SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 or 37, preferably any one of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07, 08, or 09, more preferably any one of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07, or 08, even more preferably any one of SEQ ID NOs 05, 06, 07, or 08, most preferably any one of SEQ ID NOs 01, 02, 03, or 04.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of SEQ ID No. 03, which has at least 15.0% overall sequence identity to the full length of the polypeptide of SEQ ID No. 03, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 15.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of the polypeptide of SEQ ID No. 03.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any one of SEQ ID NOs 15, 34, 35, 36 or 37, which has at least 22.0% overall sequence identity to the full length of any one of SEQ ID NOs 15, 34, 35, 36 or 37, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 22.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID NOs 15, 34, 35, 36 or 37.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 05, 08, 11, 21, 30 or 31, which has at least 30.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 05, 08, 11, 21, 30 or 31, preferably the α -1, 2-fucosyltransferase comprises an amino sequence having at least 30.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 05, 08, 11, 21, 30 or 31, preferably SEQ ID NOs 05 or 08.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 06, 07, 09, 19, 25, 27, 32 or 33 having at least 35.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 06, 07, 09, 19, 25, 27, 32 or 33, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 35.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0% amino acid sequence identity to the full length of any of the polypeptides of SEQ ID NOs 06, 07, 09, 19, 25, 27, 32 or 33, preferably SEQ ID NOs 06, 07 or 09, more preferably at least 80.0%, more preferably at least 85.0%, 90.0%, more preferably at least 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, even more preferably at least 85.0% amino acid sequence identity to the full length of any of the polypeptides of SEQ ID NOs 06, 07 or 07.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 02, 04, 14, 16, 17 or 28 having at least 40.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 02, 04, 14, 16, 17 or 28, preferably the α -1, 2-fucosyltransferase comprises an amino sequence having at least 40.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 02, 04, 14, 16, 17 or 28, preferably SEQ ID NOs 02 or 04.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 01, 10, 12, 13, 18, 20, 22, 24 or 26 having at least 45.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 01, 10, 12, 13, 18, 20, 22, 24 or 26, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 45.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 01, 10, 12, 13, 18, 20, 22, 24 or 26, preferably SEQ ID NOs 01.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of SEQ ID No. 23 that has at least 50.0% overall sequence identity to the full length of the polypeptide of SEQ ID No. 23, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence that has at least 50.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of the polypeptide of SEQ ID No. 23.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of SEQ ID No. 29 that has at least 70.0% overall sequence identity to the full length of the polypeptide of SEQ ID No. 29, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence that has at least 70.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of the polypeptide of SEQ ID No. 29.

In an alternative embodiment, the alpha-1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 10 consecutive amino acid residues from any of SEQ ID NOs 03, 05, 11, 15, 21, 31, 34, 35, 36 or 37, preferably SEQ ID NOs 03 or 05.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 13 consecutive amino acid residues from any of SEQ ID NO 06, 08, 13, 17, 19, 20, 25, 28 or 30 (preferably SEQ ID NO 06 or 08).

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 15 consecutive amino acid residues from any one of SEQ ID NOs 04, 07, 09, 10, 16, 26, 27, 32 or 33, preferably SEQ ID NOs 04, 07 or 09, more preferably SEQ ID NOs 04.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 18 consecutive amino acid residues from any one of SEQ ID NOs 01, 02, 14, 18, 22 or 24, preferably SEQ ID NOs 01 or 02.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 22 consecutive amino acid residues from any of SEQ ID NOs 12, 23 or 29.

In addition to galactose residues within the LNB, the α -1, 2-fucosyltransferase may also be fucosylated using other receptors. The additional receptors may include, but are not limited to, monosaccharides, disaccharides, and oligosaccharides such as galactose, glucose, N-acetylglucosamine (GlcNAc), lactose, lactulose, N-acetyllactosamine (LacNAc), 3 '-fucosyllactose (3' FL), lactose-N-trisaccharide (LN 3), lactose-N-tetrasaccharide (LNT), and lactose-N-neotetraose (LNnT). Alternatively, the alpha-1, 2-fucosyltransferase uses only galactose residues within the LNB for fucosylation. The term "only" means "unique". In other words, the α -1, 2-fucosyltransferase accepts only galactose residues within the LNB as receptors for fucosylation with the α -1,2 linkage, and not other receptors.

According to another embodiment of the method and/or cell of the invention, the alpha-1, 2-fucosyltransferase having galactoside alpha-1, 2-fucosyltransferase activity on galactose residues of Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (LNT, lactose-N-tetrasaccharide) has additional galactoside alpha-1, 2-fucosyltransferase activity on galactose residues of the non-reducing end of Gal-b1,3-GlcNAc-b1, 4-Glc (LNT, lactose-N-tetrasaccharide).

In one embodiment, the α -1, 2-fucosyltransferase is a polypeptide belonging to the family of gt74 fucosyltransferases and comprises the motif [ DE ] CC [ FWY ] XXX (not D, E) (Xn) [ FWY ] X [ ILMV ] [ DE ] [ DE ], wherein X can be any amino acid residue, and wherein n is 10 to 40, as shown in SEQ ID NO 39.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase comprises the polypeptide sequence of any one of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, or 18, preferably any one of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07, 08, or 09, more preferably any one of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07, or 08, even more preferably any one of SEQ ID NOs 05, 06, 07, or 08, most preferably any one of SEQ ID NOs 01, 02, 03, or 04.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 03 or 15, which has at least 20.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 03 or 15, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 20.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 03 or 15.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 05, 08 or 11, which has at least 30.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 05, 08 or 11, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 30.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 05, 08 or 11.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 02, 04, 06, 07, 09 or 17 having at least 37.50% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 02, 04, 06, 07, 09 or 17, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 37.50%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 02, 04, 06, 07, 09 or 17.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 01, 10, 12, 13, 14, 16 or 18 having at least 45.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 01, 10, 12, 13, 14, 16 or 18, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 45.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 01, 10, 12, 13, 14, 16 or 18.

In an additional and/or alternative embodiment, the alpha-1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 10 consecutive amino acid residues from any of SEQ ID NOS: 03, 05, 11 or 15 (preferably SEQ ID NOS: 03 or 05).

In an additional and/or alternative embodiment, the alpha-1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 13 consecutive amino acid residues from any one of SEQ ID NOS 06, 08, 13 or 17 (preferably SEQ ID NOS 06 or 08).

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 15 consecutive amino acid residues from any one of SEQ ID NOS: 04, 07, 09, 10, 16, preferably SEQ ID NOS: 04, 07 or 09, more preferably SEQ ID NOS: 04 or 07, most preferably SEQ ID NO: 04.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 18 consecutive amino acid residues from any of SEQ ID NO. 01, 02, 14 or 18 (preferably SEQ ID NO. 01 or 02).

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 20 consecutive amino acid residues from SEQ ID NO. 12.

According to another embodiment of the method and/or cell of the invention, the alpha-1, 2-fucosyltransferase having a galactosidase alpha-1, 2-fucosyltransferase activity on galactose residues of Gal-b1,3-GlcNAc and an additional galactosidase alpha-1, 2-fucosyltransferase activity on galactose residues of the non-reducing end of LNT has no additional galactosidase alpha-1, 2-fucosyltransferase activity on lactose. According to another alternative embodiment of the method and/or cell of the invention, said alpha-1, 2-fucosyltransferase having a galactosyl alpha-1, 2-fucosyltransferase activity on Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (LNT, lactose-N-tetraose) for galactose residues and an additional galactosyl alpha-1, 2-fucosyltransferase activity on galactose for galactose but lower than the additional galactosyl alpha-1, 2-fucosyltransferase activity on non-reducing terminal galactose residues of the LNT.

In one embodiment, the alpha-1, 2-fucosyltransferase is a polypeptide belonging to the family of gt74 fucosyltransferases comprising the motif [ DE ] CC [ FWY ] XXX (not D, E) (Xn) [ FWY ] X [ ILMV ] [ DE ] [ DE ], wherein X may be any amino acid residue, and wherein n is 10 to 40, as shown in SEQ ID NO 39.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase comprises the polypeptide sequence of any one of SEQ ID NOs 01, 02, 03, 04, 07, 09, 10, 12, 13, 14, 15, 16, 17, or 18, preferably any one of SEQ ID NOs 01, 02, 03, 04, 07, or 09, more preferably any one of SEQ ID NOs 07 or 09, and most preferably any one of SEQ ID NOs 01, 02, 03, or 04.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 02, 04, 07, 09 or 17, which has at least 37.50% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 02, 04, 06, 07, 09 or 17, preferably the α -1, 2-fucosyltransferase comprises at least 37.50%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, or even more preferably at least 99.0%, or even more preferably at least 85.0% amino acid identical to the full length of any of the polypeptides of SEQ ID NOs 02, 04, 07, 06, 07 or 09, more preferably SEQ ID NOs 02, 04, 06, 07 or 07, even more preferably at least 80.0%, more preferably at least 85.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0% or even more preferably at least 85.0% amino acid identical to the full length of any of the polypeptides of SEQ ID NOs 02, 04, 07, or even more preferably at least 04, or 04.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 01, 10, 12, 13, 14, 16 or 18 having at least 45.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 01, 10, 12, 13, 14, 16 or 18, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 45.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 01, 10, 12, 13, 14, 16 or 18, preferably SEQ ID NOs 01.

In an additional and/or alternative embodiment, the alpha-1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 10 consecutive amino acid residues from either of SEQ ID NO:03 or 15 (preferably SEQ ID NO: 03).

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 13 consecutive amino acid residues from either of SEQ ID NOs 13 or 17.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 15 consecutive amino acid residues from any one of SEQ ID NOS: 04, 07, 09, 10 or 16, preferably SEQ ID NOS: 04, 07 or 09, more preferably SEQ ID NOS: 04 or 07, most preferably SEQ ID NO: 04.

According to another embodiment of the method and/or cell of the invention, the galactose residues of Gal-b1,3-GlcNAc have a galactosidase alpha-1, 2-fucosyltransferase activity and the galactose residues of the non-reducing end of the LNT have an additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose which is higher than the additional galactosidase alpha-1, 2-fucosyltransferase activity for the galactose residues of the non-reducing end of the LNT.

In one embodiment, the alpha-1, 2-fucosyltransferase comprises the polypeptide sequence of any one of SEQ ID NOS 05, 06, 08 or 11, preferably SEQ ID NOS 05, 06 or 08.

In another and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 05, 06, 08 or 11, said functional homolog, variant or derivative having at least 35.0% overall sequence identity to the full length of any of said polypeptides of SEQ ID NOs 05, 06, 08 or 11, preferably said α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 35.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of said polypeptides of SEQ ID NOs 05, 06, 08 or 11, preferably of SEQ ID NOs 05, 06 or 08.

In an additional and/or alternative embodiment, the alpha-1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 13 consecutive amino acid residues from SEQ ID NO:05, 06, 08 or 11 (preferably SEQ ID NO:05, 06 or 08).

According to another embodiment of the method and/or cell of the invention, the alpha-1, 2-fucosyltransferase having galactoside alpha-1, 2-fucosyltransferase activity on galactose residues of Gal-b1,3-GlcNAc does not have galactoside alpha-1, 2-fucosyltransferase activity on galactose residues of the non-reducing end of LNT.

In one embodiment, the alpha-1, 2-fucosyltransferase comprises a polypeptide sequence of any one of SEQ ID NOs 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 34, 35, 36 or 37, which has at least 22.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 34, 35, 36 or 37, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 22.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 34, 35, 36 or 37.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 21, 30 or 31, which has at least 30.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 21, 30 or 31, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 35.0%, preferably at least 30.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 21, 30 or 31.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 19, 25, 27, 32 or 33, which has at least 35.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 19, 25, 27, 32 or 33, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 35.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 19, 25, 27, 32 or 33.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 20, 22, 24, 26 or 28 having at least 45.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 20, 22, 24, 26 or 28, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 45.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 20, 22, 24, 26 or 28.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 10 consecutive amino acid residues from any of SEQ ID NOs 21, 31, 34, 35, 36 or 37.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 13 consecutive amino acid residues from any of SEQ ID NOs 19, 20, 25, 28 or 30.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 15 consecutive amino acid residues from any of SEQ ID NOs 26, 27, 32 or 33.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 17 consecutive amino acid residues from either one of SEQ ID NOs 22 or 24.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 22 consecutive amino acid residues from either one of SEQ ID NOs 23 or 29.

According to another embodiment of the method and/or cell of the invention, the galactose residues of Gal-b1,3-GlcNAc have a galactosidase alpha-1, 2-fucosyltransferase activity and the galactose residues of the non-reducing end of the LNT have no galactosidase alpha-1, 2-fucosyltransferase activity and the alpha-1, 2-fucosyltransferase has no galactosidase alpha-1, 2-fucosyltransferase activity for lactose. According to another alternative embodiment of the method and/or cell of the invention, the galactose residue of Gal-b1,3-GlcNAc has a galactosidase alpha-1, 2-fucosyltransferase activity and the alpha-1, 2-fucosyltransferase having NO galactosidase alpha-1, 2-fucosyltransferase activity for the galactose residue of the non-reducing end of the LNT has an additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose but less than 3.0% of the alpha-1, 2-fucosyltransferase activity of SEQ ID NO 06 for the galactoglycoside alpha-1, 2-fucosyltransferase activity of lactose.

In one embodiment, the alpha-1, 2-fucosyltransferase comprises a polypeptide sequence of any one of SEQ ID NOs 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 33, 34, 35, 36 or 37.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 21 or 30, which has at least 30.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 21 or 30, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 30.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 21 or 30.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 19, 25, 27 or 33, which has at least 35.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 19, 25, 27 or 33, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 35.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 19, 25, 27 or 33.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 20, 22, 24 or 26, which has at least 45.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 20, 22, 24 or 26, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 45.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 20, 22, 24 or 26.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 10 consecutive amino acid residues from any of SEQ ID NOs 21, 34, 35, 36 or 37.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 13 consecutive amino acid residues from any of SEQ ID NOs 19, 20, 25 or 30.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional fragment comprising an oligopeptide sequence of at least 15 consecutive amino acid residues from any of SEQ ID NOs 26, 27 or 33.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional fragment of an oligopeptide sequence comprising at least 20 consecutive amino acid residues from SEQ ID NO. 23.

According to another embodiment of the method and/or cell of the invention, the galactose residue of Gal-b1,3-GlcNAc has a galactosidase alpha-1, 2-fucosyltransferase activity and the alpha-1, 2-fucosyltransferase of the non-reducing end of LNT has NO additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose and the alpha-1, 2-fucosyltransferase of SEQ ID NO 06 has an additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose of 4.0% to 20.0%.

In one embodiment, the alpha-1, 2-fucosyltransferase comprises a polypeptide sequence of any one of SEQ ID NOs 28, 29, 31 or 32.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of any of SEQ ID NOs 31 or 32, which has at least 35.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 31 or 32, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence having at least 35.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of any of the polypeptides of SEQ ID NOs 31 or 32.

In an additional and/or alternative embodiment, the α -1, 2-fucosyltransferase is a functional homolog, variant or derivative of SEQ ID NO. 28 that has at least 40.0% overall sequence identity to the full length of the polypeptide of SEQ ID NO. 28, preferably the α -1, 2-fucosyltransferase comprises an amino acid sequence that has at least 40.0%, preferably at least 80.0%, more preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, most preferably at least 95.0% overall sequence identity to the full length of the polypeptide of SEQ ID NO. 28.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional fragment of an oligopeptide sequence comprising at least 10 consecutive amino acid residues from SEQ ID NO. 31.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional fragment of an oligopeptide sequence comprising at least 14 consecutive amino acid residues from either of SEQ ID NOS 28 or 32.

In additional and/or alternative embodiments, the α -1, 2-fucosyltransferase is a functional fragment of an oligopeptide sequence comprising at least 22 consecutive amino acid residues from SEQ ID NO. 29.

Global alignment algorithms (e.g., NEEDLEMAN WUNSCH algorithm in program GAP (GCG Wisconsin Package, accelrys)) are used to determine overall sequence identity, preferably using default parameters, preferably using mature protein sequences (i.e., without regard to secretion signals or transit peptides). Sequence identity is generally higher when only conserved domains or motifs are considered, as compared to the overall sequence identity.

A functional fragment of an oligopeptide sequence comprising at least 10 consecutive amino acid residues from any one of SEQ ID NOs 03, 05, 11, 15, 21, 31, 34, 35, 36 or 37 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 10, 11, 12, 13, 14, 15 up to all numbers of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 03, 05, 11, 15, 21, 31, 34, 35, 36 or 37 as set forth herein and has alpha-1, 2-fucosyltransferase activity on Gal residues within the LNB.

A functional fragment comprising an oligopeptide sequence of at least 13 consecutive amino acid residues from any one of SEQ ID NOs 06, 08, 13, 17, 19, 20, 25, 28 or 30 is to be understood as any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 13, 14, 15, 16, 17 up to all numbers of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 06, 08, 13, 17, 19, 20, 25, 28 or 30 as set forth herein and has alpha-1, 2-fucosyltransferase activity on Gal residues within the LNB.

A functional fragment comprising an oligopeptide sequence of at least 15 consecutive amino acid residues from any one of SEQ ID NOs 04, 07, 09, 10, 16, 26, 27, 32 or 33 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 15, 16, 17, 18, 19, up to all numbers of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 04, 07, 09, 10, 16, 26, 27, 32 or 33 as set forth herein and has alpha-1, 2-fucosyltransferase activity on Gal residues within the LNB.

A functional fragment of an oligopeptide sequence comprising at least 18 consecutive amino acid residues from any one of SEQ ID NOs 01, 02, 14, 18, 22 or 24 is to be understood as any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 18, 19, 20, 21, 22 up to all numbers of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 01, 02, 14, 18, 22 or 24 as set forth herein and has alpha-1, 2-fucosyltransferase activity on Gal residues within the LNB.

A functional fragment of an oligopeptide sequence comprising at least 22 consecutive amino acid residues from any one of SEQ ID NOs 12, 23 or 29 is to be understood as any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 22, 23, 24, 25, 26 up to all numbers of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 12, 23 or 29 as set forth herein and has alpha-1, 2-fucosyltransferase activity on Gal residues within the LNB.

A functional fragment of an oligopeptide sequence comprising at least 10 consecutive amino acid residues from any one of SEQ ID NOs 03, 05, 11 or 15 is to be understood as any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 10, 11, 12, 13, 14, up to all numbers of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 03, 05, 11 or 15 as set forth herein, and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 13 consecutive amino acid residues from any one of SEQ ID NOs 06, 08, 13 or 17 is to be understood as any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 13, 14, 15, 16, 17, up to all number of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 06, 08, 13 or 17 as set forth herein, and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 15 consecutive amino acid residues from any one of SEQ ID NOs 04, 07, 09, 10 or 16 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 15, 16, 17, 18, 19, up to all numbers of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 04, 07, 09, 10 or 16 as set forth herein, and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 18 consecutive amino acid residues from any one of SEQ ID NOs 01, 02, 14 or 18 is to be understood as any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 18, 19, 20, 21, 22 up to all numbers of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 01, 02, 14 or 18 as set forth herein and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 20 consecutive amino acid residues from SEQ ID No. 12 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 20, 21, 22, 23, 24, up to all numbers of consecutive amino acid residues from the polypeptide of SEQ ID NO:20 as set forth herein, and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 10 consecutive amino acid residues from either one of SEQ ID NOs 03 or 15 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 10, 11, 12, 13, 14 up to all number of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 03 or 15 as given herein, has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and has additional galactosyl alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT and NO additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose or has additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose but less than its additional galactosyl alpha-1, 2-fucosyltransferase activity for non-reducing end galactose residues of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 13 consecutive amino acid residues from either one of SEQ ID NOs 13 or 17 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 13, 14, 15, 16, 17 up to all number of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 13 or 17 as given herein, and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and has additional galactosyl alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT and NO additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose or has additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose but less than its additional galactosyl alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 15 consecutive amino acid residues from any one of SEQ ID NOs 04, 07, 09, 10 or 16 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 15, 16, 17, 18, 19, up to all number of consecutive amino acid residues from any one of SEQ ID NOs 04, 07, 09, 10 or 16 as set forth herein, and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and additional galactosyl alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT and NO additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose or additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose but less than the additional galactosyl alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 18 consecutive amino acid residues from any one of SEQ ID NOs 01, 02, 14 or 18 is to be understood as any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 18, 19, 20, 21, 22 up to all number of consecutive amino acid residues from any one of SEQ ID NOs 01, 02, 14 or 18 as set forth herein, and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and has additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT and NO additional galactoside alpha-1, 2-fucosyltransferase activity for lactose or has additional galactoside alpha-1, 2-fucosyltransferase activity for lactose but less than its additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 20 consecutive amino acid residues from SEQ ID No. 12 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 20, 21, 22, 23, 24 up to all number of consecutive amino acid residues from the polypeptide of SEQ ID No. 12 as given herein and has an alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and an additional galactosidase alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT and NO additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose or an additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose but less than an additional galactosidase alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 13 consecutive amino acid residues from any one of SEQ ID NOs 05, 06, 08 or 11 is to be understood as any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 13, 14, 15, 16, 17, up to all number of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 05, 06, 08 or 11 as set forth herein, and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB, additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT, and additional galactoside alpha-1, 2-fucosyltransferase activity for lactose and higher than additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 10 consecutive amino acid residues from any one of SEQ ID NOs 21, 31, 34, 35, 36 or 37 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 10, 11, 12, 13, 14, up to all numbers of consecutive amino acid residues from any one of SEQ ID NOs 21, 31, 34, 35, 36 or 37 as set forth herein, and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and NO additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 13 consecutive amino acid residues from any one of SEQ ID NOs 19, 20, 25, 28 or 30 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 13, 14, 15, 16, 17 up to all numbers of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 19, 20, 25, 28 or 30 as set forth herein, and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and NO additional galactosyl alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 15 consecutive amino acid residues from any one of SEQ ID NOs 26, 27, 32 or 33 is to be understood as any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 15, 16, 17, 18, 19, up to all numbers of consecutive amino acid residues from any one of SEQ ID NOs 26, 27, 32 or 33 as set forth herein, and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and NO additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 17 consecutive amino acid residues from either one of SEQ ID NOs 22 or 24 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 17, 18, 19, 20, 21 up to all number of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 22 or 24 as set forth herein and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and NO additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 22 consecutive amino acid residues from either one of SEQ ID NOs 23 or 29 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 22, 23, 24, 25, 26 up to all number of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 23 or 29 as set forth herein and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB and NO additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT.

A functional fragment of an oligopeptide sequence comprising at least 10 consecutive amino acid residues from any one of SEQ ID NOs 21, 34, 35, 36 or 37 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 10, 11, 12, 13, 14 up to all number of consecutive amino acid residues from any one of SEQ ID NOs 21, 34, 35, 36 or 37 as given herein and has an alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB, NO additional galactosyl alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT and NO additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose or an additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose but less than 3.0% of the alpha-1, 2-fucosyltransferase activity of the alpha-1, 2-fucosyltransferase of SEQ ID NO 06 for lactose.

A functional fragment of an oligopeptide sequence comprising at least 13 consecutive amino acid residues from any one of SEQ ID NOs 19, 20, 25 or 30 is to be understood as any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 13, 14, 15, 16, 17, up to all number of consecutive amino acid residues from any one of SEQ ID NOs 19, 20, 25 or 30 as given herein, and has an alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB, NO additional galactosyl alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT, and NO additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose, or an additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose but less than 3.0% of the alpha-1, 2-fucosyltransferase activity of the alpha-1, 2-fucosyltransferase enzyme of SEQ ID NO 06 for lactose.

A functional fragment of an oligopeptide sequence comprising at least 15 consecutive amino acid residues from any one of SEQ ID NOs 26, 27 or 33 is to be understood as any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 15, 16, 17, 18, 19 up to all number of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 26, 27 or 33 as given herein and has an alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB, NO additional galactosyl alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT and NO additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose or an additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose but less than 3.0% of the alpha-1, 2-fucosyltransferase activity of the alpha-1, 2-fucosyltransferase of SEQ ID NO 06 for lactose.

A functional fragment of an oligopeptide sequence comprising at least 17 consecutive amino acid residues from either one of SEQ ID NOs 22 or 24 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 17, 18, 19, 20, 21 up to all number of consecutive amino acid residues from any one of the polypeptides as set forth herein in SEQ ID No. 22 or 24 and has an alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB, NO additional galactosyl alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT and NO additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose or an additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose but less than 3.0% of the alpha-1, 2-fucosyltransferase activity of the galactosyl alpha-1, 2-fucosyltransferase of SEQ ID No. 06.

A functional fragment of an oligopeptide sequence comprising at least 20 consecutive amino acid residues from SEQ ID No. 23 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 20, 21, 22, 23, 24 up to all numbers of consecutive amino acid residues from the polypeptide of SEQ ID No. 23 as given herein and has an alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB, NO additional galactosidase alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT and NO additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose or an additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose but less than 3.0% of the activity of the alpha-1, 2-fucosyltransferase of SEQ ID No. 06 for galactose.

A functional fragment of an oligopeptide sequence comprising at least 10 consecutive amino acid residues from SEQ ID No. 31 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 10, 11, 12, 13, 14 up to all numbers of consecutive amino acid residues from the polypeptide of SEQ ID No. 31 as set forth herein and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB, NO additional galactosidase alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT, and additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose and 4.0 to 20.0% of the galactosidase alpha-1, 2-fucosyltransferase activity for lactose of SEQ ID No. 06.

A functional fragment of an oligopeptide sequence comprising at least 14 consecutive amino acid residues from either one of SEQ ID NOs 28 or 32 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 14, 15, 16, 17, 18 up to all number of consecutive amino acid residues from any one of the polypeptides of SEQ ID NOs 28 or 32 as given herein and has an alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB, NO additional galactosyl alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT and an additional galactosyl alpha-1, 2-fucosyltransferase activity for lactose and is 4.0 to 20.0% of the galactosyl alpha-1, 2-fucosyltransferase activity for lactose of SEQ ID NO 06.

A functional fragment of an oligopeptide sequence comprising at least 22 consecutive amino acid residues from SEQ ID No. 29 is understood to be any such functional fragment: the functional fragment comprises an oligopeptide sequence of at least 22, 23, 24, 25, 26 up to all numbers of consecutive amino acid residues from the polypeptide of SEQ ID No. 29 as set forth herein and has alpha-1, 2-fucosyltransferase activity for Gal residues within the LNB, NO additional galactosidase alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of the LNT, and additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose and 4.0 to 20.0% of the galactosidase alpha-1, 2-fucosyltransferase activity for lactose of SEQ ID No. 06.

In preferred embodiments of the methods and/or cells of the invention, the cells express an alpha-1, 2-fucosyltransferase that preferentially uses LNB as a receptor for alpha-1, 2-fucosylation of galactose residues within the LNB relative to other receptors (e.g., galactose, glucose, glcNAc, lactose, lactulose, lacNAc, 3' FL, LN3, LNT, and LNnT). In a more preferred embodiment, at least 50% of the fucosylated compounds obtained in the mixture by the alpha-1, 2-fucosyltransferase expressed in the cell are derived from alpha-1, 2-fucosylation of the galactose residues of the LNB. In other words, at least 50% of the fucosylated compounds obtained in the mixture by the α -1, 2-fucosyltransferase expressed in the cell are α -1, 2-fucosylated LNBs or 2' flnb. At least 50% of the fucosylated compounds in the mixture should be understood as meaning that at least 50％、55％、60％、65％、70％、75％、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％、99.50％、99.60％、99.70％、99.80％、99％、90％、100％ of the fucosylated compounds in the mixture are alpha-1, 2 fucosylated LNBs or 2' flnb. Preferably, at least 60%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% of the fucosylated compounds obtained in the mixture by the α -1, 2-fucosyltransferase expressed in the cell are α -1, 2-fucosylated LNBs or 2' flnb.

In another preferred embodiment of the method and/or cell of the invention, the cell expresses an α -1, 2-fucosyltransferase as described herein, which is capable of modifying a compound comprising the structure of formula IV, V or VI produced intracellularly:

Wherein:

Preferably, the cell is capable of producing GDP-fucose, which is a donor of the alpha-1, 2-fucosyltransferase.

In another embodiment of the method and/or cell of the invention, the cell is modified in terms of expression or activity of any of said alpha-1, 2-fucosyltransferases.

In embodiments of the methods and/or cells of the invention, the cells are capable of producing one or more nucleotide activating sugars selected from the list comprising: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2, 6-dideoxy-L-arabino-4-hexulose, UDP-2-acetamido-2, 6-dideoxy-L-lyxol-4-hexulose, UDP-N-acetyl-L-rhamnose (UDP-L-RhaNAc or UDP-2-acetamido-2, 6-dideoxy-L-mannose), DP-N-acetylfucose, UDP-N-acetylfucose (UDP-L-52 or UDP-2-acetamido-2, 6-dideoxy-L-5-fucose), UDP-N-acetylgalactosamine (UDP-L-FucNAc or UDP-2-acetamido-N-5-mannosamine), UDP-N-acetylgalactosamine (UDP-N-RhaNAc or UDP-2-acetamido-N-6-acetylgalactosamine), UDP-N-acetyl-L-quiniosamine (UDP-L-QuiNAc or UDP-2-acetamido-2, 6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac)、CMP-Neu4Ac、CMP-Neu5Ac9N₃、CMP-Neu4,5Ac₂、CMP-Neu5,7Ac₂、CMP-Neu5,9Ac₂、CMP-Neu5,7(8,9)Ac₂、CMP-N- hydroxyacetyl neuraminic acid (CMP-Neu 5 Gc), GDP-rhamnose and UDP-xylose.

