PRODUCTION OF ALPHA-1,3 GLYCOSYLATED FORM OF FUC-A1,2-GAL-R

Abstract

The disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, the disclosure is in the technical field of cultivation or fermentation of metabolically engineered cells. The disclosure describes a cell metabolically engineered for production of an alpha-1,3 glycosylated form of fucose-alpha1,2-galactose-R (Fuc-a1,2-Gal-R). Furthermore, the disclosure provides a method for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R by a cell as well as the purification of the alpha-1,3 glycosylated form Fuc-a1,2-Gal-R from the cultivation.

Description

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

Pursuant to 37 C.F.R. § 1.821, a Sequence Listing ASCII text file entitled “4006-P17248US_028-PCT-US_US_SequenceListing_ST25.txt,” 293,154 bytes in size, generated Jan. 19, 2023, has been submitted via EFS-Web is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.

TECHNICAL FIELD

This disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, the disclosure is in the technical field of cultivation or fermentation of metabolically engineered cells. The disclosure describes a cell metabolically engineered for production of an alpha-1,3 glycosylated form of fucose-alpha1,2-galactose-R (Fuc-a1,2-Gal-R). Furthermore, the disclosure provides a method for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R by a cell as well as the purification of the alpha-1,3 glycosylated form Fuc-a1,2-Gal-R from the cultivation.

BACKGROUND

Oligosaccharides, often present as glyco-conjugated forms to proteins and lipids, are involved in many vital phenomena such as differentiation, development and biological recognition processes related to the development and progress of fertilization, embryogenesis, inflammation, metastasis, and host pathogen adhesion. Oligosaccharides can also be present as unconjugated glycans in body fluids and human milk wherein they also modulate important developmental and immunological processes (Bode, Early Hum. Dev. 1-4 (2015); Reily et al., Nat. Rev. Nephrol. 15, 346-366 (2019); Varki, Glycobiology 27, 3-49 (2017)). Fucose-alpha1,2-galactose-R (Fuc-a1,2-Gal-R) has been identified in multiple types of oligosaccharides and glyco-conjugated forms to proteins and lipids. It comprises the disaccharide fucose-alpha1,2-galactose epitope linked to other glycans, glycoproteins or glycolipids. The fucose-alpha1,2-galactose epitope has frequently been reported to be involved in neuronal morphology, neuronal development, learning and memory (Kalovidouris et al., J. Am. Chem. Soc. 127(5), 1340-1341 (2005); Tosh et al., Sci. Rep. 9, 18806 (2019)). Fuc-a1,2-Gal is also part of the most abundant oligosaccharide 2′-fucosyllactose (2′FL, Fuc-a1,2-Gal-b1,4-Glc) in human milk. Human milk oligosaccharides (HMOs) and specifically 2′FL have multiple function, including prebiotic, immune, gut and cognition benefits (Reverri et al., Nutrients 10(10), 1346 (2018)). Fuc-a1,2-Gal also forms the H antigen, which is the substructure of the A and B blood group antigens. Alpha-1,3 glycosylated forms of the fucose-alpha1,2-galactose epitope are part of the histo-blood group ABH carbohydrate epitopes (also called histo-blood group antigens, HBGAs). HBGAs are complex carbohydrates found on the surface of many cell types, including epithelial intestinal cells and as free oligosaccharides in biologic fluids like saliva and milk (Marionneau et al., Biochimie 83, 565-573 (2001)). The Fuc-a1,2-Gal group is also present in lacto-N-fucopentaose I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), which is a highly abundant oligosaccharide present in human milk. LNFP-I represents an important immunomodulator to prevent nursing infants from severe infectious diarrhoea by inhibition of the adhesion of pathogenic bacteria like Escherichia coli (EPEC, UPEC) and viruses. LNFP-I has also been linked to binding of pathogen toxins, growth inhibition of Group B Streptococci and selective stimulation of bifidobacterial communities (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)). LNFP-I can further be modified with a terminal galactose or N-acetylgalactosamine group resulting in an oligosaccharide structure carrying the B antigen (Gal-a1,3-(Fuc-a1,2)-Gal-beta) or the A antigen (GalNAc-a1,3-(Fuc-a1,2)-Gal-beta), respectively. There is large scientific and commercial interest in these oligosaccharide structures, yet the availability is limited as production relies on chemical or chemo-enzymatic synthesis or on purification from natural sources such as e.g., animal milk. Chemical synthesis methods are laborious and time-consuming and because of the large number of steps involved they are difficult to scale-up. Enzymatic approaches offer advantages above chemical synthesis, but stereospecificity and regioselectivity of the necessary enzymes are still a formidable challenge.

BRIEF SUMMARY

Provided are tools and methods by means of which an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R can be produced in an efficient, time and cost-effective way and, if needed, continuous process.

Further provided are a cell, a method and a new type of glycosyltransferases for the production of an alpha-1,3 glycosylated form Fuc-a1,2-Gal-R wherein the cell is genetically modified for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.

Surprisingly, it has now been found that it is possible to produce an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R by a single cell. The disclosure provides a cell and a method for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R. The method comprises the steps of providing a cell, which is capable to synthesize Fuc-a1,2-Gal-R, which expresses an alpha-1,3-glycosyltransferase and which is capable to synthesize a nucleotide-sugar, which is donor for the alpha-1,3-glycosyltransferase, and cultivating the cell under conditions permissive for producing the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R. The disclosure also provides methods to separate the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R. Furthermore, the disclosure provides a cell metabolically engineered for production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.

Definitions

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

The various embodiments and aspects of embodiments of the disclosure disclosed herein are to be understood not only in the order and context specifically described in this specification, but to include any order and any combination thereof. Whenever the context requires, all words used in the singular number shall be deemed to include the plural and vice versa. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described herein are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, purification steps are performed according to the manufacturer's specifications.

In the specification, there have been disclosed embodiments of the disclosure, and although specific terms are employed, the terms are used in a descriptive sense only and not for purposes of limitation, the scope of the disclosure 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 disclosure. It will be apparent to those skilled in the art that alterations, other embodiments, improvements, details and uses can be made consistent with the letter and spirit of the disclosure herein and within the scope of this disclosure, which is limited only by the claims, construed in accordance with the patent law, including the doctrine of equivalents. In the claims that follow, reference characters used to designate claim steps are provided for convenience of description only, and are not intended to imply any particular order for performing the steps.

In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Throughout the disclosure, the verb “to comprise” may be replaced by “to consist” or “to consist essentially of” and vice versa. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a composition as defined herein may comprise additional component(s) than the ones specifically identified, the additional component(s) not altering the unique characteristic of the disclosure. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one.”

Throughout the disclosure, unless explicitly stated otherwise, the features “synthesize,” “synthesized” and “synthesis” are interchangeably used with the features “produce,” “produced” and “production,” respectively.

Each embodiment as identified herein may be combined together unless otherwise indicated. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The full content of the priority applications, including EP20190208, EP20190198 and EP20190199 are also incorporated by reference to the same extent as if the priority application were specifically and individually indicated to be incorporated by reference.

According to the disclosure, the term “polynucleotide(s)” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotide(s)” include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions. In addition, “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term “polynucleotide(s)” also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotide(s)” according to the disclosure. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, are to be understood to be covered by the term “polynucleotides.” It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term “polynucleotide(s)” as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. The term “polynucleotide(s)” also embraces short polynucleotides often referred to as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. “Polypeptide(s)” refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. “Polypeptide(s)” include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to the skilled person. The same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Furthermore, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid sidechains, and the amino or carboxyl termini. 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, disulphide 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. Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.

The term “polynucleotide encoding a polypeptide” as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the disclosure. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions that also may contain coding and/or non-coding sequences.

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

The terms “recombinant” or “transgenic” or “metabolically engineered” or “genetically modified,” as used herein with reference to a cell or host cell are used interchangeably and indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence “foreign to the cell” or a sequence “foreign to the location or environment in the cell”). Such cells are described to be transformed with at least one heterologous or exogenous gene, or are described to be transformed by the introduction of at least one heterologous or exogenous gene. Metabolically engineered or recombinant or transgenic cells can contain genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means. The terms also encompass cells that contain a nucleic acid endogenous to the cell that has been modified or its expression or activity has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, replacement of a promoter; site-specific mutation; and related techniques. Accordingly, a “recombinant polypeptide” is one that has been produced by a recombinant cell. A “heterologous sequence” or a “heterologous nucleic acid,” as used herein, is one that originates from a source foreign to the particular cell (e.g., from a different species), or, if from the same source, is modified from its original form or place in the genome. Thus, a heterologous nucleic acid operably linked to a promoter is from a source different from which the promoter was derived, or, if from the same source, is modified from its original form or place in the genome. The heterologous sequence may be stably introduced, e.g., by transfection, transformation, conjugation or transduction, into the genome of the host microorganism cell, wherein techniques may be applied, which will depend on the cell and the sequence that is to be introduced. Various techniques are known to a person skilled in the art and are, e.g., disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The term “mutant” cell or microorganism as used within the context of the disclosure refers to a cell or microorganism that is genetically modified.

The term “endogenous,” within the context of the disclosure refers to any polynucleotide, polypeptide or protein sequence that is a natural part of a cell and is occurring at its natural location in the cell chromosome and of which the control of expression has not been altered compared to the natural control mechanism acting on its expression. The term “exogenous” refers to any polynucleotide, polypeptide or protein sequence, which originates from outside the cell under study and not a natural part of the cell or which is not occurring at its natural location in the cell chromosome or plasmid.

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

The term “modified activity” of a protein or an enzyme relates to a change in activity of the protein or the enzyme compared to the wild type, i.e., natural, activity of the protein or enzyme. The modified activity can either be an abolished, impaired, reduced or delayed activity of the protein or enzyme compared to the wild type activity of the protein or the enzyme but can also be an accelerated or an enhanced activity of the protein or the enzyme compared to the wild type activity of the protein or the enzyme. A modified activity of a protein or an enzyme is obtained by modified expression of the protein or enzyme or is obtained by expression of a modified, i.e., mutant form of the protein or enzyme. A modified activity of an enzyme further relates to a modification in the apparent Michaelis constant Km and/or the apparent maximal velocity (Vmax) of the enzyme.

The term “modified expression” of a gene relates to a change in expression compared to the wild type expression of the gene in any phase of the production process of the encoded protein. The modified expression is either a lower or higher expression compared to the wild type, wherein the term “higher expression” is also defined as “overexpression” of the gene in the case of an endogenous gene or “expression” in the case of a heterologous gene that is not present in the wild type strain. Lower expression or reduced expression is obtained by means of common well-known technologies for a skilled person (such as the usage of siRNA, CrispR, CrispRi, riboswitches, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutating genes, knocking-out genes, transposon mutagenesis, . . . ) which are used to change the genes in such a way that they are less-able (i.e., statistically significantly “less-able” compared to a functional wild-type gene) or completely unable (such as knocked-out genes) to produce functional final products. The term “riboswitch” as used herein is defined to be part of the messenger RNA that folds into intricate structures that block expression by interfering with translation. Binding of an effector molecule induces conformational change(s) permitting regulated expression post-transcriptionally.

Next to changing the gene of interest in such a way that lower expression is obtained as described above, lower expression can also be obtained by changing the transcription unit, the promoter, an untranslated region, the ribosome binding site, the Shine Dalgarno sequence or the transcription terminator. Lower expression or reduced expression can, for instance, be obtained by mutating one or more base pairs in the promoter sequence or changing the promoter sequence fully to a constitutive promoter with a lower expression strength compared to the wild type or an inducible promoter, which result in regulated expression or a repressible promoter, which results in regulated expression.

Overexpression or expression is obtained by means of common well-known technologies for a skilled person (such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids), wherein the gene is part of an “expression cassette” that relates to any sequence in which a promoter sequence, untranslated region sequence (containing either a ribosome binding sequence, Shine Dalgarno or Kozak sequence), a coding sequence and optionally a transcription terminator is present, and leading to the expression of a functional active protein. The expression is either constitutive or regulated.

The term “constitutive expression” is defined as expression that is not regulated by transcription factors other than the subunits of RNA polymerase (e.g., the bacterial sigma factors) under certain growth conditions. Non-limiting examples of such transcription factors are CRP, LacI, ArcA, Cra, IclR in E. coli. These transcription factors bind on a specific sequence and may block or enhance expression in certain growth conditions. RNA polymerase binds a specific sequence to initiate transcription, for instance, via a sigma factor in prokaryotic hosts.

The term “regulated expression” is defined as expression that is regulated by transcription factors other than the subunits of RNA polymerase (e.g., bacterial sigma factors) under certain growth conditions. Examples of such transcription factors are described above. Commonly expression regulation is obtained by means of an inducer or repressor, such as but not limited to IPTG, arabinose, rhamnose, fucose, allo-lactose or pH shifts, or temperature shifts or carbon depletion or substrates or the produced product or chemical repression.

The term “expression by a natural inducer” is defined as a facultative or regulatory expression of a gene that is only expressed upon a certain natural condition of the host (e.g., organism being in labor, or during lactation), as a response to an environmental change (e.g., including but not limited to hormone, heat, cold, pH shifts, light, oxidative or osmotic stress/signaling), or dependent on the position of the developmental stage or the cell cycle of the host cell including but not limited to apoptosis and autophagy.

The term “inducible expression upon chemical treatment” is defined as a facultative or regulatory expression of a gene that is only expressed upon treatment with a chemical inducer or repressor, wherein the inducer and repressor comprise but are not limited to an alcohol (e.g., ethanol, methanol), a carbohydrate (e.g., glucose, galactose, glycerol, lactose, arabinose, rhamnose, fucose, allo-lactose), metal ions (e.g., aluminum, copper, zinc), nitrogen, phosphates, IPTG, acetate, formate, xylene.

The term “control sequences” refers to sequences recognized by the cells transcriptional and translational systems, allowing transcription and translation of a polynucleotide sequence to a polypeptide. Such DNA sequences are thus necessary for the expression of an operably linked coding sequence in a particular cell or organism. Such control sequences can be, but are not limited to, promoter sequences, ribosome binding sequences, Shine Dalgarno sequences, Kozak sequences, transcription terminator sequences. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. DNA for a presequence or secretory leader may be operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. The control sequences can furthermore be controlled with external chemicals, such as, but not limited to, IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose via an inducible promoter or via a genetic circuit that either induces or represses the transcription or translation of the polynucleotide to a polypeptide.

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

The term “wild type” refers to the commonly known genetic or phenotypical situation as it occurs in nature.

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

As used herein, the term “mammary cell(s)” generally refers to mammary epithelial cell(s), mammary-epithelial luminal cell(s), or mammalian epithelial alveolar cell(s), or any combination thereof. As used herein, the term “mammary-like cell(s)” generally refers to cell(s) having a phenotype/genotype similar (or substantially similar) to natural mammary cell(s) but is/are derived from non-mammary cell source(s). Such mammary-like cell(s) may be engineered to remove at least one undesired genetic component and/or to include at least one predetermined genetic construct that is typical of a mammary cell. Non-limiting examples of mammary-like cell(s) may include mammary epithelial-like cell(s), mammary epithelial luminal-like cell(s), non-mammary cell(s) that exhibits one or more characteristics of a cell of a mammary cell lineage, or any combination thereof. Further non-limiting examples of mammary-like cell(s) may include cell(s) having a phenotype similar (or substantially similar) to natural mammary cell(s), or more particularly a phenotype similar (or substantially similar) to natural mammary epithelial cell(s). A cell with a phenotype or that exhibits at least one characteristic similar to (or substantially similar to) a natural mammary cell or a mammary epithelial cell may comprise a cell (e.g., derived from a mammary cell lineage or a non-mammary cell lineage) that exhibits either naturally, or has been engineered to, be capable of expressing at least one milk component.

As used herein, the term “non-mammary cell(s)” may generally include any cell of non-mammary lineage. In the context of the disclosure, a non-mammary cell can be any mammalian cell capable of being engineered to express at least one milk component. Non-limiting examples of such non-mammary cell(s) include hepatocyte(s), blood cell(s), kidney cell(s), cord blood cell(s), epithelial cell(s), epidermal cell(s), myocyte(s), fibroblast(s), mesenchymal cell(s), or any combination thereof. In some instances, molecular biology and genome editing techniques can be engineered to eliminate, silence, or attenuate myriad genes simultaneously.

Throughout the disclosure, unless explicitly stated otherwise, the expressions “capable of . . . <verb>” and “capable to . . . <verb>” are preferably replaced with the active voice of the verb and vice versa. For example, the expression “capable of expressing” is preferably replaced with “expresses” and vice versa, i.e., “expresses” is preferably replaced with “capable of expressing.”

“Variant(s)” as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to the persons skilled in the art.

The term “derivative” of a polypeptide, as used herein, is a polypeptide that 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, thus producing a functionally equivalent polypeptide. Amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; planar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Within the context of this disclosure, a derivative polypeptide as used herein, refers to a polypeptide capable of exhibiting a substantially similar in vitro and/or in vivo activity as 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 analogues can be introduced as a substitution or addition into the original polypeptide sequence.

In some embodiments, the disclosure contemplates making functional variants by modifying the structure of an enzyme as used in the disclosure. Variants can be produced by amino acid substitution, deletion, addition, or combinations thereof. For instance, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (e.g., conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Whether a change in the amino acid sequence of a polypeptide of the disclosure results in a functional homolog can be readily determined by assessing the ability of the variant polypeptide to produce a response in cells in a fashion similar to the wild-type polypeptide.

The term “functional homolog” as used herein describes those molecules that have sequence similarity (in other words, homology) and also share at least one functional characteristic such as a biochemical activity (Altenhoff et al., PLoS Comput. Biol. 8 (2012) e1002514). Functional homologs will typically give rise to the same characteristics to a similar, but not necessarily the same, degree. Functionally homologous polypeptides give the same characteristics where the quantitative measurement produced by one homolog is at least 10 percent of the other; more typically, at least 20 percent, between about 30 percent and about 40 percent; for example, between about 50 percent and about 60 percent; between about 70 percent and about 80 percent; or between about 90 percent and about 95 percent; between about 98 percent and about 100 percent, or greater than 100 percent of that produced by the original molecule. Thus, where the molecule has enzymatic activity the functional homolog will have the above-recited percent enzymatic activities compared to the original enzyme. Where the molecule is a DNA-binding molecule (e.g., a polypeptide) the homolog will have the above-recited percentage of binding affinity as measured by weight of bound molecule compared to the original molecule.

A 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 are sometimes referred to as orthologs, where “ortholog,” refers to a homologous gene or protein that is the functional equivalent of the referenced gene or protein in another species.

Orthologous genes are homologous genes in different species that originate by vertical descent from a single gene of the last common ancestor, wherein the gene and its main function are conserved. A homologous gene is a gene inherited in two species by a common ancestor.

The term “ortholog” when used in reference to an amino acid or nucleotide/nucleic acid sequence from a given species refers to the same amino acid or nucleotide/nucleic acid sequence from a different species. It should be understood that two sequences are orthologs of each other when they are derived from a common ancestor sequence via linear descent and/or are otherwise closely related in terms of both their sequence and their biological function. Orthologs will usually have a high degree of sequence identity but may not (and often will not) share 100% sequence identity.

Paralogous genes are homologous genes that originate by a gene duplication event. Paralogous genes often belong to the same species, but this is not necessary. Paralogs can be split into in-paralogs (paralogous pairs that arose after a speciation event) and out-paralogs (paralogous pairs that arose before a speciation event). Between species out-paralogs are pairs of paralogs that exist between two organisms due to duplication before speciation. Within species out-paralogs are pairs of paralogs that exist in the same organism, but whose duplication event happened after speciation. Paralogs typically have the same or similar function.

Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of the polypeptide of interest like e.g., a biomass-modulating polypeptide, a glycosyltransferase, a protein involved in nucleotide-activated sugar synthesis or a membrane transporter protein. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using amino acid sequence of a biomass-modulating polypeptide, a glycosyltransferase, a protein involved in nucleotide-activated sugar synthesis or a membrane transporter protein, respectively, as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Typically, those polypeptides in the database that have greater than 40 percent sequence identity are candidates for further evaluation for suitability as a biomass-modulating polypeptide, a glycosyltransferase, a protein involved in nucleotide-activated sugar synthesis or a membrane transporter protein, respectively. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another or substitution of one acidic amino acid for another or substitution of one basic amino acid for another, etc. Preferably, by conservative substitutions is intended combinations such as glycine by alanine and vice versa; valine, isoleucine and leucine by methionine and vice versa; aspartate by glutamate and vice versa; asparagine by glutamine and vice versa; serine by threonine and vice versa; lysine by arginine and vice versa; cysteine by methionine and vice versa; and phenylalanine and tyrosine by tryptophan and vice versa. If desired, manual inspection of such candidates can be carried out to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in productivity-modulating polypeptides, e.g., conserved functional domains.

“Fragment,” with respect to a polynucleotide, refers to a clone or any part of a polynucleotide molecule, particularly a part of a polynucleotide that retains a usable, functional characteristic of the full-length polynucleotide molecule. Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation. A “polynucleotide fragment” refers to 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 the polynucleotide SEQ ID NO (or Genbank NO.), for example, at least about 30 nucleotides or at least about 50 nucleotides of any of the polynucleotide sequences provided herein. Exemplary fragments can additionally or alternatively include fragments that comprise, consist essentially of, or consist of a region that encodes a conserved family domain of a polypeptide. Exemplary fragments can additionally or alternatively include fragments that comprise a conserved domain of a polypeptide. As such, a fragment of a polynucleotide SEQ ID NO (or Genbank NO.) preferably means a nucleotide sequence that comprises or consists of the polynucleotide SEQ ID NO (or Genbank NO.) wherein no more than 200, 150, 100, 50 or 25 consecutive nucleotides are missing, preferably no more than 50 consecutive nucleotides are missing, and which retains a usable, functional characteristic (e.g., activity) of the full-length polynucleotide molecule, which can be assessed by the skilled person through routine experimentation. Alternatively, a fragment of a polynucleotide SEQ ID NO (or Genbank NO.) preferably means a nucleotide sequence that comprises or consists of an amount of consecutive nucleotides from the polynucleotide SEQ ID NO (or Genbank NO.) and wherein the amount 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 the polynucleotide SEQ ID NO (or Genbank NO.) and retains a usable, functional characteristic (e.g., activity) of the full-length polynucleotide molecule. As such, a fragment of a polynucleotide SEQ ID NO (or Genbank NO.) preferably means a nucleotide sequence that comprises or consists of the polynucleotide SEQ ID NO (or Genbank NO.), wherein an amount of consecutive nucleotides is missing and wherein the amount is no more than 50.0%, 40.0%, 30.0% of the full-length of the polynucleotide SEQ ID NO (or Genbank NO.), preferably no 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 no more than 15.0%, even more preferably no more than 10.0%, even more preferably no more than 5.0%, most preferably no more than 2.5%, of the full-length of the polynucleotide SEQ ID NO (or Genbank NO.) and wherein the fragment retains a usable, functional characteristic (e.g., activity) of the full-length polynucleotide molecule that can be routinely assessed by the skilled person.

Fragments may additionally or alternatively include subsequences of polypeptides and protein molecules, or a subsequence of the polypeptide. In some cases, the fragment or domain is a subsequence of the polypeptide, which performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar extent, as does the intact polypeptide. A “subsequence of the polypeptide” as defined herein refers to a sequence of contiguous amino acid residues derived from the polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription. Fragments can vary in size from as few as 3 amino acid residues to the full length of the intact polypeptide, for example, at least about 20 amino acid residues in length, for example, at least about 30 amino acid residues in length. As such, a fragment of a polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) preferably means a polypeptide sequence that comprises or consists of the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) wherein no more than 80, 60, 50, 40, 30, 20 or 15 consecutive amino acid residues are missing, preferably no more than 40 consecutive amino acid residues are missing, and performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar or greater extent, as does the intact polypeptide, 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 means a polypeptide sequence that comprises or consists of an amount of consecutive amino acid residues from the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) and wherein the amount 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 the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) and which performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar or greater extent, as does the intact polypeptide, which can be routinely assessed by the skilled person. As such, a fragment of a polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) preferably means a polypeptide sequence that comprises or consists of the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.), wherein an amount of consecutive amino acid residues is missing and wherein the amount is no more than 50.0%, 40.0%, 30.0% of the full-length of the polypeptide SEQ ID NO, preferably no 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 no more than 15.0%, even more preferably no more than 10.0%, even more preferably no more than 5.0%, most preferably no more than 2.5%, of the full-length of the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) and which performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar or greater extent, as does the intact polypeptide, which can be routinely assessed by the skilled person.

Throughout the disclosure, the sequence of a polypeptide can be represented by a SEQ ID NO or alternatively by an UniProt ID or GenBank NO. Therefore, the terms “polypeptide SEQ ID NO” and “polypeptide Uniprot ID” and “polypeptide GenBank NO.” can be interchangeably used, unless explicitly stated otherwise.

Preferentially, a fragment of a polypeptide is a functional fragment that has at least one property or activity of the polypeptide from which it is derived, preferably to a similar or greater extent. A functional fragment can, for example, include a functional domain or conserved domain of a polypeptide. It is understood that a polypeptide or a fragment thereof may have conservative amino acid substitutions, which have substantially no effect on the polypeptide's activity. By conservative substitutions is intended substitutions of one hydrophobic amino acid for another or substitution of one polar amino acid for another or substitution of one acidic amino acid for another or substitution of one basic amino acid for another, etc. Preferably, by conservative substitutions is intended combinations such as glycine by alanine and vice versa; valine, isoleucine and leucine by methionine and vice versa; aspartate by glutamate and vice versa; asparagine by glutamine and vice versa; serine by threonine and vice versa; lysine by arginine and vice versa; cysteine by methionine and vice versa; and phenylalanine and tyrosine by tryptophan and vice versa.

A domain can be characterized, for example, by a Pfam (El-Gebali et al., Nucleic Acids Res. 47 (2019) D427-D432), an IPR (InterPro domain) (Mitchell et al., Nucleic Acids Res. 47 (2019) D351-D360), a protein fingerprint domain (PRINTS) (Attwood et al., Nucleic Acids Res. 31 (2003) 400-402), a SUBFAM domain (Gough et al., J. Mol. Biol. 313 (2001) 903-919), a TIGRFAM domain (Selengut et al., Nucleic Acids Res. 35 (2007) D260-D264), a Conserved Domain Database (CDD) designation (www.ncbi.nlm.nih.gov/cdd) (Lu et al., Nucleic Acids Res. 48 (2020) D265-D268), a PTHR domain (www.pantherdb.org) (Mi et al., Nucleic Acids. Res. 41 (2013) D377-D386; Thomas et al., Genome Research 13 (2003) 2129-2141) or a PATRIC identifier or PATRIC DB global family domain (/www.patricbrc.org/) (Davis et al., Nucleic Acids Res. 48(D1) (2020) D606-D612). It should be understood for those skilled in the art that for the databases used herein, comprising Pfam 32.0 (released September 2018), CDD v3.17 (released 3 Apr. 2019), eggnogdb 4.5.1 (released September 2016), InterPro 75.0 (released 4 Jul. 2019), TCDB (released 17 Jun. 2019) and PATRIC 3.6.9 (released March 2020), the content of each database is fixed at each release and is not to be changed. When the content of a specific database is changed, this specific database receives a new release version with a new release date. All release versions for each database with their corresponding release dates and specific content as annotated at these specific release dates are available and known to those skilled in the art.

Protein or polypeptide sequence information and functional information can be provided by a comprehensive resource for protein sequence and annotation data like e.g., the Universal Protein Resource (UniProt) (www.uniprot.org) (Nucleic Acids Res. 2021, 49(D1), D480-D489). UniProt comprises the expertly and richly curated protein database called the UniProt Knowledgebase (UniProtKB), together with the UniProt Reference Clusters (UniRef) and the UniProt Archive (UniParc). The UniProt identifiers (UniProt ID) are unique for each protein present in the database. UniProt IDs as used herein are the UniProt IDs in the UniProt database version of 5 May 2021. Proteins that do not have an UniProt ID are referred herein using the respective GenBank Accession number (GenBank No.) as present in the NIH genetic sequence database (www.ncbi.nlm.nih.gov/genbank/) (Nucleic Acids Res. 2013, 41(D1), D36-D42) version of 5 May 2021.

The terms “identical” or “percent identity” or “% identity” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using sequence comparison algorithms or by visual inspection. For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are inputted into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Percent identity may be calculated globally over the full-length sequence of the reference sequence, resulting in a global percent identity score. Alternatively, percent identity may be calculated over a partial sequence of the reference sequence, resulting in a local percent identity score. Using the full-length of the reference sequence in a local sequence alignment results in a global percent identity score between the test and the reference sequence.

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

The BLAST (Basic Local Alignment Search Tool)) method of alignment is an algorithm provided by the National Center for Biotechnology Information (NCBI) to compare sequences using default parameters. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance. PSI-BLAST (Position-Specific Iterative Basic Local Alignment Search Tool) derives a position-specific scoring matrix (PSSM) or profile from the multiple sequence alignment of sequences detected above a given score threshold using protein-protein BLAST (BLASTp). The BLAST method can be used for pairwise or multiple sequence alignments. Pairwise Sequence Alignment is used to identify regions of similarity that may indicate functional, structural and/or evolutionary relationships between two biological sequences (protein or nucleic acid). The web interface for BLAST is available at: blast.ncbi.nlm.nih.gov/Blast.cgi.

Clustal Omega (Clustal W) is a multiple sequence alignment program that uses seeded guide trees and HMM profile-profile techniques to generate alignments between three or more sequences. It produces biologically meaningful multiple sequence alignments of divergent sequences. The web interface for Clustal W is available at www.ebi.ac.uk/Tools/msa/clustalo/. Default parameters for multiple sequence alignments and calculation of percent identity of protein sequences using the Clustal W method are: enabling de-alignment of input sequences: FALSE; enabling mbed-like clustering guide-tree: TRUE; enabling mbed-like clustering iteration: TRUE; Number of (combined guide-tree/HIMM) iterations: default(0); Max Guide Tree Iterations: default [−1]; Max HMM Iterations: default [−1]; order: aligned.

MatGAT (Matrix Global Alignment Tool) is a computer application that generates similarity/identity matrices for DNA or protein sequences without needing pre-alignment of the data. The program performs a series of pairwise alignments using the Myers and Miller global alignment algorithm, calculates similarity and identity, and then places the results in a distance matrix. The user may specify which type of alignment matrix (e.g., BLOSUM50, BLOSUM62, and PAM250) to employ with their protein sequence examination.

EMBOSS Needle (galaxy-iuc.github.io/emboss-5.0-docs/needle.html) uses the Needleman-Wunsch global alignment algorithm to find the optimal alignment (including gaps) of two sequences when considering their entire length. The optimal alignment is ensured by dynamic programming methods by exploring all possible alignments and choosing the best. The Needleman-Wunsch algorithm is a member of the 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). The gap open penalty (default 10.0) is the score taken away when a gap is created. The default value assumes you are using the EBLOSUM62 matrix for protein sequences. The gap extension (default 0.5) penalty is added to the standard gap penalty for each base or residue in the gap. This is how long gaps are penalized.

As used herein, a polypeptide having an amino acid sequence having at least 80% sequence identity to the full-length sequence of a reference polypeptide sequence is to be understood as that the sequence has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00% 96.50% 97.00% 97.50% 98.00% 98.50% 99.00% 99.50% 99.60% 99.70%, 99.80%, 99.90%, 100% sequence identity to the full-length of the amino acid sequence of the reference polypeptide sequence. Throughout the disclosure, unless explicitly specified otherwise, a polypeptide comprising, consisting or having an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of a reference polypeptide, usually indicated with a SEQ ID NO, UniProt ID or Genbank No., preferably has 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 has at least 85.0%, even more preferably has at least 90.0%, most preferably has at least 95.0%, sequence identity to the full length reference sequence. Additionally, unless explicitly specified otherwise, a polynucleotide sequence comprising/consisting/having a nucleotide sequence having at least 80.0% sequence identity to the full-length nucleotide sequence of a reference polynucleotide sequence, usually indicated with a SEQ ID NO or Genbank No., preferably has 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 has at least 85.0%, even more preferably has at least 90.0%, most preferably has at least 95.0%, sequence identity to the full length reference sequence.

For the purposes of this disclosure, percent identity is determined using MatGAT2.01 (Campanella et al., 2003, BMC Bioinformatics 4:29). The following default parameters for protein are employed: (1) Gap cost Existence: 12 and Extension: 2; (2) The Matrix employed was 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 part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90% or 95% of the complete reference sequence.

The term “glycosyltransferase” as used herein refers to an enzyme capable to catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds.

The terms “alpha-1,3-glycosyltransferase,” “a1,3-glycosyltransferase,” “alpha 1,3 glycosyltransferase,” “a1,3 glycosyltransferase,” “a1,3-glycosyltransferase” refer to an enzyme capable to catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules in an alpha-1,3 glycosidic linkage. The term “alpha-1,3-glycosyltransferase” as used herein refers to alpha-1,3-galactosyltransferases and to alpha-1,3-N-acetylgalactosaminyltransferases.

“Alpha-1,3-galactosyltransferases,” also referred to as “a-1,3-galactosyltransferases,” “a1,3 galactosyltransferases,” “a1,3-galactosyltransferases,” are glycosyltransferases that catalyze the transfer of a galactose residue from UDP-galactose (UDP-Gal) to a specific acceptor molecule in an alpha-1,3 linkage.

Alpha-1,3-N-acetylgalactosaminyltransferases, also referred to as “a-1,3-N-acetylgalactosaminyltransferases,” “a1,3 N-acetylgalactosaminyltransferases,” “a1,3-N-acetylgalactosaminyltransferases” are glycosyltransferases that catalyze the transfer of an N-acetylgalactosamine (GalNAc) from UDP-GalNAc to a specific acceptor molecule in an alpha-1,3 linkage.

In the disclosure, polypeptide sequence stretches are being used to refer to fragments of the alpha-1,3-glycosyltransferases used in the disclosure that are common to those alpha-1,3-glycosyltransferases. Such polypeptide stretches are written in the form of a sequence of amino acids in one-letter code. In case an amino acid at a specific place in such polypeptide stretch can be several amino acids, that specific place will have amino acid code X. Unless otherwise mentioned herein, the letter “X” refers to any amino acid possible. The term [FHMQT] in a sequence refers to an F, H, M, Q or T as possible amino acid at that specific place. The term [ACG] in a sequence refers to an A, C or G as possible amino acid at that specific place. The term [ACIL] in a sequence refers to an A, C, I or L as possible amino acid at that specific place. The term [AG] in a sequence refers to an A or G as possible amino acid at that specific place.

The terms “nucleotide-sugar” or “activated sugar” as used herein refer to activated forms of monosaccharides. Examples of activated monosaccharides comprise UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-fucose (GDP-Fuc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose, CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N₃, CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂, CMP-Neu5,9Ac₂, CMP-Neu5,7(8,9)Ac₂, UDP-galacturonate, UDP-glucuronate, GDP-rhamnose, or UDP-xylose. Nucleotide-sugars act as glycosyl donors in glycosylation reactions. Those reactions are catalysed by glycosyltransferases.

“Oligosaccharide” as the term is used herein and as generally understood in the state of the art, refers to a saccharide polymer containing a small number, typically three to twenty, of simple sugars, i.e., monosaccharides. The monosaccharides as used herein are reducing sugars. The oligosaccharides can be reducing or non-reducing sugars and have a reducing and a non-reducing end. A reducing sugar is any sugar that is capable of reducing another compound and is oxidized itself, that is, the carbonyl carbon of the sugar is oxidized to a carboxyl group. The oligosaccharide as used in the disclosure can be a linear structure or can include branches. The linkage (e.g., glycosidic linkage, galactosidic linkage, glucosidic linkage, etc.) between two sugar units can be expressed, for example, as 1,4, 1->4, or (1-4), used interchangeably herein. For example, the terms “Gal-b1,4-Glc,” “b-Gal-(1->4)-Glc,” “Galbeta1-4-Glc” and “Gal-b(1-4)-Glc” have the same meaning, i.e., a beta-glycosidic bond links carbon-1 of galactose (Gal) with the carbon-4 of glucose (Glc). Each monosaccharide can be in the cyclic form (e.g., pyranose of furanose form). Linkages between the individual monosaccharide units may include alpha 1->2, alpha 1->3, alpha 1->4, alpha 1->6, alpha 2->1, alpha 2->3, alpha 2->4, alpha 2->6, beta 1->2, beta 1->3, beta 1->4, beta 1->6, beta 2->1, beta 2->3, beta 2->4, and beta 2->6. An oligosaccharide can contain both alpha- and beta-glycosidic bonds or can contain only beta-glycosidic bonds.

The term “monosaccharide” as used herein refers to a sugar that is not decomposable into simpler sugars by hydrolysis, is classed either an aldose or ketose, and contains one or more hydroxyl groups per molecule. Monosaccharides are saccharides containing only one simple sugar. Examples of monosaccharides comprise Hexose, D-Glucopyranose, D-Galactofuranose, D-Galactopyranose, L-Galactopyranose, D-Mannopyranose, D-Allopyranose, L-Altropyranose, D-Gulopyranose, L-Idopyranose, D-Talopyranose, D-Ribofuranose, D-Ribopyranose, D-Arabinofuranose, D-Arabinopyranose, L-Arabinofuranose, L-Arabinopyranose, D-Xylopyranose, D-Lyxopyranose, D-Erythrofuranose, D-Threofuranose, Heptose, L-glycero-D-manno-Heptopyranose (LDmanHep), D-glycero-D-manno-Heptopyranose (DDmanHep), 6-Deoxy-L-altropyranose, 6-Deoxy-D-gulopyranose, 6-Deoxy-D-talopyranose, 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-hexopyranose, 3,6-Dideoxy-D-arabino-hexopyranose, 3,6-Dideoxy-L-arabino-hexopyranose, 3,6-Dideoxy-D-xylo-hexopyranose, 3,6-Dideoxy-D-ribo-hexopyranose, 2,6-Dideoxy-D-ribo-hexopyranose, 3,6-Dideoxy-L-xylo-hexopyranose, 2-Amino-2-deoxy-D-glucopyranose, 2-Amino-2-deoxy-D-galactopyranose, 2-Amino-2-deoxy-D-mannopyranose, 2-Amino-2-deoxy-D-allopyranose, 2-Amino-2-deoxy-L-altropyranose, 2-Amino-2-deoxy-D-gulopyranose, 2-Amino-2-deoxy-L-idopyranose, 2-Amino-2-deoxy-D-talopyranose, 2-Acetamido-2-deoxy-D-glucopyranose, 2-Acetamido-2-deoxy-D-galactopyranose, 2-Acetamido-2-deoxy-D-mannopyranose, 2-Acetamido-2-deoxy-D-allopyranose, 2-Acetamido-2-deoxy-L-altropyranose, 2-Acetamido-2-deoxy-D-gulopyranose, 2-Acetamido-2-deoxy-L-idopyranose, 2-Acetamido-2-deoxy-D-talopyranose, 2-Acetamido-2,6-dideoxy-D-galactopyranose, 2-Acetamido-2,6-dideoxy-L-galactopyranose, 2-Acetamido-2,6-dideoxy-L-mannopyranose, 2-Acetamido-2,6-dideoxy-D-glucopyranose, 2-Acetamido-2,6-dideoxy-L-altropyranose, 2-Acetamido-2,6-dideoxy-D-talopyranose, D-Glucopyranuronic acid, D-Galactopyranuronic acid, D-Mannopyranuronic acid, D-Allopyranuronic acid, L-Altropyranuronic acid, D-Gulopyranuronic acid, L-Gulopyranuronic acid, L-Idopyranuronic acid, D-Talopyranuronic acid, sialic acid, 5-Amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid, 5-Acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid, 5-Glycolylamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid, Erythritol, Arabinitol, Xylitol, Ribitol, Glucitol, Galactitol, Mannitol, D-ribo-Hex-2-ulopyranose, D-arabino-Hex-2-ulofuranose (D-fructofuranose), D-arabino-Hex-2-ulopyranose, L-xylo-Hex-2-ulopyranose, D-lyxo-Hex-2-ulopyranose, D-threo-Pent-2-ulopyranose, D-altro-Hept-2-ulopyranose, 3-C-(Hydroxymethyl)-D-erythofuranose, 2,4,6-Trideoxy-2,4-diamino-D-glucopyranose, 6-Deoxy-3-O-methyl-D-glucose, 3-O-Methyl-D-rhamnose, 2,6-Dideoxy-3-methyl-D-ribo-hexose, 2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose, 2-Acetamido-3-O—[(R)-carboxyethyl]-2-deoxy-D-glucopyranose, 2-Glycolylamido-3-O—[(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose, 3-Deoxy-D-lyxo-hept-2-ulopyranosaric acid, 3-Deoxy-D-manno-oct-2-ulopyranosonic acid, 3-Deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, 5,7-Diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulopyranosonic acid, 5,7-Diamino-3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulopyranosonic acid, 5,7-Diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, 5,7-Diamino-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulopyranosonic acid, glucose, galactose, N-acetylglucosamine, glucosamine, mannose, xylose, N-acetylmannosamine, N-acetylneuraminic acid, N-glycolylneuraminic acid, N-acetylgalactosamine, galactosamine, fucose, rhamnose, glucuronic acid, gluconic acid, fructose and polyols.

With the term polyol is meant an alcohol containing multiple hydroxyl groups. For example, glycerol, sorbitol, or mannitol.

The term “disaccharide” as used herein refers to a saccharide composed of two monosaccharide units. Examples of disaccharides comprise lactose (Gal-b1,4-Glc), lacto-N-biose (Gal-b1,3-GlcNAc), N-acetyllactosamine (Gal-b1,4-GlcNAc), LacDiNAc (GalNAc-b1,4-GlcNAc), N-acetylgalactosaminylglucose (GalNAc-b1,4-Glc).

Preferably the oligosaccharide as described herein contains monosaccharides selected from the list as used herein above. Examples of oligosaccharides include but are not limited to Lewis-type antigen oligosaccharides, mammalian milk oligosaccharides and human milk oligosaccharides.

As used herein, “mammalian milk oligosaccharide” refers to oligosaccharides such as but not limited to 3-fucosyllactose, 2′-fucosyllactose, 6-fucosyllactose, 2′,3-difucosyllactose, 2′,2-difucosyllactose, 3,4-difucosyllactose, 6′-sialyllactose, 3′-sialyllactose, 3,6-disialyllactose, 6,6′-disialyllactose, 8,3-disialyllactose, 3,6-disialyllacto-N-tetraose, lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, monofucosylmonosialyllacto-N-tetraose c, monofucosyl para-lacto-N-hexaose, monofucosyllacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose I, sialyllacto-N-hexaose, sialyllacto-N-neohexaose II, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, difucosyllacto-N-hexaose a, difucosyllacto-N-hexaose c, galactosylated chitosan, fucosylated oligosaccharides, neutral oligosaccharide and/or sialylated oligosaccharides.

Mammalian milk oligosaccharides or MMOs comprise oligosaccharides present in milk found in any phase during lactation including colostrum milk from humans (i.e., human milk oligosaccharides or HMOs) and mammals including but not limited to cows (Bos Taurus), sheep (Ovis aries), goats (Capra aegagrus hircus), bactrian camels (Camelus bactrianus), horses (Equus ferus caballus), pigs (Sus scropha), dogs (Canis lupus familiaris), ezo brown bears (Ursus arctos yesoensis), polar bear (Ursus maritimus), Japanese black bears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asian elephants (Elephas maximus), African elephant (Loxodonta africana), giant anteater (Myrmecophaga tridactyla), common bottlenose dolphins (Tursiops truncates), northern minke whales (Balaenoptera acutorostrata), tammar wallabies (Macropus eugenii), red kangaroos (Macropus rufus), common brushtail possum (Trichosurus Vulpecula), koalas (Phascolarctos cinereus), eastern quolls (Dasyurus viverrinus), platypus (Ornithorhynchus anatinus). Human milk oligosaccharides are also known as human identical milk oligosaccharides, which are chemically identical to the human milk oligosaccharides found in human breast milk but which are biotechnologically-produced (e.g., using cell free systems or cells and organisms comprising a bacterium, a fungus, a yeast, a plant, animal, or protozoan cell, preferably genetically engineered cells and organisms). Human identical milk oligosaccharides are marketed under the name HiMO.