In a preferred embodiment of the method and/or cell of the invention, 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-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine 6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine 1-phosphate acetyltransferase, N-acetylneuraminic acid synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid 9-phosphate synthase, N-acylneuraminic acid 9-phosphate phosphatase, N-acylneuraminic acid cytidylyltransferase, galactose 1-epimerase, galactokinase, and, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase.

In a more preferred embodiment of the method and/or cell, the cell is modified in terms of expression or activity of any one of the polypeptides. Any of the polypeptides is an endogenous protein of a cell having altered expression or activity, preferably the endogenous polypeptide is overexpressed; alternatively, any of the polypeptides is a heterologous protein that is introduced and expressed, preferably overexpressed, in the cell. The endogenous polypeptide may have altered expression in a cell that also expresses the list of heterologous polypeptides.

GDP-fucose may be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such GDP-fucose producing cells may express enzymes that convert e.g. fucose to be added to the cells into GDP-fucose. Such enzymes may be, for example, bifunctional fucose kinase/fucose-1-phosphate guanyl transferase, such as Fkp from Bacteroides fragilis, or a combination of a single fucose kinase with a single fucose-1-phosphate guanyl transferase, which are known, for example, from several species including homo sapiens, wild boars and brown rats. Preferably, the cells are modified to produce GDP-fucose. More preferably, the cells are modified to enhance the production of GDP-fucose. The modification may be any one or more selected from the group consisting of: knock-out UDP-glucose: undecenyl phosphoglucose-1-phosphotransferase coding gene, over-expression of GDP-L-fucose synthase coding gene, over-expression of GDP-mannose 4, 6-dehydratase coding gene, over-expression of mannose-1-phosphogguanylate transferase coding gene, over-expression of phosphomannose mutase coding gene and over-expression of mannose-6-phosphate isomerase coding gene.

CMP-Neu5Ac can be provided by an enzyme expressed in a cell or by metabolism of a cell. Such CMP-Neu5 Ac-producing cells may express enzymes that convert sialic acid, for example, to be added to the cell, to CMP-Neu5Ac. The enzyme may be a CMP sialic acid synthetase, such as N-acyl neuraminic acid cytidylyltransferase from several species including Chile, neisseria meningitidis (NEISSERIA MENINGITIDIS) and Pasteurella spinosa (Pasteurella multocida). Preferably, the cells are modified to produce CMP-Neu5Ac. More preferably, the cells are modified to enhance the production of CMP-Neu5Ac. The modification may be any one or more selected from the group consisting of knockout of N-acetylglucosamine-6-phosphate deacetylase, knockout of glucosamine-6-phosphate deaminase, overexpression of a sialic acid synthase encoding gene, and overexpression of an N-acetylglucosamine-2-epimerase encoding gene.

UDP-GalNAc can be synthesized from UDP-GlcNAc by the action of a single step reaction using UDP-N-acetylglucosamine 4-epimerase (e.g., wbgU from Shigella dysenteriae (Plesiomonas shigelloides), gne from Yersinia enterocolitica or wbpP from Pseudomonas aeruginosa serotype O6). Preferably, the cells are modified to produce UDP-GalNAc. More preferably, the cells are modified to enhance UDP-GalNAc production.

UDP-ManNAc can be synthesized directly from UDP-GlcNAc by epimerization with UDP-GlcNAc 2-epimerase (e.g., cap5P from Staphylococcus aureus, rffE from Escherichia coli, cps19fK from Streptococcus pneumoniae and RfbC from enterococcus). Preferably, the cells are modified to produce UDP-ManNAc. More preferably, the cells are modified to enhance UDP-ManNAc production.

CMP-Neu5Gc can be synthesized directly from CMP-Neu5Ac by hydroxylation with the vertebrate CMP-Neu5Ac hydroxylase (CMAH) enzyme. Preferably, the cells are modified to produce CMP-Neu5Gc. More preferably, the cells are modified to enhance the production of CMP-Neu5Gc.

According to a preferred embodiment of the method and/or the cell of the invention, the cell expresses one or more glycosyltransferases selected from the list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosyltransferase, N-acetylmannosyltransferase, xylosyltransferase, glucuronyltransferase, galacturonate transferase, glucosaminyltransferase, N-glycolylneuraminidase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-arabinoxylan-Zhuo Tangan aminotransferase, UDP-N-acetylglucosaminenolpyruvyltransferase and fucosyltransferase.

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

In another more preferred embodiment of the method and/or cell, the sialyltransferase is selected from the list comprising alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase.

In another more preferred embodiment of the method and/or cell, 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 more preferred embodiment of the method and/or cell, 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 more preferred embodiment of the method and/or cell, the mannosyltransferase is selected from the list comprising an alpha-1, 2-mannosyltransferase, an alpha-1, 3-mannosyltransferase and an alpha-1, 6-mannosyltransferase.

In another more preferred embodiment of the method and/or cell, 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 more preferred embodiment of the method and/or cell, the N-acetylgalactosamine transferase is an alpha-1, 3-N-acetylgalactosamine transferase.

In another more preferred embodiment of the method and/or the cell of the invention, the cell is modified in terms of expression or activity of at least one of said glycosyltransferases. The glycosyltransferase is an endogenous protein of a cell having altered expression or activity, preferably the endogenous glycosyltransferase is overexpressed; alternatively, the glycosyltransferase is a heterologous protein that is introduced and expressed, preferably overexpressed, in the cell heterologous thereto. The endogenous glycosyltransferase can have altered expression in a cell that also expresses a heterologous glycosyltransferase.

According to another preferred embodiment of the method and/or the cell according to the invention, the cell uses a precursor to produce said compound comprising the structure of formula I, II or III, preferably said precursor is fed from the culture medium to the cell. According to a more preferred embodiment of the method and/or the cell, the cell uses at least two precursors to produce the compound comprising the structure of formula I, II or III, preferably the precursors are fed from the culture medium to the cell. According to another preferred embodiment of the method and/or the cell according to the invention, the cell is producing at least one precursor, preferably at least two precursors, for producing the compound comprising the structure of formula I, II or III. In preferred embodiments of the method and/or the cell, the precursor of the compound used to produce the structure comprising formula I, II or III is fully converted to the compound comprising the structure of formula I, II or III.

In another preferred embodiment of the method and/or the cell of the invention, the cell expresses a membrane transporter or a polypeptide having transport activity, thereby transporting the compound across the outer membrane of the cell wall. In another preferred embodiment of the method and/or cell of the invention, the cell expresses more than one membrane transporter or polypeptide having transport activity, thereby transporting the compound across the outer membrane of the cell wall. In a more preferred embodiment of the method and/or the cell of the invention, the cell is modified in terms of expression or activity of the membrane transporter protein or the polypeptide having transport activity. The membrane transporter or the polypeptide having transport activity is a cellular endogenous protein having altered expression or activity, preferably the endogenous membrane transporter or the polypeptide having transport activity is overexpressed; alternatively, the membrane transporter protein or the polypeptide having transport activity is a heterologous protein, which is introduced and expressed, preferably overexpressed, heterologous in the cell. The endogenous membrane transporter or a polypeptide having transport activity may have altered expression in a cell that also expresses a heterologous membrane transporter or a polypeptide having transport activity.

In a more preferred embodiment of the method and/or cell of the invention, the membrane transporter or the polypeptide having transport activity is selected from the list comprising a transporter, a P-P bond hydrolytically driven transporter, a b-barrel porin, an auxiliary transporter, a putative transporter and a phospho transport driven group translocator. In a more preferred embodiment of the method and/or cell of the invention, the transporter comprises an MFS transporter, a sugar efflux transporter, and an iron conjugate export protein. In another more preferred embodiment of the methods and/or cells of the invention, the P-P bond hydrolysis-driven transporter includes an ABC transporter and an iron conjugate exporter.

In another preferred embodiment of the method and/or cell of the invention, the membrane transporter or the polypeptide having transport activity controls the flow of the compound comprising the structure of formula I, II or III to the outer membrane of the cell wall. In alternative and/or other preferred embodiments of the methods and/or cells of the invention, the membrane transporter or a polypeptide having transport activity controls the flow of one or more precursors to the outer membrane of the cell wall, which precursors will be used in the production of the compound comprising the structure of formula I, II or III. In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, the membrane transporter or a polypeptide having transport activity controls the flow of one or more receptors to the outer membrane of the cell wall that will be used in the production of the compound comprising the structure of formula I, II or III.

In another preferred embodiment of the method and/or the cell of the invention, the membrane transporter or the polypeptide having transport activity provides improved production of the compound comprising the structure of formula I, II or III. In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, a membrane transporter or a polypeptide having transport activity enables efflux of the compound comprising the structure of formula I, II or III. In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, a membrane transporter or a polypeptide having transport activity may enhance efflux of the compound comprising the structure of formula I, II or III.

In another preferred embodiment of the method and/or the cell of the invention, the cell expresses a polypeptide selected from the group comprising: lactose transporter (e.g., lacY or lac12 permease), fucose transporter, glucose transporter, galactose transporter, nucleotide-activated sugar transporter (e.g., transporter for UDP-GlcNAc, UDP-Gal and/or GDP-Fuc).

In another preferred embodiment of the method and/or cell of the invention, the cell expresses a membrane transporter belonging to the MFS transporter family, such as MdfA polypeptides of the MdfA family of multi-drug transporter MdfA from species including escherichia coli (UniProt ID P0AEY, sequence version 1), escherichia sakazakii (Cronobacter muytjensii) (UniProt ID A0A2T7ANQ9, sequence version 1), escherichia coli (Citrobacter youngae) (UniProt ID D4BC23, sequence version 1) and Lei Jinsi fort pre-ground bacteria (Yokenella regensburgei) (UniProt ID G9Z5F4, sequence version 1). In another preferred embodiment of the method and/or cell of the invention, the cell expresses a membrane transporter belonging to the sugar efflux transporter family, e.g. SetA polypeptide from family SetA of species comprising escherichia coli (UniProt ID P31675, sequence version 3), citrobacter kei (Citrobacter koseri) (UniProt ID A0a078LM16, sequence version 1) and klebsiella pneumoniae (Klebsiella pneumoniae) (UniProt ID A0C4MGS7, sequence version 1). In another preferred embodiment of the method and/or cell of the invention, the cell expresses membrane transporters belonging to the family of iron conjugate exporters, such as E.coli entS (Unit Prot ID P24077, SEQ ID NO: 2) and E.coli iceT (Unit Prot ID A0A024L207, SEQ ID NO: 1). In another preferred embodiment of the method and/or cell of the invention, the cell expresses a membrane transporter belonging to the ABC transporter family, for example oppF from E.coli (UniProt ID P77737, SEQ ID NO: 1), lmrA from lactococcus lactis subspecies lactis (diacetyl form) (Lactococcus lactis subsp. Lactis bv. Diacetylactis) (UniProt ID A0A1V0NEL4, SEQ ID NO: 1) and Blon_2475 from bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Inntis) (UniProt ID B7GPD4, SEQ ID NO: 1). In a more preferred embodiment of the method and/or cell of the invention, the cell expresses more than one membrane transporter selected from the list comprising: lactose transporter (e.g., lacY or lac12 permease), fucose transporter, glucose transporter, galactose transporter, nucleotide-activated glucose transporter (e.g., transporter for UDP-GlcNAc, UDP-Gal and/or GDP-Fuc), mdfA protein from E.coli (UniProt ID P0AEY, SEQ ID 1), mdfA protein from E.sakazakii (Cronobacter muytjensii) (UniProt ID A0A2T7ANQ9, SEQ ID 1), mdfA protein from Citrobacter young (UniProt ID D4BC23, SEQ ID 1), mdfA protein from Lei Jinsi fort pre-ground bacteria (UniProt ID G9Z5F4, SEQ ID 1), setA protein from E.coli (UniProt ID P31675, sequence version 3), the SetA protein from Citrobacter koraiensis (UniProt ID A0a078LM16, sequence version 1), the SetA protein from klebsiella pneumoniae (UniProt ID A0C4MGS7, sequence version 1), the entS protein from escherichia coli (UniProt ID P24077, sequence version 2), the iceT protein from escherichia coli (UniProt ID A0a024L207, sequence version 1), the oppF protein from escherichia coli (UniProt ID P77737, sequence version 1), the lmrA protein from lactococcus lactis subspecies (diacetyl) protein (UniProt ID A0A1V0NEL4, sequence version 1) and the Blon 2475 protein from bifidobacterium longum subspecies infancy (UniProt ID B7GPD4, sequence version 1).

In preferred embodiments of the methods and/or cells of the invention, the cells 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 transporter, or any other protein disclosed herein. Throughout the application, the feature "plurality" means at least 2, preferably at least 3, more preferably at least 4, even more preferably at least 5.

According to another preferred embodiment of the method and/or the cell according to the invention, the cell comprises a modification of reduced acetate production. The modification may be any one or more selected from the group consisting of: overexpression of acetyl-CoA synthetase, complete or partial knockdown of pyruvate dehydrogenase or complete or partial knockdown of pyruvate dehydrogenase and lactate dehydrogenase or lactate dehydrogenase with lower functionality.

In another embodiment of the method and/or the cell of the invention, the cell is modified in terms of expression or activity of at least one acetyl-coa synthetase (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 altered expression or activity, preferably the endogenous acetyl-coa synthetase is overexpressed; alternatively, the acetyl-coa synthetase is a heterologous protein that is introduced heterologous and expressed, preferably overexpressed, in the cell. The endogenous acetyl-coa synthase can have altered expression in a cell that also expresses a heterologous acetyl-coa synthase. In a more preferred embodiment, the cell is modified in terms of expression or activity of acetyl-CoA synthetase acs (Unit Prot ID P27550, SEQ ID NO: 2) from E.coli. In another and/or additional preferred embodiment, the cell is modified in terms of expression or activity of a functional homolog, variant or derivative of acs from escherichia coli (UniProt ID P27550, sequence version 2), said functional homolog, variant or derivative having at least 80% overall sequence identity to the full length of the polypeptide of escherichia coli (UniProt ID P27550, sequence version 2), respectively, and having acetyl-coa synthetase activity.

In an alternative and/or additional further embodiment of the method and/or the cell of the invention, the cell is modified in terms of the expression or activity of at least one pyruvate dehydrogenase, such as e.g. from escherichia coli, saccharomyces cerevisiae, nootropic and phaeotropic mice. In a preferred embodiment, the cells have been modified in a manner generally known to the person skilled in the art to have at least one pyruvate dehydrogenase encoding gene which is partially or completely knocked out or mutated, so as to produce at least one protein having a lower functionality or no pyruvate dehydrogenase activity. In a more preferred embodiment, the cell has a complete knockout in the poxB encoding gene, thereby producing a cell lacking pyruvate dehydrogenase activity.

In an alternative and/or additional further embodiment of the method and/or the cell of the invention, the cell is modified in terms of expression or activity of at least one lactate dehydrogenase, such as e.g. from escherichia coli, saccharomyces cerevisiae, nootropic and phaeotropic mice. In a preferred embodiment, the cells have been modified in a manner generally known to those skilled in the art to have at least one lactate dehydrogenase-encoding gene that is partially or completely knocked out or mutated to produce at least one protein having less or no lactate dehydrogenase activity. In a more preferred embodiment, the cell has a complete knockout in the ldhA encoding gene, thereby producing a cell lacking lactate dehydrogenase activity.

According to another preferred embodiment of the method and/or the cell of the invention, the cell comprises reduced or reduced expression and/or an impaired, reduced or delayed activity of any one or more of the following proteins, said proteins comprising β -galactosidase, galactosido-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose: undecaprenyl-phosphoglucose-1-phosphotransferase, L-fucokinase, L-fucose isomerase, N-acetylneuraminic acid lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphoglycerate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphouridyltransferase, glucose-1-phosphoadenyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphoisomerase, aerobic respiration control protein, transcription repressor IclR, lon protease, glucose-specific translocated phosphotransferase IIBC component ptsG, glucose-specific translocated Phosphotransferase (PTS) IIBC component malX, IIA ^Glc, beta-glucoside specific PTS II, fructose-specific PTS polyphosphorylated proteins FruA and FruB, alcohol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphoacetyltransferase, pyruvate decarboxylase.

According to another preferred embodiment of the method and/or the cell according to the invention, the cell is capable of producing phosphoenolpyruvate (PEP). In another preferred embodiment of the method and/or the cell of the invention, the cell is modified to enhance the production and/or supply of phosphoenolpyruvate (PEP).

In preferred embodiments, as a means of enhancing PEP production and/or supply, one or more PEP-dependent sugar transport phosphotransferase systems are disrupted, such as, but not limited to: 1) N-acetyl-D-glucosamine Npi-phosphotransferase (EC 2.7.1.193), which is encoded, for example, by the nagE gene (or cluster nagABCD) in E.coli or Bacillus species, 2) ManXYZ, which encodes the enzyme ll Man complex (mannose PTS permease, protein-Npi-phosphohistidine-D-mannose phosphotransferase), which imports exogenous hexoses (mannose, glucose, glucosamine, fructose, 2-deoxyglucose, mannosamine, N-acetylglucosamine, etc.) and releases phosphate into the cytoplasm, 3) glucose-specific PTS transporter (encoded, for example, by PtsG/Crr) which ingests glucose and forms glucose-6-phosphate in the cytoplasm; 4) Sucrose-specific PTS transporter that ingests sucrose and forms sucrose-6-phosphate in the cytoplasm, 5) fructose-specific PTS transporter (e.g., encoded by genes fruA and fruB, and kinase fruK that ingests fructose and forms fructose-1-phosphate in the first step and fructose 1, 6-diphosphate in the second step, 6) lactose PTS transporter (e.g., encoded by lacE in lactobacillus casei (Lactococcus casei)) that ingests lactose and forms lactose-6-phosphate, 7) galactitol-specific PTS enzyme that ingests galactitol and/or sorbitol and forms galactitol-1-phosphate or sorbitol-6-phosphate, respectively; 8) Mannitol-specific PTS enzymes that ingest mannitol and/or sorbitol and form mannitol-1-phosphate or sorbitol-6-phosphate, respectively; and 9) trehalose-specific PTS enzyme, which ingests trehalose and forms trehalose-6-phosphate.

In another and/or additional preferred embodiment, the complete PTS system is disrupted by disruption of the PtsIH/Crr gene cluster as a means of enhancing PEP production and/or supply. PtsI (enzyme I) is a cytoplasmic protein which acts as E.coli K-12 phosphoenolpyruvate: portal (gateway) of the sugar phosphotransferase system (PTS ^sugar). PtsI is one of the components of two (PtsI and PtsH) sugar non-specific proteins of PTS ^sugar, which together with sugar-specific endomembrane permeases affect the phosphotransferase cascade, leading to phosphorylation and transport of various carbohydrate substrates. HPr (histidine-containing protein) is one of two sugar-nonspecific protein components of PTS ^sugar. It accepts a phosphate group 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). Crr or EIIA ^Glc are PEP phosphorylated in reactions requiring PtsH and PtsI.

In another and/or additional preferred embodiment, the cells are further modified to compensate for the absence of the PTS system of the carbon source by introducing and/or overexpressing the corresponding permease. These are, for example, permeases or ABC transporters, which include but are not limited to: a transport protein specifically introduced into lactose, such as a transport protein encoded by the LacY gene of E.coli, a transport protein specifically introduced into sucrose, such as a transport protein encoded by the cscB gene of E.coli, a transport protein specifically introduced into glucose, such as a transport protein encoded by the galP gene of E.coli, a transport protein specifically introduced into fructose, such as a transport protein encoded by the fruI gene of Streptococcus mutans (Streptococcus mutans), or a sorbitol/mannitol ABC transport protein, such as a transport protein encoded by the SmoEFGK cluster of rhodobacter sphaeroides (Rhodobacter sphaeroides), a trehalose/sucrose/maltose transport protein, such as a transport protein encoded by the ThuEFGK cluster of Sinorhizobium meliloti (Sinorhizobium meliloti), and an N-acetylglucosamine/galactose/glucose transport protein, such as a transport protein encoded by NagP of iron reducing bacteria (SHEWANELLA ONEIDENSIS). Examples of combinations of PTS deletions with alternative transporter overexpression are: 1) a deletion of the glucose PTS system (e.g., ptsG gene), in combination 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 fruB, fruA, fruK genes), in combination with the introduction and/or overexpression of a fructose permease (e.g., fruI), 3) a deletion of the lactose PTS system, in combination with the introduction and/or overexpression of a lactose permease (e.g., lacY), and/or 4) a deletion of the sucrose PTS system, in combination with the introduction and/or overexpression of a sucrose permease (e.g., cscB).

In another preferred embodiment, the cells are modified to compensate for the absence of the PTS system 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 proteins and kinase overexpression are: 1) A deletion of the glucose PTS system (e.g., ptsG gene), an introduction and/or overexpression of a glucose permease (e.g., galP of glcP), an introduction and/or overexpression of a glucokinase (e.g., glk), and/or 2) a deletion of the fructose PTS system, e.g., one or more of fruB, fruA, fruK genes, an introduction and/or overexpression of a fructose permease (e.g., fruI), an introduction and/or overexpression of a fructose kinase (e.g., frk or mak).

In another and/or additional preferred embodiment, the cells are modified by the introduction or modification therein of any one or more of the following list as a means of enhancing PEP production and/or supply: phosphoenolpyruvate synthase activity (EC: 2.7.9.2, for example encoded in E.coli by ppsA), phosphoenolpyruvate carboxykinase activity (EC 4.1.1.32 or EC 4.1.1.49, for example encoded by PCK in C.glutamicum or by pckA in E.coli), phosphoenolpyruvate carboxylase activity (EC 4.1.1.31, for example encoded by ppc in E.coli), oxaloacetate decarboxylase activity (EC 4.1.1.112, for example encoded by eda in E.coli), pyruvate kinase activity (EC 2.7.1.40, for example encoded by pykA and pykF in E.coli), pyruvate carboxylase activity (EC 6.4.1.1, for example encoded by pyc in E.coli) 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 cell is modified to overexpress any one or more polypeptides comprising: ppsA from E.coli (Unit Prot ID P23538, SEQ ID 5), PCK from C.glutamicum (Unit Prot ID Q6F5A5, SEQ ID 1), pcka from E.coli (Unit Prot ID P22259, SEQ ID 2), eda from E.coli (Unit Prot ID P0A955, SEQ ID 1), maeA from E.coli (Unit Prot ID P26616, SEQ ID 4) and maeB from E.coli (Unit Prot ID P76558, SEQ ID 1).

In another and/or additional preferred embodiment, the cell is modified to express 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 embodiment, the cell is modified by decreasing phosphoenolpyruvate carboxylase activity and/or pyruvate kinase activity, preferably by deleting genes encoding phosphoenolpyruvate carboxylase, pyruvate carboxylase activity and/or pyruvate kinase, as a means of enhancing PEP production and/or supply.

In exemplary embodiments, the cells are genetically modified by various modifications, such as: the overexpression of phosphoenolpyruvate synthase is combined with a deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate synthase is combined with a deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate carboxykinase is combined with a deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate carboxykinase is combined with a deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate carboxykinase is combined with a deletion of a pyruvate carboxylase gene, the overexpression of oxaloacetate decarboxylase is combined with a deletion of a pyruvate kinase gene, the overexpression of oxaloacetate decarboxylase is combined with a deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of malate dehydrogenase is combined with a deletion of a pyruvate kinase gene, the overexpression of malate dehydrogenase is combined with a deletion of a phosphoenolpyruvate carboxylase gene, and/or the overexpression of malate dehydrogenase is combined with a deletion of a pyruvate carboxylase gene.

In another exemplary embodiment, the cells are genetically modified by different modifications, such as: the overexpression of phosphoenolpyruvate synthase is combined with the overexpression of phosphoenolpyruvate carboxykinase, the overexpression of phosphoenolpyruvate synthase is combined with the overexpression of oxaloacetate decarboxylase, the overexpression of phosphoenolpyruvate carboxykinase is combined with the overexpression of malate dehydrogenase, the overexpression of oxaloacetate decarboxylase is combined with the overexpression of malate dehydrogenase, the overexpression of phosphoenolpyruvate synthase is combined with the overexpression of phosphoenolpyruvate carboxykinase and the overexpression of oxaloacetate decarboxylase, the overexpression of phosphoenolpyruvate synthase is combined with the overexpression of phosphoenolpyruvate carboxykinase and the overexpression of malate dehydrogenase, the overexpression of phosphoenolpyruvate synthase is combined with the overexpression of phosphoenolpyruvate carboxykinase, the overexpression of oxaloacetate decarboxylase and the overexpression of malate dehydrogenase, the overexpression of phosphoenolpyruvate carboxylase is combined with the overexpression of oxaloacetate decarboxylase and the overexpression of malate dehydrogenase, and the overexpression of malate dehydrogenase is combined with the overexpression of phosphoenolpyruvate carboxylase and the overexpression of malate dehydrogenase.

In another exemplary embodiment, the cells are genetically modified by different modifications, such as: the overexpression of phosphoenolpyruvate synthase binds to a deletion of a phosphoenolpyruvate carboxykinase gene, the overexpression of phosphoenolpyruvate synthase binds to a deletion of a oxaloacetate decarboxylase gene, the overexpression of phosphoenolpyruvate synthase binds to a deletion of a malate dehydrogenase gene, the overexpression of phosphoenolpyruvate carboxykinase binds to a deletion of a oxaloacetate decarboxylase gene, the overexpression of phosphoenolpyruvate carboxykinase binds to a deletion of a malate dehydrogenase gene, the overexpression of oxaloacetate decarboxylase binds to a deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate synthase binds to phosphoenolpyruvate carboxykinase and the overexpression of oxaloacetate decarboxylase binds to a deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate synthase binds to phosphoenolpyruvate carboxykinase and the overexpression of malate dehydrogenase binds to a deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate synthase binds to phosphoenolpyruvate carboxykinase and the overexpression of oxaloacetate decarboxylase and the overexpression of malate dehydrogenase binds to a deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate carboxykinase binds to oxaloacetate decarboxylase and the overexpression of malate dehydrogenase binds to a deletion of a pyruvate kinase gene, and the overexpression of phosphoenolpyruvate synthase binds to oxaloacetate decarboxylase and the overexpression of malate dehydrogenase binds to a deletion of a pyruvate kinase gene.

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

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

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

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

According to another preferred embodiment of the method and/or the cell according to the invention, the cell comprises at least partially inactivated catabolic pathways of selected mono-, di-or oligosaccharides, which are involved in and/or are necessary for said production of said compound comprising the structure of formula I, II or III.

According to another preferred embodiment of the method and/or the cell according to the invention, the cell produces 90g/L or more of said compound comprising the structure of formula I, II or III in the whole culture and/or supernatant and/or wherein said compound comprising the structure of formula I, II or III has a purity of at least 80% in the whole culture and/or supernatant, measured as the total amount of said compound comprising the structure of formula I, II or III and precursors thereof in the whole culture and/or supernatant, respectively.