As used herein the term “Lewis-type antigens” comprise the following oligosaccharides: H1 antigen, which is Fucα1-2Galβ1-3GlcNAc, or in short 2′FLNB; Lewisa, which is the trisaccharide Galβ1-3[Fucα1-4]GlcNAc, or in short 4-FLNB; Lewisb, which is the tetrasaccharide Fucα1-2Galβ1-3[Fucα1-4]GlcNAc, or in short DiF-LNB; sialyl Lewisa, which is 5-acetylneuraminyl-(2-3)-galactosyl-(1-3)-(fucopyranosyl-(1-4))-N-acetylglucosamine, orwritten in short Neu5Acα2-3Galβ1-3[Fucα1-4]GlcNAc; H2 antigen, which is Fucα1-2Galβ1-4GlcNAc, or otherwise stated 2′fucosyl-N-acetyl-lactosamine, in short 2′FLacNAc; Lewisx, which is the trisaccharide Galβ1-4[Fucα1-3]GlcNAc, or otherwise known as 3-Fucosyl-N-acetyl-lactosamine, in short 3-FLacNAc, Lewisy, which is the tetrasaccharide Fucα1-2Galβ-4[Fucα1-3]GlcNAc and sialyl Lewisx, which is 5-acetylneuraminyl-(2-3)-galactosyl-(1-4)-(fucopyranosyl-(1-3))-N-acetylglucosamine, or written in short Neu5Acα2-3Galβ1-4[Fucα1-3]GlcNAc.

The term “Fuc-a1,2-Gal-R” as used herein refers to a terminal disaccharide Fuc-alpha-1,2-Gal that is linked to an R-group. The term “Fuc-a1,2-Gal-b1,3-R” as used herein refers to a terminal disaccharide Fuc-alpha-1,2-Gal that is linked in a beta-1,3 glycosidic linkage to an R-group. The term “Fuc-a1,2-Gal-b1,3-GlcNAc-R” as used herein refers to a terminal trisaccharide Fuc-alpha-1,2-Gal-b1,3-GlcNAc that is linked to an R-group. The “R-group” or “R” as used throughout the disclosure refers to a monosaccharide, a disaccharide, an oligosaccharide, a lipid, a peptide or a protein, or a mono-, di- or oligosaccharide that is bound to a peptide, a glycopeptide, a protein, a glycoprotein, a lipid or to a glycolipid. Preferably, the “R-group” or “R” as used throughout the disclosure refers to a monosaccharide, a disaccharide or an oligosaccharide.

The terms “lacto-N-triose,” “LN3” and “LNT II” refer to the trisaccharide GlcNAc-b1,3-Gal-b1,4-Glc. The terms “lacto-N-tetraose” and “LNT” refer to the oligosaccharide Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc.

The terms “lacto-N-fucopentaose I,” “lacto-N-fucopentaose-I,” “LNFP-I,” “LNFP I,” “LNF I OH type I determinant,” “LNF I,” “LNF1,” “LNF 1” and “Blood group H antigen pentaose type 1” refer to the oligosaccharide Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc that is obtained by the catalysis of an alpha-1,2-fucosyltransferase transferring the fucose residue from GDP-L-fucose to the terminal galactose residue of LNT in an alpha-1,2-linkage as described e.g., by Miyazaki et al. (2010, Methods Enzym. 480, 511-524), Baumgartner et al. (2015, Bioorg. Med. Chem. 23, 6799-6806), Zhao et al. (2016, Chem. Commun. 52, 3899-3902), Sugiyama et al. (2016, Glycobiology 26, 1235-1247) and in patent literature (e.g., WO 2019008133, and WO 2014018596A2).

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

The terms “LNFP-II” and “lacto-N-fucopentaose II” are used interchangeably and refer to Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc.

The terms “LNFP-III” and “lacto-N-fucopentaose III” are used interchangeably and refer to Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1,3-Gal-b1,4-Glc.

The terms “LNFP-V” and “lacto-N-fucopentaose V” are used interchangeably and refer to Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc.

The terms “LNFP-VI,” “LNnFP V” and “lacto-N-neofucopentaose V” are used interchangeably and refer to Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc.

The terms “LNnFP I” and “Lacto-N-neofucopentaose I” are used interchangeably and refer to Fuc-a1,2-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc.

The terms “LNDFH I,” “Lacto-N-difucohexaose I” and “LDFH I” are used interchangeably and refer to Fuc-a1,4-(Fuc-a1,2-Gal-b1,3)-GlcNAc-b1,3-Gal-b1,4-Glc comprising the Lewisb antigen Fuc-a1,4-(Fuc-a1,2-Gal-b1,3)-GlcNAc.

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

The terms “LNnDFH,” “Lacto-N-neoDiFucohexaose” and “Lewis x hexaose” are used interchangeably and refer to Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc.

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

A “fucosylated oligosaccharide” as used herein and as generally understood in the state of the art is an oligosaccharide that is carrying a fucose-residue. Examples comprise 2′-fucosyllactose (2′FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL), lactodifucotetraose (LDFT), Lacto-N-fucopentaose I (LNFP I), Lacto-N-fucopentaose II (LNFP II), Lacto-N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-fucopentaose VI (LNFP VI), lacto-N-neofucopentaose I, lacto-N-difucohexaose I (LDFH I), lacto-N-difucohexaose II (LDFH II), Monofucosyllacto-N-hexaose III (MFLNH III), Difucosyllacto-N-hexaose (DFLNHa), difucosyl-lacto-N-neohexaose.

The terms “alpha-1,2-fucosyltransferase,” “alpha 1,2 fucosyltransferase,” “2-fucosyltransferase,” “a-1,2-fucosyltransferase,” “a 1,2 fucosyltransferase,” “2 fucosyltransferase,” “2-FT” or “2FT” as used in the disclosure, are used interchangeably and refer to a glycosyltransferase that catalyzes the transfer of fucose from the donor GDP-L-fucose, to the acceptor molecule in an alpha-1,2-linkage. The terms “2′ fucosyllactose,” “2′-fucosyllactose,” “alpha-1,2-fucosyllactose,” “alpha 1,2 fucosyllactose,” “a-1,2-fucosyllactose,” “a 1,2 fucosyllactose,” “Galβ-4(Fucα1-2)Glc,” “2FL” or “2′FL” as used in the disclosure, are used interchangeably and refer to the product obtained by the catalysis of the alpha-1,2-fucosyltransferase transferring the fucose residue from GDP-L-fucose to lactose in an alpha-1,2-linkage. The terms “difucosyllactose,” “di-fucosyllactose,” “lactodifucotetraose,” “2′,3-difucosyllactose,” “2′,3 difucosyllactose,” “a-2′,3-fucosyllactose,” “a 2′,3 fucosyllactose,” “Fucα1-2Galβ 1-4(Fucα1-3)Glc,” “DFLac,” “2′,3 diFL,” “DFL,” “DiFL” or “diFL” as used in the disclosure, are used interchangeably.

A “fucosylation pathway” as used herein is a biochemical pathway consisting of the enzymes and their respective genes, mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase and/or the salvage pathway L-fucokinase/GDP-fucose pyrophosphorylase, combined with a fucosyltransferase leading to α 1,2; α 1,3 α 1,4 or α 1,6 fucosylated oligosaccharides.

A “galactosylation pathway” as used herein is a biochemical pathway consisting of the enzymes and their respective genes, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-I-phosphate uridylyltransferase, and/or phosphoglucomutase, combined with a galactosyltransferase leading to an alpha or beta bound galactose on the 2, 3, 4, 6 hydroxyl group of an oligosaccharide.

An “N-acetylglucosamine carbohydrate pathway” as used herein is a biochemical pathway consisting of the enzymes and their respective genes, 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 uridylyltransferase, glucosamine-1-phosphate acetyltransferase, and/or glucosamine-1-phosphate acetyltransferase, combined with a glycosyltransferase leading to an alpha or beta bound N-acetylglucosamine on the 3, 4, 6 hydroxylgroup of an oligosaccharide.

The term “glycopeptide” as used herein refers to a peptide that contains one or more saccharide groups, being mono-, di-, oligo-, polysaccharides and/or glycans, that is/are covalently attached to the side chains of the amino acid residues of the peptide. Glycopeptides comprise natural glycopeptide antibiotics such as e.g., the glycosylated non-ribosomal peptides produced by a diverse group of soil actinomycetes that target Gram-positive bacteria by binding to the acyl-D-alanyl-D-alanine (D-Ala-D-Ala) terminus of the growing peptidoglycan on the outer surface of the cytoplasmatic membrane, and synthetic glycopeptide antibiotics. The common core of natural glycopeptides is made of a cyclic peptide consisting in 7 amino acids, to which are bound 2 sugars. Examples of glycopeptides comprise vancomycin, teicoplanin, oritavancin, chloroeremomycin, telavancin and dalbavancin.

The terms “glycoprotein” and “glycopolypeptide” are used interchangeably and refer to a polypeptide that contains one or more saccharide groups, being mono-, di-, oligo-, polysaccharides and/or glycans, that is/are covalently attached to the side chains of the amino acid residues of the polypeptide.

As used herein, the term “glycolipid” refers to any of the glycolipids that are generally known in the art. Glycolipids (GLs) can be subclassified into Simple (SGLs) and Complex (CGLs) glycolipids. Simple GLs, sometimes called saccharolipids, are two-component (glycosyl and lipid moieties) GLs in which the glycosyl and lipid moieties are directly linked to each other. Examples of SGLs include glycosylated fatty acids, fatty alcohols, carotenoids, hopanoids, sterols or paraconic acids. Bacterially produced SGLs can be classified into rhamnolipids, glucolipids, trehalolipids, other glycosylated (non-trehalose containing) mycolates, trehalose-containing oligosaccharide lipids, glycosylated fatty alcohols, glycosylated macro-lactones and macro-lactams, glycomacrodiolides (glycosylated macrocyclic dilactones), glyco-carotenoids and glyco-terpenoids, and glycosylated hopanoids/sterols. Complex glycolipids (CGLs) are, however, structurally more heterogeneous, as they contain, in addition to the glycosyl and lipid moieties, other residues like, for example, glycerol (glycoglycerolipids), peptide (glycopeptidolipids), acylated-sphingosine (glycosphingolipids), or other residues (lipopolysaccharides, phenolic glycolipids, nucleoside lipids).

The term “membrane transporter proteins” as used herein refers to proteins that are part of or interact with the cell membrane and control the flow of molecules and information across the cell. The membrane proteins are thus involved in transport, be it import into or export out of the cell.

Such membrane transporter proteins can be porters, P-P-bond-hydrolysis-driven transporters, β-Barrel Porins, auxiliary transport proteins, putative transport proteins and phosphotransfer-driven group translocators as defined by the Transporter Classification Database that is operated and curated by the Saier Lab Bioinformatics Group available via www.tcdb.org and providing a functional and phylogenetic classification of membrane transport proteins This Transporter Classification Database details a comprehensive IUBMB approved classification system for membrane transporter proteins known as the Transporter Classification (TC) system. The TCDB classification searches as described here are defined based on TCDB. org as released on 17 Jun. 2019.

Porters is the collective name of uniporters, symporters, and antiporters that utilize a carrier-mediated process (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). They belong to the electrochemical potential-driven transporters and are also known as secondary carrier-type facilitators. Membrane transporter proteins are included in this class when they utilize a carrier-mediated process to catalyze uniport when a single species is transported either by facilitated diffusion or in a membrane potential-dependent process if the solute is charged; antiport when two or more species are transported in opposite directions in a tightly coupled process, not coupled to a direct form of energy other than chemiosmotic energy; and/or symport when two or more species are transported together in the same direction in a tightly coupled process, not coupled to a direct form of energy other than chemiosmotic energy, of secondary carriers (Forrest et al., Biochim. Biophys. Acta 1807 (2011) 167-188). These systems are usually stereospecific. Solute:solute countertransport is a characteristic feature of secondary carriers. The dynamic association of porters and enzymes creates functional membrane transport metabolons that channel substrates typically obtained from the extracellular compartment directly into their cellular metabolism (Moraes and Reithmeier, Biochim. Biophys. Acta 1818 (2012), 2687-2706). Solutes that are transported via this porter system include but are not limited to cations, organic anions, inorganic anions, nucleosides, amino acids, polyols, phosphorylated glycolytic intermediates, osmolytes, siderophores.

Membrane transporter proteins are included in the class of P-P-bond hydrolysis-driven transporters if they hydrolyze the diphosphate bond of inorganic pyrophosphate, ATP, or another nucleoside triphosphate, to drive the active uptake and/or extrusion of a solute or solutes (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). The membrane transporter protein may or may not be transiently phosphorylated, but the substrate is not phosphorylated. Substrates that are transported via the class of P-P-bond hydrolysis-driven transporters include but are not limited to cations, heavy metals, beta-glucan, UDP-glucose, lipopolysaccharides, teichoic acid.

The β-Barrel porins membrane transporter proteins form transmembrane pores that usually allow the energy independent passage of solutes across a membrane. The transmembrane portions of these proteins consist exclusively of β-strands, which form a β-barrel (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). These porin-type proteins are found in the outer membranes of Gram-negative bacteria, mitochondria, plastids, and possibly acid-fast Gram-positive bacteria. Solutes that are transported via these β-Barrel porins include but are not limited to nucleosides, raffinose, glucose, beta-glucosides, oligosaccharides.

The auxiliary transport proteins are defined to be proteins that facilitate transport across one or more biological membranes but do not themselves participate directly in transport. These membrane transporter proteins always function in conjunction with one or more established transport systems such as but not limited to outer membrane factors (OMFs), polysaccharide (PST) porters, the ATP-binding cassette (ABC)-type transporters. They may provide a function connected with energy coupling to transport, play a structural role in complex formation, serve a biogenic or stability function or function in regulation (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). Examples of auxiliary transport proteins include but are not limited to the polysaccharide copolymerase family involved in polysaccharide transport, the membrane fusion protein family involved in bacteriocin and chemical toxin transport.

Putative transport protein comprises families that will either be classified elsewhere when the transport function of a member becomes established or will be eliminated from the Transporter Classification system if the proposed transport function is disproven. These families include a member or members for which a transport function has been suggested, but evidence for such a function is not yet compelling (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). Examples of putative transporters classified in this group under the TCDB system as released on 17 Jun. 2019 include but are not limited to copper transporters.

The phosphotransfer-driven group translocators are also known as the PEP-dependent phosphoryl transfer-driven group translocators of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). The product of the reaction, derived from extracellular sugar, is a cytoplasmic sugar-phosphate. The enzymatic constituents, catalyzing sugar phosphorylation, are superimposed on the transport process in a tightly coupled process. The PTS system is involved in many different aspects comprising in regulation and chemotaxis, biofilm formation, and pathogenesis (Lengeler, J. Mol. Microbiol. Biotechnol. 25 (2015) 79-93; Saier, J. Mol. Microbiol. Biotechnol. 25 (2015) 73-78). Membrane transporter protein families classified within the phosphotransfer-driven group translocators under the TCDB system as released on 17 Jun. 2019 include PTS systems linked to transport of glucose-glucosides, fructose-mannitol, lactose-N,N′-diacetylchitobiose-beta-glucoside, glucitol, galactitol, mannose-fructose-sorbose and ascorbate.

The major facilitator superfamily (MFS) is a superfamily of membrane transporter proteins catalyzing uniport, solute:cation (H+, but seldom Na+) symport and/or solute:H+ or solute:solute antiport. Most are of 400-600 amino acyl residues in length and possess either 12, 14, or occasionally, 24 transmembrane α-helical spanners (TMSs) as defined by the Transporter Classification Database operated by the Saier Lab Bioinformatics Group (www.tcdb.org).

“SET” or “Sugar Efflux Transporter” as used herein refers to membrane proteins of the SET family, which are proteins with InterPRO domain IPR004750 and/or are proteins that belong to the eggNOGv4.5 family ENOG410XTE9. Identification of the InterPro domain can be done by using the online tool on www.ebi.ac.uk/interpro/ or a standalone version of InterProScan (www.ebi.ac.uk/interpro/download.html) using the default values. Identification of the orthology family in eggNOGv4.5 can be done using the online version or a standalone version of eggNOG-mapperv1 (eggnogdb.embl.de/#/app/home).

The term “Siderophore” as used herein is referring to the secondary metabolite of various microorganisms, which are mainly ferric ion specific chelators. These molecules have been classified as catecholate, hydroxamate, carboxylate and mixed types. Siderophores are in general synthesized by a nonribosomal peptide synthetase (NRPS) dependent pathway or an NRPS independent pathway (NIS). The most important precursor in NRPS-dependent siderophore biosynthetic pathway is chorismate. 2, 3-DHBA could be formed from chorismate by a three-step reaction catalyzed by isochorismate synthase, isochorismatase, and 2, 3-dihydroxybenzoate-2, 3-dehydrogenase. Siderophores can also be formed from salicylate, which is formed from isochorismate by isochorismate pyruvate lyase. When ornithine is used as precursor for siderophores, biosynthesis depends on the hydroxylation of ornithine catalyzed by L-ornithine N5-monooxygenase. In the NIS pathway, an important step in siderophore biosynthesis is N(6)-hydroxylysine synthase.

A transporter is needed to export the siderophore outside the cell. Four superfamilies of membrane proteins are identified so far in this process: the major facilitator superfamily (MFS); the Multidrug/Oligosaccharidyl-lipid/Polysaccharide Flippase Superfamily (MOP); the resistance, nodulation and cell division superfamily (RND); and the ABC superfamily. In general, the genes involved in siderophore export are clustered together with the siderophore biosynthesis genes. The term “siderophore exporter” as used herein refers to such transporters needed to export the siderophore outside of the cell.

The ATP-binding cassette (ABC) superfamily contains both uptake and efflux transport systems, and the members of these two groups generally cluster loosely together. ATP hydrolysis without protein phosphorylation energizes transport. There are dozens of families within the ABC superfamily, and family generally correlates with substrate specificity. Members are classified according to class 3.A.1 as defined by the Transporter Classification Database operated by the Saier Lab Bioinformatics Group available via www.tcdb.org and providing a functional and phylogenetic classification of membrane transporter proteins.

The term “enabled efflux” means to introduce the activity of transport of a solute over the cytoplasm membrane and/or the cell wall. The transport may be enabled by introducing and/or increasing the expression of a transporter protein as described in the disclosure. The term “enhanced efflux” means to improve the activity of transport of a solute over the cytoplasm membrane and/or the cell wall. Transport of a solute over the cytoplasm membrane and/or cell wall may be enhanced by introducing and/or increasing the expression of a membrane transporter protein as described in the disclosure. “Expression” of a membrane transporter protein is defined as “overexpression” of the gene encoding the membrane transporter protein in the case the gene is an endogenous gene or “expression” in the case the gene encoding the membrane transporter protein is a heterologous gene that is not present in the wild type strain or cell.

The term “purified” refers to material that is substantially or essentially free from components that interfere with the activity of the biological molecule. For cells, saccharides, nucleic acids, polypeptides, peptides, glycoproteins, glycopeptides, lipids and glycolipids the term “purified” refers to material that is substantially or essentially free from components that normally accompany the material as found in its native state. Typically, purified saccharides, oligosaccharides, peptides, glycopeptides, proteins, glycoproteins, lipids, glycolipids or nucleic acids of the disclosure are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure as measured by band intensity on a silver stained gel or other method for determining purity. Purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized. For oligosaccharides, purity can be determined using methods such as but not limited to thin layer chromatography, gas chromatography, NMR, HPLC, capillary electrophoresis or mass spectroscopy.

The term “cultivation” refers to the culture medium wherein the cell is cultivated or fermented, the cell itself, and the alpha-1,3-glycosylated oligosaccharides that are produced by the cell of the disclosure in whole broth, i.e., inside (intracellularly) as well as outside (extracellularly) of the cell.

The terms “precursor” as used herein refers to substances that are taken up and/or synthetized by the cell for the specific production of an oligosaccharide. In this sense a precursor can be an acceptor as defined herein, but can also be another substance, metabolite, which is first modified within the cell as part of the biochemical synthesis route of the oligosaccharide. Examples of such precursors comprise the acceptors as defined herein, and/or glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, dihydroxyacetone, glucosamine, N-acetyl-glucosamine, mannosamine, N-acetyl-mannosamine, galactosamine, N-acetylgalactosamine, phosphorylated sugars like e.g., but not limited to glucose-I-phosphate, galactose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, mannose-6-phosphate, mannose-1-phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-acetyl-glucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N-acetylglucosamine-1-phosphate, N-acetyl-neuraminic acid-9-phosphate and/or nucleotide-activated sugars as defined herein like e.g., UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, CMP-sialic acid, GDP-mannose, GDP-4-dehydro-6-deoxy-α-D-mannose, GDP-fucose.

The term “acceptor” as used herein refers to a mono-, di- or oligosaccharide, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid or glycolipid that can be modified by a glycosyltransferase. Examples of such acceptors comprise glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, lacto-N-biose (LNB), lacto-N-triose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), N-acetyl-lactosamine (LacNAc), lacto-N-pentaose (LNP), lacto-N-neopentaose, para lacto-N-pentaose, para lacto-N-neopentaose, lacto-N-novopentaose I, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para lacto-N-neohexaose (pLNnH), para lacto-N-hexaose (pLNH), lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose, iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose, lacto-N-decaose, iso lacto-N-decaose, novo lacto-N-decaose, lacto-N-neodecaose, galactosyllactose, a lactose extended with 1, 2, 3, 4, 5, or a multiple of N-acetyllactosamine units and/or 1, 2, 3, 4, 5, or a multiple of, Lacto-N-biose units, and oligosaccharide containing 1 or multiple N-acetyllactosamine units and or 1 or multiple lacto-N-biose units or an intermediate into oligosaccharide, fucosylated and sialylated versions thereof, peptides, polypeptides, lipids, sphingolipids, cerebrosides, ceramide lipids, phosphatidylinositol lipids and glycosylated versions of peptides, polypeptides, lipids, sphingolipids, cerebrosides, ceramide lipids, phosphatidylinositol lipids.