Another embodiment of the invention provides a method and a cell wherein the compound comprising the structure of formula I, II or III is produced in and/or by a fungal, yeast, bacterial, insect, plant, animal or protozoal cell as described herein. The cell is selected from the list comprising bacteria, yeast or fungi, or refers to a plant, animal or protozoan cell. The aforementioned bacteria preferably belong to the Proteus (Proteus) or the Thick-walled (Firmicutes) or the cyanobacterium (Cyanobactria) phylum or the Deinococcus-Thermus (Deinococcus-Thermus) phylum. The aforementioned bacteria belonging to the Proteus phylum preferably belong to the Enterobacteriaceae family, preferably belong to the Escherichia coli (ESCHERICHIA COLI) species. The foregoing bacteria preferably relate to any strain belonging to the species escherichia coli, such as, but not limited to, escherichia coli B, escherichia coli C, escherichia coli W, escherichia coli K12, escherichia coli Nissle. More specifically, the foregoing terms relate to a cultured E.coli strain, known as E.coli K12 strain, which is well suited to laboratory environments and which, unlike the wild-type strain, has lost the ability to reproduce 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 present invention relates in particular to a mutated and/or transformed E.coli strain as described above, wherein said E.coli strain is a K12 strain. More preferably, the E.coli K12 strain is E.coli MG1655. The aforementioned bacteria belonging to the phylum firmicutes preferably belong to the genus bacillus (bacili), preferably belong to the order lactobacillus (Lactobacilliales), members of which are, for example: lactobacillus (Lactobacillus lactis), leuconostoc mesenteroides (Leuconostoc mesenteroides), or bacillus (Bacillales), members thereof, e.g. from the genus bacillus, e.g. bacillus subtilis (Bacillus subtilis) or bacillus amyloliquefaciens (b.amyloliquefaciens). The aforementioned bacterium belonging to the phylum actinomycetes (Actinobacteria) preferably belongs to the family Corynebacteriaceae (Corynebacterium), the member of which is Corynebacterium glutamicum (Corynebacterium glutamicum) or Corynebacterium nonfermentans (C. Afermannins), or belongs to the family Streptomycetaceae (Streptomycetaceae), the member of which is Streptomyces griseus (Streptomyces griseus) or Streptomyces fradiae (S.fradiae). The aforementioned yeasts preferably belong to the ascomycota (Ascomycota) or basidiomycota (Basidiomycota) or the Deuteromycota (Deuteromycota) or zygomycota (Zygomycetes). The aforementioned yeasts preferably belong to the genus Saccharomyces (members thereof, for example, saccharomyces cerevisiae (Saccharomyces cerevisiae), saccharomyces bayanus (S.bayanus), saccharomyces buticarpolylis (S.boulardii)), zygosaccharomyces (Zygosaccharomyces), pichia (Pichia) members thereof, for example, pichia pastoris (Pichia pastoris), pichia anomala (P.anomala), pichia Kluyveromyces (P.kluyveri), saccharomyces colpitis (Komagataella), hansenula (Hansenula), kluyveromyces (Kluyveromyces) members thereof, for example, kluyveromyces lactis (Kluyveromyces lactis), kluyveromyces (K.marxianus), kluyveromyces (K.thertomorro), debarycemia (Debaromyces), yarrowia (Yarrowia) (for example, yarrowia lipolytica (Yarrowia lipolytica)) or Star Mo Jiaomu (STARMERELLA) (e.g., kluyveromyces bumblebee Star Mo Jiaomu (STARMERELLA BOMBICOLA)). The yeasts are preferably selected from Pichia pastoris, yarrowia lipolytica (Yarrowia lipolitica), saccharomyces cerevisiae and Kluyveromyces lactis the fungi preferably belong to the genus Rhizopus, pelargonium (Dictyostelium), penicillium (Penicillium), mucor or Aspergillus flavus (Aspergillus), plant cells include cells of flowering plants and non-flowering plants, and algal cells such as Chlamydomonas (Chlamydomonas), chlorella (Chlorella) and the like. Preferably, the plants are tobacco, alfalfa, rice, tomato, cotton, canola, soybean, maize or corn plants. 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. flagellins, salmon, tuna, bass, trout, catfish), invertebrates (e.g. lobsters, crabs, shrimps, clams, oysters, mussels, sea urchins), reptiles (e.g. snakes, crocodiles, sea turtles), amphibians (e.g. frogs) or insects (e.g. flies, nematodes) or genetically modified cell lines derived from human cells (excluding embryonic stem cells). Human and non-human mammalian cells are preferably selected from the list comprising epithelial cells (e.g. mammary epithelial cells), embryonic kidney cells (e.g. HEK293 or HEK 293T cells), fibroblasts, COS cells, chinese Hamster Ovary (CHO) cells, murine myeloma cells (e.g. N20, SP2/0 or YB2/0 cells), NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, as described in WO 21067641. The aforementioned insect cells are preferably derived from cells similar to Spodoptera frugiperda (Spodoptera frugiperda), such as Sf9 or Sf21 cells, silkworm (Bombyxmori), cabbage looper (Mamestra brassicae), trichoplusia ni (Trichoplusia ni) like cells, such as BTI-TN-5B1-4 cells, or Drosophila melanogaster (Drosophila melanogaster) like cells, such as Drosophila S2 cells. The aforementioned protozoan cells are preferably Leishmania tarabica (LEISHMANIA TARENTOLAE) cells.

According to a preferred embodiment of the method and/or the cell of the invention, the compound comprising the structure of formula I, II or III is produced in and/or by a cell which is a living gram-negative bacterium comprising a reduced or eliminated synthesis of poly-N-acetylglucosamine (PNAG), enterobacter Common Antigen (ECA), cellulose, capsular polysaccharide acid, core oligosaccharide, osmoregulation Periplasmic Glucan (OPG), glyceroglucoside, glycan and/or trehalose.

In a more preferred embodiment of the method and/or cell, the reduced or eliminated synthesis of the poly-N-acetylglucosamine (PNAG), enterobacter co-antigen (ECA), cellulose, capsular polygluconic acid, core oligosaccharide, osmoregulation Periplasmic Glucan (OPG), glyceroglucoside, glycan and/or trehalose is provided by any one or more glycosyltransferase mutations involved in the synthesis of any one of the poly-N-acetylglucosamine (PNAG), enterobacter co-antigen (ECA), cellulose, capsular polygluconic acid, core oligosaccharide, osmoregulation Periplasmic Glucan (OPG), glyceroglucoside, glycan and/or trehalose, wherein the mutation provides for a deletion or lower expression of any one of the glycosyltransferases. The glycosyltransferase comprises: encoding: a glycosyltransferase gene for a poly-N-acetyl-D-glucosamine synthase subunit, UDP-N-acetylglucosamine-undecenyl-phosphate N-acetylglucosamine phosphotransferase, fuc4NAc (4-acetamido-4, 6-dideoxy-D-galactose) transferase, UDP-N-acetyl-D-mannuronate transferase (UDP-N-acetyl-D-mannosaminuronic ACID TRANSFERASE), a glycosyltransferase gene encoding a cellulose synthase catalytic subunit, a cellulose biosynthetic protein, a capsular polysaccharide acid biosynthetic glucuronyl transferase, a capsular polysaccharide acid biosynthetic galactosyltransferase, a capsular polysaccharide acid biosynthetic fucosyltransferase, UDP-glucose: undecenyl phosphoglucose-1-phosphotransferase, putative capsular polysaccharide acid biosynthesis 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-galactofuranosyltransferase, undecenyl phosphate 4-deoxy-4-carboxamide-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 glycerophosphate phosphorylase, glycogen synthase, 1, 4-alpha-glucan branching enzyme, 4-alpha-glucan transferase and trehalose-6-phosphate synthase. In exemplary embodiments, the cell is mutated in a glycosyltransferase comprising any one or more of 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 reduced 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 overexpression of a carbon storage modulator-encoding gene, deletion of a na+/h+ antiporter regulator-encoding gene, and/or deletion of a sensor histidine kinase-encoding gene.

Another embodiment provides cells stably cultured in a medium, wherein the medium may be any type of growth medium well known to those skilled in the art, including minimal medium, complex medium, or growth medium enriched with certain compounds such as, but not limited to, vitamins, trace elements, amino acids, and/or precursors and/or receptors as defined herein.

The cells used herein are capable of growing on complex media of monosaccharides, disaccharides, oligosaccharides, polysaccharides, polyols, glycerol, including molasses, corn steep liquor, peptone, tryptone, yeast extract or mixtures thereof (e.g. mixed raw materials), preferably mixed monosaccharide raw materials (e.g. hydrolyzed sucrose) as the main carbon source. The term "complex medium" refers to a medium of indefinite exact composition. The term "predominantly" refers to the most important carbon source from which the cells produce the di-and/or oligosaccharides of interest, biomass formation, carbon dioxide and/or byproducts (e.g., acids and/or alcohols such as acetic acid, lactic acid and/or ethanol), i.e., 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% of all the desired carbon is derived from the above carbon source. In one embodiment of the invention, the carbon source is the sole carbon source of the organism, i.e. 100% of all required carbon comes from the carbon sources mentioned above. Common primary carbon sources include, but are not limited to, glucose, glycerol, fructose, sucrose, maltose, lactose, arabinose, maltooligosaccharide, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn steep liquor, high fructose syrup, acetate, citrate, lactate and ketonate. As used herein, the precursors defined herein cannot be used as a carbon source to produce the compounds comprising the structure of formula I, II or III.

According to another embodiment of the method of the invention, the conditions allowing the production of the compound comprising the structure of formula I, II or III comprise the use of a medium comprising at least one precursor and/or acceptor to produce the compound comprising the structure of formula I, II or III. Preferably, the medium contains at least one precursor selected from lactose, galactose, fucose, sialic acid, glcNAc, galNAc, lactose-N-disaccharide (LNB), N-acetyllactosamine (LacNAc).

According to alternative and/or additional embodiments of the method of the invention, the conditions allowing the production of the compound comprising the structure of formula I, II or III comprise adding at least one precursor and/or acceptor feed to the culture medium to produce a compound comprising the structure of formula I, II or III.

According to an alternative embodiment of the method of the invention, the conditions allowing the production of the compound comprising the structure of formula I, II or III comprise culturing the cell of the invention using a medium to produce the compound comprising the structure of formula I, II or III, wherein the medium lacks any precursors and/or acceptors for producing the compound comprising the structure of formula I, II or III and is combined with at least one precursor and/or acceptor feed further added to the medium to produce the compound comprising the structure of formula I, II or III.

In a preferred embodiment, the method for producing the compound comprising the structure of formula I, II or III as described herein comprises at least one of the following steps:

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

ii) adding at least one precursor and/or acceptor feed to the medium in the reactor, wherein the total reactor volume ranges from 250mL (milliliters) to 10.000m ³ (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium is no more than 3 times, preferably no more than 2 times, more preferably less than 2 times the volume of the medium prior to the addition of the precursor and/or acceptor feed;

iii) Adding at least one precursor and/or acceptor feed to the medium in the reactor, wherein the total reactor volume ranges from 250mL (milliliters) to 10.000m ³ (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium is no more than 3 times, preferably no more than 2 times, more preferably less than 2 times the volume of the medium prior to adding the precursor and/or acceptor feed, and wherein preferably the pH of the precursor and/or acceptor feed is set at 3 to 7, and wherein preferably the temperature of the precursor and/or acceptor feed is maintained at 20 ℃ to 80 ℃;

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

v) feeding at least one precursor and/or acceptor feed to the culture medium in a continuous manner by means of a feed solution for 1,2,3, 4, 5 days, and wherein preferably the pH of the feed solution is set at 3 to 7, and wherein preferably the temperature of the feed solution is maintained at 20 to 80 ℃;

The method produces the compound comprising the structure of formula I, II or III at 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 embodiment, the method for producing the compound comprising the structure of formula I, II or III as described herein comprises at least one of the following steps:

i) Using a medium comprising an initial reactor volume of at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams lactose/liter, wherein the reactor volume ranges from 250mL to 10.000m ³ (cubic meters);

ii) adding at least one precursor and/or acceptor to the culture medium in a single pulse or in a discontinuous (pulsed) manner, wherein the total reactor volume is in the range of 250mL (milliliter) to 10.000m ³ (cubic meter), preferably such that the final volume of the culture medium is no more than 3 times, preferably no more than 2 times, more preferably less than 2 times the volume of the culture medium prior to the addition of the precursor and/or acceptor feed pulse;

iii) Adding at least one precursor and/or acceptor feed to the medium in the reactor in a single pulse or in a discontinuous (pulsed) manner, wherein the total reactor volume ranges from 250mL (milliliter) to 10.000m ³ (cubic meter), preferably such that the final volume of the medium is no more than 3 times, preferably no more than 2 times, more preferably less than 2 times the volume of the medium before the precursor and/or acceptor feed pulse is added, and wherein preferably the pH of the precursor and/or acceptor feed pulse is set at 3 to 7, and wherein preferably the temperature of the precursor and/or acceptor feed pulse is maintained at 20 ℃ to 80 ℃;

iv) adding at least one precursor and/or acceptor feed to the culture 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;

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

In another more preferred embodiment, the method for producing the compound comprising the structure of formula I, II or III as described herein comprises at least one of the following steps:

ii) adding a lactose feed to the 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 lactose per liter of initial reactor volume, wherein the total reactor volume ranges from 250mL (milliliters) to 10.000m ³ (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium is no more than 3 times, preferably no more than 2 times, more preferably less than 2 times the volume of the medium prior to the addition of the lactose feed;

iii) Adding a lactose feed to the 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 lactose per liter of initial reactor volume, wherein the reactor volume ranges from 250mL to 10.000m ³ (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium is no more than 3 times, preferably no more than 2 times, more preferably less than 2 times the volume of the medium before adding the lactose feed, and wherein preferably the pH of the lactose feed is set at 3 to 7, and wherein preferably the temperature of the lactose feed is maintained at 20 ℃ to 80 ℃;

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

v) adding lactose feed to the medium in a continuous manner by means of a lactose feed solution for 1 day, 2 days, 3 days, 4 days, 5 days, and wherein the concentration of the 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 the pH of the feed solution is preferably set at 3 to 7, and wherein the temperature of the feed solution is preferably maintained at 20 ℃ to 80 ℃;

Preferably, lactose feeding is accomplished 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 feeding is accomplished 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.

In another preferred embodiment, the cells are cultured for at least about 60, 80, 100 or about 120 hours or in a continuous manner.

In a preferred embodiment, a carbon source, preferably sucrose, is provided in the medium for 3 days or more, preferably up to 7 days; and/or providing an initial culture volume of at least 100, advantageously at least 105, more advantageously at least 110, even more advantageously at least 120 grams sucrose per liter in said medium in a continuous manner such that the final volume of said medium is no more than 3 times, advantageously no more than 2 times, more advantageously less than 2 times the volume of the medium prior to culturing.

Preferably, when carrying out the methods described herein, the first stage of exponential cell growth is provided by adding a carbon source (preferably glucose or sucrose) to the medium, and then lactose is added to the medium in the second stage.

In another preferred embodiment of the method of the invention, the first stage of exponential cell growth is provided by adding a carbon-based substrate (preferably glucose or sucrose) to a medium comprising a precursor (preferably lactose), followed by a second stage, wherein only the carbon-based substrate (preferably glucose or sucrose) is added to the medium.

In another preferred embodiment of the method of the invention, the first stage of exponential cell growth is provided by adding a carbon-based substrate (preferably glucose or sucrose) to a medium comprising a precursor (preferably lactose), followed by a second stage, wherein the carbon-based substrate (preferably glucose or sucrose) and the precursor (preferably lactose) are added to the medium.

In an alternative preferred embodiment, in the methods described herein lactose is added with the carbon-based substrate in the first stage of exponential growth.

According to the invention, the method described herein preferably comprises the step of isolating the compound comprising the structure of formula I, II or III from the culture.

The term "isolated from the culture" refers to harvesting, collecting, or recovering the compound comprising the structure of formula I, II or III from the cells and/or their growth medium.

The compounds comprising the structures of formula I, II or III can be isolated from the aqueous medium in which the cells are grown in a conventional manner. If the compound comprising the structure of formula I, II or III is still present in the cell producing the compound comprising the structure of formula I, II or III, the compound comprising the structure of formula I, II or III may be dissociated or extracted from the cell using conventional methods, e.g., disrupting the cell using high pH, heat shock, sonication, french press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergent, hydrolysis, etc., and then the culture medium and/or cell extract may be used together and separately to isolate the compound comprising the structure of formula I, II or III.

This preferably involves clarifying the compound comprising the structure of formula I, II or III to remove suspended particles and contaminants, particularly cells, cell components, insoluble metabolites and debris produced by culturing genetically modified cells. In this step, the compound comprising the structure of formula I, II or III may be clarified by conventional means. Preferably, the compound comprising the structure of formula I, II or III is clarified by centrifugation, flocculation, decantation and/or filtration. The further step of isolating the compound comprising the structure of formula I, II or III preferably comprises removing substantially all proteins, peptides, amino acids, RNA and DNA, any endotoxins and glycolipids from the compound comprising the structure of formula I, II or III that may interfere with the subsequent isolation steps, preferably after it has been clarified. In this step, proteins and related impurities may be removed from the compound comprising the structure of formula I, II or III in a conventional manner. Preferably, proteins, salts, byproducts, colors, endotoxins and other related impurities are removed from the compounds comprising the structure of I, II or III by ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated carbon or carbon treatment, treatment with a nonionic surfactant, enzymatic digestion, tangential flow high efficiency filtration, tangential flow ultrafiltration, electrophoresis (e.g. using plate-polyacrylamide or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE)), affinity chromatography (using affinity ligands, including, for example, DEAE-Sepharose, poly-L-lysine and polymyxin-B, endotoxin selective adsorbent matrices), ion exchange chromatography (such as, but not limited to, cation exchange, anion exchange, mixed bed ion exchange, varus-valgus ligand attachment (inside-out LIGAND ATTACHMENT)), hydrophobic interaction chromatography and/or gel filtration (i.e. size exclusion chromatography), in particular by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography. In addition to size exclusion chromatography, the remaining proteins and related impurities are retained by the chromatographic medium or selected membranes.

In a further preferred embodiment, the process as described herein also provides for further purification of the compound of the invention comprising the structure of formula I, II or III. Further purification of the compound comprising the structure of formula I, II or III may be accomplished, for example, as follows: use of (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange to remove any residual DNA, proteins, LPS, endotoxins or other impurities. Alcohols (such as ethanol) and aqueous alcohol mixtures may also be used. Another purification step is accomplished by crystallization, evaporation or precipitation of the compound comprising the structure of formula I, II or III. Another purification step is drying, such as spray drying, lyophilization, spray freeze drying, freeze spray drying, belt drying (belt dry), vacuum belt drying, drum drying (drum dry), roller drying (roller dry), vacuum drum drying or vacuum roller drying of the resulting compound comprising the structure of formula I, II or III.

In an exemplary embodiment, the isolation and purification of the compound comprising the structure of formula I, II or III is performed in a process comprising the following steps in any order:

a) Contacting the culture or clarified form thereof with a nanofiltration membrane having a molecular weight cut-off (MWCO) of 600-3500Da, ensuring entrapment of the produced compound comprising the structure of formula I, II or III and allowing at least a portion of proteins, salts, byproducts, colors and other related impurities to pass through,

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

C) And collecting a retentate enriched in the compound comprising the structure of formula I, II or III in the form of a salt of the cation of the electrolyte.

In an alternative exemplary embodiment, the isolation and purification of the compound comprising the structure of formula I, II or III is performed in a process comprising the following steps in any order: the culture or clarified form thereof is subjected to two membrane filtration steps using different membranes, wherein-one membrane has a molecular weight cutoff of about 300 to about 500 daltons and-the other membrane has a molecular weight cutoff of about 600 to about 800 daltons.

In an alternative exemplary embodiment, the isolation and purification of the compound comprising the structure of formula I, II or III is performed in a process comprising the following steps in any order: comprising the step 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 alternative exemplary embodiments, the isolation and purification of the compound comprising the structure of formula I, II or III is performed as follows. The culture comprising the resulting compound comprising the structure of formula I, II or III, biomass, media components, and contaminants is applied to the following purification steps:

i) Separating the biomass from the culture,

Ii) treatment with a cation exchanger to remove positively charged species,

Iii) The anion exchanger is treated to remove negatively charged species,

Iv) a nanofiltration step and/or an electrodialysis step,

Wherein a purified solution is provided comprising the resulting compound comprising the structure of formula I, II or III in greater than or equal to 80% purity. Optionally, drying the purified solution by any one or more drying steps selected from the list consisting of spray drying, lyophilization, spray freeze drying, freeze spray drying, tape drying, vacuum tape drying, drum drying, vacuum drum drying, and vacuum drum drying.

In an alternative exemplary embodiment, the isolation and purification of the compound comprising the structure of formula I, II or III is performed in a process comprising the following steps in any order: enzyme treatment of the culture; removing biomass from the culture; ultrafiltration; nano-filtration; and a column chromatography step. Preferably, such column chromatography is a single column or multiple columns. Further preferably, the column chromatography step is simulated moving bed chromatography. Such simulated moving bed chromatography preferably comprises: i) At least 4 columns, wherein at least one column comprises a weak or strong cation exchange resin; and/or II) four zones I, II, III and IV with different flow rates; and/or iii) an eluent comprising water; and/or iv) an operating temperature of 15 degrees celsius to 60 degrees celsius.

In a specific embodiment, the present invention provides a resulting compound comprising a structure of formula I, II or III that is dried to a powder by any one or more drying steps selected from the list consisting of spray drying, lyophilization, spray freeze drying, freeze spray drying, belt drying, vacuum belt drying, drum drying, vacuum drum drying and vacuum drum drying, wherein the dried powder contains <15 wt% water, preferably <10 wt% water, more preferably <7 wt% water, most preferably <5 wt% water.

Another aspect of the invention provides the use of a cell as defined herein in a method of producing the compound comprising a structure of formula I, II or III, preferably in a method of producing the compound comprising a structure of formula I, II or III according to the invention. An alternative and/or additional aspect of the invention provides the use of a cell as defined herein in a method of producing a di-and oligosaccharide mixture comprising at least one compound comprising a structure of formula I, II or III, wherein R1 (when present) is a mono-, di-or oligosaccharide. An alternative and/or additional aspect of the invention provides the use of a cell as defined herein in a method of producing a charged and/or neutral disaccharide and oligosaccharide mixture comprising at least one compound comprising a structure of formula I, II or III wherein R1 (when present) is a monosaccharide, disaccharide or oligosaccharide. A preferred aspect of the invention provides the use of a cell as defined herein in a method of producing a sialylated and/or neutral di-and oligosaccharide mixture comprising at least one compound comprising a structure of formula I, II or III, wherein R1 (when present) is a mono-, di-or oligosaccharide. An alternative and/or additional aspect of the invention provides the use of a cell as defined herein in a method of producing an oligosaccharide mixture comprising at least one compound comprising a structure of formula I, II or III, wherein R1 (when present) is a monosaccharide, disaccharide or oligosaccharide. An alternative and/or additional aspect of the invention provides the use of a cell as defined herein in a method of producing a charged and/or neutral oligosaccharide mixture comprising at least one compound comprising a structure of formula I, II or III, wherein R1 (when present) is a monosaccharide, disaccharide or oligosaccharide. A preferred aspect of the invention provides the use of a cell as defined herein in a method of producing a sialylated and/or neutral oligosaccharide mixture comprising at least one compound comprising a structure of formula I, II or III, wherein R1 (when present) is a monosaccharide, disaccharide or oligosaccharide. A preferred aspect provides the use of a cell of the invention in a method of producing a mixture of Mammalian Milk Oligosaccharides (MMOs) comprising at least one compound comprising a structure of formula I, II or III, wherein R1 (when present) is a monosaccharide, disaccharide or oligosaccharide. Another aspect of the invention provides the use of a method as defined herein to produce the compound comprising a structure of formula I, II or III.

Furthermore, the invention also relates to the compounds comprising the structure of formula I, II or III obtained by the method according to the invention, and to the use of a polynucleotide, vector, host cell or polypeptide as described above for producing the compounds comprising the structure of formula I, II or III. The compounds comprising the structure of formula I, II or III may be used as food additives, prebiotics, synbiotics (symbiotic), for supplementing infant food, adult food or feed, adult animal feed, or as therapeutically or pharmaceutically active compounds or in cosmetic applications. With the novel method, the compound comprising the structure of formula I, II or III can be easily and efficiently provided without requiring a complicated, time-consuming and cost-effective synthesis process.

To identify the compounds comprising the structure of formula I, II or III produced in the cells as described herein, monomeric building blocks (e.g., monosaccharide or glycan unit composition), the anomeric configuration of the side chains, the presence and position of substituents, the degree of polymerization/molecular weight, and the mode of attachment can be identified by standard methods known in the art, such as, for example, methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas chromatography-mass spectrometry), MALDI-MS (matrix assisted laser desorption/ionization-mass spectrometry), ESI-MS (electrospray ionization-mass spectrometry), HPLC (high performance liquid chromatography with ultraviolet or refractive index detection), HPAEC-PAD (high performance anion exchange chromatography with pulsed amperometric detection), CE (capillary electrophoresis), IR (infrared)/raman spectroscopy, and NMR (nuclear magnetic resonance) spectroscopy techniques. The crystal structure can be analyzed using, for example, solid state NMR, FT-IR (fourier transform infrared spectroscopy), WAXS (wide angle X-ray scattering), and the like. The Degree of Polymerization (DP), DP distribution and polydispersity can be determined by, for example, viscometry and SEC (SEC-HPLC, high performance size exclusion chromatography). For the identification of the monomeric components of di-and/or oligosaccharides, methods such as acid-catalyzed hydrolysis, HPLC (high performance liquid chromatography) or GLC (gas-liquid chromatography) can be used (after conversion to the polyol acetate (alditol acetates)). To determine glycosidic linkages, the compound comprising the structure of formula I, II or III was methylated with methyl iodide and a strong base in DMSO, hydrolyzed to effect reduction of the partially methylated polyol, acetylated to methylated polyol acetate, and analyzed by GLC/MS (gas-liquid chromatography combined with mass spectrometry). To determine the glycan sequence, partial depolymerization is performed using acids or enzymes to determine structure. To identify the anomeric configuration, the compound comprising the structure of formula I, II or III is subjected to an enzymatic analysis, e.g., contacting it with an enzyme specific for a particular type of bond (e.g., β -galactosidase or α -glucosidase, etc.), and the product can be analyzed using NMR.

The isolated and preferably also purified compounds comprising the structure of formula I, II or III described herein are incorporated into a food (e.g., a human food or feed), dietary supplement, pharmaceutical ingredient, cosmetic ingredient, or pharmaceutical. In certain embodiments, the compound comprising the structure of formula I, II or III is mixed with one or more ingredients suitable for use in food, feed, dietary supplements, pharmaceutical ingredients, cosmetic ingredients, or pharmaceuticals.

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

"Prebiotics" are substances that promote the growth of microorganisms beneficial to the host, particularly microorganisms in the gastrointestinal tract. In certain embodiments, the dietary supplement provides a variety of prebiotics, including as prebiotics the compounds comprising the structure of formula I, II or III produced and/or purified by the methods disclosed in the present specification to promote the growth of one or more beneficial microorganisms. Examples of prebiotic ingredients for dietary supplements include other prebiotic molecules (e.g., HMO) and plant polysaccharides (such as inulin, pectin, b-glucan, and xylooligosaccharides). "probiotic" products typically contain viable microorganisms that replace or are added to the gastrointestinal microbial flora to benefit the recipient. Examples of such microorganisms include Lactobacillus species (e.g., lactobacillus acidophilus (L. Acidophilus) and Lactobacillus bulgaricus (L. Bulgaricum)), bifidobacterium species (e.g., bifidobacterium animalis (B. Animalis), bifidobacterium longum (B. Longum) and Bifidobacterium infantis (B. Infartis) (e.g., bi-26)) and Saccharomyces boulardii (Saccharomyces boulardii). In certain embodiments, the compounds comprising the structure of formula I, II or III produced and/or purified by the methods of the present description are administered orally in combination with such microorganisms.

Examples of other ingredients for dietary supplements include oligosaccharides (such as 2' -fucosyllactose, 3' -sialyllactose, 6' -sialyllactose), disaccharides (such as lactose), monosaccharides (such as glucose, galactose, L-fucose, sialic acid, glucosamine and N-acetylglucosamine), thickeners (such as gum arabic), acidity regulators (such as trisodium citrate), water, skim milk and flavoring agents.

In certain embodiments, the compound comprising the structure of formula I, II or III is incorporated into a human infant food (e.g., an infant formula). Infant formulas are generally processed foods for feeding infants as a complete or partial replacement for human breast milk. In certain embodiments, the infant formula is sold as a powder and is prepared for feeding to an infant with a bottle or cup by mixing with water. Infant formulas are often designed to have a composition that substantially mimics human breast milk. In certain embodiments, the compound comprising the structure of formula I, II or III produced and/or purified by the methods in this specification is included in an infant formula to provide a nutritional benefit similar to that provided by oligosaccharides in human breast milk. In certain embodiments, the compound comprising the structure of formula I, II or III 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, lactose-N-trisaccharide II, LNT, LNnT, lactose-N-fucopentaose I, lactose-N-neofucopentaose, lactose-N-fucopentaose II, lactose-N-fucopentaose III, lactose-N-fucopentaose V, lactose-N-neofucopentaose V, lactose-N-disaccharide hexaose I, lactose-N-disaccharide hexaose II, 6 '-galactosyl lactose, 3' -galactosyl lactose, lactose-N-hexaose, and lactose-N-neohexaose.

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

In certain embodiments, the concentration of oligosaccharides in the infant formula is about the same as the concentration of oligosaccharides typically present in human breast milk.

In certain embodiments, the compound comprising the structure of formula I, II or III is incorporated into a feed preparation, wherein the feed is selected from the list comprising: pet food, animal milk substitute, veterinary product, post-weaning feed or young animal feed.

As shown in the examples herein, the methods and cells of the invention preferably provide at least one of the following surprising advantages when compared to a host lacking the α -1, 2-fucosyltransferase of the invention (which has the galactosyl α -1, 2-fucosyltransferase activity for the galactose residues of LNB) for producing the compound comprising the structure of formula I, II or III:

higher titres (g/L) of the compounds comprising the structure of formula I, II or III,

Higher productivity r (g said compound comprising the structure of formula I, II or III per L/h),

A higher cell performance index CPI (g the compound comprising the structure of formula I, II or III/g X),

Higher specific productivity Qp (g the compound comprising the structure of formula I, II or III/g X/h),

Higher yields Ys calculated as sucrose (g of the compound comprising the structure of formula I, II or III per g of sucrose),

Higher sucrose uptake/conversion Qs (g sucrose/g X/h),

Higher lactose conversion/consumption rate rs (g lactose/h),

-Higher secretion of said compound comprising the structure of formula I, II or III, and/or

Higher growth rate of the production host.

In the context of the present invention, "X" means biomass, "g" means gram, "L" means liter, and "h" means hour. The "g-oligosaccharides" may be measured throughout the culture broth and/or supernatant.

Preferably, the methods and cells of the invention preferably provide at least one of the following surprising advantages when compared to a host lacking the α -1, 2-fucosyltransferase of the invention (which has the galactoside α -1, 2-fucosyltransferase activity for the galactose residues of LNB) for producing the compound comprising the structure of formula I, II or III:

A higher cell performance index CPI (g of the compound comprising the structure of formula I, II or III/g X), a higher specific productivity Qp (g of the compound comprising the structure of formula I, II or III/g X/h),

Higher sucrose uptake/conversion Qs (g sucrose/g X/h),

Higher lactose conversion/consumption rate rs (g lactose/h), and/or

-Higher secretion of the compound comprising the structure of formula I, II or III.

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 in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described above and below are those well known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Typically, the purification step is performed according to manufacturer specifications.

Other advantages may be derived from the specific embodiments and implementations. 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 alone without departing from the scope of the invention.

Furthermore, the invention relates to the following specific embodiments:

1. a method for producing a compound comprising a structure of formula I, II or III by a cell, preferably a single cell:

Wherein:

R ¹ is a monosaccharide, disaccharide, oligosaccharide, protein, glycoprotein, peptide, glycopeptide, lipid or glycolipid; r ² is a monosaccharide, disaccharide or oligosaccharide;

Wherein the method comprises the steps of:

i. Providing a cell expressing an alpha-1, 2-fucosyltransferase, and

Culturing and/or incubating the cells under conditions allowing expression of the compound comprising the structure of formula I, II or III,

Preferably, isolating the compound comprising the structure of formula I, II or III from the culture,

Characterized in that the alpha-1, 2-fucosyltransferase has a galactosylα -1, 2-fucosyltransferase activity on galactose residues of Gal-b1,3-GlcNAc (LNB, lactose-N-disaccharide), and

-A polypeptide belonging to the family of gt11 fucosyltransferases and comprising the motif X (not M) X (not F) XXXGNX (not N) [ ILMV ] X (not E, S) X (not E) XXXX (not F, S) X (not Y) XXXXX (not H, S, Y) shown in SEQ ID NO 38, wherein X may be any amino acid residue, or

-Is a polypeptide belonging to the family of gt74 fucosyltransferases, or

-Comprising a polypeptide sequence as indicated in any of SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 or 37, preferably any of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07, 08 or 09, more preferably any of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07 or 08, even more preferably any of SEQ ID NOs 05, 06, 07 or 08, most preferably any of SEQ ID NOs 01, 02, 03 or 04, or

-Is a functional homolog, variant or derivative of SEQ ID No. 03, said functional homolog, variant or derivative having at least 15.0% overall sequence identity to the full length of said polypeptide of SEQ ID No. 03, or

-Is a functional homolog, variant or derivative of any one of SEQ ID NOs 15, 34, 35, 36 or 37 having at least 22.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID NOs 15, 34, 35, 36 or 37, or

-Is any one of SEQ ID NOs 05, 08, 11, 21, 30 or 31, preferably a functional homolog, variant or derivative of SEQ ID NO 05 or 08, said functional homolog, variant or derivative having at least 30.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID NO 05, 08, 11, 21, 30 or 31, preferably SEQ ID NO 05 or 08, or

-Is any one of SEQ ID NOs 06, 07, 09, 19, 25, 27, 32 or 33, preferably any one of SEQ ID NOs 06, 07 or 09, more preferably a functional homolog, variant or derivative of SEQ ID NOs 06 or 07 having at least 35.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID NOs 06, 07, 09, 19, 25, 27, 32 or 33, preferably any one of SEQ ID NOs 06, 07 or 09, more preferably SEQ ID NOs 06 or 07, or

-Is any one of SEQ ID NOs 02, 04, 14, 16, 17 or 28, preferably a functional homolog, variant or derivative of SEQ ID NO 02 or 04, said functional homolog, variant or derivative having at least 40.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID NO 02, 04, 14, 16, 17 or 28, preferably SEQ ID NO 02 or 04, or

-Is any one of SEQ ID No. 01, 10, 12, 13, 18, 20, 22, 24 or 26, preferably a functional homolog, variant or derivative of SEQ ID No. 01, said functional homolog, variant or derivative having at least 45.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID No. 01, 10, 12, 13, 18, 20, 22, 24 or 26, preferably SEQ ID No. 01, or

-Is a functional homolog, variant or derivative of SEQ ID No. 23 having at least 50.0% overall sequence identity to the full length of said polypeptide of SEQ ID No. 23, or

-Is a functional homolog, variant or derivative of SEQ ID No. 29 having at least 70.0% overall sequence identity to the full length of said polypeptide of SEQ ID No. 29, or

-Is a functional fragment comprising an oligopeptide sequence of at least 10 consecutive amino acid residues from any one of SEQ ID NOs 03, 05, 11, 15, 21, 31, 34, 35, 36 or 37, preferably SEQ ID NOs 03 or 05, more preferably SEQ ID NO 03; or (b)

-Is a functional fragment comprising an oligopeptide sequence of at least 13 consecutive amino acid residues from any one of SEQ ID NOs 06, 08, 13, 17, 19, 20, 25, 28 or 30, preferably SEQ ID NOs 06 or 08; or (b)

-Is a functional fragment comprising an oligopeptide sequence of at least 15 consecutive amino acid residues from any one of SEQ ID NOs 04, 07, 09, 10, 16, 26, 27, 32 or 33, preferably SEQ ID NOs 04, 07 or 09, more preferably SEQ ID NOs 04 or 07, most preferably SEQ ID NOs 04; or (b)

-Is a functional fragment comprising an oligopeptide sequence of at least 18 consecutive amino acid residues from any one of SEQ ID NOs 01, 02, 14, 18, 22 or 24, preferably SEQ ID NOs 01 or 02; or (b)

-Is a functional fragment of an oligopeptide sequence comprising at least 22 consecutive amino acid residues from any one of SEQ ID NOs 12, 23 or 29.

2. The method of embodiment 1, wherein the α -1, 2-fucosyltransferase has additional galactosidase α -1, 2-fucosyltransferase activity for galactose residues of the non-reducing end of Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (LNT, lactose-N-tetrasaccharide), and wherein the α -1, 2-fucosyltransferase:

-a polypeptide belonging to the family of gt74 fucosyltransferases and comprising the motif [ DE ] CC [ FWY ] XXX (not D, E) (Xn) FWY ] X [ ILMV ] [ DE ] [ DE ] shown in SEQ ID NO:39, wherein X may be any amino acid residue and wherein n is 10-40, or

-Comprising a polypeptide sequence as set forth in any of SEQ ID NO:01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 or 18, preferably any of SEQ ID NO:01, 02, 03, 04, 05, 06, 07, 08 or 09, more preferably any of SEQ ID NO:01, 02, 03, 04, 05, 06, 07 or 08, even more preferably any of SEQ ID NO:05, 06, 07 or 08, most preferably any of SEQ ID NO:01, 02, 03 or 04, or

-Is any one of SEQ ID No. 03 or 15, preferably a functional homolog, variant or derivative of SEQ ID No. 03, said functional homolog, variant or derivative having at least 20.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID No. 03 or 15, preferably SEQ ID No. 03, or

-Is any one of SEQ ID No. 05, 08 or 11, preferably a functional homolog, variant or derivative of SEQ ID No. 05 or 08, said functional homolog, variant or derivative having at least 30.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID No. 05, 08 or 11, preferably SEQ ID No. 05 or 08, or

-Is any one of SEQ ID No. 02, 04, 06, 07, 09 or 17, preferably any one of SEQ ID No. 02, 04, 06, 07 or 09, more preferably any one of SEQ ID No. 02, 04, 06 or 07, even more preferably any one of SEQ ID No. 02 or 04, a functional homolog, variant or derivative thereof having at least 37.50% overall sequence identity to the full length of any one of said polypeptides of SEQ ID No. 02, 04, 06, 07 or 17, preferably any one of SEQ ID No. 02, 04, 06, 07 or 09, more preferably any one of SEQ ID No. 02, 04, 06 or 07, even more preferably any one of SEQ ID No. 02 or 04, or

-Is any one of SEQ ID No. 01, 10, 12, 13, 14, 16 or 18, preferably a functional homolog, variant or derivative of SEQ ID No. 01, said functional homolog, variant or derivative having at least 45.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID No. 01, 10, 12, 13, 14, 16 or 18, preferably SEQ ID No. 01, or

-Is a functional fragment comprising an oligopeptide sequence of at least 10 consecutive amino acid residues from any one of SEQ ID NOs 03, 05, 11 or 15, preferably SEQ ID NOs 03 or 05, or

-Is a functional fragment comprising an oligopeptide sequence of at least 13 consecutive amino acid residues from any one of SEQ ID NOs 06, 08, 13 or 17, preferably SEQ ID NOs 06 or 08, or

-Is a functional fragment comprising an oligopeptide sequence of at least 15 consecutive amino acid residues from any one of SEQ ID NOs 04, 07, 09, 10, 16, preferably SEQ ID NOs 04, 07 or 09, more preferably SEQ ID NOs 04 or 07, even more preferably SEQ ID NOs 04, or

-Is a functional fragment comprising an oligopeptide sequence of at least 18 consecutive amino acid residues from any one of SEQ ID NO 01, 02, 14 or 18, preferably SEQ ID NO 01 or 02, or

-Is a functional fragment of an oligopeptide sequence comprising at least 20 consecutive amino acid residues from SEQ ID No. 12.

3. The method of embodiment 2, wherein the alpha-1, 2-fucosyltransferase has no additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose or has additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose but less than the additional galactosidase alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of LNT, and

-Comprising a polypeptide sequence as set forth in any of SEQ ID NOs 01, 02, 03, 04, 07, 09, 10, 12, 13, 14, 15, 16, 17 or 18, preferably any of SEQ ID NOs 01, 02, 03, 04, 07 or 09, more preferably any of SEQ ID NOs 01, 02, 03, 04 or 07, even more preferably any of SEQ ID NOs 01, 02, 03 or 04, or

-Is any one of SEQ ID NOs 02, 04, 07, 09 or 17, preferably any one of SEQ ID NOs 02, 04, 07 or 09, more preferably SEQ ID NOs 02, 04 or 07, even more preferably a functional homolog, variant or derivative of SEQ ID NOs 02 or 04 having at least 37.50% overall sequence identity to the full length of any one of said polypeptides of SEQ ID NOs 02, 04, 06, 07, 09 or 17, preferably SEQ ID NOs 02, 04, 07 or 09, more preferably SEQ ID NOs 02, 04 or 07, even more preferably SEQ ID NOs 02 or 04, or

-Is a functional fragment of an oligopeptide sequence comprising at least 10 consecutive amino acid residues from either of SEQ ID NO:03 or 15, preferably SEQ ID NO:03, or

-Is a functional fragment of an oligopeptide sequence comprising at least 13 consecutive amino acid residues from either of SEQ ID NO 13 or 17, or

-Is a functional fragment comprising an oligopeptide sequence of at least 15 consecutive amino acid residues from any one of SEQ ID NOs 04, 07, 09, 10 or 16, preferably SEQ ID NOs 04, 07 or 09, more preferably SEQ ID NOs 04 or 07, even more preferably SEQ ID NOs 04, or

4. The method of embodiment 2, wherein the alpha-1, 2-fucosyltransferase has additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose and additional galactosidase alpha-1, 2-fucosyltransferase activity that is higher than its galactose residue for the non-reducing end of LNT, and

-A polypeptide sequence comprising any of SEQ ID NO 05, 06, 08 or 11, preferably SEQ ID NO 05, 06 or 08, or

-Is any one of SEQ ID No. 05, 06, 08 or 11, preferably a functional homolog, variant or derivative of SEQ ID No. 05, 06 or 08, said functional homolog, variant or derivative having at least 35.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID No. 05, 06, 08 or 11, preferably SEQ ID No. 05, 06 or 08, or

-Is a functional fragment comprising an oligopeptide sequence of at least 13 consecutive amino acid residues from SEQ ID No. 05, 06, 08 or 11, preferably SEQ ID No. 05, 06 or 08.

5. The method of embodiment 1, wherein the α -1, 2-fucosyltransferase has no galactosidase α -1, 2-fucosyltransferase activity for galactose residues of the non-reducing end of the LNT, and

-A polypeptide sequence comprising any one of SEQ ID NOs 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37, or

-Is a functional homolog, variant or derivative of any one of SEQ ID NOs 34, 35, 36 or 37 having at least 22.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID NOs 34, 35, 36 or 37, or

-Is a functional homolog, variant or derivative of any of SEQ ID NOs 21, 30 or 31 having at least 30.0% overall sequence identity to the full length of any of said polypeptides of EQ ID NOs 21, 30 or 31, or

-Is a functional homolog, variant or derivative of any one of SEQ ID NOs 19, 25, 27, 32 or 33 having at least 35.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID NOs 19, 25, 27, 32 or 33, or

-Is a functional homolog, variant or derivative of any one of SEQ ID NOs 20, 22, 24, 26 or 28 having at least 45.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID NOs 20, 22, 24, 26 or 28, or

-Is a functional fragment of an oligopeptide sequence comprising at least 10 consecutive amino acid residues from any one of SEQ ID NOs 21, 31, 34, 35, 36 or 37, or

-Is a functional fragment of an oligopeptide sequence comprising at least 13 consecutive amino acid residues from any one of SEQ ID NOs 19, 20, 25, 28 or 30, or

-Is a functional fragment of an oligopeptide sequence comprising at least 15 consecutive amino acid residues from any one of SEQ ID NOs 26, 27, 32 or 33, or

-Is a functional fragment of an oligopeptide sequence comprising at least 17 consecutive amino acid residues from either one of SEQ ID NO. 22 or 24, or

-Is a functional fragment of an oligopeptide sequence comprising at least 22 consecutive amino acid residues from either of SEQ ID NOs 23 or 29.

6. The method of embodiment 5, wherein the alpha-1, 2-fucosyltransferase has NO galactosylα -1, 2-fucosyltransferase activity on lactose or has additional galactosylα -1, 2-fucosyltransferase activity on lactose but less than 3.0% of the galactosylα -1, 2-fucosyltransferase activity on lactose of the alpha-1, 2-fucosyltransferase of SEQ ID NO 06, and

-A polypeptide sequence comprising any one of SEQ ID NOs 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 33, 34, 35, 36 or 37, or

-Is a functional homolog, variant or derivative of either of SEQ ID No. 21 or 30 having at least 30.0% overall sequence identity to the full length of either of said polypeptides of SEQ ID No. 21 or 30, or

-Is a functional homolog, variant or derivative of any one of SEQ ID NOs 19, 25, 27 or 33 having at least 35.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID NOs 19, 25, 27 or 33, or

-Is a functional homolog, variant or derivative of any one of SEQ ID NOs 20, 22, 24 or 26 having at least 45.0% overall sequence identity to the full length of any one of said polypeptides of SEQ ID NOs 20, 22, 24 or 26, or

-Is a functional fragment of an oligopeptide sequence comprising at least 10 consecutive amino acid residues from any one of SEQ ID NOs 21, 34, 35, 36 or 37, or

-Is a functional fragment of an oligopeptide sequence comprising at least 13 consecutive amino acid residues from any one of SEQ ID NOs 19, 20, 25 or 30, or

-Is a functional fragment of an oligopeptide sequence comprising at least 15 consecutive amino acid residues from any one of SEQ ID NO 26, 27 or 33, or

-Is a functional fragment of an oligopeptide sequence comprising at least 17 consecutive amino acid residues from SEQ ID NO. 22 or 24, or

-Is a functional fragment of an oligopeptide sequence comprising at least 20 consecutive amino acid residues from SEQ ID No. 23.

7. The method of embodiment 5, wherein the alpha-1, 2-fucosyltransferase has additional galactosylα -1, 2-fucosyltransferase activity on lactose and is 4.0 to 20.0% of the galactosylα -1, 2-fucosyltransferase activity on lactose of the alpha-1, 2-fucosyltransferase of SEQ ID NO 06, and

-Comprising a polypeptide sequence as set forth in any one of SEQ ID NOs 28, 29, 31 or 32, or

-Is a functional homolog, variant or derivative of either of SEQ ID No. 31 or 32 having at least 35.0% overall sequence identity to the full length of either of said polypeptides of SEQ ID No. 31 or 32, or

-Is a functional homolog, variant or derivative of SEQ ID No. 28 having at least 40.0% overall sequence identity to the full length of said polypeptide of SEQ ID No. 28, or

-Is a functional fragment of an oligopeptide sequence comprising at least 10 consecutive amino acid residues from SEQ ID NO. 31, or

-Is a functional fragment of an oligopeptide sequence comprising at least 14 consecutive amino acid residues from either one of SEQ ID NO 28 or 32, or

-Is a functional fragment of an oligopeptide sequence comprising at least 22 consecutive amino acid residues from SEQ ID No. 29.

8. The method according to any one of the preceding embodiments, wherein the cell is modified in terms of expression or activity of any one of the alpha-1, 2-fucosyltransferases.

9. The method of any one of the preceding embodiments, wherein the cell is capable of producing one or more nucleotide-activating sugars selected from the list comprising: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2, 6-dideoxy-L-arabino-4-hexulose, UDP-2-acetamido-2, 6-dideoxy-L-lyxol-4-hexulose, UDP-N-acetyl-L-rhamnose (UDP-L-RhaNAc or UDP-2-acetamido-2, 6-dideoxy-L-mannose), DP-N-acetylfucose, UDP-N-acetylfucose (UDP-L-52 or UDP-2-acetamido-2, 6-dideoxy-L-5-fucose), UDP-N-acetylgalactosamine (UDP-L-FucNAc or UDP-2-acetamido-N-5-mannosamine), UDP-N-acetylgalactosamine (UDP-N-RhaNAc or UDP-2-acetamido-N-6-acetylgalactosamine), UDP-N-acetyl-L-quiniosamine (UDP-L-QuiNAc or UDP-2-acetamido-2, 6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac)、CMP-Neu4Ac、CMP-Neu5Ac9N₃、CMP-Neu4,5Ac₂、CMP-Neu5,7Ac₂、CMP-Neu5,9Ac₂、CMP-Neu5,7(8,9)Ac₂、CMP-N- hydroxyacetyl neuraminic acid (CMP-Neu 5 Gc), GDP-rhamnose and UDP-xylose.

10. The method of any one of the preceding embodiments, 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-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine 6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine 1-phosphate acetyltransferase, N-acetylneuraminic acid synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid 9-phosphate synthase, N-acylneuraminic acid 9-phosphate phosphatase, N-acylneuraminic acid cytidylyltransferase, galactose 1-epimerase, galactokinase, and, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein said cell is modified in terms of expression or activity of any of said polypeptides.

11. The method according to any one of the preceding embodiments, wherein the cells express one or more glycosyltransferases selected from the list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosyltransferase, N-acetylmannosyltransferase, xylosyltransferase, glucuronyltransferase, galacturonate transferase, glucosaminyltransferase, N-glycolylneuraminidase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-arabinoxylan-Zhuo Tangan aminotransferase, UDP-N-acetylglucosaminenolpyruvylase and fucosyltransferase,

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

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 the list comprising an alpha-1, 2-mannosyltransferase, an alpha-1, 3-mannosyltransferase and an 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 an alpha-1, 3-N-acetylgalactosamine transferase,

Preferably, the cell is modified in terms of expression or activity of any of the glycosyltransferases.

12. The method according to any one of the preceding embodiments, wherein the compound comprising the structure of formula I, II or III is an oligosaccharide, preferably the oligosaccharide is a Mammalian Milk Oligosaccharide (MMO), more preferably a Human Milk Oligosaccharide (HMO).

13. The method according to any of the preceding embodiments, wherein the compound comprising the structure of formula I, II or III is a charged, preferably sialylated compound or is a neutral compound,

Preferably wherein the compound comprising the structure of formula I, II or III is a charged, preferably sialylated oligosaccharide or a neutral oligosaccharide.

14. The method of any one of the preceding embodiments, wherein the compound comprising the structure of formula I, II or III is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R comprising one R group selected from the list comprising monosaccharides, disaccharides, or oligosaccharides,

Preferably wherein the compound comprising the structure of formula I, II or III is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R comprising an R group selected from the list comprising monosaccharides, disaccharides or oligosaccharides,

More preferably wherein the compound comprising the structure of formula I, II or III is lactose-N-fucopentaose I (LNFP-I, fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc).

15. The method of any one of the preceding embodiments, wherein the cells use one or more precursors to produce the compound comprising the structure of formula I, II or III, the precursors being fed to the cells from a culture medium.

16. The method of any one of the preceding embodiments, wherein the cells produce one or more precursors for producing the compound comprising the structure of formula I, II or III.

17. The method of any one of embodiments 15 or 16, wherein the precursor for producing the compound comprising the structure of formula I, II or III is fully converted to the compound comprising the structure of formula I, II or III.

18. The method of any one of the preceding embodiments, wherein the cell is capable of producing a compound comprising a structure of formula IV, V or VI:

Wherein:

19. The method of any one of the preceding embodiments, wherein the cell produces the compound comprising the structure of formula I, II or III inside the cell, and wherein a portion or substantially all of the produced compound comprising the structure of formula I, II or III remains inside the cell and/or is excreted outside the cell by passive or active transport.

20. The method according to any one of the preceding embodiments, wherein the cell expresses a membrane transporter protein or a polypeptide having a transport activity, whereby the compound is transported across the outer membrane of the cell wall,

Preferably, the cell is modified in terms of expression or activity of the membrane transporter protein or a peptide having transport activity.

21. The method of embodiment 20, wherein the membrane transporter or a polypeptide having translocator activity is selected from the list comprising: transporter, P-P bond hydrolysis-driven transporter, b-barrel porin, auxiliary transporter, putative transporter and phosphate transport-driven group translocator,

Preferably, the transporter includes an MFS transporter, a sugar efflux transporter and an iron conjugate export protein,

Preferably, the P-P bond hydrolytically driven transporter includes ABC transporter and iron conjugate exporter.

22. The method of any one of embodiments 20 or 21, wherein the membrane transporter or a polypeptide having transport activity controls the flow of the compound comprising the structure of formula I, II or III and/or one or more precursors and/or receptors for the production of the compound comprising the structure of formula I, II or III to the outer membrane of the cell wall.

23. The method according to any one of embodiments 20 to 22, wherein the membrane transporter or the polypeptide having transport activity provides improved production and/or allowable and/or enhanced efflux of the compound comprising the structure of formula I, II or III.

24. The method of any one of the preceding embodiments, wherein the cell is a genetically engineered cell.

25. The method of embodiment 24, wherein the cell is modified with one or more gene expression modules, wherein expression from any one of the expression modules is constitutive or produced by a natural inducer.

26. The method of any one of embodiments 24 or 25, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein.

27. The method of any one of embodiments 24-26, wherein the cell comprises a modification that reduces acetate production.

28. The method according to any one of embodiments 24 to 27, wherein the cell comprises reduced or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the following proteins, said proteins comprising β -galactosidase, galactosido-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphase, EIICBA-Nag, UDP-glucose: undecaprenyl-phosphoglucose-1-phosphotransferase, L-fucokinase, L-fucose isomerase, N-acetylneuraminic acid lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phospho2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphouridyltransferase, glucose-1-phosphoadenyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphoisomerase, aerobic respiration control protein, transcription repressor IclR, lon protease, glucose-specific translocated phosphotransferase IIBC component ptsG, glucose-specific translocated Phosphotransferase (PTS) IIBC component malX, enzyme IIA ^Glc, beta-glucosidase II, phosphoryl transferase specific PTS protein 3525 and phosphoPTS protein 383824 Alcohol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphoacetyltransferase, pyruvate decarboxylase.

29. The method according to any one of the preceding embodiments, wherein the cell is capable of producing phosphoenolpyruvate (PEP).

30. The method according to any one of the preceding embodiments, wherein the cell is modified to enhance the production and/or supply of phosphoenolpyruvate (PEP).

31. The method of any one of the preceding embodiments, wherein the cell comprises an at least partially inactivated catabolic pathway of a selected monosaccharide, disaccharide or oligosaccharide that is involved in and/or necessary for the production of the compound comprising the structure of formula I, II or III.

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

33. The method according to any one of the preceding embodiments, wherein the cells produce 90g/L or more of the compound comprising the structure of formula I, II or III in the whole culture and/or supernatant, and/or wherein the compound comprising the structure of formula I, II or III has a purity of at least 80% in the whole culture and/or supernatant, measured as the total amount of the compound comprising the structure of formula I, II or III and its precursors in the whole culture and/or supernatant, respectively.

34. The method according to any one of the preceding embodiments, wherein the cell is a bacterium, fungus, yeast, plant cell, animal cell or protozoan cell,

Preferably, the bacterium is an E.coli strain, more preferably an E.coli strain as K-12 strain, even more preferably the E.coli K-12 strain is E.coli MG1655,

Preferably, the fungus belongs to a genus selected from the group comprising: rhizopus (Rhizopus), pelargonium (Dictyostelium), penicillium (Penicillium), mucor (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), star Mo Jiaomu (STARMERELLA), kluyveromyces (Kluyveromyces) or Debaromyces (Debaromyces),

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

Preferably, the animal cells are derived from non-human mammals, birds, fish, invertebrates, reptiles, amphibians or insects, or genetically modified cell lines derived from human cells other than embryonic stem cells, more preferably, the human and non-human mammalian cells are epithelial cells, embryonic kidney cells, fibroblasts, COS cells, 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 (Bombyx mori), cabbage looper (Mamestra brassicae), trichoplusia ni (Trichoplusia ni) or Drosophila melanogaster (Drosophila melanogaster),

Preferably, the protozoan cell is a leishmania tarabica (LEISHMANIA TARENTOLAE) cell.

35. The method of embodiment 34, wherein the cell is a living gram-negative bacterium comprising reduced or eliminated synthesis of: poly-N-acetyl-glucosamine (PNAG), enterobacter Common Antigen (ECA), cellulose, capsular polysaccharide, core oligosaccharide, osmoregulation Periplasmic Glucan (OPG), glycerol glucoside, glycans and/or trehalose.

36. The method according to any one of the preceding embodiments, wherein the cells are stably cultured in a medium.

37. The method of any one of the preceding embodiments, wherein the conditions comprise:

-using a medium comprising at least one precursor and/or acceptor for producing the compound comprising the structure of formula I, II or III, and/or

-Adding at least one precursor and/or acceptor feed to the culture medium, said precursor and/or acceptor feed being used to produce said compound comprising the structure of formula I, II or III.

38. The method according to any of the preceding embodiments, comprising at least one of the following steps:

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

iii) Adding at least one precursor and/or acceptor feed to the medium in the reactor, wherein the total reactor volume ranges from 250mL (milliliters) to 10.000m ³ (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium is no more than 3 times, preferably no more than 2 times, more preferably less than 2 times the volume of the medium prior to adding the precursor and/or acceptor feed, and wherein the pH of the precursor and/or acceptor feed is preferably set at 3 to 7, and wherein the temperature of the precursor and/or acceptor feed is preferably maintained at 20 to 80 ℃;

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

v) adding at least one precursor and/or acceptor feed to the culture medium in a continuous manner by means of a feed solution for 1,2, 3, 4, 5 days, and wherein the pH of the feed solution is preferably set at 3 to 7, and wherein the temperature of the pre-feed solution is preferably maintained at 20 to 80 ℃;

The method produces the compound comprising the structure of formula I, II or III having 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.

39. The method according to any one of embodiments 1 to 37, 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 lactose per liter of initial reactor volume, wherein the reactor volume ranges from 250mL to 10.000m ³ (cubic meters);

ii) adding a lactose feed to the 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 lactose per liter of initial reactor volume, wherein the reactor volume ranges from 250mL to 10.000m ³ (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium is no more than 3 times, preferably no more than 2 times, more preferably less than 2 times the volume of the medium prior to adding the lactose feed;

iii) Adding a lactose feed to 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 lactose per liter of initial reactor volume, wherein the reactor volume ranges from 250mL to 10.000m ³ (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium is no more than 3 times, preferably no more than 2 times, more preferably less than 2 times the volume of the medium before adding the lactose feed, and wherein preferably the pH of the lactose feed is set at 3 to 7, and wherein preferably the temperature of the pre-lactose feed is maintained at 20 ℃ to 80 ℃;

iii) Adding lactose feed to the medium in a continuous manner by means of a feed solution for 1 day, 2 days, 3 days, 4 days, 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 the pH of the feed solution is preferably set at 3 to 7, and wherein the temperature of the feed solution is preferably maintained at 20 ℃ to 80 ℃;

40. The method according to embodiment 39, wherein lactose feeding is accomplished 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.

41. The method according to embodiment 39 or 40, wherein the lactose feeding is done by adding lactose to the culture in a concentration such that a lactose concentration of at least 5mM, preferably 10mM or 30mM is obtained throughout the production phase of the culture.

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

43. The method according to any one of the preceding embodiments, wherein the cells are 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, maltooligosaccharide, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn steep liquor, high fructose syrup, acetate, citrate, lactate and pyruvate.

44. The method according to any one of the preceding embodiments, wherein the medium contains at least one precursor selected from the group consisting of: lactose, galactose, fucose, sialic acid, glcNAc, galNAc, lactose-N-disaccharide (LNB), N-acetyllactosamine (LacNAc).

45. The method according to any of the preceding embodiments, wherein the first stage of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to a medium comprising a precursor, preferably lactose, followed by a second stage, wherein only the carbon-based substrate, preferably glucose or sucrose, is added to the medium.

46. The method according to any one of embodiments 1 to 44, wherein the first stage of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to a medium comprising a precursor, preferably lactose, preferably glucose or sucrose, followed by a second stage, wherein the carbon-based substrate, preferably glucose or sucrose, and the precursor, preferably lactose, are added to the medium.

47. The method according to any one of the preceding embodiments, wherein the cell produces a mixture of charged, preferably sialylated and/or neutral di-and oligosaccharides, said mixture comprising at least one compound comprising a structure of formula I, II or III, wherein R1 is mono-, di-or oligosaccharide when present.