DETAILED DESCRIPTION

According to a first aspect, the disclosure provides a method for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, wherein the alpha-1,3 glycosylation occurs at the terminal “fucose-a1,2-galactose”-group of fucose-alpha-1,2-galactose R (Fuc-a1,2-Gal-R). The method comprises the steps of:

i) providing a cell, preferably a single cell, that is capable to synthesize Fuc-a1,2-Gal-R, that expresses an alpha-1,3-glycosyltransferase and that is capable to synthesize a nucleotide-sugar, which is donor for the alpha-1,3-glycosyltransferase, and

ii) cultivating the cell under conditions permissive to synthesize the Fuc-a1,2-Gal-R, to express the alpha-1,3-glycosyltransferase, to synthesize the nucleotide-sugar and to synthesize the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R,

iii) preferably, separating the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R from the cultivation.

In an embodiment, the disclosure provides a method for the production of a mixture of alpha-1,3 glycosylated forms of Fuc-a1,2-Gal-R. The method comprises the steps of:

i) providing a cell, preferably a single cell, that is capable to synthesize at least two different Fuc-a1,2-Gal-R, that expresses an alpha-1,3-glycosyltransferase and that is capable to synthesize a nucleotide-sugar, which is donor for the alpha-1,3-glycosyltransferase, and

ii) cultivating the cell under conditions permissive to synthesize the at least two different Fuc-a1,2-Gal-R, to express the alpha-1,3-glycosyltransferase, to synthesize the nucleotide-sugar and to synthesize the alpha-1,3 glycosylated form of each Fuc-a1,2-Gal-R,

iii) preferably, separating the alpha-1,3 glycosylated form of each Fuc-a1,2-Gal-R from the cultivation.

According to the disclosure, the mixture comprises or consists of at least two different “alpha-1,3 glycosylated forms of Fuc-a1,2-Gal-R,” preferably at least three different “alpha-1,3 glycosylated forms of Fuc-a1,2-Gal-R,” more preferably at least four different “alpha-1,3 glycosylated forms of Fuc-a1,2-Gal-R.” Preferably the at least two, more preferably at least three, even more preferably at least four, different Fuc-a1,2-Gal-R are synthesized by the cell. In a further and/or alternative embodiment, a mixture of alpha-1,3 glycosylated forms of Fuc-a1,2-Gal-R can be obtained by a method as disclosed herein, wherein the cell expresses multiple alpha-1,3-glycosyltransferases (preferably an alpha-1,3-galactosyltransferase and an alpha-1,3-N-acetylgalactosaminyltransferase). In a further and/or alternative embodiment, a mixture of alpha-1,3 glycosylated forms of Fuc-a1,2-Gal-R can be obtained by a method as disclosed herein, wherein multiple different acceptors as disclosed herein are provided.

In a second aspect, the disclosure provides a cell metabolically engineered for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R as described herein. In the context of the disclosure, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R as described herein preferably does not occur in the wild type progenitor of the cell.

A metabolically engineered cell, preferably a single cell, is provided, which is capable to synthesize Fuc-a1,2-Gal-R, which expresses an alpha-1,3-glycosyltransferase and which is capable to synthesize a nucleotide-sugar, which is donor for the alpha-1,3-glycosyltransferase.

According to the disclosure, the method for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R can make use of a non-metabolically engineered cell or can make use of a metabolically engineered cell as disclosed herein.

In the context of the disclosure, it should be understood that the Fuc-a1,2-Gal-R, nucleotide-sugar and alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R are preferably synthesized intracellularly. The skilled person will further understand that a fraction or substantially all of the synthesized alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R remains intracellularly and/or is excreted outside the cell either passively or through active transport.

Throughout the disclosure, unless explicitly stated otherwise, a “genetically modified cell” or “metabolically engineered cell” preferably means a cell that is genetically modified or metabolically engineered, respectively, for the production of an alpha-1,3-glycosylated form of Fuc-a1,2-Gal-R according to the disclosure.

In the context of the disclosure, the term “alpha-1,3 glycosylated form” of Fuc-a1,2-Gal-R (or derived structures thereof as recited herein) preferably means that a sugar moiety (e.g., a monosaccharide) is bound through an alpha-1,3-glycosidic bond with the galactose residue of the “Fuc-a1,2-Gal”-group of fucose-alpha-1,2-galactose-R (Fuc-a1,2-Gal-R), i.e., the sugar moiety is not linked directly to another residue comprised in Fuc-a1,2-Gal-R such as the a1,2-linked fucose or any residue comprised in the R moiety.

In a preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is (i) Gal-a1,3-(Fuc-a1,2)-Gal-R, preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-R, more preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-R, even more preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-R, even more preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-R, most preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc; or (ii) GalNAc-a1,3-(Fuc-a1,2)-Gal-R, preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-R, more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-R, even more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-R, even more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-R, most preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc.

In other words, in a preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3 galactose or an alpha-1,3 GalNAc modified form of Fuc-a1,2-Gal-R wherein the galactose or GlcNAc is bound to the galactose residue of the “Fuc-a1,2-Gal”-group of fucose-alpha-1,2-galactose-R (Fuc-a1,2-Gal-R) in an alpha-1,3-glycosidic linkage.

In a more preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is Gal-a1,3-(Fuc-a1,2)-Gal-R, preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-R, more preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-R, even more preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-R, even more preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-R, most preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc. In other words, in a more preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3 galactose modified Fuc-a1,2-Gal-R.

In another more preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is GalNAc-a1,3-(Fuc-a1,2)-Gal-R, preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-R, more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-R, even more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-R, even more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-R, most preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc. In other words, in a more preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3 GalNAc modified Fuc-a1,2-Gal-R.

Throughout the disclosure, unless explicitly stated otherwise, the Fuc-a1,2-Gal-R is preferably Fuc-a1,2-Gal-b1,3-R, more preferably the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-R, even more preferably the Fuc-a1,2-Gal-R is lacto-N-fucopentaose I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc).

In one embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is a structure of the histo blood group antigen (HBGA) system. In a preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-R. In a more preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc. In an alternative preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-R. In a more preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc.

In another embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, wherein the glucose can be optionally fucosylated (preferably a1,3-fucosylated). Preferably, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc.

In another embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc.

In another embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is the alpha-tetrasaccharide GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc.

In another embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3 GalNAc or an alpha-1,3 galactose modified Fuc-a1,2-Gal-GlcNAc, wherein the galactose in Fuc-a1,2-Gal-GlcNAc is bound to the GlcNAc via either a beta-1,3 or via a beta-1,4 linkage. In a preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is GalNAc-a1,3-(Fuc-a1,2)-Gal-GlcNAc, wherein the galactose is bound to the GlcNAc via either a beta-1,3 or via a beta-1,4 linkage. In a more preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc. In another more preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc. In a preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is an Gal-a1,3-(Fuc-a1,2)-Gal-GlcNAc, wherein the galactose is bound to the GlcNAc via either a beta-1,3 or via a beta-1,4 linkage. In a more preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc. In another more preferred embodiment, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc.

In the scope of the disclosure, the wording “permissive conditions” is to be understood to be conditions relating to physical or chemical parameters including but not limited to temperature, pH, pressure, osmotic pressure and product/precursor/acceptor concentration.

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

According to a preferred embodiment of the disclosure, the cell is modified with one or more expression modules. The expression modules are also known as transcriptional units and comprise polynucleotides for expression of recombinant genes including coding gene sequences and appropriate transcriptional and/or translational control signals that are operably linked to the coding genes. The control signals comprise promoter sequences, untranslated regions, ribosome binding sites, terminator sequences. The expression modules can contain elements for expression of one single recombinant gene but can also contain elements for expression of more recombinant genes or can be organized in an operon structure for integrated expression of two or more recombinant genes. The polynucleotides may be produced by recombinant DNA technology using techniques well-known in the art. Methods that are well known to those skilled in the art to construct expression modules include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989 and yearly updates).

The expression of each of the expression modules can be constitutive or is created by a natural or chemical inducer. As used herein, constitutive expression should be understood as expression of a gene that is transcribed continuously in an organism. Expression that is created by a natural inducer should be understood as a facultative or regulatory expression of a gene that is only expressed upon a certain natural condition of the host (e.g., organism being in labor, or during lactation), as a response to an environmental change (e.g., including but not limited to hormone, heat, cold, pH shifts, light, oxidative or osmotic stress/signaling), or dependent on the position of the developmental stage or the cell cycle of the cell including but not limited to apoptosis and autophagy. Expression that is created by a chemical inducer should be understood as a facultative or regulatory expression of a gene that is only expressed upon sensing of external chemicals (e.g., IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose) via an inducible promoter or via a genetic circuit that either induces or represses the transcription or translation of the polynucleotide to a polypeptide.

The expression modules can be integrated in the genome of the cell or can be presented to the cell on a vector. The vector can be present in the form of a plasmid, cosmid, phage, liposome, or virus, which is to be stably transformed/transfected into the metabolically engineered cell. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. These vectors may contain selection markers such as but not limited to antibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNA sense/antisense markers. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., see above. For recombinant production, cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the disclosure. Introduction of a polynucleotide into the cell can be effected 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 polynucleotides for expression of at least one recombinant gene. The recombinant gene is involved in the expression of a polypeptide acting in the synthesis of LNFP-I or an alpha-1,3 glycosylated form of LNFP-I; or the recombinant gene is linked to other pathways in the cell that are not involved in the synthesis of LNFP-I or an alpha-1,3 glycosylated form of LNFP-I. The recombinant genes encode endogenous proteins with a modified expression or activity, preferably the endogenous proteins are overexpressed; or the recombinant genes encode heterologous proteins that are heterogeneously introduced and expressed in the modified cell, preferably overexpressed. The endogenous proteins can have a modified expression in the cell, which also expresses a heterologous protein.

In an embodiment of the method and/or cell according to the disclosure, the cell is capable to synthesize lacto-N-fucopentaose I (LNFP-I). LNFP-I is a fucosylated pentasaccharide that is derived from lacto-N-tetraose (LNT), wherein the LNT is modified with a fucose group a1,2-linked to its terminal galactose residue. In a preferred embodiment, the cell is capable to synthesize LNT and expresses an alpha-1,2-fucosyltransferase, which transfers a fucose residue from a GDP-fucose donor to the acceptor LNT to produce LNFP-I. In a more preferred embodiment, the cell is capable to synthesize LNT, expresses an alpha-1,2-fucosyltransferase that uses LNT as acceptor for alpha-1,2-fucosylation and is capable to synthesize GDP-fucose, which is donor for the alpha-1,2-fucosyltransferase.

LNT production in a cell can be obtained by over-expression of a galactoside beta-1,3-N-acetylglucosaminyltransferase gene and an N-acetylglucosamine beta-1,3-galactosyltransferase gene, which respectively transfers a GlcNAc residue from UDP-GlcNAc to lactose to form LN3 and that transfers a Gal residue from UDP-Gal to LN3 to form LNT. Preferably, the cell does not have an active galactosidase like e.g., lacZ that degrades lactose into glucose and galactose. The lactose that is needed by the galactoside beta-1,3-N-acetylglucosaminyltransferase can be fed to the cultivation or can be synthesized by the metabolism of the cell. The UDP-GlcNAc and UDP-Gal that are needed by the enzymes can be provided by enzymes expressed in the cell or by the metabolism of the cell.

A cell using lactose as acceptor in glycosylation reactions preferably has a transporter for the uptake of lactose from the cultivation. More preferably, the cell is optimized for lactose uptake. The optimization can be over-expression of a lactose transporter like a lactose permease from E. coli or Kluyveromyces lactis.

In a preferred embodiment of the method and/or cell according to the disclosure, the cell resists the phenomenon of lactose killing when grown in an environment in which lactose is combined with one or more other carbon source(s). With the term “lactose killing” is meant the hampered growth of the cell in medium in which lactose is present together with another carbon source. In a preferred embodiment, the cell is genetically modified such that it retains at least 50% of the lactose influx without undergoing lactose killing, even at high lactose concentrations, as is described in WO 2016/075243. The genetic modification comprises expression and/or over-expression of an exogenous and/or an endogenous lactose transporter gene by a heterologous promoter that does not lead to a lactose killing phenotype and/or modification of the codon usage of the lactose transporter to create an altered expression of the lactose transporter that does not lead to a lactose killing phenotype. The content of WO 2016/075243 in this regard is incorporated by reference. In the context of the disclosure, lactose is preferably taken up by a cell as disclosed herein, wherein the lactose is further glycosylated by a glycosyltransferase as disclosed herein to synthesize a MMO, preferably a HMO.

Alternatively, a cell producing lactose can be obtained by expression of a beta-1,4-galactosyltransferase and an UDP-glucose 4-epimerase. More preferably, the cell is modified for enhanced lactose production. The modification can be any one or more chosen from the group comprising over-expression of a beta-1,4-galactosyltransferase, over-expression of an UDP-glucose 4-epimerase.

A cell producing an UDP-GlcNAc can express enzymes converting, e.g., GlcNAc, which is to be added to the cell, to UDP-GlcNAc. These enzymes may be an N-acetyl-D-glucosamine kinase, an N-acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, and an N-acetylglucosamine-1-phosphate uridylyltransferase/glucosamine-1-phosphate acetyltransferase from several species including Homo sapiens, and Escherichia coli. Preferably, the cell is modified to produce UDP-GlcNAc. More preferably, the cell is modified for enhanced UDP-GlcNAc production. The modification can be any one or more chosen from the group comprising knock-out of an N-acetylglucosamine-6-phosphate deacetylase, over-expression of an L-glutamine-D-fructose-6-phosphate aminotransferase, over-expression of a phosphoglucosamine mutase, and over-expression of an N-acetylglucosamine-1-phosphate uridylyltransferase/glucosamine-1-phosphate acetyltransferase.

A cell producing UDP-Gal can express an enzyme converting, e.g., UDP-glucose, to UDP-Gal. This enzyme may be, e.g., the UDP-glucose 4-epimerase GalE like as known from several species including Homo sapiens, Escherichia coli, and Rattus norvegicus. Preferably, the cell is modified to produce UDP-Gal. More preferably, the cell is modified for enhanced UDP-Gal production. The modification can be any one or more chosen from the group comprising knock-out of an bifunctional 5′-nucleotidase/UDP-sugar hydrolase encoding gene, knock-out of a galactose-1-phosphate uridylyltransferase encoding gene and over-expression of an UDP-glucose 4-epimerase encoding gene.

GDP-fucose can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing GDP-fucose can express an enzyme converting, e.g., fucose, which is to be added to the cell, to GDP-fucose. This enzyme may be, e.g., a bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase, like Fkp from Bacteroides fragilis, or the combination of one separate fucose kinase together with one separate fucose-1-phosphate guanylyltransferase like they are known from several species including Homo sapiens, Sus scrofa and Rattus norvegicus.

Preferably, the cell is modified to produce GDP-fucose. More preferably, the cell is modified for enhanced GDP-fucose production. The modification can be any one or more chosen from the group comprising knock-out of an UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase encoding gene, over-expression of a GDP-L-fucose synthase encoding gene, over-expression of a GDP-mannose 4,6-dehydratase encoding gene, over-expression of a mannose-1-phosphate guanylyltransferase encoding gene, over-expression of a phosphomannomutase encoding gene and over-expression of a mannose-6-phosphate isomerase encoding gene.

The alpha-1,2-fucosyltransferase that transfers the fucose residue from GDP-fucose to the intracellularly synthesized LNT is an alpha-1,2-fucosyltransferase that accepts the terminal galactose residue of LNT as acceptor for fucosylation. The alpha-1,2-fucosyltransferase can also use other acceptors in addition to LNT for fucosylation. The additional acceptor can include but is not limited to a mono-, di- and oligosaccharide like e.g., galactose, glucose, N-acetylglucosamine (GlcNAc), lactose, lactulose, lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), 3′-fucosyllactose (3′FL), lacto-N-triose (LN3) and lacto-N-neotetraose (LNnT). The alpha-1,2-fucosyltransferase can be e.g., the alpha-1,2-fucosyltransferase from H. pylori as exemplified herein.

In a preferred embodiment of the method and/or cell of the disclosure, the alpha-1,2-fucosyltransferase is chosen from the list comprising the polypeptide from Brachyspira pilosicoli (UniProt ID AOA2N5RQ26), the polypeptide from Dysgonomonas mossii (UniProt ID F8X274), the polypeptide from Dechlorosoma suillum (UniProt ID G8QLF4), the polypeptide from Desulfovibrio alaskensis (UniProt ID Q316B5) and the polypeptide from Polaribacter vadi (UniProt ID AOA1B8TNT0).

In an alternative preferred embodiment, the alpha-1,2-fucosyltransferase is a functional fragment of any one of the polypeptide from B. pilosicoli (UniProt ID AOA2N5RQ26), the polypeptide from D. mossii (UniProt ID F8X274), the polypeptide from D. suillum (UniProt ID G8QLF4), the polypeptide from D. alaskensis (UniProt ID Q316B5) and the polypeptide from P. vadi (UniProt ID A0A1B8TNT0) having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of LNT.

In an alternative preferred embodiment, the alpha-1,2-fucosyltransferase is a functional homolog, variant or derivative of any one of the polypeptide from B. pilosicoli (UniProt ID A0A2N5RQ26), the polypeptide from D. mossii (UniProt ID F8X274), the polypeptide from D. suillum (UniProt ID G8QLF4), the polypeptide from D. alaskensis (UniProt ID Q316B5) and the polypeptide from P. vadi (UniProt ID A0A1B8TNT0) having at least 80% overall sequence identity to the full length of any one of the polypeptide from B. pilosicoli (UniProt ID A0A2N5RQ26), the polypeptide from D. mossii (UniProt ID F8X274), the polypeptide from D. suillum (UniProt ID G8QLF4), the polypeptide from D. alaskensis (UniProt ID Q316B5) and the polypeptide from P. vadi (UniProt ID A0A1B8TNT0), respectively, and having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of LNT.

In an alternative preferred embodiment, the alpha-1,2-fucosyltransferase is a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any one of the polypeptide from B. pilosicoli (UniProt ID A0A2N5RQ26), the polypeptide from D. mossii (UniProt ID F8X274), the polypeptide from D. suillum (UniProt ID G8QLF4), the polypeptide from D. alaskensis (UniProt ID Q316B5) or the polypeptide from P. vadi (UniProt ID A0A1B8TNT0) and having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of LNT.

In another preferred embodiment of the method and/or cell of the disclosure, the cell expresses an alpha-1,2-fucosyltransferase that preferably uses LNT as acceptor for alpha-1,2-fucosylation over other acceptors like e.g., galactose, glucose, N-acetylglucosamine (GlcNAc), lactose, lactulose, lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), 3′-fucosyllactose (3′FL), lacto-N-triose (LN3) and lacto-N-neotetraose (LNnT). In a more preferred embodiment, at least 50% of the fucosylated compound obtained in a mixture by the alpha-1,2-fucosyltransferase expressed in the cell is derived from alpha-1,2-fucosylation of LNT. In other words, at least 50% of the fucosylated compound obtained in a mixture by the alpha-1,2-fucosyltransferase expressed in the cell is fucosylated LNT. At least 50% of the fucosylated compound in a mixture should be understood as 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%, 99,60%, 99,70%, 99,80%, 99,90%, 100% of the fucosylated compound in a mixture is fucosylated LNT. 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 compound obtained in a mixture by the alpha-1,2-fucosyltransferase expressed in the cell is fucosylated LNT.

In an even more preferred embodiment, the alpha-1,2-fucosyltransferase solely uses LNT as acceptor for alpha-1,2-fucosylation. With the term “solely” is meant only. In other words, the alpha-1,2-fucosyltransferase only accepts LNT as acceptor for fucosylation in an alpha-1,2-linkage of the terminal galactose residue of the LNT and no other acceptors.

According to an embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase that is capable to produce an alpha-1,3-glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3-galactosyltransferase, which is a glycosyltransferase with the ability to transfer a galactose residue from UDP-Gal to a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R wherein the R comprises a monosaccharide, a disaccharide, an oligosaccharide, a peptide, a glycopeptide, a protein, a glycoprotein, a lipid or a glycolipid as defined earlier herein.

In one embodiment of the method and/or cell of the disclosure, the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain and comprises the motif YX[FHMQQT]XAXX[ACG][ACG] with SEQ ID NO:01 wherein X can be any amino acid residue.

In an alternative embodiment, the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain and comprises the motif YXQXCXX[ACG][ACG] with SEQ ID NO:02 wherein X can be any amino acid residue.

In an alternative embodiment, the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain and comprises a polypeptide sequence according to any one of SEQ ID NOs: 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.

In an alternative embodiment, the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain and is a functional homolog, variant or derivative of any one of SEQ ID NO: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 having at least 80% overall sequence identity to the full length of any one of the a-1,3-galactosyltransferase polypeptide with SEQ ID Nos: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 having a-1,3-galactosyltransferase activity on a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R.

In an alternative embodiment, the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain and is a functional fragment of any one of SEQ ID NOs 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 having a-1,3-galactosyltransferase activity on a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R.

In a preferred alternative embodiment, the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain and comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs 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 having a-1,3-galactosyltransferase activity on a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R.

In an alternative embodiment, the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain and comprises or consists of a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any one of SEQ ID NOs 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 having a-1,3-galactosyltransferase activity on a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R.

According to another embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase that is capable to produce an alpha-1,3-glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3-N-acetylgalactosaminyltransferase, which is a glycosyltransferase with the ability to transfer an N-acetylgalactosamine residue from UDP-GalNAc to a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R wherein the R comprises a monosaccharide, a disaccharide, an oligosaccharide, a peptide, a glycopeptide, a protein, a glycoprotein, or a lipid or a glycolipid as defined earlier herein.

In one embodiment of the method and/or cell of the disclosure, the alpha-1,3-N-acetylgalactosaminyltransferase has a PFAM PF03414 domain and comprises the motif YX[ACIL]XGXX[ACG][ACG] with SEQ ID NO:38 wherein X can be any amino acid residue.

In an alternative embodiment, the alpha-1,3-N-acetylgalactosaminyltransferase has a PFAM PF03414 domain and comprises the motif YX[AG]XAXX[ACG][ACG] with SEQ ID NO:39 wherein X can be any amino acid residue.

In an alternative embodiment, the alpha-1,3-N-acetylgalactosaminyltransferase has a PFAM PF03414 domain and comprises a polypeptide sequence according to any one of SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102.