48. The method according to any one of the preceding embodiments, wherein the cells produce a mixture of charged, preferably sialylated and/or neutral oligosaccharides, said mixture comprising at least one compound comprising a structure of formula I, II or III, wherein R1 is a monosaccharide, disaccharide or oligosaccharide when present.

49. The method of any one of the preceding embodiments, wherein the isolating comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated carbon or carbon treatment, treatment with nonionic surfactants, enzymatic digestion, tangential flow high efficiency filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography.

50. The method of any one of the preceding embodiments, further comprising purifying the compound comprising the structure of formula I, II or III from the cell.

51. The method of embodiment 50, 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, using alcohols, using aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying, lyophilization, spray freeze drying, freeze spray drying, belt drying, vacuum belt drying, drum drying, vacuum drum drying or vacuum drum drying.

52. A cell genetically engineered to produce a compound comprising a structure of formula I, II or III:

Wherein:

Wherein the cell is capable of expressing, preferably expressing, an alpha-1, 2-fucosyltransferase,

-Is a polypeptide belonging to the family of gt74 fucosyltransferases, or

53. The cell of embodiment 52, wherein the α -1, 2-fucosyltransferase has additional galactosidase α -1, 2-fucosyltransferase activity for galactose residues of the non-reducing end of Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (LNT, lactose-N-tetrasaccharide), and wherein the α -1, 2-fucosyltransferase:

54. The cell of embodiment 53, wherein the α -1, 2-fucosyltransferase has no additional galactosidase α -1, 2-fucosyltransferase activity for lactose or has additional galactosidase α -1, 2-fucosyltransferase activity for lactose but less than the additional galactosidase α -1, 2-fucosyltransferase activity for galactose residues of the non-reducing end of LNT, and

55. The cell of embodiment 53, wherein the α -1, 2-fucosyltransferase has additional galactosidase α -1, 2-fucosyltransferase activity for lactose and additional galactosidase α -1, 2-fucosyltransferase activity that is higher than that for galactose residues of the non-reducing end of LNT, and

56. The cell of embodiment 52, wherein the alpha-1, 2-fucosyltransferase has no galactosidase alpha-1, 2-fucosyltransferase activity for galactose residues of the non-reducing end of an LNT, and

57. The cell of embodiment 56, wherein the α -1, 2-fucosyltransferase has NO galactosylα -1, 2-fucosyltransferase activity on lactose or has additional galactosylα -1, 2-fucosyltransferase activity on lactose but less than 3.0% of the α -1, 2-fucosyltransferase activity on lactose of α -1, 2-fucosyltransferase of SEQ ID NO 06, and

58. The cell of embodiment 56, wherein the α -1, 2-fucosyltransferase has additional galactosylα -1, 2-fucosyltransferase activity on lactose and is 4.0 to 20.0% of the galactosylα -1, 2-fucosyltransferase activity on lactose of α -1, 2-fucosyltransferase of SEQ ID NO 06, and

59. The cell of any one of embodiments 52 to 58, wherein the cell is modified with one or more gene expression modules, wherein expression from any one of the expression modules is constitutive or produced by a natural inducer.

60. The cell according to any one of embodiments 52 to 59, wherein the cell is modified in expression or activity of any one of the α -1, 2-fucosyltransferases.

61. The cell of any one of embodiments 52 to 60, wherein the cell is capable of producing one or more nucleotide-activating sugars selected from the list comprising: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2, 6-dideoxy-L-arabino-4-hexulose, UDP-2-acetamido-2, 6-dideoxy-L-lyxol-4-hexulose, UDP-N-acetyl-L-rhamnose (UDP-L-RhaNAc or UDP-2-acetamido-2, 6-dideoxy-L-mannose), DP-N-acetylfucose, UDP-N-acetylfucose (UDP-L-52 or UDP-2-acetamido-2, 6-dideoxy-L-5-fucose), UDP-N-acetylgalactosamine (UDP-L-FucNAc or UDP-2-acetamido-N-5-mannosamine), UDP-N-acetylgalactosamine (UDP-N-RhaNAc or UDP-2-acetamido-N-6-acetylgalactosamine), UDP-N-acetyl-L-quiniosamine (UDP-L-QuiNAc or UDP-2-acetamido-2, 6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac)、CMP-Neu4Ac、CMP-Neu5Ac9N₃、CMP-Neu4,5Ac₂、CMP-Neu5,7Ac₂、CMP-Neu5,9Ac₂、CMP-Neu5,7(8,9)Ac₂、CMP-N- hydroxyacetyl neuraminic acid (CMP-Neu 5 Gc), GDP-rhamnose and UDP-xylose.

62. The cell of any one of embodiments 52 to 61, 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-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine 6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine 1-phosphate acetyltransferase, N-acetylneuraminic acid synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid 9-phosphate synthase, N-acylneuraminic acid 9-phosphate phosphatase, N-acylneuraminic acid cytidylyltransferase, galactose 1-epimerase, galactokinase, and, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein said cell is modified in terms of expression or activity of any of said polypeptides.

63. The cell according to any one of embodiments 52 to 62, wherein the cell expresses one or more glycosyltransferases selected from the list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosyltransferase, N-acetylmannosyltransferase, xylosyltransferase, glucuronyltransferase, galacturonate transferase, glucosaminyltransferase, N-glycolylneuraminidase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-arabinoxylan-Zhuo Tangan aminotransferase, UDP-N-acetylglucosaminenolpyruvylase and fucosyltransferase,

64. The cell according to any one of embodiments 52 to 63, wherein the compound comprising the structure of formula I, II or III is an oligosaccharide, preferably the oligosaccharide is a Mammalian Milk Oligosaccharide (MMO), more preferably a Human Milk Oligosaccharide (HMO).

65. The cell according to any of embodiments 52 to 64, wherein the compound comprising the structure of formula I, II or III is a charged, preferably sialylated compound or a neutral compound,

66. The cell of any one of embodiments 52-65, wherein the compound comprising the structure of formula I, II or III is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R comprising one R group selected from the list comprising monosaccharides, disaccharides, or oligosaccharides,

67. The cell of any one of embodiments 52-66, wherein the cell uses one or more precursors to produce the compound comprising the structure of formula I, II or III, the precursors being fed to the cell from a culture medium.

68. The cell of any one of embodiments 52-67, wherein the cell produces one or more precursors for producing the compound comprising the structure of formula I, II or III.

69. The cell of any one of embodiments 67 or 68, wherein the precursor for producing the compound comprising the structure of formula I, II or III is fully converted to the compound comprising the structure of formula I, II or III.

70. The cell of any one of embodiments 52 to 69, wherein the cell is capable of producing a compound comprising a structure of formula IV, V or VI:

Wherein:

71. The cell of any one of embodiments 52-70, wherein the cell produces the compound comprising the structure of formula I, II or III inside the cell, and wherein a portion or substantially all of the produced compound comprising the structure of formula I, II or III remains inside the cell and/or is excreted outside the cell by passive or active transport.

72. The cell according to any one of embodiments 52 to 71, wherein the cell expresses a membrane transporter or a polypeptide having a transport activity, whereby a compound is transported across the outer membrane of a cell wall,

73. The cell of embodiment 72, wherein the membrane transporter or a polypeptide having a transport activity is selected from the list comprising: transporter, P-P bond hydrolysis-driven transporter, b-barrel porin, auxiliary transporter, putative transporter and phosphate transport-driven group translocator,

74. The cell of any one of embodiments 72 or 73, wherein the membrane transporter or a polypeptide having transport activity controls the flow of the compound comprising the structure of formula I, II or III and/or one or more precursors and/or receptors for the production of the compound comprising the structure of formula I, II or III to the outer membrane of the cell wall.

75. The cell of any one of embodiments 72 to 74, wherein the membrane transporter or a polypeptide having transport activity provides improved production and/or permissive and/or enhanced efflux of the compound comprising the structure of formula I, II or III.

76. The cell according to any one of embodiments 52 to 75, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein.

77. The cell of any one of embodiments 52-76, wherein the cell comprises a modification that reduces acetate production.

78. The cell according to any one of embodiments 52 to 77, wherein the cell comprises reduced or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the following proteins, said proteins comprising β -galactosidase, galactosido-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphase, EIICBA-Nag, UDP-glucose: undecaprenyl-phosphoglucose-1-phosphotransferase, L-fucokinase, L-fucose isomerase, N-acetylneuraminic acid lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phospho2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphouridyltransferase, glucose-1-phosphoadenyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphoisomerase, aerobic respiration control protein, transcription repressor IclR, lon protease, glucose-specific translocated phosphotransferase IIBC component ptsG, glucose-specific translocated Phosphotransferase (PTS) IIBC component malX, enzyme IIA ^Glc, beta-glucosidase II, phosphoryl transferase specific PTS protein 3525 and phosphoPTS protein 383824 Alcohol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphoacetyltransferase, pyruvate decarboxylase.

79. The cell according to any one of embodiments 52 to 78, wherein the cell is capable of producing phosphoenolpyruvate (PEP).

80. The cell according to any one of embodiments 52 to 79, wherein the cell is modified to enhance the production and/or supply of phosphoenolpyruvate (PEP).

81. The cell according to any one of embodiments 52 to 80, wherein the cell comprises an at least partially inactivated catabolic pathway of a selected monosaccharide, disaccharide or oligosaccharide that is involved in the production of the compound comprising the structure of formula I, II or III and/or is necessary for the production of the compound comprising the structure of formula I, II or III.

82. The cell of any one of embodiments 52-81, wherein the cell is resistant to lactose killing when grown in an environment where lactose is combined with one or more other carbon sources.

83. The cell according to any one of embodiments 52 to 82, wherein the cell produces 90g/L or more of the compound comprising the structure of formula I, II or III in the whole culture and/or supernatant, and/or wherein the compound comprising the structure of formula I, II or III has a purity of at least 80% in the whole culture and/or supernatant, as measured by the total amount of the compound comprising the structure of formula I, II or III and its precursors in the whole culture and/or supernatant, respectively.

84. The cell according to any one of embodiments 52 to 83, wherein the cell is a bacterium, a fungus, a yeast, a plant cell, an animal cell, or a protozoan cell,

85. The cell of embodiment 84, wherein the cell is a living gram-negative bacterium comprising reduced or eliminated synthesis of: poly-N-acetyl-glucosamine (PNAG), enterobacter Common Antigen (ECA), cellulose, capsular polysaccharide, core oligosaccharide, osmoregulation Periplasmic Glucan (OPG), glycerol glucoside, glycans and/or trehalose.

86. The cell according to any one of embodiments 52 to 85, wherein the cell produces a mixture of charged, preferably sialylated and/or neutral di-and oligosaccharides, said mixture comprising at least one compound comprising a structure of formula I, II or III, wherein R1, when present, is a mono-, di-or oligosaccharide.

87. The cell according to any one of embodiments 52 to 86, wherein the cell produces a mixture of charged, preferably sialylated and/or neutral oligosaccharides, said mixture comprising at least one compound comprising a structure of formula I, II or III, wherein R1 when present is a monosaccharide, disaccharide or oligosaccharide.

88. Use of the cell of any one of embodiments 52 to 87 or the method of any one of embodiments 1 to 51 for producing a compound comprising a structure of formula I, II or III.

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

Examples

Example 1 calculation of percent identity between nucleotide or polypeptide sequences

Sequence alignment methods for alignment are well known in the art and include GAP, BESTFIT, BLAST, FASTA and tfast a. GAP uses the algorithms of Needleman and Wunsch (J.mol. Biol. (1970) 48:443-453) to find an overall (i.e., trans-full length sequence) alignment of two sequences that maximizes the number of matches and minimizes the number of GAPs. The BLAST algorithm (Altschul et al, J.mol.biol. (1990) 215:403-10) calculates the percent overall sequence identity (i.e., over the full length sequence) and performs a statistical analysis of the similarity between the two sequences. Software for performing BLAST analysis is publicly available through the national center for biotechnology information (National Centre for Biotechnology Information, NCBI). Homologs can be readily identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), using default pairwise alignment parameters and a percent scoring method. The overall percentage of similarity and identity (i.e., across the full-length sequence) can also be determined using one of the methods available in MatGAT software package (Campanella et al, BMC Bioinformatics (2003) 4:29). As will be appreciated by those skilled in the art, small manual edits may be made to optimize alignment between conserved motifs. Furthermore, instead of using full-length sequences to identify homologs, specific domains may also be used to determine so-called local sequence identity. Using the above procedure and using default parameters, the sequence identity value can be determined over the entire nucleic acid or amino acid sequence (=local sequence identity search over the full length sequence, yielding an overall sequence identity score) or over a selected domain or conserved motif (=local sequence identity search over a partial sequence, yielding a local sequence identity score). For local alignment, the Smith-Waterman algorithm is particularly useful (Smith TF, waterman MS (1981) J.mol. Biol 147 (1); 195-7).

Example 2 materials and methods E.coli

Culture medium

The Luria Broth (LB) medium consists of 1% tryptone peptone (Difco, eremmbotegem, belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR, leuven, belgium). The minimal medium used for the culture experiments in 96-well plates or shake flasks contained 2.00g/L NH4Cl、5.00g/L(NH4)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 molybdate solution and 1mL/L selenium solution. As specified in the various examples, 0.30g/L sialic acid, 0.30g/L GlcNAc, 20g/L lactose, 20g/L LacNAc and/or 20g/L LNB were additionally added to the medium as precursors. The minimal medium was set to pH 7 with 1M KOH. The vitamin solution consists of 3.6g/L FeCl2.4H2O、5g/L CaCl2.2H2O、1.3g/LMnCl2.2H2O、0.38g/L CuCl2.2H2O、0.5g/L CoCl2.6H2O、0.94g/L ZnCl2、0.0311g/L H3BO4、0.4g/L Na2EDTA.2H2O and 1.01g/L thiamine 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 various examples, 0.30g/L sialic acid, 0.30g/L GlcNAc, 20g/L lactose, 20g/L LacNAc and/or 20g/L LNB were additionally added to the medium as precursors.

The complex medium was sterilized by autoclaving (121 ℃ C., 21 min) and the minimal medium was sterilized by filtration (0.22 μm Sartorius). If necessary, the medium is made selective by the addition of antibiotics: such as chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L), and/or kanamycin (50 mg/L).

Plasmid(s)

PKD46 (Red helper plasmid, ampicillin resistance), pKD3 (containing the chloramphenicol resistance (cat) gene flanking FRT), pKD4 (containing the kanamycin resistance (kan) gene flanking FRT) and pCP20 (expressing FLP recombinase activity) plasmids were obtained from the professor r.cunin (university of brussel free belgium (Vrije Universiteit Brussel), obtained in 2007). The plasmid was maintained in host E.coli DH5α(F^-,phi80dlacZΔM15,Δ(lacZYA-argF)U169,deoR,recA1,endA1,hsdR17(rk^-,mk⁺),phoA,supE44,λ^-,thi-1,gyrA96,relA1) purchased from Invitrogen.

Strains and mutations

Coli K12 MG1655[ lambda ^-,F^-, rph-1] was obtained from the Escherichia coli genetic collection (U.S.) at month 3 of 2007, CGSC strain # 7740. Gene disruption, gene introduction and gene replacement were performed using the techniques published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). The technique is based on antibiotic selection after homologous recombination by lambda Red recombinase. Subsequent catalysis of the invertase recombinase will ensure removal of the antibiotic selection cassette in the final production strain. Transformants harboring Red helper plasmid pKD46 were grown to an OD ₆₀₀ nm of 0.6 at 30℃in 10mL of LB medium containing ampicillin (100 mg/L) and L-arabinose (10 mM). Cells were placed in an inductive receptive state by washing the cells a first time with 50mL ice-cold water and a second time with 1mL ice-cold water. Then, the cells were resuspended in 50. Mu.L ice-cold water. Electroporation was performed using Gene Pulser ^TM (BioRad) (600Ω, 25 μFD and 250 volts) with 50 μL cells and 10-100ng of linear double stranded-DNA product. Following electroporation, cells were added to 1mL of LB medium, incubated at 37℃for 1h, and finally plated on LB agar containing 25mg/L chloramphenicol or 50mg/L kanamycin to select for antibiotic-resistant transformants. Selected mutants were verified by PCR using primers upstream and downstream of the modified region and grown in LB agar at 42 ℃ to lose helper plasmids. The mutants were tested for ampicillin sensitivity. The linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and derivatives thereof as templates. A part of the sequence of the primer used is complementary to the template and another part is complementary to the side of the chromosomal DNA on which recombination must take place. For genomic knockouts, the homology region is designed 50 nucleotides upstream and 50 nucleotides downstream of the start and stop codons of the gene of interest. For genome knock-in, the transcription start point (+1) must be respected. The PCR product was PCR purified, digested with Dpnl, re-purified from agarose gel and suspended in elution buffer (5 mM Tris, pH 8.0). Selected mutants were transformed with pCP20 plasmid, an ampicillin and chloramphenicol resistant plasmid, showing temperature sensitive replication and thermal induction of FLP synthesis. Ampicillin resistant transformants were selected at 30℃and several colonies were purified in LB at 42℃and tested for all antibiotic resistance and loss of FLP helper plasmid. Gene knockouts and knockins were checked using control primers.

In one example of GDP-fucose production, the mutant strain is derived from escherichia coli K12MG1655, comprising escherichia coli wcaJ and thyA gene knockouts and genome knock-in of a constitutive transcription unit comprising: sucrose transporters, such as, for example, cscB (UniProt ID E0IXR1, sequence version 1) from e.coli W; fructokinase such as, for example, frk (UniProt ID Q03417, sequence version 1) from zymomonas mobilis (Zymomonas mobilis); and sucrose phosphorylases, such as BaSP (UniProt ID A0 zh6, sequence version 1) from bifidobacterium adolescentis (Bifidobacterium adolescentis), for example. GDP-fucose production can be further optimized in E.coli mutants by genomic knockout of any one or more of the 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 be additionally optimized, including genome knock-in for the following constitutive transcription units: mannose-6-phosphate isomerase, such as, for example, manA from E.coli (UniProt ID P00946, sequence version 1); phosphomannomutases, such as manB from e.coli (UniProt ID P24175, sequence version 1); mannose-1-phosphate guanylate transferases, such as, for example, manC from E.coli (UniProt ID P24174, sequence version 3); GDP-mannose 4, 6-dehydratase, such as, for example, gmd from E.coli (UniProt ID P0AC88, sequence version 1); and GDP-L-fucose synthase such as fcl from E.coli (UniProt ID P32055, sequence version 2). GDP-fucose production can also be obtained by genomic knock-out of the E.coli fucK and fucI genes and by genomic knock-in of constitutive transcription units containing a fucose permease such as, for example, fucP from E.coli (UniProt ID P11551, SEQ ID NO: 3)) and a bifunctional fucose kinase/fucose-1-phosphate guanyl transferase activity such as, for example, fkp (UniProt ID SUV40286.1, SEQ ID NO: 1) from Bacteroides fragilis (Bacteroides fragilis). All mutants can be modified additionally by: the genome knock-out of the LacZ, lacY and LacA genes of E.coli, and the constitutive transcription units of the genome knock-in lactose permeases such as, for example, lacY (UniProt ID P02920, sequence version 1) from E.coli.

For the production of fucosylated oligosaccharides, GDP-fucose producing mutants are additionally modified with an expression plasmid comprising constitutive transcription units for α -1, 2-fucosyltransferases (such as e.g. any one or more of SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 or 37) and/or α -1, 3-fucosyltransferases (such as e.g. HpFucT (UniProt ID O30511, sequence version 1) from helicobacter pylori (h.pyri)) and constitutive transcription units for escherichia coli thyA (UniProt ID P0a884, sequence version 1) are used as selectable markers. Additionally, and/or alternatively, constitutive transcription units of the fucosyltransferase gene may be present in the E.coli mutant by genome knock-in.

Alternatively, and/or additionally, GDP-fucose and/or fucosylated oligosaccharides production in an e.coli mutant strain may be further optimized by genome knock-in of a constitutive transcription unit comprising: membrane transporters such as, for example, mdfA (UniProt ID A0A2T7ANQ9, sequence version 1) from enterobacter sakazakii (Cronobacter muytjensii), mdfA (UniProt ID D4BC23, sequence version 1) from citric acid bacillus (Citrobacter youngae), mdfA (UniProt ID P0AEY, sequence version 1) from escherichia coli, mdfA (UniProt ID G9Z5F4, sequence version 1) from Lei Jinsi fort pre-roll (Yokenella regensburgei), iceT (UniProt ID A0a024L207, sequence version 1) from escherichia coli or iceT (UniProt ID D4B8A6, sequence version 1) from citric acid bacillus yang (Citrobacter youngae).

In an example for the production of lactose-N-disaccharide (LNB, gal-b1, 3-GlcNAc), a strain is modified by genome knock-in or using an expression plasmid comprising constitutive transcription units for glucosamine-6-phosphate N-acetyltransferase such as GNA1 (UniProt ID P43577, SEQ ID 1) from Saccharomyces cerevisiae (S.cerevisiae) and N-acetylglucosamine beta-1, 3-galactosyltransferase such as WbgO (Uniprot ID D3QY14, SEQ ID 1) from E.coli O55:H7, for example.

In the examples where lactose-N-trisaccharide (LN 3, glcNAc-b1,3-Gal-b1, 4-Glc) is produced, the mutant strain is derived from E.coli K12 MG1655 and is modified by knocking out E.coli lacZ, lacY, lacA and nagB genes and the genome knock-in constitutive transcription unit for: lactose permeases such as, for example, e.coli LacY (UniProt ID P02920, sequence version 1); and galactoside beta-1, 3-N-acetylglucosaminyl transferases such as lgtA (UniProt ID Q9JXQ6, sequence version 1) from Neisseria meningitidis (N.menningitidis), for example.

In the examples of the production of LN3 derived oligosaccharides such as lactose-N-tetrasaccharides (LNT, gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc), mutant LN 3-producing strains are further modified using constitutive transcription units delivered to the strain by genome knock-in or from expression plasmids for N-acetylglucosamine beta-1, 3-galactosyltransferases such as wbgO (Uniprot ID D3QY14, sequence version 1) from E.coli O55:H 7.

LNB, LN3 and/or LNT production in the e.coli mutant can be further optimized by genomic knockout of e.coli genes including any one or more of galT, ushA, ldhA and agp.

Mutant LNB, LN3 and LNT production strains may also optionally be further modified to increase UDP-GlcNAc production by genome knock-in of constitutive transcription units used for L-glutamine-D-fructose-6-phosphate aminotransferases, such as for example the mutant glmS 54 from e.coli (differing from wild-type e.coli glmS protein (UniProt ID P17169, sequence version 4) by the a39T, R C and G472S mutations, as described by Deng et al (Biochimie 2006, 88:419-429).

The E.coli mutant strain may also be altered optionally by genome knock-in of a constitutive transcription unit for: UDP-glucose-4-epimerase such as galE (UniProt ID P09147, sequence version 1) from E.coli; phosphoglucomutase, such as, for example, glmM from E.coli (UniProt ID P31120, sequence version 3); and N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase, such as, for example, glmU from E.coli (UniProt ID P0ACC7, sequence version 1).

Coli mutants can also be altered for growth under sucrose, optionally by genome knock-in of a constitutive transcription unit comprising: sucrose transporters, such as, for example, cscB (UniProt ID E0IXR1, sequence version 1) from e.coli W; fructokinase such as, for example, frk (UniProt ID Q03417, sequence version 1) from zymomonas mobilis (Zymomonas mobilis); and sucrose phosphorylases, such as BaSP from bifidobacterium adolescentis (UniProt ID A0ZZH6, sequence version 1), for example.

Alternatively, and/or additionally, the production of LNB, LN3, LNT and their derived oligosaccharides in the e.coli mutant strain can be further optimized by genome knock-in constitutive transcription units comprising membrane transporters such as e.g. MdfA (UniProt ID A0A2T7ANQ9, sequence version 1) from e.sakazakii (Cronobacter muytjensii), mdfA (UniProt ID D4BC23, sequence version 1) from e.yang (Citrobacter youngae), mdfA (UniProt ID P0AEY, sequence version 1) from e.coli, mdfA (UniProt ID G9Z5F4, sequence version 1) from Lei Jinsi fort pre-ground bacteria (Yokenella regensburgei), iceT (UniProt ID A0a024L207, sequence version 1) from e.coli (Citrobacter youngae) or iceT (prot ID D4B8A6, sequence version 1) from e.yang (Citrobacter youngae).

In an example of sialic acid production, the mutant is derived from E.coli K12 MG1655, comprising a genomic knock-in constitutive transcription unit containing one or more copies of glucosamine 6-phosphate N-acetyltransferase such as, for example, GNA1 (UniProt ID P43577, SEQ ID 1) from Saccharomyces cerevisiae (Saccharomyces cerevisiae), N-acetylglucosamine 2-epimerase such as, for example, AGE (UniProt ID A7LVG6, SEQ ID 1) from Bacteroides ovalis (Bacteroides ovatus) and N-acetylneuraminic acid synthase such as, for example, from Neisseria meningitidis (UniProt ID E0NCD4, SEQ ID 1) or Campylobacter jejuni (Campylobacter jejuni) (UniProt ID Q93MP9, SEQ ID 1).

Alternatively, and/or additionally, sialic acid production may be obtained by genomic knock-in of a constitutive transcription unit comprising UDP-N-acetylglucosamine 2-epimerase (as for example NeuC from campylobacter jejuni (UniProt ID Q93MP8, sequence version 1)) and N-acetylneuraminic acid synthase (as for example from neisseria meningitidis (UniProt ID E0NCD4, sequence version 1) or campylobacter jejuni (UniProt ID Q93MP9, sequence version 1)).

Alternatively and/or additionally, sialic acid production may be obtained by genome knock-in of a constitutive transcription unit comprising phosphoglucosamine mutase (such as e.g. glmM (UniProt ID P31120, SEQ ID NO: 3)), N-acetylglucosamine-1-phosphoglucosamide transferase/glucosamine-1-phosphoglycerate acetyltransferase (such as e.g. glmU (UniProt ID P0ACC7, SEQ ID NO: 1)) from E.coli, UDP-N-acetylglucosamine 2-epimerase (such as e.g. NeuC (UniProt ID Q93MP8, SEQ ID NO: 1)) from E.jejunum) and N-acetylneuraminic acid synthase (such as e.g. from Neisseria meningitidis (UniProt ID E0NCD4, SEQ ID NO: 1)) or Campylobacter jejuni (UniProt ID Q93MP9, SEQ ID NO: 1).

Alternatively, and/or additionally, sialic acid production may be obtained by genomic knock-in of a constitutive transcription unit comprising: bifunctional UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase such as, for example, from mice (Mus musculus) (strain C57 BL/6J) (UniProt ID Q91WG8, sequence version 1); n-acyl neuraminic acid-9-phosphate synthetases, such as, for example, from Pseudomonas species UW4 (UniProt ID K9NPH9, sequence version 1); and N-acyl neuraminic acid-9-phosphatases, such as, for example, HK-1 (UniProt ID KPA15328.1, SEQ ID NO: 1) from Candida magnetotactic (Candidatus Magnetomorum sp.) or Bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron) (UniProt ID Q8A712, SEQ ID NO: 1).

Alternatively, and/or additionally, sialic acid production may be obtained by genomic knock-in of a constitutive transcription unit comprising: phosphoglucomutase, such as, for example, glmM from E.coli (UniProt ID P31120, sequence version 3); n-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase, such as, for example, glmU from E.coli (UniProt ID P0ACC7, SEQ ID NO: 1); bifunctional UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase such as, for example, from mice (strain C57 BL/6J) (UniProt ID Q91WG8, sequence version 1); n-acyl neuraminic acid-9-phosphate synthetases, such as, for example, from Pseudomonas species UW4 (UniProt ID K9NPH9, sequence version 1); and N-acyl neuraminic acid-9-phosphatases, such as, for example, from Candida magnetotactic HK-1 (UniProt ID KPA15328.1, SEQ ID NO: 1) or from Bacteroides thetaiotaomicron (UniProt ID Q8A712, SEQ ID NO: 1).

Sialic acid production in E.coli mutants can be further optimized by genomic knockout of E.coli genes comprising any one or more of nagA, nagB, nagC, nagD, nagE, nanA, nanE, nanK, manX, manY and manZ, as described in WO18122225, and/or genomic knockout of E.coli genes comprising any one or more of nanT, poxB, ldhA, adhE, aldB, pflA, pflC, ybiY, ackA and/or pta, and genomic knockout of a constitutive transcription unit comprising one or more copies of L-glutamine-D-fructose-6-phosphate aminotransferase (such as for example mutant glmS 54 from E.coli (differing from wild-type E.coli glmS protein (UniProt ID P17169, SEQ ID 4) by A39T, R C and G472S mutations, as described in Deng et al (Biochie 88,419-29 (2006)), preferably phosphatase such as for example any one of 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 (Pseudomonas putida), scDOG from Bacillus cereus or from Bacillus subtilis (such as described in WO 35 E.coli, SEQ ID No. 25, and acetyl 96 E.sp.sp.sp.fumonis (SEQ ID 2) and F.sp.sp.fumosi.fumosi (F.fumose.fumosi.fumose.No. F.5, SEQ ID No. 35).

For sialylated oligosaccharide production, the sialic acid producing strain may be further modified to express N-acyl neuraminic acid cytidylyltransferase, such as for example NeuA enzyme from campylobacter jejuni (UniProt ID Q93MP7, sequence version 1), neuA enzyme from haemophilus influenzae (Haemophilus influenzae) (GenBank No. agv 11798.1) or NeuA enzyme from pasteurella multocida (Pasteurella multocida) (GenBank No. amk 07891.1), and β -galactosidase α -2, 3-sialyltransferase expressing one or more copies, such as for example PmultST3 from pasteurella multocida (UniProt ID Q9CLP3, sequence version 1) or PmultST-like polypeptide having β -galactosidase activity (consisting of amino acid residues 1 to GenBank ID Q9CLP3, sequence version 1), genBank NmeniST (GenBank No. d 9CLP 9) or a strain of multiple neisseria multocida (pinaco No. 62.0298.62, a. Mu.0270); beta-galactoside alpha-2, 6-sialyltransferase, such as, for example, pdST (UniProt ID O66375, sequence version 1) from a mermaid light emitting bacterium (Photobacterium damselae) or PdST-like polypeptide (consisting of UniProt ID O66375, amino acid residues 108 to 497 of sequence version 1) having beta-galactoside alpha-2, 6-sialyltransferase activity or P-JT-ISH-224-ST6 (UniProt ID A8QYL1, sequence version 1) from a light emitting bacterium species JT-ISH-224 or P-JT-ISH-224-ST 6-like polypeptide (consisting of UniProt ID A8QYL1, amino acid residues 18 to 514) having beta-galactoside alpha-2, 6-sialyltransferase activity; and/or alpha-2, 8-sialyltransferase, such as, for example, from mice (UniProt ID Q64689, sequence version 2). The constitutive transcription units of N-acyl neuraminic acid cytidylyltransferase and sialyltransferase can be delivered into the mutant by genomic knock-in or by expression plasmids. If the sialic acid and CMP-sialic acid producing mutants are intended to produce sialylated lactose structures, the strain is additionally modified by genomic knock-out of the E.coli LacZ, lacY and LacA genes and genomic knock-in of the constitutive transcription units of lactose permeases such as e.coli LacY (UniProt ID P02920, sequence version 1). All saliva, CMP-sialic acid and/or sialylated oligosaccharides producing mutants can optionally be altered by genome knock-in of a constitutive transcription unit comprising: sucrose transporters, such as, for example, cscB (UniProt ID E0IXR1, sequence version 1) from e.coli W; fructokinase such as, for example, frk from zymomonas mobilis (UniProt ID Q03417, sequence version 1); and sucrose phosphorylases, such as BaSP from bifidobacterium adolescentis (UniProt ID A0ZZH6, sequence version 1), for example.

Alternatively, and/or additionally, sialic acid and/or sialylated oligosaccharide production in E.coli mutants can be further optimized by genome knock-in of a constitutive transcription unit comprising: membrane transporters, such as, for example, sialic acid transporters, such as, for example, nanT (UniProt ID P41036, SEQ ID 2), nanT (UniProt ID Q8FD59, SEQ ID 2) from E.coli O6:H1, nanT (UniProt ID Q8X9G8, SEQ ID 2) from E.coli O157:H7 or nanT (UniProt ID B1EFH1, SEQ ID 1) from E.albertii; or a transporter (porter), such as, for example, entS (UniProt ID A0a378GQ13, sequence version 1) from escherichia coli (UniProt ID P24077, sequence version 2), entS (UniProt ID A0a378GQ13, sequence version 1) from escherichia coli (Kluyvera ascorbata) or EntS (UniProt ID A0A6Y2K4E8, sequence version 1) from salmonella, such as, for example, mdfA (UniProt ID A0A2T7ANQ9, sequence version 1) from escherichia coli (Cronobacter muytjensii), mdfA (UniProt ID D4BC23, sequence version 1) from escherichia coli (Citrobacter youngae), mdfA (UniProt ID P0AEY8, sequence version 1) from escherichia coli (Yokenella regensburgei), mdfA (UniProt ID G9Z5F4, sequence version 1) from escherichia coli (Lei Jinsi fort), mdfA (UniProt ID A0A2 a 7ANQ9, sequence version 1) from escherichia coli (Cronobacter muytjensii), mdfA (UniProt ID 4BC23, sequence version 1) from escherichia coli (Citrobacter youngae), mdfA (UniProt ID 4B 4, sequence version 3 from escherichia coli (6726), and sequence version 3 from escherichia coli (678); or ABC transporters such as, for example, oppF from E.coli (UniProt ID P77737, SEQ ID 1), lmrA from lactococcus lactis subspecies lactis (diacetyl type) (Lactococcus lactis subsp.lacti bv. Diacetylactis) (UniProt ID A0A1V0NEL4, SEQ ID 1), or Blon_2475 from Bifidobacterium longum subsp.infantis (UniProt ID B7GPD4, SEQ ID 1).

Preferably, but not necessarily, any one or more of the glycosyltransferases (i.e., proteins involved in nucleotide-activated sugar synthesis) and/or membrane transporters are fused at the N-and/or C-terminus to a solubilising tag, such as, for example, a SUMO-tag, MBP-tag, his, FLAG, strep-II, halo-tag, nusA, thioredoxin, GST and/or Fh 8-tag, to enhance its solubility (Costa 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 E.coli mutant is modified by genomic knock-in of a constitutive transcription unit encoding a chaperone protein, such as for example DnaK, dnaJ, grpE or GroEL/ES chaperone system (Baneyx F.,Palumbo J.L.(2003)Improving Heterologous Protein Folding via Molecular Chaperone and Foldase Co-Expression.In:Vaillancourt P.E.() E.coli Gene Expression protocols.methods in Molecular Biology ^TM, vol 205.Humana Press.

Optionally, the E.coli mutant is modified to produce a sugar minimized (glycominimized) E.coli strain comprising a genomic knockout of any one or more of the nonessential 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 constitutive promoter, UTR and terminator sequences are derived from libraries described by Cambray et al (Nucleic Acids Res.2013,41 (9), 5139-5148), dunn et al (Nucleic Acids Res.1980,8, 2119-2132), edens et al (Nucleic Acids Res.1975, 2, 1811-1820), kim and Lee (FEBS Letters 1997,407,353-356) and Mutalik et al (Nat.methods 2013, no.10, 354-360).

The SEQ ID NOs described in the present invention are summarized in Table 1.

All genes were ordered synthetically on a Twist Bioscience (twistbioscience. Com) or IDT (eu. Idtna. Com) and the codon usage was modified using the vendor's tools.

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

TABLE 1 summary of SEQ ID NOs according to the invention

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Culture conditions

Pre-incubation for 96-well microtiter plate experiments starts from frozen vials and is incubated overnight at 37℃in 150. Mu.L LB at 800rpm on an orbital shaker. The culture was used as an inoculum for 96 Kong Fangxing microtiter plates and 400. Mu.L of minimal medium was used by dilution 400-fold. These final 96-well 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 were taken from each well and the broth was boiled at 60 ℃ for 15 minutes (=average of intracellular and extracellular sugar concentrations) before centrifuging the cells.

The pre-culture of the bioreactor was started from the whole 1mL frozen vial of the particular strain, inoculated into 250mL or 500mL minimal medium in 1L or 2.5L shake flasks and incubated at 37 ℃ on an orbital shaker at 200rpm for 24h. Then a 5L bioreactor (with 5L working volume) (250 mL inoculum in 2L batch medium) was inoculated; the process is controlled by MFCS control software (Sartorius Stedim Biotech, melsungen, germany). The 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 H2SO4 and 20% NH4 OH. The exhaust gas is cooled. When foam is generated during fermentation, a 10% silicone defoamer solution is added.

Optical density

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

Analytical analysis

Standards such as, but not limited to, sucrose, lactose-N-disaccharide (LNB, gal-b1, 3-GlcNAc), fucosylated LNB (2 'FLNB,4' FLNB), lactose-N-trisaccharide II (LN 3), lactose-N-tetrasaccharide (LNT), lactose-N-neo-tetrasaccharide (LNnT), LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI are available from Carbosynth (UK), elicityl (France) and IsoSep (Sweden). Other compounds were analyzed using internally prepared standards.

Neutral oligosaccharides were analyzed on a Waters acquisition H-like UPLC with Evaporative Light Scattering Detector (ELSD) or Refractive Index (RI) detection. A sample of 0.7. Mu.L was injected into a column with Acquity UPLC BEH Amide Van guard @A Waters Acquity UPLC BEH Amide column (2.1 x 100mm; /(I)1.7 Μm) was applied to the column. The column temperature was 50 ℃. The mobile phase consisted of 1/4 water and 3/4 acetonitrile solution to which 0.2% triethylamine was added. The process was isocratic with a flow rate of 0.130mL/min. The ELS detector has a drift tube temperature of 50℃and a nitrogen pressure of 50psi, a gain of 200, and a data rate of 10pps. The temperature of the RI detector was set at 35 ℃.

Sialylated oligosaccharides were analyzed on a Waters Acquity H-like UPLC with Refractive Index (RI) detection. A 0.5 μl volume of sample was injected onto Waters Acquity UPLC BEH Amide column (2.1x100mm; 1.7 μm). The column temperature was 50 ℃. The mobile phase consisted of a mixture of 70% acetonitrile, 26% ammonium acetate buffer (150 mM) and 4% methanol to which was added 0.05% pyrrolidine. The process was isocratic with a flow rate of 0.150mL/min. The temperature of the RI detector was set at 35 ℃.

Neutral and sialylated saccharides were analysed on a Waters Acquity H-like UPLC with Refractive Index (RI) detection. A 0.5 μl volume of sample was injected onto Waters Acquity UPLC BEH Amide column (2.1 x 100mm; 1.7 μm). The column temperature was 50 ℃. The mobile phase consisted of a mixture of 72% acetonitrile and 28% ammonium acetate buffer (100 mM) to which was added 0.1% triethylamine. The process was isocratic with a flow rate of 0.260mL/min. The temperature of the RI detector was set at 35 ℃.

For analysis on a mass spectrometer, waters Xevo TQ-MS with electrospray ionization (ESI) was used, the desolvation temperature was 450 ℃, the desolvation nitrogen flow was 650L/h, and the cone voltage was 20V. For all oligosaccharides, the MS was run in a Selective Ion Monitoring (SIM) in negative mode. The separation was performed on Waters Acquity UPLC with Thermo Hypercarb columns (2.1X 100mm;3 μm) at 35 ℃. A gradient was used in which eluent a was ultrapure water containing 0.1% formic acid, and in which eluent B was acetonitrile containing 0.1% formic acid. The oligosaccharides were isolated within 55 minutes using the following gradient: first increasing from 2% to 12% eluent B over 21 minutes, second increasing from 12% to 40% eluent B over 11 minutes, and third increasing from 40% to 100% eluent B over 5 minutes. As a washing step, 100% of eluent B was used for 5 minutes. For column equilibration, 2% of the initial conditions of eluent B were restored within 1 minute and maintained for 12 minutes.

Low concentrations (below 50 mg/L) of neutral and sialylated saccharides were analyzed on a Dionex HPAEC system with Pulse Amperometric Detection (PAD). A5 μl volume of sample was injected onto a Dionex CarboPac PA column (4 x 250 mm) with Dionex CarboPac PA200,200 guard columns (4 x 50 mm). The column temperature was set at 30 ℃. A gradient was used, wherein eluent a was deionized water, wherein eluent B was 200mM sodium hydroxide, and wherein eluent C was 500mM sodium acetate. Oligosaccharides were separated within 60 minutes while maintaining a constant proportion of 25% eluent B using the following gradient: the initial isocratic step maintained 75% of eluent a for 10 minutes, first increased from 0% to 4% of eluent C over 8 minutes, the second isocratic step maintained 71% of eluent a and 4% of eluent C for 6 minutes, the second increased from 4% to 12% of eluent C over 2.6 minutes, the third isocratic step maintained 63% of eluent a and 12% of eluent C for 3.4 minutes, and the third increased from 12% to 48% of eluent C over 5 minutes. As a washing step 48% of eluent C was used for 3 minutes. For column equilibration, initial conditions of 75% eluent a and 0% eluent C were restored within 1 minute and maintained for 11 minutes. The flow rate applied was 0.5mL/min.

Example 3 materials and methods-Saccharomyces cerevisiae

Culture medium

The strain was grown on (SYNTHETIC DEFINED) yeast medium (SD CSM) containing the determined synthetic composition of the complete supplementation mixture or on CSM-deficient medium (SD CSM-Ura, SD CSM-Trp, SD CSM-His) containing 6.7g/L of amino acid-free yeast nitrogen source basal medium (Yeast Nitrogen Base, amino acid-free YNB, 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).

Strain

Saccharomyces cerevisiae BY4742, which is available at Euroscarf culture collection, established BY Brachmann et al (Yeast (1998) 14:115-32) was used. All mutants were established by homologous recombination or plasmid transformation using the method of Gietz (Yeast 11:355-360, 1995).

Plasmid(s)

To produce GDP-fucose, a yeast expression plasmid such as p2a_2μ_Fuc (Chan 2013, plasmid70, 2-17) can be used to express foreign genes in Saccharomyces cerevisiae. The plasmid contains the ampicillin resistance gene and bacterial origin of replication for selection and maintenance in E.coli, and contains 2. Mu. Yeast ori and Ura3 selectable markers for selection and maintenance in yeast. The plasmid was further modified with the following constitutive transcription units: lactose permeases (such as, for example, LAC12 (UniProt ID P07921, sequence version 1) from kluyveromyces lactis), GDP-mannose 4, 6-dehydratases (such as, for example, gmd (UniProt ID P0AC88, sequence version 1) from escherichia coli) and GDP-L-fucose synthases (such as, for example, fcl (UniProt ID P32055, sequence version 2) from escherichia coli). Yeast expression plasmid p2a_2μ_Fuc2 can be used as a surrogate expression plasmid for the p2a_2μ_Fuc plasmid, which contains bacterial ori next to the ampicillin resistance gene, the 2 μ yeast ori and the Ura3 selectable marker for the following constitutive transcription unit: lactose permease (such as LAC12 (UniProt ID P07921, sequence version 1), for example from kluyveromyces lactis), fucose permease (such as fucP (UniProt ID P11551, sequence version 3), for example from escherichia coli) and bifunctional enzymes with fucose kinase/fucose-1-phosphate guanyl transferase activity (such as fkp (UniProt ID SUV40286.1, sequence version 1), for example) from bacteroides fragilis. To further produce fucosylated oligosaccharides, p2a_2μ_fuc2 and its variants p2a_2μ_fuc2 additionally contain constitutive transcription units for one or more fucosyltransferases such as e.g. α -1, 2-fucosyltransferases and/or α -1, 3-fucosyltransferases selected from the list ：SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 or 37 comprising.

To produce UDP-galactose, the yeast expression plasmid may be derived from the pRS 420-plasmid series (Christianson et al 1992,Gene 110:119-122) containing the HIS3 selection marker and constitutive transcription units of UDP-glucose-4-epimerase (such as galE (Unit ID P09147, SEQ ID NO: 1) from E.coli). The plasmid may be further modified with constitutive transcriptional units of lactose permeases such as, for example, LAC12 (UniProt ID P07921, sequence version 1) from kluyveromyces lactis and galactoside beta-1, 3-N-acetylglucosaminyl transferase activities such as, for example, lgtA (UniProt ID Q9JXQ6, sequence version 1) from neisseria meningitidis to produce LN3. To further produce LN 3-derived oligosaccharides, such as LNT, the mutant LN3 production strain is further modified with a constitutive transcription unit of N-acetylglucosamine beta-1, 3-galactosyltransferase, such as WbgO (Uniprot ID D3QY14, sequence version 1) from E.coli O55:H7, for example.

In lactose-N-disaccharide-producing examples, the yeast expression plasmid may be derived from the pRS 420-plasmid series (Christianson et al 1992,Gene 110:119-122) containing the TRP1 selection marker and constitutive transcription units for: one or more copies of L-glutamine-D-fructose-6-phosphate aminotransferase, such as, for example, mutant glmS 54 from e.coli (differing from wild-type e.coli glmS protein (UniProt ID P17169, sequence version 4) in the a39T, R C and G472S mutations, as described by Deng et al (Biochimie 88,419-29 (2006)); phosphatase, such as, for example, any one of the following: coli genes including aphA、Cof、HisB、OtsB、SurE、Yaed、YcjU、YedP、YfbT、YidA、YigB、YihX、YniC、YqaB、YrbL、AppA、Gph、SerB、YbhA、YbiV、YbjL、Yfb、YieH、YjgL、YjjG、YrfG and YbiU, or PsMupP from pseudomonas putida, scDOG from saccharomyces cerevisiae or BsAraL from bacillus subtilis, as described in WO 18122225; glucosamine 6-phosphate N-acetyltransferases, such as, for example, GNA1 from Saccharomyces cerevisiae (UniProt ID P43577, SEQ ID NO: 1); and N-acetylglucosamine beta-1, 3-galactosyltransferase, such as WbgO (Uniprot ID D3QY14, sequence version 1) from E.coli O55:H7, for example.

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

Optionally, the yeast mutant is modified by genomic knock-in of a constitutive transcription unit encoding a chaperone protein, such as for example Hsp31、Hsp32、Hsp33、Sno4、Kar2、Ssb1、Sse1、Sse2、Ssa1、Ssa2、Ssa3、Ssa4、Ssb2、Ecm10、Ssc1、Ssq1、Ssz1、Lhs1、Hsp82,Hsc82、Hsp78、Hsp104、Tcp1、Cct4、Cct8、Cct2、Cct3、Cct5、Cct6 or Cct7 (Gong et al 2009, mol. Syst. Biol. 5:275). The plasmid was maintained in host E.coli DH5α(F^-,phi80dlacZδM15,δ(lacZYA-argF)U169,deoR,recA1,endA1,hsdR17(rk^-,mk⁺),phoA,supE44,λ^-,thi-1,gyrA96,relA1) purchased from Invitrogen.

Heterologous and homologous expression

The genes to be expressed (whether from plasmids or from the genome) are synthesized 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 individual colonies. These plates were grown at 30℃for 2-3 days. Starting from a single colony, the preculture was grown overnight at 30℃in 5mL with shaking at 200 rpm. 125mL shake flask experiments were then inoculated with 2% of this preculture in 25mL of medium. These flasks were incubated at 30℃with orbital shaking at 200 rpm.

Gene expression promoter

The genes were expressed using synthetic constitutive promoters as described in Blazeck (Biotechnology and Bioengineering, vol.109, no.11,2012).

EXAMPLE 4 production of 2' FLNB Using modified E.coli Strain

The E.coli mutant modified to produce GDP-fucose and LNB (Gal-b 1, 3-GlcNAc) described in example 2 was transformed with an expression plasmid comprising constitutive transcription units for one alpha-1, 2-fucosyltransferase selected from SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37. The production of 2' FLNB (Fuc-a 1,2-Gal-b1, 3-GlcNAc) of the new strain was evaluated in a growth experiment according to the culture conditions provided in example 2, wherein the strain was incubated in minimal medium containing 30g/L sucrose. These strains were grown in four biological replicates in 96-well plates. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC. For each strain detected with a particular a1, 2-fucosyltransferase, the measured 2'flnb concentration was averaged over all biological replicates and then normalized to the average 2' flnb concentration for the reference strain expressing the a-1, 2-fucosyltransferase shown in SEQ ID No. 06. As shown in table 2, all new strains showed production of 2' flnb. The strains expressing SEQ ID NO 31, 32, 33 or 36 produced less 2'FLNB than the reference strain, while the other strains tested produced more 2' FLNB than the reference strain. Strains expressing SEQ ID NO:05, 09, 12, 16, 13, 19, 21, 22, 24, 25, 26, 27, 29 or 30 produced more than two times more 2'FLNB than the reference strain, while strains expressing SEQ ID NO:01, 02, 07, 10, 14, 17, 18, 20, 23 or 28 produced more than three times more 2' FLNB than the reference strain.

Table 2. Relative yields of 2' FLNB (Fuc-a 1,2-Gal-b1, 3-GlcNAc) (%) in E.coli mutants expressing alpha-1, 2-fucosyltransferase and producing GDP-fucose and LNB were evaluated in growth experiments conducted under the culture conditions provided in accordance with example 2, in which the medium contained 30g/L sucrose as a carbon source and compared with a reference strain expressing the alpha-1, 2-fucosyltransferase shown in SEQ ID NO: 06.

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EXAMPLE 5 evaluation of mutant E.coli 2' FLNB production strains in fed-batch fermentation

The E.coli mutants described in example 4 were evaluated during fed-batch fermentation. Bioreactor-scale fed-batch fermentation was performed as described in example 2. Sucrose is used as a carbon source. Lactose is not added in the fermentation process. Unlike the culture experiments described herein, in which the final samples were collected only at the end of the culture (i.e., 72 hours as described herein), regular broth samples were collected at several time points during the fermentation, and the production of LNB and 2' flnb (Fuc-a 1,2-Gal-b1, 3-GlcNAc) at each of these time points was measured using UPLC as described in example 2. Fermentation of E.coli mutant 06 expressing the alpha-1, 2-fucosyltransferase shown in SEQ ID NO. 06 as described in example 4 showed equal titers of LNB and 2' FLNB in all broth samples collected at the end of the fermentation.

EXAMPLE 6 production of LNFP-I Using modified E.coli Strain

The E.coli mutant modified for GDP-fucose production described in example 2 was further modified by: a genome knock-in constitutive transcription unit for galactoside beta-1, 3-N-acetylglucosaminyl transferase lgtA (UniProt ID Q9JXQ6, sequence version 1) from neisseria meningitidis and N-acetylglucosaminyl beta-1, 3-galactosyltransferase wbgO (UniProt ID D3QY14, sequence version 1) from e.coli 55:h7, and transforming an expression plasmid comprising a constitutive transcription unit for one alpha-1, 2-fucosyltransferase selected from SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37. LNFP-I (Fuc-a 1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc) and 2' -FL (Fuc-a 1,2-Gal-b1, 4-Glc) production of the new strains were evaluated in a growth experiment conducted in accordance with the culture conditions provided in example 2, in which the strains were incubated in minimal medium containing 30g/L sucrose and 20g/L lactose. These strains were grown in four biological replicates in 96-well plates. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC. For each strain tested with a particular a1, 2-fucosyltransferase, the measured LNFP-I and 2'-FL concentrations were averaged repeatedly for all organisms and then normalized to the average LNFP-I or 2' -FL concentration, respectively, for the reference strain expressing the alpha-1, 2-fucosyltransferase shown in SEQ ID NO. 06. In this experiment, it was shown that strains expressing the alpha-1, 2-fucosyltransferases shown in SEQ ID NO 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 or 18 produced LNFP-I (see Table 3). Strains expressing the alpha-1, 2-fucosyltransferases shown in SEQ ID NO 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 did not produce LNFP-I (results not shown). Strains expressing the alpha-1, 2-fucosyltransferases shown in SEQ ID NOS: 01, 02, 04, 05, 06, 08, 11, 17 and 18 showed additional 2' -FL production in the cells, next to LNFP-I production (Table 3). Strains expressing SEQ ID NOS: 01, 02, 04, 17 and 18 showed low 2' -FL production, less than 50% of the amount of LNFP-I formed in the cells. The strain expressing SEQ ID NO 05, 06, 08 or 11 is the best 2'-FL producer and the 2' -FL produced is 4 to 8 times that of LNFP-I.

TABLE 3 LNFP-I (Fuc-a 1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc) (%) and 2'-FL (%) relative yields in E.coli mutants expressing alpha-1, 2-fucosyltransferase and producing GDP-fucose were evaluated in growth experiments conducted under the culture conditions provided in example 2, in which the culture medium contained 30g/L sucrose as a carbon source and 20g/L lactose as a precursor, and compared with LNFP-I or 2' -FL yields of the reference strain expressing alpha-1, 2-fucosyltransferase shown in SEQ ID NO:06, respectively.

Strain	Expressed alpha-1, 2-fucosyltransferase	LNFP-I(％)	2’-FL(％)	2’-FL/LNFP-I(％)
					38	SEQ ID NO:06	100	100	650
39	SEQ ID NO:18	9.0	0.70	50
					40	SEQ ID NO:17	12.0	0.70	40
41	SEQ ID NO:16	16.0	0	0
					42	SEQ ID NO:15	25.0	0	0
43	SEQ ID NO:14	30.8	0	0
					44	SEQ ID NO:13	46.8	0	0
45	SEQ ID NO:12	56.7	0	0
					46	SEQ ID NO:11	67.1	84.4	803
47	SEQ ID NO:10	76.8	0	0
					48	SEQ ID NO:09	94.7	0	0
49	SEQ ID NO:08	96.1	57.1	388
					50	SEQ ID NO:07	99.0	0	0
51	SEQ ID NO:05	131	90.7	454
					52	SEQ ID NO:04	229	6.70	18.8
53	SEQ ID NO:03	261	0	0
					54	SEQ ID NO:02	318	10.5	21.4
55	SEQ ID NO:01	355	0.1	0

EXAMPLE 7 production of 2' -FL Using modified E.coli Strain

The E.coli mutant modified for GDP-fucose production described in example 2 was further transformed with an expression plasmid comprising constitutive transcription units for one alpha-1, 2-fucosyltransferase selected from SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37. The production of 2' -FL (Fuc-a 1,2-Gal-b1, 4-Glc) by the new strain was evaluated in a growth experiment carried out according to the culture conditions provided in example 2, wherein the strain was incubated in minimal medium containing 30g/L sucrose and 20g/L lactose. These strains were grown in four biological replicates in 96-well plates. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC. For each strain detected with a specific alpha 1, 2-fucosyltransferase, the measured 2'-FL concentration was averaged over all biological replicates and then normalized to the average 2' -FL concentration of the reference strain expressing the alpha 1, 2-fucosyltransferase shown in SEQ ID NO. 06.

The strain expressing the alpha-1, 2-fucosyltransferase shown in SEQ ID No. 02, 03, 04, 07, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 30, 33, 34, 35, 36 or 37 did not produce 2' -FL (results not shown), and the strain expressing the alpha-1, 2-fucosyltransferase shown in SEQ ID No. 01, 09, 10, 12, 15, 24, 25, 28, 29, 31 or 32 showed a low 2' -FL titer of less than 20% of the 2' FL produced by the reference strain 56 expressing the alpha-1, 2-fucosyltransferase shown in SEQ ID No. 06. The strain expressing the alpha-1, 2-fucosyltransferase shown in SEQ ID NO. 05, 08 or 11 produced 80% or more of 2'FL compared to the 2' FL titer measured in the reference strain having SEQ ID NO. 06.

Table 4. Relative yields of 2' -FL (Fuc-a 1,2-Gal-b1, 4-Glc) (%) in E.coli mutants expressing alpha-1, 2-fucosyltransferase and producing GDP-fucose were evaluated in a growth experiment conducted under the culture conditions provided in accordance with example 2, in which the medium contained 30g/L sucrose as a carbon source and 20g/L lactose as a precursor, and compared with a reference strain expressing the alpha-1, 2-fucosyltransferase shown in SEQ ID NO: 06.

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EXAMPLE 8 evaluation of E.coli mutant in fed-batch fermentation

The E.coli mutants described in example 6 were evaluated during fed-batch fermentation. Bioreactor-scale fed-batch fermentation was performed as described in example 2. Sucrose is used as a carbon source and lactose is added as a precursor in the batch medium during fermentation. Unlike the culture experiments described herein, in which the final samples were collected only at the end of the culture (i.e., 72 hours as described herein), regular broth samples were collected at several time points during the fermentation, and the production of LNFP-I and/or 2' fl at each of these time points was measured using UPLC as described in example 2. The relative yields of 2' -FL or LNFP-I obtained in the broth samples of each strain were calculated as follows: the yield of 2'-FL or LNFP was divided by the sum of the 2' FL and LNFP-I yields titer produced by the strain. Fermentation using E.coli mutants 38 and 54 (expressing the. Alpha. -1, 2-fucosyltransferase shown as SEQ ID NO:06 or 02, respectively) showed that both 2' -FL and LNFP-I were produced in different ratios in the whole broth samples collected at the end of the fermentation. When calculated for the sum of 2'-FL and LNFP-I produced, E.coli mutant 38 showed a relative yield of 55%2' -FL and 45% LNFP-I in the total broth sample collected at the end of the fermentation. Coli mutant 54 showed relative yields of 3.60%2'-FL and 96.7% LNFP-I in the total broth samples collected at the end of the fermentation when calculated for the sum of 2' -FL and LNFP-I produced. A similar fermentation was performed using E.coli mutant 53 (expressing the alpha-1, 2-fucosyltransferase shown as SEQ ID NO: 03). Here, a high production titer of LNFP-I was obtained and no 2' FL was detected in the medium.

Example 9 production of 2'FLNB, 2' -FL and LNFP-I in modified E.coli strains

The GDP-fucose producing, α -1, 2-fucosyltransferase expressing a selected from the list comprising SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37 and the LNB and 2' -FLNB producing escherichia coli mutant strain described in example 4 was further modified by genome knock-in of a constitutive transcriptional unit for the galactoside β -1, 3-N-acetylglucosaminyl transferase lgtA (UniProt ID Q9JXQ6, sequence version 1) from neisseria meningitidis. The novel strains were evaluated for 2'FL, LN3, LNT, LNFP-I, and 2' -FLNB production in a growth experiment according to the culture conditions provided in example 1, wherein the medium contained 30g/L sucrose and 20g/L lactose. These strains were grown in four biological replicates in 96-well plates. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC.