In an alternative embodiment, the alpha-1,3-N-acetylgalactosaminyltransferase has a PFAM PF03414 domain and is a functional homolog, variant or derivative of any one of SEQ ID NOs 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 having at least 80% overall sequence identity to the full length of any one of the a-1,3-N-acetylgalactosyltransferase polypeptide with SEQ ID NOs 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and having a-1,3-N-acetylgalactosyltransferase activity on a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R.

In an alternative embodiment, the alpha-1,3-N-acetylgalactosaminyltransferase has a PFAM PF03414 domain and is a functional fragment of any one of SEQ ID NOs 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and having a-1,3-N-acetylgalactosyltransferase activity on a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R.

In a preferred alternative embodiment, the alpha-1,3-N-acetylgalactosaminyltransferase has a PFAM PF03414 domain and comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and having a-1,3-N-acetylgalactosyltransferase activity on a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R.

In an alternative embodiment, the alpha-1,3-N-acetylgalactosaminyltransferase has a PFAM PF03414 domain and comprises or consists of a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any one of SEQ ID NOs 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and having a-1,3-N-acetylgalactosyltransferase activity on a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R.

The PFAM PF03414 domain as used herein refers to the PF03414 domain as present in the Pfam 32.0 database as released in September 2018 and present within the glycosyltransferase 6 (GT6) family. The a-1,3-galactosyltransferases and a-1,3-N-acetylgalactosyltransferases both belong to the GT6 family.

The overall sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably with default parameters and preferably with sequences of mature proteins (i.e., without taking into account secretion signals or transit peptides). Compared to overall sequence identity, the sequence identity will generally be higher when only conserved domains or motifs are considered.

An oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of the polypeptides with SEQ ID Nos: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 having a-1,3-galactosyltransferase activity on a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R should be understood as any one of oligopeptide sequences of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 up to the total number of consecutive amino acid residues from any one of the polypeptides with SEQ ID NOs: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 as given herein, preferably wherein the oligopeptide does not fully overlap with a PFAM domain if present, more preferably wherein the oligopeptide does not overlap with a PFAM domain if present, and having a-1,3-galactosyltransferase activity on a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R.

An oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID Nos:40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and having a-1,3-N-acetylgalactosyltransferase activity on a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R should be understood as any one of oligopeptide sequences of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 up to the total number of consecutive amino acid residues from any one of the polypeptides with SEQ ID Nos:40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 as given herein, preferably wherein the oligopeptide does not fully overlap with a PFAM domain if present, more preferably wherein the oligopeptide does not overlap with a PFAM domain if present, and having a-1,3-N-acetylgalactosyltransferase activity on a terminal “fucose-a1,2-galactose” group of Fuc-a1,2-Gal-R.

In a preferred embodiment of the method and/or cell of the disclosure, the cell expresses an alpha-1,3-glycosyltransferase that is capable to modify the intracellularly synthesized Fuc-a1,2-Gal-R and hence forming an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R as disclosed earlier herein. Preferably, the cell is capable to synthesize a nucleotide-sugar, which is donor for the alpha-1,3-glycosyltransferase.

In a more preferred embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase as described herein with the ability to transfer a galactose residue from UDP-Gal to the terminal “fucose-a1,2-galactose” of Fuc-a1,2-Gal-R as described herein, the nucleotide-sugar is UDP-Gal, resulting in an alpha-1,3 galactose modified Fuc-a1,2-Gal-R as described herein.

In another more preferred embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase as described herein with the ability to transfer a GalNAc residue from UDP-GalNAc to the terminal “fucose-a1,2-galactose” of Fuc-a1,2-Gal-R as described herein, the nucleotide-sugar is UDP-GalNAc, resulting in an alpha-1,3 GalNAc modified Fuc-a1,2-Gal-R as described herein.

In a more preferred embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase as described herein with the ability to transfer a galactose residue from UDP-Gal to the terminal “fucose-a1,2-galactose” of Fuc-a1,2-Gal-b1,3-R as described herein, the nucleotide-sugar is UDP-Gal, resulting in an alpha-1,3 galactose modified Fuc-a1,2-Gal-b1,3-R as described herein.

In another more preferred embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase as described herein with the ability to transfer a GalNAc residue from UDP-GalNAc to the terminal “fucose-a1,2-galactose” of Fuc-a1,2-Gal-b1,3-R as described herein, the nucleotide-sugar is UDP-GalNAc, resulting in an alpha-1,3 GalNAc modified Fuc-a1,2-Gal-b1,3-R as described herein.

In a more preferred embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase as described herein with the ability to transfer a galactose residue from UDP-Gal to the terminal “fucose-a1,2-galactose” of Fuc-a1,2-Gal-b1,3-GlcNAc-R as described herein, the nucleotide-sugar is UDP-Gal, resulting in an alpha-1,3 galactose modified Fuc-a1,2-Gal-b1,3-GlcNAc-R as described herein.

In another more preferred embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase as described herein with the ability to transfer a GalNAc residue from UDP-GalNAc to the terminal “fucose-a1,2-galactose” of Fuc-a1,2-Gal-b1,3-GlcNAc-R as described herein, the nucleotide-sugar is UDP-GalNAc, resulting in an alpha-1,3 GalNAc modified Fuc-a1,2-Gal-b1,3-GlcNAc-R as described herein.

In a more preferred embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase as described herein with the ability to transfer a galactose residue from UDP-Gal to the terminal “fucose-a1,2-galactose” of Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R as described herein, the nucleotide-sugar is UDP-Gal, resulting in an alpha-1,3 galactose modified Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R as described herein.

In another more preferred embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase as described herein with the ability to transfer a GalNAc residue from UDP-GalNAc to the terminal “fucose-a1,2-galactose” of Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R as described herein, the nucleotide-sugar is UDP-GalNAc, resulting in an alpha-1,3 GalNAc modified Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R as described herein.

In a more preferred embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase as described herein with the ability to transfer a galactose residue from UDP-Gal to the terminal “fucose-a1,2-galactose” of Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R as described herein, the nucleotide-sugar is UDP-Gal, resulting in an alpha-1,3 galactose modified Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R as described herein.

In another more preferred embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase as described herein with the ability to transfer a GalNAc residue from UDP-GalNAc to the terminal “fucose-a1,2-galactose” of Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R as described herein, the nucleotide-sugar is UDP-GalNAc, resulting in an alpha-1,3 GalNAc modified Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R as described herein.

In a more preferred embodiment of the method and/or cell of the disclosure, the cell expresses an alpha-1,3-glycosyltransferase that is capable to modify the intracellularly synthesized LNFP-I into an alpha-1,3 glycosylated form of LNFP-I. In another additional embodiment of the method and/or cell of the disclosure, the cell is capable to synthesize a nucleotide-sugar, which is donor for the alpha-1,3-glycosyltransferase.

In a more preferred embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase as described herein with the ability to transfer a galactose residue from UDP-Gal to the terminal “fucose-a1,2-galactose”-group of LNFP-I, the nucleotide-sugar is UDP-Gal and the alpha-1,3 glycosylated form of lacto-N-fucopentaose I (LNFP-I) is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (Gal-a1,3-LNFP-I).

In another more preferred embodiment of the method and/or cell of the disclosure, the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase as described herein with the ability to transfer a GalNAc residue from UDP-GalNAc to the terminal “fucose-a1,2-galactose”-group of LNFP-I, the nucleotide-sugar is UDP-GalNAc and the alpha-1,3 glycosylated form of lacto-N-fucopentaose I (LNFP-I) is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (GalNAc-a1,3-LNFP-I).

In a further embodiment of the method and/or cell of the disclosure, the cell is modified in the expression or activity of at least one glycosyltransferase. In one embodiment, the glycosyltransferases comprise galactosyltransferases (e.g., beta-1,3-galactosyltransferases, beta-1,4-galactosyltransferases, alpha-1,3-galactosyltransferases, alpha-1,4-galactosyltransferases), N-acetylgalactosyltransferases, fucosyltransferases (e.g., alpha-1,2-fucosyltransferases, alpha-1,3/1,4-fucosyltransferases, alpha-1,6-fucosyltransferases), N-acetylglucosaminyltransferases, mannosyltransferases, N-acetylmannosaminyltransferases, glucosyltransferases. In a preferred embodiment, the glycosyltransferase comprises alpha-1,3-galactosyltransferases with a-1,3-galactosyltransferase activity on a terminal “fucose-a1,2-galactose-R” group and alpha-1,3-N-acetylgalactosyltransferases with a-1,3-N-acetylgalactosyltransferase activity on a terminal “fucose-a1,2-galactose-R” group as described herein.

In an embodiment, the glycosyltransferase is an endogenous protein of the cell with a modified expression or activity, preferably the endogenous glycosyltransferase is overexpressed; alternatively the glycosyltransferase is a heterologous protein that is heterogeneously introduced and expressed in the cell, preferably overexpressed. The endogenous glycosyltransferase can have a modified expression in the cell, which also expresses a heterologous glycosyltransferase.

In an embodiment of the method of the disclosure, the cultivation is fed with a precursor for the synthesis of fucose-a1,2-galactose-R and/or an alpha-1,3 glycosylated form of fucose-a1,2-galactose-R. Precursors that could be fed to the cultivation for synthesis of fucose-a1,2-galactose-R and/or an alpha-1,3 glycosylated form of fucose-a1,2-galactose-R comprise lactose, lacto-N-triose (LN3, LNT II), fucose, glucose, galactose, GlcNAc, GDP-fucose, UDP-galactose and UDP-GlcNAc or any other precursor as defined herein.

In one embodiment of the method of the disclosure, the cultivation is fed with a precursor for the synthesis of LNFP-I and/or an alpha-1,3 glycosylated form of LNFP-I. Precursors that could be fed to the cultivation for synthesis of LNFP-I and/or an alpha-1,3 glycosylated form of LNFP-I comprise lactose, lacto-N-triose (LN3, LNT II), fucose, glucose and galactose.

In an embodiment of the method and/or cell according to the disclosure, the cell expresses a membrane transporter protein or a polypeptide having transport activity hereby transporting compounds across the outer membrane of the cell wall. In a preferred embodiment of the method and/or cell of the disclosure, the cell expresses more than one membrane transporter protein or polypeptide having transport activity hereby transporting compounds across the outer membrane of the cell wall. In a more preferred embodiment of the method and/or cell of the disclosure, the cell is modified in the expression or activity of the membrane transporter protein or polypeptide having transport activity. The membrane transporter protein or polypeptide having transport activity is an endogenous protein of the cell with a modified expression or activity, preferably the endogenous membrane transporter protein or polypeptide having transport activity is overexpressed; alternatively the membrane transporter protein or polypeptide having transport activity is a heterologous protein that is heterogeneously introduced and expressed in the cell, preferably overexpressed. The endogenous membrane transporter protein or polypeptide having transport activity can have a modified expression in the cell, which also expresses a heterologous membrane transporter protein or polypeptide having transport activity.

In another preferred embodiment of the method and/or cell of the disclosure, the membrane transporter protein or polypeptide having transport activity is chosen from the list comprising porters, P-P-bond-hydrolysis-driven transporters, b-barrel porins, auxiliary transport proteins, putative transport proteins and phosphotransfer-driven group translocators. In a more preferred embodiment of the method and/or cell of the disclosure, the porters comprise MFS transporters, sugar efflux transporters and siderophore exporters. In another more preferred embodiment of the method and/or cell of the disclosure, the P-P-bond-hydrolysis-driven transporters comprise ABC transporters and siderophore exporters.

In another preferred embodiment of the method and/or cell of the disclosure, the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of an alpha-1,3 glycosylated form of fucose-alpha1,2-galactose-R as described herein. In an alternative and/or additional preferred embodiment of the method and/or cell of the disclosure, the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of one or more precursor(s) to be used in the production of an alpha-1,3 glycosylated form of fucose-alpha1,2-galactose-R. In an alternative and/or additional preferred embodiment of the method and/or cell of the disclosure, the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of one or more acceptor(s) to be used in the production of an alpha-1,3 glycosylated form of fucose-alpha1,2-galactose-R.

In another preferred embodiment of the method and/or cell of the disclosure, the cell expresses a membrane transporter protein belonging to the family of MFS transporters like e.g., an MdfA polypeptide of the multidrug transporter MdfA family from species comprising E. coli (UniProt ID P0AEY8), Cronobacter muytjensii (UniProt ID AOA2T7ANQ9), Citrobacter youngae (UniProt ID D4BC23) and Yokenella regensburgei (UniProt ID G9Z5F4). In another preferred embodiment of the method and/or cell of the disclosure, the cell expresses a membrane transporter protein belonging to the family of sugar efflux transporters like e.g., a SetA polypeptide of the SetA family from species comprising E. coli (UniProt ID P31675), Citrobacter koseri (UniProt ID AOA078LM16) and Klebsiella pneumoniae (UniProt ID A0A0C4MGS7). In another preferred embodiment of the method and/or cell of the disclosure, the cell expresses a membrane transporter protein belonging to the family of siderophore exporters like e.g., the E. coli entS (UniProt ID P24077) and the E. coli iceT (UniProt ID A0A024L207). In another preferred embodiment of the method and/or cell of the disclosure, the cell expresses a membrane transporter protein belonging to the family of ABC transporters like e.g., oppF from E. coli (UniProt ID P77737), lmrA from Lactococcus lactis subsp. lactis bv. diacetylactis (UniProt ID AOA1V0NEL4) and Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4). In a more preferred embodiment of the method and/or cell of the disclosure, the cell expresses more than one membrane transporter protein chosen from the list comprising a lactose transporter like e.g., the LacY or lac12 permease, a fucose transporter, a glucose transporter, a galactose transporter, a transporter for a nucleotide-activated sugar like, for example, a transporter for UDP-GlcNAc, UDP-Gal and/or GDP-Fuc, the MdfA protein from E. coli (UniProt ID P0AEY8), the MdfA protein from Cronobacter muytjensii (UniProt ID AOA2T7ANQ9), the MdfA protein from Citrobacter youngae (UniProt ID D4BC23), the MdfA protein from Yokenella regensburgei (UniProt ID G9Z5F4), the SetA protein from E. coli (UniProt ID P31675), the SetA protein from Citrobacter koseri (UniProt ID AOA078LM16), the SetA protein from Klebsiella pneumoniae (UniProt ID A0A0C4MGS7), the entS protein from E. coli (UniProt ID P24077), the iceT protein from E. coli (UniProt ID A0A024L207), the oppF protein from E. coli (UniProt ID P77737), the lmrA protein from Lactococcus lactis subsp. lactis bv. diacetylactis (UniProt ID AOA1V0NEL4) and Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4). Preferably, the cell is transformed to comprise at least one nucleic acid sequence encoding a protein selected from the group comprising a lactose transporter like e.g., the LacY or lac12 permease, a fucose transporter, a glucose transporter, a galactose transporter, a transporter for a nucleotide-activated sugar like, for example, a transporter for UDP-GlcNAc, UDP-GalNAc and/or GDP-Fuc. As such, the transporter internalizes a to the medium added precursor and/or acceptor for the synthesis of the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R of disclosure.

In an additional and/or alternative embodiment of the method and/or cell according to the disclosure, the cell is genetically modified to export the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R of disclosure over the membrane. Such transporter is, for example, a membrane transporter protein belonging to the siderophore exporter family, the major facilitator superfamily (MFS), the ATP-binding cassette (ABC) transporter family or the sugar efflux transporter family.

In a further embodiment of the method and/or cell of the disclosure, the cell preferably comprises multiple copies of the same coding DNA sequence encoding for one protein. In the context of the disclosure, the protein can be a glycosyltransferase, a membrane transporter protein or any other protein as disclosed herein. Throughout the disclosure, the feature “multiple” means at least 2, preferably at least 3, more preferably at least 4, even more preferably at least 5.

In another embodiment of the method and/or cell of the disclosure, the cell comprises a modification for reduced production of acetate. The modification can be any one or more chosen from the group comprising overexpression of an acetyl-coenzyme A synthetase, a full or partial knock-out or rendered less functional pyruvate dehydrogenase and a full or partial knock-out or rendered less functional lactate dehydrogenase.

In a further embodiment of the method and/or cell of the disclosure, the cell is modified in the expression or activity of at least one acetyl-coenzyme A synthetase like e.g., acs from E. coli, S. cerevisiae, H. sapiens, M. musculus. In a preferred embodiment, the acetyl-coenzyme A synthetase is an endogenous protein of the cell with a modified expression or activity, preferably the endogenous acetyl-coenzyme A synthetase is overexpressed; alternatively, the acetyl-coenzyme A synthetase is a heterologous protein that is heterogeneously introduced and expressed in the cell, preferably overexpressed. The endogenous acetyl-coenzyme A synthetase can have a modified expression in the cell, which also expresses a heterologous acetyl-coenzyme A synthetase. In a more preferred embodiment, the cell is modified in the expression or activity of the acetyl-coenzyme A synthetase acs from E. coli (UniProt ID P27550). In another and/or additional preferred embodiment, the cell is modified in the expression or activity of a functional homolog, variant or derivative of acs from E. coli (UniProt ID P27550) having at least 800% overall sequence identity to the full-length of the polypeptide from E. coli (UniProt ID P27550) and having acetyl-coenzyme A synthetase activity.

In an alternative and/or additional further embodiment of the method and/or cell of the disclosure, the cell is modified in the expression or activity of at least one pyruvate dehydrogenase like e.g., from E. coli, S. cerevisiae, H. sapiens and R. norvegicus. In a preferred embodiment, the cell has been modified to have at least one partially or fully knocked out or mutated pyruvate dehydrogenase encoding gene by means generally known by the person skilled in the art resulting in at least one protein with less functional or being disabled for pyruvate dehydrogenase activity. In a more preferred embodiment, the cell has a full knock-out in the poxB encoding gene resulting in a cell lacking pyruvate dehydrogenase activity.

In an alternative and/or additional further embodiment of the method and/or cell of the disclosure, the cell is modified in the expression or activity of at least one lactate dehydrogenase like e.g., from E. coli, S. cerevisiae, H. sapiens and R. norvegicus. In a preferred embodiment, the cell has been modified to have at least one partially or fully knocked out or mutated lactate dehydrogenase encoding gene by means generally known by the person skilled in the art resulting in at least one protein with less functional or being disabled for lactate dehydrogenase activity. In a more preferred embodiment, the cell has a full knock-out in the ldhA encoding gene resulting in a cell lacking lactate dehydrogenase activity.

According to another preferred embodiment of the method and/or cell of the disclosure, the cell comprises a lower or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the proteins comprising beta-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose undecaprenyl-phosphate glucose-1-phosphatetransferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridylyltransferase, glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IclR, lon protease, glucose-specific translocating phosphotransferase enzyme IIBC component ptsG, glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIA^Glc, beta-glucoside specific PTS enzyme II, fructose-specific PTS multiphosphoryl transfer protein FruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, pyruvate decarboxylase.

According to another preferred embodiment of the method and/or cell of the disclosure, the cell is capable to produce phosphoenolpyruvate (PEP). In another preferred embodiment of the method and/or cell of the disclosure, the cell is modified for enhanced production and/or supply of phosphoenolpyruvate (PEP).

In a preferred embodiment and as a means for enhanced production and/or supply of PEP, one or more PEP-dependent, sugar-transporting phosphotransferase system(s) is/are disrupted such as but not limited to: 1) the N-acetyl-D-glucosamine Npi-phosphotransferase (EC 2.7.1.193), which is, for instance, encoded by the nagE gene (or the cluster nagABCD) in E. coli or Bacillus species, 2) ManXYZ, which encodes the Enzyme 11 Man complex (mannose PTS permease, protein-Npi-phosphohistidine-D-mannose phosphotransferase) that imports exogenous hexoses (mannose, glucose, glucosamine, fructose, 2-deoxyglucose, mannosamine, N-acetylglucosamine, etc.) and releases the phosphate esters into the cell cytoplasm, 3) the glucose-specific PTS transporter (for instance, encoded by PtsG/Crr) which takes up glucose and forms glucose-6-phosphate in the cytoplasm, 4) the sucrose-specific PTS transporter, which takes up sucrose and forms sucrose-6-phosphate in the cytoplasm, 5) the fructose-specific PTS transporter (for instance, encoded by the genes fruA and fruB and the kinase fruK, which takes up fructose and forms in a first step fructose-1-phosphate and in a second step fructose1,6 bisphosphate, 6) the lactose PTS transporter (for instance, encoded by lacE in Lactococcus casei) which takes up lactose and forms lactose-6-phosphate, 7) the galactitol-specific PTS enzyme, which takes up galactitol and/or sorbitol and forms galactitol-1-phosphate or sorbitol-6-phosphate, respectively, 8) the mannitol-specific PTS enzyme, which takes up mannitol and/or sorbitol and forms mannitol-1-phosphate or sorbitol-6-phosphate, respectively, and 9) the trehalose-specific PTS enzyme, which takes up trehalose and forms trehalose-6-phosphate.

In another and/or additional preferred embodiment and as a means for enhanced production and/or supply of PEP, the full PTS system is disrupted by disrupting the PtsIH/Crr gene cluster. PtsI (Enzyme I) is a cytoplasmic protein that serves as the gateway for the phosphoenolpyruvate:sugar phosphotransferase system (PTS^sugar) of E. coli K-12. PtsI is one of two (PtsI and PtsH) sugar non-specific protein constituents of the PTS^sugar, which along with a sugar-specific inner membrane permease effects a phosphotransfer cascade that results in the coupled phosphorylation and transport of a variety of carbohydrate substrates. HPr (histidine containing protein) is one of two sugar-non-specific protein constituents of the PTS^sugar. It accepts a phosphoryl group from phosphorylated Enzyme I (PtsI-P) and then transfers it to the EIIA domain of any one of the many sugar-specific enzymes (collectively known as Enzymes II) of the PTS^sugar Crr or EIIA^Glc is phosphorylated by PEP in a reaction requiring PtsH and PtsI.

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

In a further preferred embodiment, the cell is modified to compensate for the deletion of a PTS system of a carbon source by the introduction of carbohydrate kinases, 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 overexpression of alternative transporters and a kinase are: 1) the deletion of the glucose PTS system, e.g., ptsG gene, combined with the introduction and/or overexpression of a glucose permease (e.g., galP of glcP), combined with the introduction and/or overexpression of a glucokinase (e.g., glk), and/or 2) the deletion of the fructose PTS system, e.g., one or more of the fruB, fruA, fruK genes, combined with the introduction and/or overexpression of fructose permease, e.g., fruI, combined with the introduction and/or overexpression of a fructokinase (e.g., frk or mak).