Example 10 production of 2'FLNB, 2' -FL and LNFP-I in modified E.coli strains

The GDP-fucose LN3 and LNT-producing and a mutant strain of escherichia coli expressing an alpha-1, 2-fucosyltransferase selected from the list comprising SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37 as described in example 6 was further modified by genomic knock-in of constitutive transcription units for: mutant glmS 54 from E.coli (compared to wild-type E.coli glmS protein (UniProt ID P17169, SEQ ID 4) differs in the A39T, R C and G472S mutations, as described by Deng et al (Biochimie 88,419-29 (2006)), glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577, SEQ ID 1) and one phosphatase selected from the list comprising any one or more of the following: 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 and BsAraL from Bacillus subtilis. According to the culture conditions provided in WO 18122225. 2'FL, LN3, LNFP-I, LNB and 2' -FLNB of the new strain were evaluated in a growth experiment, wherein the medium contained 30G/L sucrose and 20G/L lactose were grown in four biological replicates in 96 and analyzed on a UPLC well plate as described in WO 18122225.

EXAMPLE 11 production of 2' FLNB Using modified Saccharomyces cerevisiae Strain

Modifying a saccharomyces cerevisiae strain with a first yeast expression plasmid and a second yeast expression plasmid to produce GDP-fucose and LNB and express alpha-1, 2-fucosyltransferase as described in example 3, the first yeast expression plasmid comprising constitutive transcription units for: lactose permease LAC12 from kluyveromyces lactis (UniProt ID P07921, sequence version 1), GDP-mannose 4, 6-dehydratase gmd from escherichia coli (UniProt ID P0AC88, sequence version 1), GDP-L-fucose synthase fcl from escherichia coli (UniProt ID P32055, sequence version 2), and one alpha 1, 2-fucosyltransferase selected from the list comprising SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37, the second yeast expression plasmid comprising constitutive transcription units for: UDP-glucose 4-epimerase galE from E.coli (UniProt ID P09147, sequence version 1) and N-acetylglucosamine beta-1, 3-galactosyltransferase WbgO from E.coli O55:H7 (UniProt ID D3QY14, sequence version 1). The production of 2' -FLNB by the new strain was evaluated in a growth experiment according to the culture conditions provided in example 3, wherein SD CSM-Ura-His auxotrophic medium comprises glucose as carbon source and GlcNAc as precursor. These strains were grown in four biological replicates in 96-well plates. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC.

EXAMPLE 12 production of 2' FLNB Using modified Saccharomyces cerevisiae Strain

The s.cerevisiae mutant for producing GDP-fucose and LNB and for expressing an α -1, 2-fucosyltransferase selected from the list comprising SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37 described in example 11 was further modified by genome knock-in of a constitutive transcription unit for producing GlcNAc: mutant glmS.times.54 from E.coli (differing from the wild-type E.coli glmS protein (UniProt ID P17169, SEQ ID NO: 4) by the A39T, R C and G472S mutations, as described by Deng et al (Biochimie 88,419-29 (2006)), additional copies of glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577, SEQ ID NO: 1) and one phosphatase selected from the list comprising any one or more of the following: coli genes including aphA、Cof、HisB、OtsB、SurE、Yaed、YcjU、YedP、YfbT、YidA、YigB、YihX、YniC、YqaB、YrbL、AppA、Gph、SerB、YbhA、YbiV、YbjL、Yfb、YieH、YjgL、YjjG、YrfG and YbiU, or PsMupP from pseudomonas putida, scDOG from saccharomyces cerevisiae and BsAraL from bacillus subtilis, as described in WO 18122225. The production of 2' -FLNB by the new strain was evaluated in a growth experiment according to the culture conditions provided in example 3, wherein SD CSM-Ura-His auxotrophic medium contains glucose as carbon source, without precursor. These strains were grown in four biological replicates in 96-well plates. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC.

EXAMPLE 13 production of LNFP-I Using modified Saccharomyces cerevisiae Strain

Modifying a saccharomyces cerevisiae strain with a first yeast expression plasmid and a second yeast expression plasmid to produce GDP-fucose and LNT and express alpha-1, 2-fucosyltransferase as described in example 3, the first yeast expression plasmid comprising constitutive transcription units for: lactose permease LAC12 from kluyveromyces lactis (UniProt ID P07921, sequence version 1), GDP-mannose 4, 6-dehydratase gmd from escherichia coli (UniProt ID P0AC88, sequence version 1), GDP-L-fucose synthase fcl from escherichia coli (UniProt ID P32055, sequence version 2), and one alpha 1, 2-fucosyltransferase selected from the list comprising SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37, the second yeast expression plasmid comprising constitutive transcription units for: UDP-glucose 4-epimerase galE from E.coli (UniProt ID P09147, sequence version 1), galactoside beta-1, 3-N-acetylglucosaminyl transferase lgtA from Neisseria meningitidis (UniProt ID Q9JXQ6, sequence version 1), and N-acetylglucosaminyl beta-1, 3-galactosyltransferase WbgO from E.coli O55: H7 (UniProt ID D3QY14, sequence version 1). The LNFP-I production of the new strain was evaluated in a growth experiment according to the culture conditions provided in example 3, wherein SD CSM-Ura-His auxotrophic medium contained glucose as carbon source and lactose as precursor. These strains were grown in four biological replicates in 96-well plates. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC. LNB and 2' flnb production of this mutant was also assessed when GlcNAc was added as additional precursor.

Example 14 materials and methods-Bacillus subtilis

Culture medium

Two different media were used, namely Luria Broth (LB) rich and minimal medium for shake flasks (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/LCuCl2.2H2O、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/LFECl3.6H2O, 1g/L sodium citrate (Hoch 1973PMC 1212887).

The Luria Broth (LB) medium consists of 1% tryptone peptone (Difco, eremmbotegem, belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR, leuven, belgium). Luria Broth Agar (LBA) plates consisted of LB medium supplemented with 12g/L agar (Difco, eremmbotegem, belgium).

Minimal medium (MMfs) for shake flask experiments contained 2.00g/L(NH₄)2SO₄、7.5g/LKH₂PO₄、17.5g/L K₂HPO₄、1.25g/L sodium citrate, 0.25g/L MgSO ₄.7H₂ O, 0.05g/L tryptophan, 10 to 30g/L glucose or another carbon source, 10ml/L trace element mixture, and 10ml/L ferric citrate solution, the other carbon sources including but not limited to: fructose, maltose, sucrose, glycerol and maltotriose will be indicated in the examples. The medium was set to pH 7 with 1M KOH. According to the experiment, lactose, glcNAc, LNB or LacNAc may be added as a precursor.

The complex medium (e.g. LB) is sterilized by autoclaving (121 ℃,21 min) and the minimal medium is sterilized by filtration (0.22 μm Sartorius). The medium is made selective by the addition of antibiotics (e.g., bleomycin (20 mg/L)) if necessary.

Strains, plasmids and mutations

Bacillus subtilis 168, available from the Bacillus genetic collection center (Bacillus Genetic Stock Center, ohio, USA).

Plasmids were constructed for gene deletion by Cre/lox as described in Yan et al (Appl. & environm. Microbial., sept 2008, p 5556-5562). Gene disruption was performed by homologous recombination with linear DNA and transformation by electroporation as described by Xue et al (J. Microb. Meth.34 (1999) 183-191). Methods for gene knockout are described in Liu et al (Metab. Engine.24 (2014) 61-69). The method uses 1000bp homologs upstream and downstream of the target gene.

The integration vector described by Popp et al (sci.rep., 2017,7,15158) is used as an expression vector and may further be used for genomic integration, if desired. Promoters suitable 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 Gibson assembly, golden Gate assembly, cliva assembly, LCR, or restriction ligation.

In an embodiment for producing an LNB, a bacillus subtilis mutant strain is modified by a genomic knock-in comprising a constitutive transcription unit for: mutant glmS.times.54 from E.coli (differing from wild-type E.coli glmS protein (UniProt ID P17169, SEQ ID NO: 4) by the A39T, R C and G472S mutations, as described by Deng et al (Biochimie 88,419-29 (2006)), glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577, SEQ ID NO: 1), a phosphatase selected from the list comprising any one or more of the following: coli genes including aphA、Cof、HisB、OtsB、SurE、Yaed、YcjU、YedP、YfbT、YidA、YigB、YihX、YniC、YqaB、YrbL、AppA、Gph、SerB、YbhA、YbiV、YbjL、Yfb、YieH、YjgL、YjjG、YrfG and YbiU, or PsMupP from Pseudomonas putida, DOG1 from Saccharomyces cerevisiae and AraL from Bacillus subtilis, as described in WO18122225, and N-acetylglucosamine beta-1, 3-galactosyltransferase WbgO from E.coli O55: H7 (UniProt ID D3QY14, SEQ ID NO: 1). To further fucosylate the LNB to a 2' flnb, the mutant strain is further modified by a constitutive transcription unit for an α -1, 2-fucosyltransferase selected from the list comprising SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37.

In an example for the production of lactose-based oligosaccharides, a bacillus subtilis mutant strain was established that comprises a gene encoding a lactose input, such as the e.coli lacY gene (UniProt ID P02920, sequence version 1).

In an embodiment for the production of lactose-N-trisaccharide (LNT-II, LN3, glcNAc-b1,3-Gal-b1, 4-Glc), a strain of Bacillus subtilis is modified by genomic knock-in of a constitutive transcription unit comprising: lactose importants such as E.coli lacY (UniProt ID P02920, sequence version 1) and galactoside beta-1, 3-N-acetylglucosaminyl transferase such as LgtA (GenBank: AAM 33849.1) from Neisseria meningitidis, for example. For LNT production, LN3 production strains were further modified using a WbgO (UniProt ID D3QY14, sequence version 1) constitutive transcription unit for N-acetylglucosamine β -1, 3-galactosyltransferase, such as, for example, from E.coli O55:H 7. N-acetylglucosamine β -1, 3-galactosyltransferase can be delivered to the strain by genomic knock-in or via an expression plasmid. For LNFP-I production, the LNT production strain is further modified using constitutive transcription units for the alpha-1, 2-fucosyltransferase (as e.g.SEQ ID NO:01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12,13, 14, 15, 16, 17 or 18).

For growth under sucrose, the mutant strain may additionally be modified by genomic knock-in of a constitutive transcription unit comprising sucrose transporter (CscB) from e.coli W (UniProt ID E0IXR1, sequence version 1), fructokinase (Frk) from zymomonas mobilis (UniProt ID Q03417, sequence version 1) and sucrose phosphorylase (BaSP) from bifidobacterium adolescentis (UniProt ID A0 zh6, sequence version 1).

Heterologous and homologous expression

The genes to be expressed (whether from plasmids or from the genome) are synthesized by one of the following companies: DNA2.0, gen9, twist Bioscience 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-incubation of 96 well microtiter plates was started from frozen vials or individual colonies from LB plates in 150. Mu.L LB and incubated overnight at 37℃on an orbital shaker at 800 rpm. The culture was used as an inoculum for 96 Kong Fangxing microtiter plates and 400 μ L MMsf medium was used by dilution 400-fold. Each strain was grown as a biological replicate in multiple wells of a 96-well culture plate. These final 96-well 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 incubation experiment, samples were taken from each well to measure the supernatant concentration (extracellular sugar concentration, after centrifugation of the cells for 5 minutes) or by boiling the culture broth at 90 ℃ for 15 minutes or at 60 ℃ for 15 minutes (=whole broth concentration, intracellular and extracellular sugar concentrations, as defined herein) prior to centrifugation of the cells.

In addition, culture dilutions were performed to measure optical density at 600 nm. The cell performance index or CPI is determined by dividing the oligosaccharide concentration measured throughout the broth by the biomass, expressed as a relative percentage compared to the reference strain. The biomass was empirically determined to be about one third (1/3) of the optical density measured at 600 nm.

EXAMPLE 15 production of 2' FLNB Using modified Bacillus subtilis Strain

First, bacillus subtilis strains were modified to produce LNBs by genomic knockout of nagB, glmS, and gamA genes and genomic knockout of constitutive transcription units comprising genes encoding: native fructose-6-P-aminotransferase (UniProt ID P0CI73, sequence version 1), mutant glmS 54 from escherichia coli (differing from wild-type escherichia coli glmS protein (UniProt ID P17169, sequence version 4) by mutations a39T, R C and G472S, as described by Deng et al (biochimie 88,419-29 (2006)), glucosamine 6-phosphate N-acetyltransferase GNA1 from saccharomyces cerevisiae (UniProt ID P43577, sequence version 1), phosphatase AraL from bacillus subtilis (UniProt ID P94526, sequence version 1), N-acetylglucosamine β -1, 3-galactosyltransferase WbgO from escherichia coli O55: H7 (UniProt ID D3QY14, sequence version 1), sucrose transporter from escherichia coli W (CscB) (prot ID E0i 1, sequence version 1), kinase enzyme from zymomonas mobilis (prot) (sequence version 24, sequence version BaSP) and sucrose phosphorylase (zq 4) from bifidobacterium sp (prot ID 4). In a next step, the LNB production strain is transformed with an expression plasmid comprising constitutive transcription units for α -1, 2-fucosyltransferases selected from the list comprising SEQ ID 01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37. The production of 2' flnb by the new strain was evaluated in a growth experiment performed in MMsf medium lacking the precursor according to the culture conditions provided in example 14. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC.

EXAMPLE 16 production of LNFP-I Using modified Bacillus subtilis Strain

First, bacillus subtilis strains were modified for LN3 production and growth under sucrose by genomic knock-out nagB, glmS and gamA genes and genomic knock-in constitutive transcription units comprising genes encoding: lactose permease (LacY) from E.coli (UniProt ID P02920, sequence version 1), native fructose-6-P-aminotransferase (UniProt ID P0CI73, sequence version 1), galactoside beta-1, 3-N-acetylglucosaminyltransferase LgtA from Neisseria meningitidis (GenBank: AAM33849.1, sequence version 1), sucrose transporter (CscB) from E.coli W (UniProt ID E0IXR1, sequence version 1), fructokinase (Frk) from Zymomonas mobilis (UniProt ID Q03417, sequence version 1) and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (UniProt ID A0ZZH6, sequence version 1). In the next step, the mutant was further modified by genome knock-in of a constitutive transcription unit comprising N-acetylglucosamine β -1, 3-galactosyltransferase WbgO (UniProt ID D3QY14, sequence version 1) from E.coli O55: H7 to produce LNT. In a subsequent step, the LNT production strain is transformed with an expression plasmid comprising constitutive transcription units for an α -1, 2-fucosyltransferase selected from the list comprising SEQ ID 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 and 18. LNFP-I and 2' -FL production of the new strain was evaluated in a growth experiment performed in MMsf medium containing lactose as a precursor according to the culture conditions provided in example 14. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC.

Example 17 materials and methods-Corynebacterium glutamicum

Culture medium

Two different media were used: namely, rich tryptone-yeast extract (TY) medium and minimal medium. Minimal medium stock was used with 1000x trace element mixture.

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.

The minimal medium used in the shake flask (MMsf) experiments contained 20g/L (NH ₄)₂SO₄, 5g/L urea, 1g/L KH2PO4, 1g/L K HPO4, 0.25g/L MgSO4.7H2O, 42g/L MOPS, 10 to 30g/L glucose or another carbon source (including but not limited to fructose, maltose, sucrose, glycerol and maltotriose, as will be indicated in the examples) and 1ml/L trace element mixture depending on the experiment lactose, glcNAc, LNB or LacNAc may be added as precursors.

TY medium consisted of 1.6% tryptone (Difco, eremmbotegem, belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, belgium). TY agar (TYA) plates consisted of TY medium supplemented with 12g/L agar (Difco, eremmbotegem, belgium).

The complex medium (e.g. TY) is sterilized by autoclaving (121 ℃,21 min) and the minimal medium is sterilized by filtration (0.22 μm Sartorius). If necessary, the medium is made selective by the addition of antibiotics (e.g., kanamycin, ampicillin).

Strains and mutations

Corynebacterium glutamicum ATCC 13032, which is available from the American type culture Collection, was used.

Integration vectors based on the Cre/loxP technology described by Suzuki et al (appl. Microbiol. Biotechnol.,2005Apr,67 (2): 225-33) and temperature sensitive shuttle vectors described by Okibe et al (Journal of Microbiological methods85,2011, 155-163) were constructed for gene deletion, mutation and insertion. Promoters suitable for (heterologous) gene expression may be derived from YIm et al (Biotechnol. Bioeng.,2013nov,110 (11): 2959-69). Cloning can be performed using Gibson assembly, golden Gate assembly, cliva assembly, LCR, or restriction ligation.

In an embodiment for producing an LNB, a corynebacterium glutamicum strain is modified by genomic knock-in of a constitutive expression unit comprising: mutant glmS.times.54 from E.coli (differing from wild-type E.coli glmS protein (UniProt ID P17169, SEQ ID NO: 4) by the A39T, R C and G472S mutations, as described by Deng et al (Biochimie 88,419-29 (2006)), glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577, SEQ ID NO: 1), a phosphatase selected from the list comprising any one or more of the following: coli genes including aphA、Cof、HisB、OtsB、SurE、Yaed、YcjU、YedP、YfbT、YidA、YigB、YihX、YniC、YqaB、YrbL、AppA、Gph、SerB、YbhA、YbiV、YbjL、Yfb、YieH、YjgL、YjjG、YrfG and YbiU, or PsMupP from Pseudomonas putida, scDOG from Saccharomyces cerevisiae and BsAraL from Bacillus subtilis, as described in WO18122225, and N-acetylglucosamine beta-1, 3-galactosyltransferase WbgO from E.coli O55: H7 (UniProt ID D3QY14, SEQ ID NO: 1). To further fucosylate the LNB to a 2' flnb, the mutant strain is further modified by a constitutive transcription unit for an α -1, 2-fucosyltransferase selected from the list comprising SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37.

In an example of the production of lactose-based oligosaccharides, a corynebacterium glutamicum mutant strain was created, which comprises a gene encoding a lactose donor (e.g. escherichia coli LacY (UniProt ID P02920, sequence version 1)).

In an example of the production of lactose-N-trisaccharides (LNT-II, LN3, glcNAc-b1,3-Gal-b1, 4-Glc), a strain of Corynebacterium glutamicum is modified by genomic knock-in of a constitutive expression unit comprising a lactose input (e.g.E.coli lacY (UniProt ID P02920, SEQ ID NO: 1)) and a galactoside beta-1, 3-N-acetylglucosaminyl transferase (e.g. LgtA from Neisseria meningitidis (GenBank: AAM 33849.1)). For LNT production, the LN3 production strain is further modified by constitutive transcription units for N-acetylglucosamine beta-1, 3-galactosyltransferase, such as WbgO (UniProt ID D3QY14, sequence version 1) from E.coli O55:H7, for example. N-acetylglucosamine β -1, 3-galactosyltransferase can be delivered to the strain by genomic knock-in or via an expression plasmid. For LNFP-I production, LNT-producing strains were further modified using an alpha-1, 2-fucosyltransferase expression construct selected from the list comprising SEQ ID NOs: 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 and 18.

Heterologous and homologous expression

Culture conditions

Pre-incubation of 96 well microtiter plates was started from frozen vials or individual colonies from TY plates in 150. Mu.L TY and incubated overnight at 37℃on an orbital shaker at 800 rpm. The culture was used as an inoculum for 96 Kong Fangxing microtiter plates and 400. Mu.L of minimal medium was used by dilution 400-fold. Each strain was grown as a biological replicate in multiple wells of a 96-well culture plate. These final 96-well 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 centrifugation of the cells for 5 minutes) or by boiling the culture broth at 60 ℃ for 15 minutes (=whole broth concentration, intracellular and extracellular sugar concentrations, as defined herein) prior to centrifugation of the cells.

EXAMPLE 18 production of 2' FLNB Using modified Corynebacterium glutamicum Strain

First, the corynebacterium glutamicum strain was modified for LNB production and growth under sucrose by genomic knock-out of the ldh, cgl2645 and nagB genes and genomic knock-in of a constitutive transcription unit comprising genes encoding: mutation gmS from E.coli (differing from wild-type E.coli glmS protein (UniProt ID P17169, SEQ ID 4) in the A39T, R C and G472S mutations, as described by Deng et al (Biochimie 88,419-29 (2006)), glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577, SEQ ID 1), phosphatase AraL from Bacillus subtilis (UniProt ID P94526, SEQ ID 1), N-acetylglucosamine beta-1, 3-galactosyltransferase WbgO from E.coli O55: H7 (UniProt ID D3QY14, SEQ ID 1), sucrose transporter from E.coli W (CscB) (UniProt ID E0IXR1, SEQ ID 1), fructose kinase from Zymomonas mobilis (Frk) (UniProt ID Q03417, SEQ ID 1) and sucrose phosphorylase from Bifide (UniProt H0) ZH 0. In a next step, the LNB production strain is transformed with an expression plasmid comprising constitutive transcription units for α -1, 2-fucosyltransferases selected from the list comprising SEQ ID 01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37. The production of 2' flnb by the new strain was evaluated in a growth experiment performed in MMsf medium lacking the precursor according to the culture conditions provided in example 17. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC.

EXAMPLE 19 production of LNFP-I Using modified Corynebacterium glutamicum Strain

First, the corynebacterium glutamicum strain was modified for LN3 production and growth under sucrose by genomic knock-out of the ldh, cgl2645 and nagB genes and genomic knock-in of a constitutive transcription unit comprising genes encoding: lactose permease (LacY) from E.coli (UniProt ID P02920, sequence version 1), native fructose-6-P-aminotransferase (UniProt ID P0CI73, sequence version 1), galactoside beta-1, 3-N-acetylglucosaminyltransferase LgtA from Neisseria meningitidis (GenBank: AAM 33849.1), sucrose transporter (CfcB) from E.coli W (UniProt ID E0IXR1, sequence version 1), fructokinase (Frk) from Zymomonas mobilis (UniProt ID Q03417, sequence version 1) and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (UniProt ID A0ZZH6, sequence version 1). In the next step, the mutant was further modified by genome knock-in of a constitutive transcription unit comprising N-acetylglucosamine β -1, 3-galactosyltransferase WbgO (UniProt ID D3QY14, sequence version 1) from E.coli O55: H7 to produce LNT. In a subsequent step, the LNT production strain is transformed with an expression plasmid comprising constitutive transcription units for an α -1, 2-fucosyltransferase selected from the list comprising SEQ ID 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 and 18. LNFP-I and 2' -FL production of the new strain was evaluated in a growth experiment performed in MMsf medium containing lactose as a precursor according to the culture conditions provided in example 17. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC.

Example 20 materials and methods-Chlamydomonas reinhardtii

Culture medium

Chlamydomonas reinhardtii (C.reinhardtii) cells were cultured in Tris-acetate phosphate (TAP) medium (pH 7.0). TAP medium was stock-stored using 1000x Hutner trace element mixtures. The Hutner trace element mixture consisted of ：50g/L Na2EDTA.H2O(Titriplex III),22g/L ZnSO4.7H2O,11.4g/L H3BO3,5g/L MnCl2.4H2O,5g/L FeSO4.7H2O,1.6g/L CoCl2.6H2O,1.6g/L CuSO4.5H2O and 1.1g/L (NH 4) 6MoO3.

TAP medium contained 2.42g/L Tris (Tris (hydroxymethyl) aminomethane), 25mg/L saline stock solution, 0.108g/L K ₂HPO₄、0.054g/L KH₂PO₄ and 1.0mL/L glacial acetic acid. The saline stock solution consisted of 15g/LNH4CL, 4g/L MgSO4.7H2O and 2g/L CaCl2.2H2O. Regarding precursors for sugar synthesis, precursors may be added, such as galactose, glucose, fructose, fucose, glcNAc, LNB and/or LacNAc. The medium was sterilized by autoclaving (121 ℃,21 min). For storage cultures on agar slants, TAP medium containing 1% agar (highly purified, 1000g/cm 2) was used.

Algae strain, plasmid and mutation

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, https:// www.chlamycollection.org, university of Minnesota, U.S.A.).

The expression plasmid was derived from pSI103, which is available from the C.sp.resource center. Cloning can be performed using Gibson assembly, golden Gate assembly, cliva assembly, LCR, or restriction ligation. Promoters suitable for (heterologous) gene expression may be derived, for example, from Scandon et al (Algal Res.2016, 15:135-142). Targeted gene modification (e.g., gene knockout or gene replacement) can be performed using, for example, the Crispr-Cas technique described 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 (light intensity 8000 Lx) to a cell density of 1.0-2.0X10 ⁷ cells/mL. However, cells were inoculated into fresh liquid TAP medium at a concentration of 1.0X10 ⁶ cells/mL and grown under continuous light for 18-20 hours until the cell density reached 4.0X10 ⁶ cells/mL. Cells were then collected by centrifugation at 1250g for 5 min at room temperature, washed and resuspended in chilled liquid TAP medium containing 60mM sorbitol (Sigma, u.s.a.) for 10 min on ice. Then, 250. Mu.L of the cell suspension (corresponding to 5.0X10 ⁷ cells) was placed in a pre-chilled 0.4cm electroporation cuvette with 100ng plasmid DNA (400 ng/mL). Electroporation was performed using a BTX ECM830 electroporation device (1575 Ω,50 μfd) with 500v 6 pulses each of 4ms in length and 100ms in pulse interval. Immediately after electroporation, the electroporation cuvette was placed on ice for 10 minutes. Finally, the cell suspension was transferred to a 50mL conical centrifuge tube containing 10mL of fresh liquid TAP medium containing 60mM sorbitol and slowly shaken overnight under dim light. After overnight resuscitation, cells were collected again and placed on an ampicillin (100 mg/L) or chloramphenicol (100 mg/L) selective 1.5% (w/v) agar-TAP plate using starch embedding. The plates were then incubated at 23+ -0.5℃under continuous irradiation with light intensity 8000 Lx. Cells were analyzed after 5-7 days.

In the example of UDP-galactose production, chlamydomonas reinhardtii cells were modified with transcription units comprising a gene encoding galactokinase (KIN, uniProt ID Q9SEE5, SEQ ID NO: 2) from Arabidopsis thaliana (Arabidopsis thaliana) and a gene encoding UDP-sugar pyrophosphorylase (USP) (UniProt ID Q9C5I1, SEQ ID NO: 1) from Arabidopsis thaliana.

In the LNB-producing example, the Chlamydomonas reinhardtii cells modified for UDP-galactose production were further modified with an expression plasmid containing transcriptional units related to N-acetylglucosamine beta-1, 3-galactosyltransferase WbgO (UniProt ID D3QY14, SEQ ID NO: 1) from E.coli O55: H7. Alternatively, mutant Chlamydomonas reinhardtii cells can be modified with an expression plasmid comprising a transcriptional unit for an alpha-1, 2-fucosyltransferase (selected from the list comprising SEQ ID NO:ID 01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37).

Heterologous and homologous expression

Culture conditions

Chlamydomonas reinhardtii cells were cultured on selective TAP-agar plates at 23+/-0.5℃under 14/10h light/dark cycles (light intensity 8000 Lx). Cells were analyzed after 5-7 days of culture.

For high density culture, cells may be cultured in closed systems such as vertical or horizontal tube, stirred tank or plate photobioreactors as described in Chen et al (bioresouur. Technol.2011, 102:71-81) and Johnson et al (biotechnol. Prog.2018, 34:811-827).

Example 21 production of 2' FLNB in modified Chlamydomonas reinhardtii cells

Chlamydomonas reinhardtii cells were engineered to produce UDP-Gal by genomic knock-in of a constitutive transcription unit comprising galactokinase (KIN, unit ID Q9SEE5, SEQ ID NO: 2) from Arabidopsis thaliana and UDP-sugar pyrophosphorylase (USP) (Unit ID Q9C5I1, SEQ ID NO: 1) from Arabidopsis thaliana as described in example 20. In the next step, the mutant cells are transformed with an expression plasmid comprising a transcription unit comprising N-acetylglucosamine β -1, 3-galactosyltransferase WbgO from E.coli O55: H7 (Unit Prot ID D3QY14, sequence version 1) and an α -1, 2-fucosyltransferase selected from the list comprising SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37. New algal strains were evaluated in a culture experiment on TAP-agar plates containing galactose and GlcNAc as precursors according to the culture conditions provided in example 20. After 5 days of incubation, cells were collected and analyzed for 2' flnb production on UPLC.

Example 22 materials and methods-animal cells

Isolation of mesenchymal Stem cells from adipose tissue of different animals

Fresh adipose tissue is obtained from slaughterhouses (e.g. cattle, pigs, sheep, chickens, ducks, catfish, snakes, frog slaughterhouses) or liposuction departments (e.g. in the case of humans, after informed consent) and kept in phosphate buffer supplemented with antibiotics. The adipose tissues were subjected to enzymatic digestion, and then mesenchymal stem cells were isolated by centrifugation. Isolated mesenchymal stem cells were transferred to cell culture flasks 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. After the first passage, the medium was replaced with 10% fbs (fetal bovine serum) -supplemented medium. For example, ahmad and Shakoori (2013,Stem Cell Regen.Med.9 (2): 29-36), which are incorporated herein by reference 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 other mammal as described herein. An equal volume of phosphate buffer was added to the diluted milk followed by centrifugation for 20 minutes. The cell pellet was washed three times with phosphate buffer and cells were inoculated under standard culture conditions in DMEM-F12, RPMI and alpha-MEM medium supplemented with 10% fetal bovine serum and 1% antibiotics in cell culture flasks. For example, hassiotou et al (2012,Stem Cells.30 (10): 2164-2174), which is incorporated herein by reference in its entirety for all purposes, describe certain variations of the methods described in this example.

Stem cell differentiation Using 2D and 3D culture systems

The isolated mesenchymal stem 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 ASPECTS, springer, dordrecht.141-145; williams et al 2009,Breast Cancer Res 11 (3): 26-43; and Arevalo et al 2015,Am J Physiol Cell Physiol.310 (5): C348-C356, each of which is incorporated herein by reference in its entirety for all purposes.

For 2D culture, the isolated cells were initially inoculated in growth medium supplemented with 10ng/mL of epidermal growth factor and 5pg/mL of insulin in culture plates. At confluence, the cells were fed growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100U/mL penicillin, 100ug/mL streptomycin) and 5pg/mL insulin for 48 hours. To induce differentiation, cells were fed with complete growth medium containing 5pg/mL insulin, 1pg/mL cortisol, 0.65ng/mL triiodothyronine, 100nM dexamethasone, and 1pg/mL prolactin. After 24 hours, serum was removed from the complete induction medium.

For 3D culture, the isolated cells were trypsinized and cultured in Matrigel (Matrigel), hyaluronic acid or ultra low adhesion surface plates for six days and induced to differentiate and lactation by addition of growth medium supplemented with 10ng/mL epidermal growth factor and 5pg/mL insulin. At confluence, the cells were fed growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100U/mL penicillin, 100ug/mL streptomycin) and 5pg/mL insulin for 48 hours. To induce differentiation, cells were fed with complete growth medium containing 5pg/mL insulin, 1pg/mL cortisol, 0.65ng/mL triiodothyronine, 100nM dexamethasone, and 1pg/mL prolactin. After 24 hours, serum was removed from the complete induction medium.

Method for preparing breast-like cells

Mammalian cells were rendered induced pluripotent by reprogramming with viral vectors encoding Oct4, sox2, klf4 and c-Myc. The resulting reprogrammed cells are then cultured in Mammocult medium (obtained from Stem Cell Technologies) or mammary cell enrichment medium (DMEM, 3% fbs, estrogens, progesterone, heparin, cortisol, insulin, EGF) to make them mammary-like cells from which the expression of the selected milk component can be induced. Alternatively, epigenetic engineering is performed using engineering systems (e.g., CRISPR/Cas 9) to activate selected genes of interest, such as casein, a-lactalbumin, are opened constitutively to allow expression of their respective proteins, and/or down-regulated and/or knockout of selected endogenous genes, as described, for example, in WO21067641, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

Culturing

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 cortisol. Complete lactation medium included high glucose DMEM/F12, 1% NEAA, 1% pen/strep, 1% ITS-X, 1%F-Glu, 10ng/mL EGF, 5pg/mL cortisol and 1pg/mL prolactin (5 ug/mL, hyunh 1991). Cells were seeded and adhered on collagen-coated flasks at a density of 20000 cells/cm ² in complete growth medium, expanded in complete growth medium for 48 hours, after which the medium was replaced with complete lactation medium. Upon exposure to lactation medium, the cells begin to differentiate and stop growing. Within about one week, the cells begin to secrete one or more milk-secreting products (e.g., milk lipids, lactose, casein, and whey) into the culture medium. The desired concentration of the lactation medium can be achieved by concentration or by dilution by ultrafiltration. The desired salt balance of the lactation medium may be achieved by dialysis, e.g. removal of unwanted metabolites from the medium. The hormones and other growth factors used may be selectively extracted by resin purification, for example using nickel resin to remove His-tagged growth factors, thereby further reducing the level of contaminants in the lactation products.

Example 23 evaluation of 2'FL, LNFP-I, and 2' FLNB production in non-mammary adult Stem cells

The isolated mesenchymal cells and reprogrammed breast-like cells of example 22 were modified by CRISPR-CAS to overexpress: GDP-fucose synthase GFUS from Chile (UniProt ID Q13630, SEQ ID 1), and a codon-optimized alpha-1, 2-fucosyltransferase selected from the list comprising SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 and 37, from Chile (Homo sapiens) beta-1, 4-galactosyltransferase 1B4GalT1 (Uniprot ID P15291, SEQ ID 5). Cells were seeded and adhered on collagen-coated flasks at a density of 20000 cells/cm ² in complete growth medium, expanded in complete growth medium for 48 hours, after which the medium was replaced with complete lactation medium for about 7 days. After incubation as described in example 22, cells were subjected to UPLC to analyze the production of 2'FL, LNFP-I, and 2' FLNB.

EXAMPLE 24 production LNDFHI Using modified E.coli Strain

The E.coli mutant modified to produce GDP-fucose and LNFP-I described in example 6 (expressing one alpha-1, 2-fucosyltransferase selected from SEQ ID NOs: 03, 07, 09, 10, 12, 13, 14, 15 and 16, respectively) was further modified using an expression plasmid comprising a constitutive transcription unit for a second fucosyltransferase from helicobacter pylori (UniProt ID O30511, sequence version 1). The production of LNDFH I (Fuc-a 1,2-Gal-b1,3- [ Fuc-a1,4] -GlcNAc-b1,3-Gal-b1, 4-Glc) of the new strain was evaluated in a growth experiment according to the culture conditions provided in example 1, wherein the medium contained 30g/L sucrose and 20g/L lactose. These strains were grown in four biological replicates in 96-well plates. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC.

EXAMPLE 25 evaluation of alpha-1, 2-fucosyltransferase Activity

Another embodiment provides an assessment of the alpha-1, 2-fucosyltransferase activity of the enzymes of the invention SEQ ID NO:01、02、03、04、05、06、07、08、09、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36 or 37. These enzymes may be produced in a cell-free expression system such as, but not limited to PURExpress system (NEB), such as, but not limited to E.coli or Saccharomyces cerevisiae, which may then be isolated and optionally further purified each of the above-listed enzymes together with GDP-fucose and buffer components such as Tris-HCl or HEPES and a substrate such as, for example, lactose-N-disaccharide (LNB) or lactose-N-tetrasaccharide (LNT) may then be added to the reaction mixture at a specific temperature (e.g., 37 ℃) Wen Yote for a defined period of time (e.g., 24 hours) during which the enzymes convert lactose, LNB or LNT to 2 '-fucosyllactose, 2' FLNB or LNT, respectively, which is then separated from the reaction mixture by methods known in the art.

EXAMPLE 26 production of LNDFH-I using E.coli mutant in fed-batch fermentation

In another experiment, E.coli mutant 53 (which expresses the alpha 1, 2-fucosyltransferase of SED ID NO: 03) described in example 6 was transformed with a second plasmid expressing fucosyltransferase (uniprot ID A A0G4K5H1 (SEQ ID NO:03, 16. 2015)). The strain was evaluated during fed-batch fermentation. Bioreactor-scale fed-batch fermentation was performed as described in example 2. Sucrose is used as a carbon source and lactose is added as a precursor to the batch medium during fermentation. Unlike the culture experiments described herein, in which the final samples were collected only at the end of the culture (i.e., 72 hours as described herein), regular broth samples were collected at several time points during the fermentation, and the production of LN3, LNT, LNFP-I, LNFP-II, LNDFH-I, and/or LNDFH-II at each of these time points was measured using UPLC as described in example 2. Fermentation of E.coli mutants expressing fucosyltransferases (uniprot ID A A0G4K5H1 (SEQ ID NO:03, 16 th. 2015)) and alpha-1, 2-fucosyltransferases of LNT, LNFP-I, LNFP-II, LNDFH-I or LNDFH-II showed relative yields of 17.0% LNT, 48.0% LNFP-I, 10.5% LNFP-II, 20.7% LNDFH-I and 3.8% LNDFH-II in the total broth samples collected at the end of the fermentation when calculated by dividing the titres of LNT, LNFP-I, LNFP-II, LNDFH-I or LNDFH-II yield by the sum of the yields of LNT, LNFP-I, LNFP-II, LNDFH-I and LNDFH-II produced by the strain.

Claims

Wherein:

Wherein the method comprises the steps of:

i. Providing a cell expressing an alpha-1, 2-fucosyltransferase, and

-Is a polypeptide belonging to the family of gt74 fucosyltransferases, or

2. The method of claim 1, wherein the α -1, 2-fucosyltransferase has additional galactosidase α -1, 2-fucosyltransferase activity for galactose residues of the non-reducing end of Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (LNT, lactose-N-tetrasaccharide), and wherein the α -1, 2-fucosyltransferase:

3. The method of claim 2, wherein the alpha-1, 2-fucosyltransferase has no additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose or has additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose but less than the additional galactosidase alpha-1, 2-fucosyltransferase activity for galactose residues on the non-reducing end of LNT, and

4. The method of claim 2, wherein the alpha-1, 2-fucosyltransferase has additional galactosidase alpha-1, 2-fucosyltransferase activity on lactose but greater than the additional galactosidase alpha-1, 2-fucosyltransferase activity on galactose residues of the non-reducing end of LNT, and

5. The method of claim 1, wherein the alpha-1, 2-fucosyltransferase has no galactosidase alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of an LNT, and

6. The method of claim 5, wherein the alpha-1, 2-fucosyltransferase has NO galactosylα -1, 2-fucosyltransferase activity on lactose or has additional galactosylα -1, 2-fucosyltransferase activity on lactose but less than 3.0% of the galactosylα -1, 2-fucosyltransferase activity on lactose of the alpha-1, 2-fucosyltransferase of SEQ ID NO 06, and

7. The method of claim 5, wherein the alpha-1, 2-fucosyltransferase has additional galactosylα -1, 2-fucosyltransferase activity on lactose and is 4.0 to 20.0% of the galactosylα -1, 2-fucosyltransferase activity on lactose of the alpha-1, 2-fucosyltransferase of SEQ ID NO 06, and

8. The method of any one of the preceding claims, wherein the cell is modified in terms of expression or activity of any one of the alpha-1, 2-fucosyltransferases.

9. The method of any one of the preceding claims, wherein the cell is capable of producing one or more nucleotide-activating sugars selected from the list comprising: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2, 6-dideoxy-L-arabino-4-hexulose, UDP-2-acetamido-2, 6-dideoxy-L-lyxol-4-hexulose, UDP-N-acetyl-L-rhamnose (UDP-L-RhaNAc or UDP-2-acetamido-2, 6-dideoxy-L-mannose), DP-N-acetylfucose, UDP-N-acetylfucose (UDP-L-52 or UDP-2-acetamido-2, 6-dideoxy-L-5-fucose), UDP-N-acetylgalactosamine (UDP-L-FucNAc or UDP-2-acetamido-N-5-mannosamine), UDP-N-acetylgalactosamine (UDP-N-RhaNAc or UDP-2-acetamido-N-6-acetylgalactosamine), UDP-N-acetyl-L-quiniosamine (UDP-L-QuiNAc or UDP-2-acetamido-2, 6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac)、CMP-Neu4Ac、CMP-Neu5Ac9N₃、CMP-Neu4,5Ac₂、CMP-Neu5,7Ac₂、CMP-Neu5,9Ac₂、CMP-Neu5,7(8,9)Ac₂、CMP-N- hydroxyacetyl neuraminic acid (CMP-Neu 5 Gc), GDP-rhamnose and UDP-xylose.

10. The method of any one of the preceding claims, 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-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine 6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine 1-phosphate acetyltransferase, N-acetylneuraminic acid synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid 9-phosphate synthase, N-acylneuraminic acid 9-phosphate phosphatase, N-acylneuraminic acid cytidylyltransferase, galactose 1-epimerase, galactokinase, and, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein said cell is modified in terms of expression or activity of any of said polypeptides.

11. The method of any one of the preceding claims, wherein the cell expresses one or more glycosyltransferases selected from the list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosyltransferase, N-acetylmannosyltransferase, xylosyltransferase, glucuronyltransferase, galacturonate transferase, glucosaminyltransferase, N-glycolylneuraminidase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-arabinoxylan-Zhuo Tangan aminotransferase, UDP-N-acetylglucosaminenolpyruvylase and fucosyltransferase,

12. The method according to any of the preceding claims, wherein the compound comprising the structure of formula I, II or III is an oligosaccharide, preferably the oligosaccharide is a Mammalian Milk Oligosaccharide (MMO), more preferably a Human Milk Oligosaccharide (HMO).

13. The method according to any of the preceding claims, wherein the compound comprising the structure of formula I, II or III is a charged, preferably sialylated compound or is a neutral compound,

14. The method of any one of the preceding claims, wherein the compound comprising the structure of formula I, II or III is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R comprising one R group selected from the list comprising monosaccharides, disaccharides or oligosaccharides,

15. The method of any one of the preceding claims, wherein the cells use one or more precursors to produce the compound comprising the structure of formula I, II or III, the precursors being fed to the cells from a culture medium.

16. The method of any one of the preceding claims, wherein the cells produce one or more precursors for producing the compound comprising the structure of formula I, II or III.

17. The method of any one of claims 15 or 16, wherein the precursor for producing the compound comprising the structure of formula I, II or III is fully converted to the compound comprising the structure of formula I, II or III.

18. The method of any one of the preceding claims, wherein the cell is capable of producing a compound comprising a structure of formula IV, V or VI:

Wherein:

19. The method of any one of the preceding claims, wherein the cell produces the compound comprising the structure of formula I, II or III inside the cell, and wherein a portion or substantially all of the produced compound comprising the structure of formula I, II or III remains inside the cell and/or is excreted outside the cell by passive or active transport.

20. The method according to any of the preceding claims, wherein the cell expresses a membrane transporter or a polypeptide having transport activity, whereby the compound is transported across the outer membrane of the cell wall,

21. The method of claim 20, wherein the membrane transporter or a polypeptide having translocator activity is selected from the list comprising: transporter, P-P bond hydrolysis-driven transporter, b-barrel porin, auxiliary transporter, putative transporter and phosphate transport-driven group translocator,

22. The method of any one of claims 20 or 21, wherein the membrane transporter or a polypeptide having transport activity controls the flow of the compound comprising the structure of formula I, II or III and/or one or more precursors and/or receptors for the production of the compound comprising the structure of formula I, II or III to the outer membrane of the cell wall.

23. The method according to any one of claims 20 to 22, wherein the membrane transporter or a polypeptide having transport activity provides improved production and/or allowable and/or enhanced efflux of the compound comprising the structure of formula I, II or III.

24. The method of any one of the preceding claims, wherein the cell is a genetically engineered cell.

25. The method of claim 24, wherein the cells are modified with one or more gene expression modules, wherein expression from any one of the expression modules is constitutive or produced by a natural inducer.

26. The method of any one of claims 24 or 25, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein.

27. The method of any one of claims 24-26, wherein the cell comprises a modification that reduces acetate production.

28. The method of any one of claims 24 to 27, wherein the cell comprises reduced or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the following proteins, the proteins comprising β -galactosidase, galactosido-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphase, EIICBA-Nag, UDP-glucose: undecaprenyl-phosphoglucose-1-phosphotransferase, L-fucokinase, L-fucose isomerase, N-acetylneuraminic acid lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phospho2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphouridyltransferase, glucose-1-phosphoadenyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphoisomerase, aerobic respiration control protein, transcription repressor IclR, lon protease, glucose-specific translocated phosphotransferase IIBC component ptsG, glucose-specific translocated Phosphotransferase (PTS) IIBC component malX, enzyme IIA ^Glc, beta-glucosidase II, phosphoryl transferase specific PTS protein 3525 and phosphoPTS protein 383824 Alcohol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphoacetyltransferase, pyruvate decarboxylase.

29. The method of any one of the preceding claims, wherein the cell is capable of producing phosphoenolpyruvate (PEP).

30. The method of any one of the preceding claims, wherein the cell is modified to enhance production and/or supply of phosphoenolpyruvate (PEP).

31. The method of any one of the preceding claims, wherein the cell comprises an at least partially inactivated catabolic pathway of a selected monosaccharide, disaccharide or oligosaccharide that is involved in and/or necessary for the production of the compound comprising the structure of formula I, II or III.

32. The method of any one of the preceding claims, wherein the cells resist lactose killing when grown in an environment where lactose is combined with one or more other carbon sources.

33. The method of any one of the preceding claims, wherein the cells produce 90g/L or more of the compound comprising the structure of formula I, II or III in the whole culture and/or supernatant, and/or wherein the compound comprising the structure of formula I, II or III has a purity of at least 80% in the whole culture and/or supernatant, as measured by the total amount of the compound comprising the structure of formula I, II or III and its precursors in the whole culture and/or supernatant, respectively.

34. The method according to any one of the preceding claims, wherein the cell is a bacterium, fungus, yeast, plant cell, animal cell or protozoan cell,

35. The method of claim 34, wherein the cell is a living gram-negative bacterium comprising reduced or eliminated synthesis of: poly-N-acetyl-glucosamine (PNAG), enterobacter Common Antigen (ECA), cellulose, capsular polysaccharide, core oligosaccharide, osmoregulation Periplasmic Glucan (OPG), glycerol glucoside, glycans and/or trehalose.

36. The method of any one of the preceding claims, wherein the cells are stably cultured in a medium.

37. The method of any one of the preceding claims, wherein the conditions comprise:

38. The method according to any of the preceding claims, comprising at least one of the following steps:

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

39. The method according to any one of claims 1 to 37, comprising at least one of the following steps:

40. The method according to claim 39, wherein lactose feeding is accomplished by adding lactose from the beginning of the cultivation 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.

41. The method according to claim 39 or 40, wherein the lactose feeding is done by adding lactose to the culture in a concentration such that a lactose concentration of at least 5mM, preferably 10mM or 30mM is obtained throughout the production phase of the culture.

42. The method of any one of the preceding claims, wherein the cells are cultured for at least about 60, 80, 100, or about 120 hours or in a continuous manner.

43. The method of any one of the preceding claims, wherein the cells are 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, maltooligosaccharide, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn steep liquor, high fructose syrup, acetate, citrate, lactate and pyruvate.

44. The method according to any one of the preceding claims, wherein the medium contains at least one precursor selected from the group comprising: lactose, galactose, fucose, sialic acid, glcNAc, galNAc, lactose-N-disaccharide (LNB), N-acetyllactosamine (LacNAc).

45. The method according to any of the preceding claims, wherein the first stage of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to a medium comprising a precursor, preferably lactose, followed by a second stage, wherein only the carbon-based substrate, preferably glucose or sucrose, is added to the medium.

46. The method according to any one of claims 1 to 44, wherein the first stage of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to a medium comprising a precursor, preferably lactose, preferably glucose or sucrose, followed by a second stage, wherein the carbon-based substrate, preferably glucose or sucrose, and the precursor, preferably lactose, are added to the medium.

47. The method according to any one of the preceding claims, wherein the cells produce a mixture of charged, preferably sialylated and/or neutral di-and oligosaccharides, said mixture comprising at least one compound comprising a structure of formula I, II or III, wherein R1 when present is a mono-, di-or oligosaccharide.

48. The method according to any one of the preceding claims, wherein the cells produce a mixture of charged, preferably sialylated and/or neutral oligosaccharides, said mixture comprising at least one compound comprising a structure of formula I, II or III, wherein R1 when present is a monosaccharide, disaccharide or oligosaccharide.

49. The method of any one of the preceding claims, wherein the separating comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated carbon or carbon treatment, treatment with nonionic surfactants, enzymatic digestion, tangential flow high efficiency filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography.

50. The method of any one of the preceding claims, further comprising purifying the compound comprising the structure of formula I, II or III from the cell.

51. The method of claim 50, 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, using alcohols, using aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying, lyophilization, spray freeze drying, freeze spray drying, belt drying, vacuum belt drying, drum drying, vacuum drum drying or vacuum drum drying.

Wherein:

-Is a polypeptide belonging to the family of gt74 fucosyltransferases, or

53. The cell of claim 52, wherein the α -1, 2-fucosyltransferase has additional galactosidase α -1, 2-fucosyltransferase activity for galactose residues of the non-reducing end of Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (LNT, lactose-N-tetrasaccharide), and wherein the α -1, 2-fucosyltransferase:

54. The cell of claim 53, wherein the alpha-1, 2-fucosyltransferase has no additional galactoside alpha-1, 2-fucosyltransferase activity for lactose or has additional galactoside alpha-1, 2-fucosyltransferase activity for lactose but less than the additional galactoside alpha-1, 2-fucosyltransferase activity for galactose residues on the non-reducing end of LNT, and

55. The cell of claim 53, wherein the alpha-1, 2-fucosyltransferase has additional galactosidase alpha-1, 2-fucosyltransferase activity for lactose and additional galactosidase alpha-1, 2-fucosyltransferase activity that is higher than its galactose residue for the non-reducing end of LNT, and

56. The cell of claim 52, wherein the alpha-1, 2-fucosyltransferase has no galactosidase alpha-1, 2-fucosyltransferase activity for galactose residues at the non-reducing end of an LNT, and

57. The cell according to claim 56, wherein the alpha-1, 2-fucosyltransferase has NO galactosylα -1, 2-fucosyltransferase activity on lactose or has additional galactosylα -1, 2-fucosyltransferase activity on lactose but less than 3.0% of the galactosylα -1, 2-fucosyltransferase activity on lactose of the alpha-1, 2-fucosyltransferase of SEQ ID NO 06, and

58. The cell according to claim 56, wherein the alpha-1, 2-fucosyltransferase has additional galactosylα -1, 2-fucosyltransferase activity on lactose and is 4.0 to 20.0% of the galactosylα -1, 2-fucosyltransferase activity on lactose of the alpha-1, 2-fucosyltransferase of SEQ ID NO 06, and

59. The cell of any one of claims 52 to 58, wherein the cell is modified by one or more gene expression modules, wherein expression from any one of the expression modules is constitutive or produced by a natural inducer.

60. The cell of any one of claims 52 to 59, wherein the cell is modified in expression or activity of any one of the alpha-1, 2-fucosyltransferases.

61. The cell of any one of claims 52 to 60, wherein the cell is capable of producing one or more nucleotide-activating sugars selected from the list comprising: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2, 6-dideoxy-L-arabino-4-hexulose, UDP-2-acetamido-2, 6-dideoxy-L-lyxol-4-hexulose, UDP-N-acetyl-L-rhamnose (UDP-L-RhaNAc or UDP-2-acetamido-2, 6-dideoxy-L-mannose), DP-N-acetylfucose, UDP-N-acetylfucose (UDP-L-52 or UDP-2-acetamido-2, 6-dideoxy-L-5-fucose), UDP-N-acetylgalactosamine (UDP-L-FucNAc or UDP-2-acetamido-N-5-mannosamine), UDP-N-acetylgalactosamine (UDP-N-RhaNAc or UDP-2-acetamido-N-6-acetylgalactosamine), UDP-N-acetyl-L-quiniosamine (UDP-L-QuiNAc or UDP-2-acetamido-2, 6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac)、CMP-Neu4Ac、CMP-Neu5Ac9N₃、CMP-Neu4,5Ac₂、CMP-Neu5,7Ac₂、CMP-Neu5,9Ac₂、CMP-Neu5,7(8,9)Ac₂、CMP-N- hydroxyacetyl neuraminic acid (CMP-Neu 5 Gc), GDP-rhamnose and UDP-xylose.

62. The cell of any one of claims 52 to 61, 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-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine 6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine 1-phosphate acetyltransferase, N-acetylneuraminic acid synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid 9-phosphate synthase, N-acylneuraminic acid 9-phosphate phosphatase, N-acylneuraminic acid cytidylyltransferase, galactose 1-epimerase, galactokinase, and, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein said cell is modified in terms of expression or activity of any of said polypeptides.

63. The cell of any one of claims 52 to 62, wherein the cell expresses one or more glycosyltransferases selected from the list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosyltransferase, N-acetylmannosyltransferase, xylosyltransferase, glucuronyltransferase, galacturonate transferase, glucosaminyltransferase, N-glycolylneuraminidase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-arabinoxylan-Zhuo Tangan aminotransferase, UDP-N-acetylglucosaminenolpyruvylase and fucosyltransferase,

64. The cell according to any one of claims 52 to 63, wherein the compound comprising the structure of formula I, II or III is an oligosaccharide, preferably the oligosaccharide is a Mammalian Milk Oligosaccharide (MMO), more preferably a Human Milk Oligosaccharide (HMO).

65. The cell according to any one of claims 52 to 64, wherein the compound comprising the structure of formula I, II or III is a charged, preferably sialylated compound or a neutral compound,

66. The cell of any one of claims 52 to 65, wherein the compound comprising the structure of formula I, II or III is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R comprising one R group selected from the list comprising monosaccharides, disaccharides or oligosaccharides,

67. The cell of any one of claims 52 to 66, wherein the cell uses one or more precursors to produce the compound comprising the structure of formula I, II or III, the precursors being fed to the cell from a culture medium.

68. The cell of any one of claims 52 to 67, wherein the cell produces one or more precursors for producing the compound comprising the structure of formula I, II or III.

69. The cell of any one of claims 67 or 68, wherein the precursor for producing the compound comprising the structure of formula I, II or III is fully converted to the compound comprising the structure of formula I, II or III.

70. The cell of any one of claims 52 to 69, wherein the cell is capable of producing a compound comprising a structure of formula IV, V or VI:

Wherein:

71. The cell of any one of claims 52 to 70, wherein the cell produces the compound comprising the structure of formula I, II or III inside the cell, and wherein a portion or substantially all of the produced compound comprising the structure of formula I, II or III remains inside the cell and/or is excreted outside the cell by passive or active transport.

72. The cell of any one of claims 52 to 71, wherein the cell expresses a membrane transporter or a polypeptide having transport activity, thereby transporting a compound across the outer membrane of a cell wall,

73. The cell of claim 72, wherein the membrane transporter or a polypeptide having translocator activity is selected from the list comprising: transporter, P-P bond hydrolysis-driven transporter, b-barrel porin, auxiliary transporter, putative transporter and phosphate transport-driven group translocator,

74. The cell of any one of claims 72 or 73, wherein the membrane transporter or a polypeptide having transport activity controls the flow of the compound comprising the structure of formula I, II or III and/or one or more precursors and/or receptors for the production of the compound comprising the structure of formula I, II or III to the outer membrane of the cell wall.

75. The cell of any one of claims 72 to 74, wherein the membrane transporter or a polypeptide having transport activity provides improved production and/or permissive and/or enhanced efflux of the compound comprising the structure of formula I, II or III.

76. The cell of any one of claims 52 to 75, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein.

77. The cell of any one of claims 52 to 76, wherein the cell comprises a modification that reduces acetate production.

78. The cell of any one of claims 52 to 77, wherein the cell comprises reduced or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the following proteins, the proteins comprising β -galactosidase, galactosido-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphase, EIICBA-Nag, UDP-glucose: undecaprenyl-phosphoglucose-1-phosphotransferase, L-fucokinase, L-fucose isomerase, N-acetylneuraminic acid lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phospho2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphouridyltransferase, glucose-1-phosphoadenyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphoisomerase, aerobic respiration control protein, transcription repressor IclR, lon protease, glucose-specific translocated phosphotransferase IIBC component ptsG, glucose-specific translocated Phosphotransferase (PTS) IIBC component malX, enzyme IIA ^Glc, beta-glucosidase II, phosphoryl transferase specific PTS protein 3525 and phosphoPTS protein 383824 Alcohol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphoacetyltransferase, pyruvate decarboxylase.

79. The cell of any one of claims 52-78, wherein the cell is capable of producing phosphoenolpyruvate (PEP).

80. The cell of any one of claims 52 to 79, wherein the cell is modified to enhance synthesis and/or supply of phosphoenolpyruvate (PEP).

81. The cell of any one of claims 52 to 80, wherein the cell comprises an at least partially inactivated catabolic pathway of a selected monosaccharide, disaccharide or oligosaccharide that is involved in and/or necessary for the production of the compound comprising the structure of formula I, II or III.

82. The cell of any one of claims 52 to 81, wherein the cell is resistant to lactose killing when grown in an environment where lactose is combined with one or more other carbon sources.

83. The cell of any one of claims 52-82, wherein the cell produces 90g/L or more of the compound comprising the structure of formula I, II or III in the whole culture and/or supernatant, and/or wherein the compound comprising the structure of formula I, II or III has a purity of at least 80% in the whole culture and/or supernatant, as measured by the total amount of the compound comprising the structure of formula I, II or III and its precursors in the whole culture and/or supernatant, respectively.

84. The cell of any one of claims 52 to 83, wherein the cell is a bacterium, a fungus, a yeast, a plant cell, an animal cell, or a protozoan cell,

85. The cell of claim 84, wherein the cell is a living gram-negative bacterium comprising reduced or eliminated synthesis of: poly-N-acetyl-glucosamine (PNAG), enterobacter Common Antigen (ECA), cellulose, capsular polysaccharide, core oligosaccharide, osmoregulation Periplasmic Glucan (OPG), glycerol glucoside, glycans and/or trehalose.

86. The cell of any one of claims 52 to 85, wherein the cell produces a mixture of charged, preferably sialylated and/or neutral di-and oligosaccharides, said mixture comprising at least one compound comprising a structure of formula I, II or III, wherein R1, when present, is a mono-, di-or oligosaccharide.

87. The cell of any one of claims 52 to 86, wherein the cell produces a mixture of charged, preferably sialylated and/or neutral oligosaccharides, said mixture comprising at least one compound comprising a structure of formula I, II or III, wherein R1, when present, is a monosaccharide, disaccharide or oligosaccharide.

88. Use of the cell of any one of claims 52 to 87 or the method of any one of claims 1 to 51 for producing a compound comprising a structure of formula I, II or III.