In another and/or additional preferred embodiment and as a means for enhanced production and/or supply of PEP, the cell is modified by the introduction of or modification in any one or more of the list comprising phosphoenolpyruvate synthase activity (EC: 2.7.9.2 encoded, for instance, in E. coli by ppsA), phosphoenolpyruvate carboxykinase activity (EC 4.1.1.32 or EC 4.1.1.49 encoded, for instance, in Corynebacterium glutamicum by PCK or in E. coli by pckA, resp.), phosphoenolpyruvate carboxylase activity (EC 4.1.1.31 encoded, for instance, in E. coli by ppc), oxaloacetate decarboxylase activity (EC 4.1.1.112 encoded, for instance, in E. coli by eda), pyruvate kinase activity (EC 2.7.1.40 encoded, for instance, in E. coli by pykA and pykF), pyruvate carboxylase activity (EC 6.4.1.1 encoded, for instance, in B. subtilis by pyc) and malate dehydrogenase activity (EC 1.1.1.38 or EC 1.1.1.40 encoded, for instance, in E. coli by maeA or maeB, resp.).

In a more preferred embodiment, the cell is modified to overexpress any one or more of the polypeptides comprising ppsA from E. coli (UniProt ID P23538), PCK from C. glutamicum (UniProt ID Q6F5A5), pcka from E. coli (UniProt ID P22259), eda from E. coli (UniProt ID P0A955), maeA from E. coli (UniProt ID P26616) and maeB from E. coli (UniProt ID P76558).

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

In another and/or additional preferred embodiment and as a means for enhanced production and/or supply of PEP, the cell is modified by a reduced activity of phosphoenolpyruvate carboxylase activity, and/or pyruvate kinase activity, preferably a deletion of the genes encoding for phosphoenolpyruvate carboxylase, the pyruvate carboxylase activity and/or pyruvate kinase.

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

In another exemplary embodiment, the cell is genetically modified by different adaptations such as the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of an oxaloacetate decarboxylase, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a malate dehydrogenase, the overexpression of phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase, the overexpression of phosphoenolpyruvate carboxykinase combined with the overexpression of a malate dehydrogenase, the overexpression of oxaloacetate decarboxylase combined with the overexpression of a malate dehydrogenase, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of a malate dehydrogenase, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase, the overexpression of phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase and/or the overexpression of phosphoenolpyruvate synthase combined with the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase.

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

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

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

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

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

According to another preferred embodiment of the method and/or cell of the disclosure, the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides, which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the production of an alpha-1,3 glycosylated form of fucose-a1,2-galactose-R as described herein.

According to another preferred embodiment of the method and/or cell of the disclosure, the cell produces 90 g/L or more of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R in the whole broth and/or supernatant and/or wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R in the whole broth and/or supernatant has a purity of at least 80% measured on the total amount of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R and its precursor(s) in the whole broth and/or supernatant, respectively.

According to another embodiment of the method of the disclosure, the conditions permissive to produce the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R comprise the use of a culture medium comprising at least one precursor and/or acceptor for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R. Preferably, the culture medium contains at least one precursor selected from the group comprising lactose, galactose, fucose, sialic acid, GlcNAc, GalNAc, lacto-N-biose (LNB), N-acetyllactosamine (LacNAc).

According to an alternative and/or additional embodiment of the method of the disclosure, the conditions permissive to produce the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R comprise adding to the culture medium at least one precursor and/or acceptor feed for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.

According to an alternative embodiment of the method of the disclosure, the conditions permissive to produce the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R comprise the use of a culture medium to cultivate a cell of disclosure for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R wherein the culture medium lacks any precursor and/or acceptor for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R and is combined with a further addition to the culture medium of at least one precursor and/or acceptor feed for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.

In a preferred embodiment, the method for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R as described herein comprises at least one of the following steps:

• • i) Use of a culture medium comprising at least one precursor and/or acceptor;
  • ii) Adding to the culture medium in a reactor at least one precursor and/or acceptor feed wherein the total reactor volume ranges from 250 mL (milliliter) to 10,000 m³ (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the culture medium before the addition of the precursor and/or acceptor feed;
  • iii) Adding to the culture medium in a reactor at least one precursor and/or acceptor feed wherein the total reactor volume ranges from 250 mL (millilitre) to 10,000 m³ (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the culture medium before the addition of the precursor and/or acceptor feed and wherein preferably, the pH of the precursor and/or acceptor feed is set between 3 and 7 and wherein preferably, the temperature of the precursor and/or acceptor feed is kept between 20° C. and 80° C.;
  • iv) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution;
  • v) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein preferably, the pH of the feeding solution is set between 3 and 7 and wherein preferably, the temperature of the feeding solution is kept between 20° C. and 80° C.;
  • the method resulting in an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R with a concentration of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L in the final cultivation.

In another and/or additional preferred embodiment, the method for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R as described herein comprises at least one of the following steps:

• • i) Use of a culture 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 gram of lactose per liter of initial reactor volume wherein the reactor volume ranges from 250 mL to 10,000 m³ (cubic meter);
  • ii) Adding to the culture medium at least one precursor and/or acceptor in one pulse or in a discontinuous (pulsed) manner wherein the total reactor volume ranges from 250 mL (milliliter) to 10,000 m³ (cubic meter), preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the culture medium before the addition of the precursor and/or acceptor feed pulse(s);
  • iii) Adding to the culture medium in a reactor at least one precursor and/or acceptor feed in one pulse or in a discontinuous (pulsed) manner wherein the total reactor volume ranges from 250 mL (milliliter) to 10,000 m³ (cubic meter), preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the culture medium before the addition of the precursor and/or acceptor feed pulse(s) and wherein preferably, the pH of the precursor and/or acceptor feed pulse(s) is set between 3 and 7 and wherein preferably, the temperature of the precursor and/or acceptor feed pulse(s) is kept between 20° C. and 80° C.;
  • iv) Adding at least one precursor and/or acceptor feed in a discontinuous (pulsed) manner to the culture medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution;
  • v) Adding at least one precursor and/or acceptor feed in a discontinuous (pulsed) manner to the culture medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein preferably, the pH of the feeding solution is set between 3 and 7 and wherein preferably, the temperature of the feeding solution is kept between 20° C. and 80° C.;
  • the method resulting in an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R with a concentration of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L in the final cultivation.

In a further, more preferred embodiment, the method for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R as described herein comprises at least one of the following steps:

• • i) Use of a culture 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 gram of lactose per liter of initial reactor volume wherein the reactor volume ranges from 250 mL to 10,000 m³ (cubic meter);
  • ii) Adding to the culture medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per liter of initial reactor volume wherein the total reactor volume ranges from 250 mL (milliliter) to 10,000 m³ (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture medium before the addition of the lactose feed;
  • iii) Adding to the culture medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per liter of initial reactor volume wherein the reactor volume ranges from 250 mL to 10,000 m³ (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture medium before the addition of the lactose feed and wherein preferably the pH of the lactose feed is set between 3 and 7 and wherein preferably the temperature of the lactose feed is kept between 20° C. and 80° C.;
  • iv) Adding a lactose feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution;
  • v) Adding a lactose feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein the concentration of the lactose feeding solution is 50 g/L, preferably 75 g/L, more preferably 100 g/L, more preferably 125 g/L, more preferably 150 g/L, more preferably 175 g/L, more preferably 200 g/L, more preferably 225 g/L, more preferably 250 g/L, more preferably 275 g/L, more preferably 300 g/L, more preferably 325 g/L, more preferably 350 g/L, more preferably 375 g/L, more preferably, 400 g/L, more preferably 450 g/L, more preferably 500 g/L, even more preferably, 550 g/L, most preferably 600 g/L; and wherein preferably the pH of the feeding solution is set between 3 and 7 and wherein preferably the temperature of the feeding solution is kept between 20° C. and 80° C.; the method resulting in an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R with a concentration of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L in the final cultivation.

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

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

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

In a preferred embodiment, a carbon source is provided, preferably sucrose, in the culture medium for 3 or more days, preferably up to 7 days; and/or provided, in the culture medium, at least 100, advantageously at least 105, more advantageously at least 110, even more advantageously at least 120 grams of sucrose per liter of initial culture volume in a continuous manner, so that the final volume of the culture medium is not more than three-fold, advantageously not more than two-fold, more advantageously less than two-fold of the volume of the culturing medium before the culturing.

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

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

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

In an alternative preferable embodiment, in the method as described herein, the lactose is added already in the first phase of exponential growth together with the carbon-based substrate.

In an embodiment, the method as described herein preferably comprises a step of separating the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R as described herein.

In a preferred embodiment, the method as described herein preferably comprises a step of separating the alpha-1,3 glycosylated form of LNFP-I.

The terms “separating from the cultivation” means harvesting, collecting, or retrieving the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R as described herein or the alpha-1,3 glycosylated form of LNFP-I from the cell and/or the medium of its growth.

The alpha-1,3 glycosylated form of fucose-a1,2-galactose-R as described herein or alpha-1,3 glycosylated form of LNFP-I can be separated in a conventional manner from the aqueous culture medium, in which the cell was grown. In case the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I is still present in the cells producing the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or the alpha-1,3 glycosylated form of LNFP-I, conventional manners to free or to extract the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I out of the cells can be used, such as cell destruction using high pH, heat shock, sonication, French press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergent, hydrolysis, . . . The culture medium and/or cell extract together and separately can then be further used for separating the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I. This preferably involves clarifying the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I containing mixture to remove suspended particulates and contaminants, particularly cells, cell components, insoluble metabolites and debris produced by culturing the genetically modified cell. In this step, the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I containing mixture can be clarified in a conventional manner. Preferably, the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I containing mixture is clarified by centrifugation, flocculation, decantation and/or filtration.

Another step of separating the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I from the alpha-1,3 glycosylated form of LNFP-I containing mixture preferably involves removing substantially all the proteins, as well as peptides, amino acids, RNA and DNA and any endotoxins and glycolipids that could interfere with the subsequent separation step, from the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I containing mixture, preferably after it has been clarified. In this step, proteins and related impurities can be removed from the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I containing mixture in a conventional manner. Preferably, proteins, salts, by-products, color, endotoxins and other related impurities are removed from the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I containing mixture by ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, electrophoresis (e.g., using slab-polyacrylamide or sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE)), affinity chromatography (using affinity ligands including e.g., DEAE-SEPHAROSE®, poly-L-lysine and polymyxin-B, endotoxin-selective adsorber matrices), ion exchange chromatography (such as but not limited to cation exchange, anion exchange, mixed bed ion exchange, inside-out ligand attachment), hydrophobic interaction chromatography and/or gel filtration (i.e., size exclusion chromatography), particularly by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography. With the exception of size exclusion chromatography, proteins and related impurities are retained by a chromatography medium or a selected membrane, the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I remains in the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I containing mixture.

In a further preferred embodiment, the methods as described herein also provide for a further purification of an alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I from the mixture. A further purification of the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I may be accomplished, for example, by use of (activated) charcoal or carbon, nanofiltration, ultrafiltration or ion exchange to remove any remaining DNA, protein, LPS, endotoxins, or other impurity. Alcohols, such as ethanol, and aqueous alcohol mixtures can also be used. Another purification step is accomplished by crystallization, evaporation or precipitation of the product. Another purification step is to dry, e.g., spray dry, lyophilize, spray freeze dry, freeze spray dry, band dry, belt dry, vacuum band dry, vacuum belt dry, drum dry, roller dry, vacuum drum dry or vacuum roller dry the produced alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I.

In an exemplary embodiment, the separation and purification of alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I is made in a process, comprising the following steps in any order:

• • a) contacting the cultivation or a clarified version thereof with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da ensuring the retention of the produced alpha-1,3 glycosylated form of LNFP-I and allowing at least a part of the proteins, salts, by-products, color and other related impurities to pass,
  • b) conducting a diafiltration process on the retentate from step a), using the membrane, with an aqueous solution of an inorganic electrolyte, followed by optional diafiltration with pure water to remove excess of the electrolyte,
  • c) and collecting the retentate enriched in the alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I, respectively in the form of a salt from the cation of the electrolyte.

In an alternative exemplary embodiment, the separation and purification of an alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I is made in a process, comprising the following steps in any order: subjecting the cultivation or a clarified version thereof to two membrane filtration steps using different membranes, wherein—one membrane has a molecular weight cut-off of between about 300 Dalton to about 500 Dalton, and—the other membrane as a molecular weight cut-off of between about 600 Dalton to about 800 Dalton.

In an alternative exemplary embodiment, the separation and purification of an alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I is made in a process, comprising the following steps in any order comprising the step of treating the cultivation or a clarified version thereof with a strong cation exchange resin in H+-form and a weak anion exchange resin in free base form.

In an alternative exemplary embodiment, the separation and purification of an alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I is made in the following way.

The cultivation comprising the produced alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I, biomass, medium components and contaminants is applied to the following purification steps:

• • i) separation of biomass from the cultivation,
  • ii) cationic ion exchanger treatment for the removal of positively charged material,
  • iii) anionic ion exchanger treatment for the removal of negatively charged material,
  • iv) nanofiltration step and/or electrodialysis step,
  • wherein a purified solution comprising the produced alpha-1,3 glycosylated form of LNFP-I at a purity of greater than or equal to 80 percent is provided. Optionally the purified solution is dried by any one or more drying steps chosen from the list comprising spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying and vacuum roller drying.

In an alternative exemplary embodiment, the separation and purification of an alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I is made in a process, comprising the following steps in any order: enzymatic treatment of the cultivation; removal of the biomass from the cultivation; ultrafiltration; nanofiltration; and a column chromatography step. Preferably such column chromatography is a single column or a multiple column. Further preferably the column chromatography step is simulated moving bed chromatography. Such simulated moving bed chromatography preferably comprises i) at least 4 columns, wherein at least one column comprises a weak or strong cation exchange resin; and/or ii) four zones I, II, III and IV with different flow rates; and/or iii) an eluent comprising water; and/or iv) an operating temperature of 15 degrees to 60 degrees centigrade. Preferably, the process further comprises a step of spray drying.

In one embodiment, the disclosure provides the produced alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I, which is dried to powder by any one or more drying steps chosen from the list comprising spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying and vacuum roller drying, wherein the dried powder contains <15 percent-wt. of water, preferably <10 percent-wt. of water, more preferably <7 percent-wt. of water, most preferably <5 percent-wt. of water.

Another embodiment of the disclosure provides for a method and a cell wherein an alpha-1,3 glycosylated form of fucose-a1,2-galactose-R as described herein, preferably an alpha-1,3 glycosylated form of LNFP-I, is produced in and/or by a fungal, yeast, bacterial, insect, plant, animal or protozoan cell as described herein. The cell is chosen from the list comprising a bacterium, a yeast, or a fungus, or, refers to a plant, animal, or protozoan cell. The latter bacterium preferably belongs to the phylum of the Proteobacteria or the phylum of the Firmicutes or the phylum of the Cyanobacteria or the phylum Deinococcus-Thermus. The latter bacterium belonging to the phylum Proteobacteria belongs preferably to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacterium preferably relates to any strain belonging to the species Escherichia coli such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to cultivated Escherichia coli strains—designated as E. coli K12 strains—which are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. Well-known examples of the E. coli K12 strains are K12 Wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Hence, the disclosure specifically relates to a mutated and/or transformed Escherichia coli cell or strain as indicated above wherein the E. coli strain is a K12 strain. More preferably, the Escherichia coli K12 strain is E. coli MG1655. The latter bacterium belonging to the phylum Firmicutes belongs preferably to the Bacilli, preferably Lactobacilliales, with members such as Lactobacillus lactis, Leuconostoc mesenteroides, or Bacillales with members such as from the genus Bacillus, such as Bacillus subtilis or, B. amyloliquefaciens. The latter Bacterium belonging to the phylum Actinobacteria, preferably belonging to the family of the Corynebacteriaceae, with members Corynebacterium glutamicum or C. afermentans, or belonging to the family of the Streptomycetaceae with members Streptomyces griseus or S. fradiae. The latter yeast preferably belongs to the phylum of the Ascomycota or the phylum of the Basidiomycota or the phylum of the Deuteromycota or the phylum of the Zygomycetes. The latter yeast belongs preferably to the genus Saccharomyces (with members like e.g., Saccharomyces cerevisiae, S. bayanus, S. boulardii), Zygosaccharomyces, Pichia (with members like e.g., Pichia pastoris, P. anomala, P. kluyveri), Komagataella, Hansenula, Kluyveromyces (with members like e.g., Kluyveromyces lactis, K. marxianus, K. thermotolerans), Debaromyces, Yarrowia (like e.g., Yarrowia lipolytica) or Starmerella (like e.g., Starmerella bombicola). The latter yeast is preferably selected from Pichia pastoris, Yarrowia lipolitica, Saccharomyces cerevisiae and Kluyveromyces lactis. The latter fungus belongs preferably to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus. Plant cells include cells of flowering and non-flowering plants, as well as algal cells, for example, Chlamydomonas, Chlorella, etc. Preferably, the plant is a tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy, maize or corn plant. The latter animal cell is preferably derived from non-human mammals (e.g., cattle, buffalo, pig, sheep, mouse, rat), birds (e.g., chicken, duck, ostrich, turkey, pheasant), fish (e.g., swordfish, salmon, tuna, sea bass, trout, catfish), invertebrates (e.g., lobster, crab, shrimp, clams, oyster, mussel, sea urchin), reptiles (e.g., snake, alligator, turtle), amphibians (e.g., frogs) or insects (e.g., fly, nematode) or is a genetically modified cell line derived from human cells excluding embryonic stem cells. Both human and non-human mammalian cells are preferably chosen from the list comprising an epithelial cell like e.g., a mammary epithelial cell, an embryonic kidney cell (e.g., HEK293 or HEK 293T cell), a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell like e.g., an N20, SP2/0 or YB2/0 cell, an NIH-3T3 cell, a non-mammary adult stem cell or derivatives thereof such as described in WO21067641. The latter insect cell is preferably derived from Spodoptera frugiperda like e.g., Sf9 or Sf21 cells, Bombyx mori, Mamestra brassicae, Trichoplusia ni like e.g., BTI-TN-5B1-4 cells or Drosophila melanogaster like e.g., Drosophila S2 cells. The latter protozoan cell preferably is a Leishmania tarentolae cell.

According to a preferred embodiment of the method and/or cell of the disclosure, an alpha-1,3 glycosylated form of fucose-a1,2-galactose-R is produced in and/or by a cell, which is a viable Gram-negative bacterium that comprises a reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose.

In a more preferred embodiment of the method and/or cell, the reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose is provided by a mutation in any one or more glycosyltransferases involved in the synthesis of any one of the poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose, wherein the mutation provides for a deletion or lower expression of any one of the glycosyltransferases. The glycosyltransferases comprise glycosyltransferase genes encoding poly-N-acetyl-D-glucosamine synthase subunits, UDP-N-acetylglucosamine-undecaprenyl-phosphate N-acetylglucosaminephosphotransferase, Fuc4NAc (4-acetamido-4,6-dideoxy-D-galactose) transferase, UDP-N-acetyl-D-mannosaminuronic acid transferase, the glycosyltransferase genes encoding the cellulose synthase catalytic subunits, the cellulose biosynthesis protein, colanic acid biosynthesis glucuronosyltransferase, colanic acid biosynthesis galactosyltransferase, colanic acid biosynthesis fucosyltransferase, UDP-glucose:undecaprenyl-phosphate glucose-I-phosphate transferase, putative colanic biosynthesis glycosyl transferase, UDP-glucuronate:LPS(HepIII) glycosyltransferase, ADP-heptose-LPS heptosyltransferase 2, ADP-heptose:LPS heptosyltransferase 1, putative ADP-heptose:LPS heptosyltransferase 4, lipopolysaccharide core biosynthesis protein, UDP-glucose:(glucosyl)LPS a-1,2-glucosyltransferase, UDP-D-glucose:(glucosyl)LPS a-1,3-glucosyltransferase, UDP-D-galactose:(glucosyl)lipopolysaccharide-1,6-D-galactosyltransferase, lipopolysaccharide glucosyltransferase I, lipopolysaccharide core heptosyltransferase 3, β-1,6-galactofuranosyltransferase, undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase, lipid IVA 4-amino-4-deoxy-L-arabinosyltransferase, bactoprenol glucosyl transferase, putative family 2 glycosyltransferase, the osmoregulated periplasmic glucans (OPG) biosynthesis protein G, OPG biosynthesis protein H, glucosylglycerate phosphorylase, glycogen synthase, 1,4-α-glucan branching enzyme, 4-α-glucanotransferase and trehalose-6-phosphate synthase. In an exemplary embodiment, the cell is mutated in any one or more of the glycosyltransferases comprising pgaC, pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcaI, wcaJ, wcaL, waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbI, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA and yaiP, wherein the mutation provides for a deletion or lower expression of any one of the glycosyltransferases.

In an alternative and/or additional preferred embodiment of the method and/or cell, the reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG) is provided by over-expression of a carbon storage regulator encoding gene, deletion of a Na+/H+ antiporter regulator encoding gene and/or deletion of the sensor histidine kinase encoding gene.

The microorganism or cell as used herein is capable to grow on a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone, yeast extract or a mixture thereof like e.g., a mixed feedstock, preferably a mixed monosaccharide feedstock like e.g., hydrolyzed sucrose as the main carbon source. With the term “main” is meant the most important carbon source for the bioproducts of interest, biomass formation, carbon dioxide and/or by-products formation (such as acids and/or alcohols, such as acetate, lactate, and/or ethanol), i.e., 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99% of all the required carbon is derived from the above-indicated carbon source. In one embodiment of the disclosure, the carbon source is the sole carbon source for the organism, i.e., 100% of all the required carbon is derived from the above-indicated carbon source. Common main carbon sources comprise but are not limited to glucose, glycerol, fructose, maltose, lactose, arabinose, malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, sucrose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvate. With the term complex medium is meant a medium for which the exact constitution is not determined. Examples are molasses, corn steep liquor, peptone, tryptone or yeast extract. As used herein, a precursor as defined herein cannot be used as a carbon source for the production of an alpha-1,3 glycosylated form of fucose-a1,2-galactose-R.

In a further preferred embodiment, the microorganism or cell described herein is using a split metabolism having a production pathway and a biomass pathway as described in WO 2012/007481, which is herein incorporated by reference. The organism can, for example, be genetically modified to accumulate fructose-6-phosphate by altering the genes selected from the phosphoglucoisomerase gene, phosphofructokinase gene, fructose-6-phosphate aldolase gene, fructose isomerase gene, and/or fructose:PEP phosphotransferase gene.

In a third aspect, the disclosure provides for the use of a metabolically engineered cell as described herein for the production of alpha-1,3 glycosylated form of fucose-a1,2-galactose-R, preferably an alpha-1,3 glycosylated form of LNFP-I. In a preferred embodiment of the third aspect, a metabolically engineered cell as described herein is used for the production of (i) Gal-a1,3-(Fuc-a1,2)-Gal-R, preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-R, more preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-R, even more preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-R, even more preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-R, most preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc; or (ii) GalNAc-a1,3-(Fuc-a1,2)-Gal-R, preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-R, more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-R, even more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-R, even more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-R, most preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc. In a more preferred embodiment of the third aspect, a metabolically engineered cell as described herein is used for the production of a structure of the histo blood group antigen (HBGA) system as disclosed herein. In another more preferred embodiment of the third aspect, a metabolically engineered cell as described herein is used for the production of alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, wherein the glucose can be optionally fucosylated (preferably a1,3-fucosylated) as disclosed herein. In another more preferred embodiment of the third aspect, a metabolically engineered cell as described herein is used for the production of alpha-1,3 GalNAc modified or an alpha-1,3 galactose modified Fuc-a1,2-Gal-GlcNAc, wherein the galactose in Fuc-a1,2-Gal-GlcNAc is bound to the GlcNAc via either a beta-1,3 or via a beta-1,4 linkage as disclosed herein.

For identification of an alpha-1,3 glycosylated form of fucose-a1,2-galactose-R or alpha-1,3 glycosylated form of LNFP-I as described herein, the monomeric building blocks (e.g., the monosaccharide or glycan unit composition), the anomeric configuration of side chains, the presence and location of substituent groups, degree of polymerization/molecular weight and the linkage pattern can be identified by standard methods known in the art, such as, e.g., methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas 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 solved using, e.g., solid-state NMR, FT-IR (Fourier transform infrared spectroscopy), and WAXS (wide-angle X-ray scattering). The degree of polymerization (DP), the DP distribution, and polydispersity can be determined by, e.g., viscosimetry and SEC (SEC-HPLC, high performance size-exclusion chromatography). To identify the monomeric components of the saccharide methods such as, e.g., acid-catalyzed hydrolysis, HPLC (high performance liquid chromatography) or GLC (gas-liquid chromatography) (after conversion to alditol acetates) may be used. To determine the glycosidic linkages, the saccharide is methylated with methyl iodide and strong base in DMSO, hydrolysis is performed, a reduction to partially methylated alditols is achieved, an acetylation to methylated alditol acetates is performed, and the analysis is carried out by GLC/MS (gas-liquid chromatography coupled with mass spectrometry). To determine the oligosaccharide sequence, a partial depolymerization is carried out using an acid or enzymes to determine the structures. To identify the anomeric configuration, the oligosaccharide is subjected to enzymatic analysis, e.g., it is contacted with an enzyme that is specific for a particular type of linkage, e.g., beta-galactosidase, or alpha-glucosidase, etc., and NMR may be used to analyze the products comprising the produced alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, preferably an alpha-1,3 glycosylated form of LNFPI.

In some embodiments, an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, wherein the R is a monosaccharide, a disaccharide or an oligosaccharide and which is produced as described herein, is incorporated into a food (e.g., human food or feed), dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine. In some embodiments, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, wherein the R is a monosaccharide, a disaccharide or an oligosaccharide, is mixed with one or more ingredients suitable for food, feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredient, or medicine.

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

A “prebiotic” is a substance that promotes growth of microorganisms beneficial to the host, particularly microorganisms in the gastrointestinal tract. In some embodiments, a dietary supplement provides multiple prebiotics, including the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, wherein the R is a monosaccharide, a disaccharide or an oligosaccharide and which is produced and/or purified by a process disclosed in this specification, to promote growth of one or more beneficial microorganisms. Examples of prebiotic ingredients for dietary supplements include other prebiotic molecules (such as HMOs) and plant polysaccharides (such as inulin, pectin, b-glucan and xylooligosaccharide). A “probiotic” product typically contains live microorganisms that replace or add to gastrointestinal microflora, to the benefit of the recipient. Examples of such microorganisms include Lactobacillus species (for example, L. acidophilus and L. bulgaricus), Bifidobacterium species (for example, B. animalis, B. longum and B. infantis (e.g., Bi-26)), and Saccharomyces boulardii. In some embodiments, an oligosaccharide produced and/or purified by a process of this specification is orally administered in combination with such microorganism.

Examples of further ingredients for dietary supplements include disaccharides (such as lactose), monosaccharides (such as glucose and galactose), thickeners (such as gum arabic), acidity regulators (such as trisodium citrate), water, skimmed milk, and flavorings.

In some embodiments, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, wherein the R is a monosaccharide, a disaccharide or an oligosaccharide, is incorporated into a human baby food (e.g., infant formula). Infant formula is generally a manufactured food for feeding to infants as a complete or partial substitute for human breast milk. In some embodiments, infant formula is sold as a powder and prepared for bottle- or cup-feeding to an infant by mixing with water. The composition of infant formula is typically designed to be roughly mimic human breast milk. In some embodiments, an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, wherein the R is a monosaccharide, a disaccharide or an oligosaccharide and which is purified by a process in this specification is included in infant formula to provide nutritional benefits similar to those provided by the oligosaccharides in human breast milk. In some embodiments, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, wherein the R is a monosaccharide, a disaccharide or an oligosaccharide, is mixed with one or more ingredients of the infant formula. Examples of infant formula ingredients include non-fat milk, carbohydrate sources (e.g., lactose), protein sources (e.g., whey protein concentrate and casein), fat sources (e.g., vegetable oils—such as palm, high oleic safflower oil, rapeseed, coconut and/or sunflower oil; and fish oils), vitamins (such as vitamins A, B6, B12, C and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate) and possibly human milk oligosaccharides (HMOs). Such HMOs may include, for example, DiFL, lacto-N-triose II, LNT, LNnT, lacto-N-fucopentaose I, lacto-N-neofucopentaose, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6′-galactosyllactose, 3′-galactosyllactose, lacto-N-hexaose and lacto-N-neohexaose.

In some embodiments, the one or more infant formula ingredients comprise non-fat milk, a carbohydrate source, a protein source, a fat source, and/or a vitamin and mineral.

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

In some embodiments, the concentration of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, wherein the R is a monosaccharide, a disaccharide or an oligosaccharide, in the infant formula is approximately the same concentration as the oligosaccharide's concentration generally present in human breast milk.

In some embodiments, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, wherein the R is a monosaccharide, a disaccharide or an oligosaccharide, is incorporated into a feed preparation, wherein the feed is chosen from the list comprising petfood, animal milk replacer, veterinary product, post weaning feed, or creep feed.

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

Throughout the disclosure, unless explicitly stated otherwise, the articles “a” and “an” are preferably replaced by “at least two,” more preferably by “at least three,” even more preferably by “at least four,” even more preferably by “at least five,” even more preferably by “at least six,” most preferably by “at least two.”

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described above and below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, purification steps are performed according to the manufacturer's specifications.

Further advantages follow from the specific embodiments and the examples. It goes without saying that the abovementioned features and the features that are still to be explained below can be used not only in the respectively specified combinations, but also in other combinations or on their own, without departing from the scope of the disclosure.

The disclosure relates to following specific embodiments:

• • 1. A method to produce an alpha-1,3 glycosylated form of fucose-alpha-1,2-galactose-R (Fuc-a1,2-Gal-R) by a cell, preferably a single cell, wherein the alpha-1,3 glycosylation occurs at the terminal “fucose-a1,2-galactose”-group of fucose-alpha-1,2-galactose R (Fuc-a1,2-Gal-R), wherein the method comprises the steps of:
  • i. providing a cell capable to synthesize Fuc-a1,2-Gal-R, expressing an alpha-1,3-glycosyltransferase and capable to synthesize a nucleotide-sugar, which is donor for the alpha-1,3-glycosyltransferase, and
  • ii. cultivating the cell under conditions permissive to synthesize the Fuc-a1,2-Gal-R, to express the alpha-1,3-glycosyltransferase, to synthesize the nucleotide-sugar and to synthesize the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R,
  • iii. preferably, separating the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R from the cultivation.
  • 2. Method according to embodiment 1, wherein the galactose (Gal) residue within the Fuc-a1,2-Gal-R is bound to R via a beta-1,3 or a beta-1,4 glycosidic linkage.
  • 3. Method according to any one of embodiment 1 or 2, wherein the R comprises a monosaccharide, a disaccharide, an oligosaccharide, a peptide, a protein, a glycopeptide, a glycoprotein, a lipid or a glycolipid.
  • 4. Method according to any one of embodiments 1 to 3, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-R, preferably wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-R.
  • 5. Method according to embodiment 4, wherein the N-acetylglucosamine (GlcNAc) residue within the Fuc-a1,2-Gal-b1,3-GlcNAc-R is bound to R via a beta-1,3 or a beta-1,4 glycosidic linkage.
  • 6. Method according to any one of embodiment 4 or 5, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R, preferably wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R, more preferably wherein the Fuc-a1,2-Gal-R is lacto-N-fucopentaose I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc).
  • 7. Method according to any one of embodiments 1 to 3, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,4-R, preferably wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,4-Glc, optionally wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc.
  • 8. Method according to any one of embodiments 1 to 7, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is a structure of the histo blood group antigen (HBGA) system.
  • 9. Method according to any one of embodiments 1 to 8, wherein the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase, which is a glycosyltransferase with the ability to transfer a galactose (Gal) residue from UDP-galactose (UDP-Gal) to the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R).
  • 10. Method according to any one of embodiments 1 to 6, 8 or 9, wherein the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase, which is a glycosyltransferase with the ability to transfer a galactose (Gal) residue from UDP-Gal to the terminal “fucose-a1,2-galactose”-group of LNFP-I.
  • 11. Method according to any one of embodiments 1 to 3, 7, 9 or 10, wherein the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase, which is a glycosyltransferase with the ability to transfer a galactose (Gal) residue from UDP-Gal to the terminal “fucose-a1,2-galactose”-group of Fuc-a1,2-Gal-b1,4-Glc, optionally the glucose residue in the Fuc-a1,2-Gal-b1,4-Glc is fucosylated, preferably alpha-1,3-fucosylated.
  • 12. Method according to any one of embodiments 1 to 6 or 8 to 10, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3 glycosylated form of lacto-N-fucopentaose I (LNFP-I) which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (Gal-a1,3-LNFP-I), the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase and the nucleotide-sugar is UDP-galactose (UDP-Gal).
  • 13. Method according to any one of embodiments 1 to 3, 7, or 9 to 11, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-Glc, which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, optionally an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc, which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc, the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase and the nucleotide-sugar is UDP-galactose (UDP-Gal).
  • 14. Method according to any one of embodiments 9 to 13, wherein the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain, and
  • a. comprises the motif YX[FHMQT]XAXX[ACG][ACG] with SEQ ID NO:01 wherein X can be any amino acid residue, or
  • b. comprises the motif YXQXCXX[ACG][ACG] with SEQ ID NO:02 wherein X can be any amino acid residue, or
  • c. comprises a polypeptide sequence according to any one of SEQ ID NOs: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, or
  • d. is a functional homolog, variant or derivative of any one of SEQ ID NOs 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 having at least 80% overall sequence identity to the full length of any one of the a-1,3-galactosyltransferase polypeptide with SEQ ID NOs 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 having a-1,3-galactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R), or
  • e. is a functional fragment comprising an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs 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 having a-1,3-galactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R).
  • 15. Method according to any one of embodiments 1 to 8, wherein the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase, which is a glycosyltransferase with the ability to transfer an N-acetylgalactosamine (GalNAc) residue from UDP-N-acetylgalactosamine (UDP-GalNAc) to the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R).
  • 16. Method according to any one of embodiments 1 to 6, 8 or 15, wherein the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase, which is a glycosyltransferase with the ability to transfer an N-acetylgalactosamine (GalNAc) residue from UDP-N-acetylgalactosamine (UDP-GalNAc) to the terminal “fucose-a1,2-galactose”-group of LNFP-I.
  • 17. Method according to any one of embodiments 1 to 3, 7 or 15, wherein the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase, which is a glycosyltransferase with the ability to transfer an N-acetylgalactosamine (GalNAc) residue from UDP-GalNAc to the terminal “fucose-a1,2-galactose”-group of Fuc-a1,2-Gal-b1,4-Glc, optionally the glucose residue in the Fuc-a1,2-Gal-b1,4-Glc is fucosylated, preferably alpha-1,3-fucosylated.
  • 18. Method according to any one of embodiments 1 to 6, 8, 15 or 16, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3 glycosylated form of lacto-N-fucopentaose I (LNFP-I) which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (GalNAc-a1,3-LNFP-I), the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase and the nucleotide-sugar is UDP-N-acetylgalactosamine (UDP-GalNAc).
  • 19. Method according to any one of embodiments 1 to 3, 7, 15 or 17, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-Glc, which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc (alpha-tetrasaccharide or A-tetrasaccharide), optionally an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc, which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc, the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase and the nucleotide-sugar is UDP-N-acetylgalactosamine (UDP-GalNAc).
  • 20. Method according to any one of embodiments 15 to 19, wherein the alpha-1,3-N-acetylgalactosaminyltransferase has a PFAM PF03414 domain, and
  • a. comprises the motif YX[ACIL]XGXX[ACG][ACG] with SEQ ID NO:38 wherein X can be any amino acid residue, or
  • b. comprises the motif YX[AG]XAXX[ACG][ACG] with SEQ ID NO:39 wherein X can be any amino acid residue, or
  • c. comprises a polypeptide sequence according to any one of SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102, or
  • d. is a functional homolog, variant or derivative of any one of SEQ ID NOs 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 having at least 80% overall sequence identity to the full length of any one of the a-1,3-N-acetylgalactosyltransferase polypeptide with SEQ ID NOs 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and having a-1,3-N-acetylgalactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R), or
  • e. is a functional fragment comprising an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and having a-1,3-N-acetylgalactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R).
  • 21. Method according to any one of embodiments 6, 10, 12, 14, 16, 18 or 20, wherein the LNFP-I is synthesized in the cell by the transfer of fucose from GDP-fucose to the terminal galactose residue of lacto-N-tetraose (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) by action of a glycosyltransferase, which is:
  • a. an alpha-1,2-fucosyltransferase chosen from the list comprising the polypeptides from Brachyspira pilosicoli with UniProt ID AOA2N5RQ26, Dysgonomonas mossii with UniProt ID F8X274, Dechlorosoma suillum with UniProt ID G8QLF4, Desulfovibrio alaskensis with UniProt ID Q316B5 and Polaribacter vadi with UniProt ID AOA1B8TNT0, or
  • b. a functional fragment of any one of the polypeptide from B. pilosicoli (UniProt ID AOA2N5RQ26), the polypeptide from D. mossii (UniProt ID F8X274), the polypeptide from D. suillum (UniProt ID G8QLF4), the polypeptide from D. alaskensis (UniProt ID Q316B5) and the polypeptide from P. vadi (UniProt ID AOA1B8TNT0) having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of LNT, or
  • c. a functional homolog, variant or derivative of any one of the polypeptides from B. pilosicoli with UniProt ID AOA2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, D. alaskensis with UniProt ID Q316B5 and P. vadi with UniProt ID AOA1B8TNT0 having at least 80% overall sequence identity to the full length of any one of the polypeptides from B. pilosicoli with UniProt ID AOA2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, D. alaskensis with UniProt ID Q316B5 and P. vadi with UniProt ID AOA1B8TNT0, respectively, and having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of lacto-N-tetraose (LNT), or
  • d. a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any one of the polypeptide from B. pilosicoli (UniProt ID AOA2N5RQ26), the polypeptide from D. mossii (UniProt ID F8X274), the polypeptide from D. suillum (UniProt ID G8QLF4), the polypeptide from D. alaskensis (UniProt ID Q316B5) or the polypeptide from P. vadi (UniProt ID AOA1B8TNT0) and having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of LNT.
  • 22. Method according to any one of embodiments 1 to 21, wherein the cell is modified in the expression or activity of a glycosyltransferase.
  • 23. Method according to any one of previous embodiments, wherein the cell expresses a membrane transporter protein or a polypeptide having transport activity hereby transporting compounds across the outer membrane of the cell wall.
  • 24. Method according to embodiment 23, wherein the membrane transporter protein or polypeptide having transport activity is chosen from the list comprising porters, P-P-bond-hydrolysis-driven transporters, b-barrel porins, auxiliary transport proteins, putative transport proteins and phosphotransfer-driven group translocators,
  • preferably, the porters comprise MFS transporters, sugar efflux transporters and siderophore exporters,
  • preferably, the P-P-bond-hydrolysis-driven transporters comprise ABC transporters and siderophore exporters.
  • 25. Method according to any one of embodiment 23 or 24, wherein the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R and/or of one or more precursor(s) and/or acceptor(s) to be used in the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.
  • 26. Method according to any one of embodiment 23 to 25, wherein the membrane transporter protein or polypeptide having transport activity provides improved production and/or enabled and/or enhanced efflux of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.
  • 27. Method according to any one of previous embodiments, wherein the cell is a metabolically engineered cell.
  • 28. Method according to embodiment 27, wherein the cell is modified with gene expression modules, characterized in that the expression from any of the expression modules is either constitutive or is created by a natural inducer.
  • 29. Method according to any one of embodiment 27 or 28, wherein the cell comprises multiple copies of the same coding DNA sequence encoding for one protein.
  • 30. Method according to any one of embodiment 27 to 29, wherein the cell comprises a modification for reduced production of acetate.
  • 31. Method according to any one of embodiments 27 to 29, wherein the cell comprises a lower or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the proteins comprising beta-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose undecaprenyl-phosphate glucose-I-phosphate transferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridylyltransferase, glucose-I-phosphate adenylyltransferase, glucose-I-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IclR, lon protease, glucose-specific translocating phosphotransferase enzyme IIBC component ptsG, glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIA^Glc, beta-glucoside specific PTS enzyme II, fructose-specific PTS multiphosphoryl transfer protein FruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, pyruvate decarboxylase.
  • 32. Method according to any one of previous embodiments, wherein the cell is capable to produce phosphoenolpyruvate (PEP).
  • 33. Method according to any one of previous embodiments, wherein the cell is modified for enhanced production and/or supply of phosphoenolpyruvate (PEP).
  • 34. Method according to any one of previous embodiments, wherein the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides, which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.
  • 35. Method according to any one of the previous embodiments, wherein the cell resists the phenomenon of lactose killing when grown in an environment in which lactose is combined with one or more other carbon source(s).
  • 36. Method according to any one of the previous embodiments, wherein the cell produces 90 g/L or more of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R in the whole broth and/or supernatant and/or wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R in the whole broth and/or supernatant has a purity of at least 80% measured on the total amount of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R and its precursor(s) in the whole broth and/or supernatant, respectively.
  • 37. Method according to any one of previous embodiments, wherein the cell is stably cultured in a medium.
  • 38. Method according to any one of previous embodiments, wherein the conditions comprise:
  • (i) use of a culture medium comprising at least one precursor and/or acceptor for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, and/or
  • (ii) adding to the culture medium at least one precursor and/or acceptor feed for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.
  • 39. Method according to any one of previous embodiments, the method comprising at least one of the following steps:
  • i) Use of a culture medium comprising at least one precursor and/or acceptor;
  • ii) Adding to the culture medium in a reactor at least one precursor and/or acceptor feed wherein the total reactor volume ranges from 250 mL (milliliter) to 10,000 m³ (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the culture medium before the addition of the precursor and/or acceptor feed;
  • iii) Adding to the culture medium in a reactor at least one precursor and/or acceptor feed wherein the total reactor volume ranges from 250 mL (milliliter) to 10,000 m³ (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the culture medium before the addition of the precursor and/or acceptor feed and wherein preferably, the pH of the precursor and/or acceptor feed is set between 3 and 7 and wherein preferably, the temperature of the precursor and/or acceptor feed is kept between 20° C. and 80° C.;
  • iv) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution;
  • v) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein preferably, the pH of the feeding solution is set between 3 and 7 and wherein preferably, the temperature of the feeding solution is kept between 20° C. and 80° C.;
  • the method resulting in an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R with a concentration of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L in the final cultivation.
  • 40. Method according to any one of embodiments 1 to 38, the method comprising at least one of the following steps:
  • i) Use of a culture 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 gram of lactose per liter of initial reactor volume wherein the reactor volume ranges from 250 mL to 10,000 m³ (cubic meter);
  • ii) Adding to the culture medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per liter of initial reactor volume wherein the reactor volume ranges from 250 mL to 10,000 m³ (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture medium before the addition of the lactose feed;
  • iii) Adding to the culture medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per liter of initial reactor volume wherein the reactor volume ranges from 250 mL to 10,000 m³ (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture medium before the addition of the lactose feed and wherein preferably the pH of the lactose feed is set between 3 and 7 and wherein preferably the temperature of the lactose feed is kept between 20° C. and 80° C.;
  • iv) Adding a lactose feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution;
  • v) Adding a lactose feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein the concentration of the lactose feeding solution is 50 g/L, preferably 75 g/L, more preferably 100 g/L, more preferably 125 g/L, more preferably 150 g/L, more preferably 175 g/L, more preferably 200 g/L, more preferably 225 g/L, more preferably 250 g/L, more preferably 275 g/L, more preferably 300 g/L, more preferably 325 g/L, more preferably 350 g/L, more preferably 375 g/L, more preferably, 400 g/L, more preferably 450 g/L, more preferably 500 g/L, even more preferably, 550 g/L, most preferably 600 g/L; and wherein preferably the pH of the feeding solution is set between 3 and 7 and wherein preferably the temperature of the feeding solution is kept between 20° C. and 80° C.;
  • the method resulting in an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R with a concentration of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L in the final volume of the cultivation.
  • 41. Method according to embodiment 39, wherein the lactose feed is accomplished by adding lactose from the beginning of the cultivation in a concentration of at least 5 mM, preferably in a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably in a concentration >300 mM.
  • 42. Method according to any one of embodiment 39 or 40, wherein the lactose feed is accomplished by adding lactose to the cultivation in a concentration, such, that throughout the production phase of the cultivation a lactose concentration of at least 5 mM, preferably 10 mM or 30 mM is obtained.
  • 43. Method according to any one of previous embodiments, wherein the cells are cultivated for at least about 60, 80, 100, or about 120 hours or in a continuous manner.
  • 44. Method according to any one of previous embodiments, wherein the cell is cultivated in culture medium comprising a carbon source comprising a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract; preferably, wherein the carbon source is chosen from the list comprising glucose, glycerol, fructose, sucrose, maltose, lactose, arabinose, malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvate.
  • 45. Method according to any one of previous embodiments, wherein the culture medium contains at least one precursor selected from the group comprising lactose, galactose, fucose, sialic acid, GlcNAc, GalNAc, lacto-N-biose (LNB), N-acetyllactosamine (LacNAc).
  • 46. Method according to any one of previous embodiments, wherein a first phase of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the culture medium comprising a precursor, preferably lactose, followed by a second phase wherein only a carbon-based substrate, preferably glucose or sucrose, is added to the culture medium.
  • 47. Method according to any one of embodiments 1 to 45, wherein a first phase of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the culture medium comprising a precursor, preferably lactose, followed by a second phase wherein a carbon-based substrate, preferably glucose or sucrose, and a precursor, preferably lactose, are added to the culture medium.
  • 48. Method according to any one of previous embodiments, wherein the cell produces a mixture of charged, preferentially sialylated, and/or neutral, di- and oligosaccharides comprising an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.
  • 49. Method according to any one of previous embodiments, wherein the cell produces a mixture of charged, preferentially sialylated, and/or neutral oligosaccharides comprising an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.
  • 50. A cell metabolically engineered for the production of an alpha-1,3 glycosylated form of fucose-alpha-1,2-galactose-R (Fuc-a1,2-Gal-R), wherein the alpha-1,3 glycosylation occurs at the terminal “fucose-a1,2-galactose”-group of fucose-alpha-1,2-galactose R (Fuc-a1,2-Gal-R), and wherein the cell
    • synthesizes Fuc-a1,2-Gal-R, and
    • expresses an alpha-1,3-glycosyltransferase, and
    • is capable to produce a nucleotide-sugar, wherein the nucleotide-sugar is donor for the alpha-1,3-glycosyltransferase.
  • 51. Cell according to embodiment 50, wherein the galactose (Gal) residue within the Fuc-a1,2-Gal-R is bound to R via a beta-1,3 or a beta-1,4 glycosidic linkage.
  • 52. Cell according to any one of embodiment 50 or 51, wherein the R comprises a monosaccharide, a disaccharide, an oligosaccharide, a peptide, a protein, a glycopeptide, a glycoprotein, a lipid or a glycolipid.
  • 53. Cell according to any one of embodiments 50 to 52, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-R, preferably wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-R.
  • 54. Cell according to embodiment 53, wherein the N-acetylglucosamine (GlcNAc) residue within the Fuc-a1,2-Gal-b1,3-GlcNAc-R is bound to R via a beta-1,3 or a beta-1,4 glycosidic linkage.
  • 55. Cell according to any one of embodiment 53 or 54, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R, preferably wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R, more preferably wherein the Fuc-a1,2-Gal-R is lacto-N-fucopentaose I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc).
  • 56. Cell according to any one of embodiments 50 to 52, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,4-R, preferably wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,4-Glc, optionally wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc.
  • 57. Cell according to any one of embodiments 50 to 56, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is a structure of the histo blood group antigen (HBGA) system.
  • 58. Cell according to any one of embodiments 50 to 57, wherein the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase, which is a glycosyltransferase with the ability to transfer a galactose (Gal) residue from UDP-galactose (UDP-Gal) to the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R).
  • 59. Cell according to any one of embodiments 50 to 55, 57 or 58, wherein the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase, which is a glycosyltransferase with the ability to transfer a galactose (Gal) residue from UDP-Gal to the terminal “fucose-a1,2-galactose”-group of LNFP-I.
  • 60. Cell according to any one of embodiments 50 to 52, 56, 58 or 59, wherein the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase, which is a glycosyltransferase with the ability to transfer a galactose (Gal) residue from UDP-Gal to the terminal “fucose-a1,2-galactose”-group of Fuc-a1,2-Gal-b1,4-Glc, optionally the glucose residue in the Fuc-a1,2-Gal-b1,4-Glc is fucosylated, preferably alpha-1,3-fucosylated.
  • 61. Cell according to any one of embodiments 50 to 55 or 57 to 59, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3 glycosylated form of lacto-N-fucopentaose I (LNFP-I) which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (Gal-a1,3-LNFP-I), the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase and the nucleotide-sugar is UDP-galactose (UDP-Gal).
  • 62. Cell according to any one of embodiments 50 to 52, 56, or 58 to 60, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-Glc, which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, optionally an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc, which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc, the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase and the nucleotide-sugar is UDP-galactose (UDP-Gal).
  • 63. Cell according to any one of embodiments 58 to 62, wherein the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain, and
  • a. comprises the motif YX[FHMQT]XAXX[ACG][ACG] with SEQ ID NO:01 wherein X can be any amino acid residue, or
  • b. comprises the motif YXQXCXX[ACG][ACG] with SEQ ID NO:02 wherein X can be any amino acid residue, or
  • c. comprises a polypeptide sequence according to any one of SEQ ID NOs: 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, or
  • d. is a functional homolog, variant or derivative of any one of SEQ ID NOs 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 having at least 80% overall sequence identity to the full length of any one of the a-1,3-galactosyltransferase polypeptide with SEQ ID NOs 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 having a-1,3-galactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R), or
  • e. is a functional fragment comprising an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs 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 having a-1,3-galactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R).
  • 64. Cell according to any one of embodiments 50 to 57, wherein the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase, which is a glycosyltransferase with the ability to transfer an N-acetylgalactosamine (GalNAc) residue from UDP-N-acetylgalactosamine (UDP-GalNAc) to the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R).
  • 65. Cell according to any one of embodiments 50 to 55, 57 or 64, wherein the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase, which is a glycosyltransferase with the ability to transfer an N-acetylgalactosamine (GalNAc) residue from UDP-N-acetylgalactosamine (UDP-GalNAc) to the terminal “fucose-a1,2-galactose”-group of LNFP-I.
  • 66. Cell according to any one of embodiments 50 to 52, 56 or 64, wherein the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase, which is a glycosyltransferase with the ability to transfer an N-acetylgalactosamine (GalNAc) residue from UDP-GalNAc to the terminal “fucose-a1,2-galactose”-group of Fuc-a1,2-Gal-b1,4-Glc, optionally the glucose residue in the Fuc-a1,2-Gal-b1,4-Glc is fucosylated, preferably alpha-1,3-fucosylated.
  • 67. Cell according to any one of embodiments 50 to 55, 57, 64 or 65, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3 glycosylated form of lacto-N-fucopentaose I (LNFP-I) which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (GalNAc-a1,3-LNFP-I), the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase and the nucleotide-sugar is UDP-N-acetylgalactosamine (UDP-GalNAc).
  • 68. Cell according to any one of embodiments 50 to 52, 56, 64 or 66, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-Glc, which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc (alpha-tetrasaccharide or A-tetrasaccharide), optionally an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc, which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc, the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase and the nucleotide-sugar is UDP-N-acetylgalactosamine (UDP-GalNAc).
  • 69. Cell according to any one of embodiments 64 to 68, wherein the alpha-1,3-N-acetylgalactosaminyltransferase has a PFAM PF03414 domain, and
  • a. comprises the motif YX[ACIL]XGXX[ACG][ACG] with SEQ ID NO:38 wherein X can be any amino acid residue, or
  • b. comprises the motif YX[AG]XAXX[ACG][ACG] with SEQ ID NO:39 wherein X can be any amino acid residue, or
  • c. comprises a polypeptide sequence according to any one of SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102, or
  • d. is a functional homolog, variant or derivative of any one of SEQ ID NOs 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 having at least 80% overall sequence identity to the full-length of any one of the a-1,3-N-acetylgalactosyltransferase polypeptide with SEQ ID NOs 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and having a-1,3-N-acetylgalactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R), or
  • e. is a functional fragment comprising an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and having a-1,3-N-acetylgalactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R).
  • 70. Cell according to any one of embodiments 55, 59, 61, 63, 65, 67 or 69, wherein the LNFP-I is synthesized in the cell by the transfer of fucose from GDP-fucose to the terminal galactose residue of lacto-N-tetraose (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) by action of a glycosyltransferase, which is:
  • a. an alpha-1,2-fucosyltransferase chosen from the list comprising the polypeptides from Brachyspira pilosicoli with UniProt ID AOA2N5RQ26, Dysgonomonas mossii with UniProt ID F8X274, Dechlorosoma suillum with UniProt ID G8QLF4, Desulfovibrio alaskensis with UniProt ID Q316B5 and Polaribacter vadi with UniProt ID AOA1B8TNT0, or
  • b. a functional fragment of any one of the polypeptide from B. pilosicoli (UniProt ID AOA2N5RQ26), the polypeptide from D. mossii (UniProt ID F8X274), the polypeptide from D. suillum (UniProt ID G8QLF4), the polypeptide from D. alaskensis (UniProt ID Q316B5) and the polypeptide from P. vadi (UniProt ID AOA1B8TNT0) having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of LNT, or
  • c. a functional homolog, variant or derivative of any one of the polypeptides from B. pilosicoli with UniProt ID AOA2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, D. alaskensis with UniProt ID Q316B5 and P. vadi with UniProt ID AOA1B8TNT0 having at least 80% overall sequence identity to the full length of any one of the polypeptides from B. pilosicoli with UniProt ID A0A2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, D. alaskensis with UniProt ID Q316B5 and P. vadi with UniProt ID A0A1B8TNT0, respectively, and having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of lacto-N-tetraose (LNT), or
  • d. a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any one of the polypeptide from B. pilosicoli (UniProt ID A0A2N5RQ26), the polypeptide from D. mossii (UniProt ID F8X274), the polypeptide from D. suillum (UniProt ID G8QLF4), the polypeptide from D. alaskensis (UniProt ID Q316B5) or the polypeptide from P. vadi (UniProt ID A0A1B8TNT0) and having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of LNT.
  • 71. Cell according to any one of embodiments 50 to 70, wherein the cell is modified in the expression or activity of a glycosyltransferase.
  • 72. Cell according to any one of embodiments 50 to 71, wherein the cell expresses a membrane transporter protein or a polypeptide having transport activity hereby transporting compounds across the outer membrane of the cell wall.
  • 73. Cell according to embodiment 72, wherein the membrane transporter protein or polypeptide having transport activity is chosen from the list comprising porters, P-P-bond-hydrolysis-driven transporters, b-barrel porins, auxiliary transport proteins, putative transport proteins and phosphotransfer-driven group translocators,
  • preferably, the porters comprise MFS transporters, sugar efflux transporters and siderophore exporters,
  • preferably, the P-P-bond-hydrolysis-driven transporters comprise ABC transporters and siderophore exporters.
  • 74. Cell according to any one of embodiment 72 or 73, wherein the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R and/or of one or more precursor(s) and/or acceptor(s) to be used in the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.
  • 75. Cell according to any one of embodiment 72 to 74, wherein the membrane transporter protein or polypeptide having transport activity provides improved production and/or enabled and/or enhanced efflux of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.
  • 76. Cell according to any one of embodiments 50 to 75, wherein the cell is modified with gene expression modules, characterized in that the expression from any of the expression modules is either constitutive or is created by a natural inducer.
  • 77. Cell according to any one of embodiment 50 to 76, wherein the cell comprises multiple copies of the same coding DNA sequence encoding for one protein.
  • 78. Cell according to any one of embodiments 50 to 77, wherein the cell comprises a modification for reduced production of acetate.
  • 79. Cell according to any one of embodiments 50 to 78, wherein the cell comprises a lower or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the proteins comprising beta-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose undecaprenyl-phosphate glucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridylyltransferase, glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IclR, lon protease, glucose-specific translocating phosphotransferase enzyme IIBC component ptsG, glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIA^Glc, beta-glucoside specific PTS enzyme II, fructose-specific PTS multiphosphoryl transfer protein FruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, pyruvate decarboxylase.
  • 80. Cell according to any one of embodiments 50 to 79, wherein the cell is capable to produce phosphoenolpyruvate (PEP).
  • 81. Cell according to any one of embodiments 50 to 80, wherein the cell is modified for enhanced production and/or supply of phosphoenolpyruvate (PEP).
  • 82. Cell according to any one of embodiments 50 to 81, wherein the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides, which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.
  • 83. Cell according to any one of embodiments 50 to 82, wherein the cell resists the phenomenon of lactose killing when grown in an environment in which lactose is combined with one or more other carbon source(s).
  • 84. Cell according to any one of embodiments 50 to 83, wherein the cell produces 90 g/L or more of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R in the whole broth and/or supernatant and/or wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R in the whole broth and/or supernatant has a purity of at least 80% measured on the total amount of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R and its precursor(s) in the whole broth and/or supernatant, respectively.
  • 85. Cell according to any one of embodiments 50 to 84, wherein the cell produces a mixture of charged, preferentially sialylated, and/or neutral, di- and oligosaccharides comprising an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.
  • 86. Cell according to any one of embodiments 50 to 85, wherein the cell produces a mixture of charged, preferentially sialylated, and/or neutral oligosaccharides comprising an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R
  • 87. Method according to any one of embodiments 1 to 49 or cell according to any one of embodiments 50 to 86, wherein the cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell,
    • preferably the bacterium is an Escherichia coli strain, more preferably an Escherichia coli strain, which is a K-12 strain, even more preferably the Escherichia coli K-12 strain is E. coli MG1655,
    • preferably the fungus belongs to a genus chosen from the group comprising Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus,
    • preferably the yeast belongs to a genus chosen from the group comprising Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces or Debaromyces,
    • preferably the plant cell is an algal cell or is derived from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant,
    • preferably the animal cell is derived from non-human mammals, birds, fish, invertebrates, reptiles, amphibians or insects or is a genetically modified cell line derived from human cells excluding embryonic stem cells, more preferably the human and non-human mammalian cell is an epithelial cell, an embryonic kidney cell, a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell, an NIH-3T3 cell, a non-mammary adult stem cell or derivatives thereof, more preferably the insect cell is derived from Spodoptera frugiperda, Bombyx mori, Mamestra brassicae, Trichoplusia ni or Drosophila melanogaster,
    • preferably the protozoan cell is a Leishmania tarentolae cell.
  • 88. Method according to any one of embodiments 1 to 49 and 87, or cell according to any one of embodiments 50 to 87, wherein the cell is a cell of a bacterium, preferably of an Escherichia coli strain, more preferably of an Escherichia coli strain, which is a K-12 strain, even more preferably the Escherichia coli K-12 strain is E. coli MG1655.
  • 89. Method according to embodiment 88, or cell according to embodiment 88, wherein the cell is a viable Gram-negative bacterium that comprises a reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose.
  • 90. Method according to any one of embodiments 1 to 49 and 87, or cell according to any one of embodiments 50 to 87, wherein the cell is a yeast cell.
  • 91. Method according to any one of embodiments 1 to 49 and 87 to 90, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography.
  • 92. Method according to any one of embodiments 1 to 49 and 87 to 91 further comprising purification of any one of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R from the cell, preferably alpha-1,3 glycosylated forms of LNFP-I from the cell.
  • 93. Method according to any one of embodiments 1 to 49 and 87 to 92 wherein the purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying or vacuum roller drying.
  • 94. Use of a cell according to any one of embodiments 50 to 90, or method according to any one of embodiment 1 to 49, or 87 to 93 for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, preferably an alpha-1,3 glycosylated form of LNFP-I.

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

EXAMPLES

Example 1. Materials and Methods Escherichia coli

Media

The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). The minimal medium used in the cultivation experiments in 96-well plates or in shake flasks contained 2.00 g/L NH₄Cl, 5.00 g/L (NH₄)₂SO₄, 2.993 g/L KH₂PO₄, 7.315 g/L K₂HPO₄, 8.372 g/L MOPS, 0.5 g/L NaCl, 0.5 g/L MgSO₄·7H₂O, 30 g/L sucrose or 30 g/L glycerol, 1 ml/L vitamin solution, 100 μl/L molybdate solution, and 1 mL/L selenium solution. As specified in the respective examples, 0.30 g/L sialic acid, 20 g/L lactose, 20 g/L LacNAc and/or 20 g/L LNB were additionally added to the medium as precursor(s). The minimal medium was set to a pH of 7 with 1M KOH. Vitamin solution consisted of 3.6 g/L FeCl₂·4H₂O, 5 g/L CaCl₂)·2H₂O, 1.3 g/L MnCl₂·2H₂O, 0.38 g/L CuCl₂·2H₂O, 0.5 g/L COCl₂·6H₂O, 0.94 g/L ZnCl₂, 0.0311 g/L H₃BO₄, 0.4 g/L Na₂EDTA·2H₂O and 1.01 g/L thiamine·HCl. The molybdate solution contained 0.967 g/L NaMoO₄·2H₂O. The selenium solution contained 42 g/L SeO₂.

The minimal medium for fermentations contained 6.75 g/L NH₄Cl, 1.25 g/L (NH₄)₂SO₄, 2.93 g/L KH₂PO₄ and 7.31 g/L KH₂PO₄, 0.5 g/L NaCl, 0.5 g/L MgSO₄·7H₂O, 30 g/L sucrose or 30 g/L glycerol, 1 mL/L vitamin solution, 100 μL/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above. As specified in the respective examples, 0.30 g/L sialic acid, 20 g/L lactose, 20 g/L LacNAc and/or 20 g/L LNB were additionally added to the medium as precursor(s).

Complex medium was sterilized by autoclaving (121° C., 21 min) and minimal medium by filtration (0.22 μm Sartorius). When necessary, the medium was made selective by adding an antibiotic: e.g., chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L).

Plasmids

pKD46 (Red helper plasmid, Ampicillin resistance), pKD3 (contains an FRT-flanked chloramphenicol resistance (cat) gene), pKD4 (contains an FRT-flanked kanamycin resistance (kan) gene), and pCP20 (expresses FLP recombinase activity) plasmids were obtained from Prof. R. Cunin (Vrije Universiteit Brussel, Belgium in 2007). Plasmids were maintained in the host E. coli DH5alpha (F⁻, phi80dlacZΔM15, Δ(lacZYA-argF) U169, deoR, recA1, endA1, hsdR17(rk⁻, mk⁺), phoA, supE44, lambda⁻, thi-1, gyrA96, relA1) bought from Invitrogen.

Strains and Mutations

Escherichia coli K12 MG1655 [λ⁻, F⁻, rph-1] was obtained from the Coli Genetic Stock Center (US), CGSC Strain #: 7740, in March 2007. Gene disruptions, gene introductions and gene replacements were performed using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). This technique is based on antibiotic selection after homologous recombination performed by lambda Red recombinase. Subsequent catalysis of a flippase recombinase ensures removal of the antibiotic selection cassette in the final production strain. Transformants carrying a Red helper plasmid pKD46 were grown in 10 mL LB media with ampicillin, (100 mg/L) and L-arabinose (10 mM) at 30° C. to an OD₆₀₀ nm of 0.6. The cells were made electrocompetent by washing them with 50 mL of ice-cold water, a first time, and with 1 mL ice cold water, a second time. Then, the cells were resuspended in 50 μL of ice-cold water. Electroporation was done with 50 μL of cells and 10-100 ng of linear double-stranded-DNA product by using a Gene Pulser™ (BioRad) (600 Ω, 25 μFD, and 250 volts). After electroporation, cells were added to 1 mL LB media incubated 1 h at 37° C., and finally spread onto LB-agar containing 25 mg/L of chloramphenicol or 50 mg/L of kanamycin to select antibiotic resistant transformants. The selected mutants were verified by PCR with primers upstream and downstream of the modified region and were grown in LB-agar at 42° C. for the loss of the helper plasmid. The mutants were tested for ampicillin sensitivity. The linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and their derivates as template. The primers used had a part of the sequence complementary to the template and another part complementary to the side on the chromosomal DNA where the recombination must take place. For the genomic knock-out, the region of homology was designed 50-nt upstream and 50-nt downstream of the start and stop codon of the gene of interest. For the genomic knock-in, the transcriptional starting point (+1) had to be respected. PCR products were PCR-purified, digested with DpnI, re-purified from an agarose gel, and suspended in elution buffer (5 mM Tris, pH 8.0). Selected mutants were transformed with pCP20 plasmid, which is an ampicillin and chloramphenicol resistant plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis. The ampicillin-resistant transformants were selected at 30° C., after which a few were colony purified in LB at 42° C. and then tested for loss of all antibiotic resistance and of the FLP helper plasmid. The gene knock-outs and knock-ins are checked with control primers.

In one example for GDP-fucose production, the mutant strain was derived from E. coli K12 MG1655 comprising knock-outs of the E. coli wcaJ and thyA genes and genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g., CscB from E. coli W (UniProt ID E0IXR1), a fructose kinase like e.g., Frk originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g., BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6). GDP-fucose production can further be optimized in the mutant E. coli strain by genomic knock-outs of any one or more of the E. coli genes comprising glgC, agp, pfkA, pfkB, pgi, arcA, icR, pgi and Ion as described in WO 2016075243 and WO 2012007481. GDP-fucose production can additionally be optimized comprising genomic knock-ins of constitutive transcriptional units for a mannose-6-phosphate isomerase like e.g., manA from E. coli (UniProt ID P00946), a phosphomannomutase like e.g., manB from E. coli (UniProt ID P24175), a mannose-1-phosphate guanylyltransferase like e.g., manC from E. coli (UniProt ID P24174), a GDP-mannose 4,6-dehydratase like e.g., gmd from E. coli (UniProt ID P0AC88) and a GDP-L-fucose synthase like e.g., fcl from E. coli (UniProt ID P32055). GDP-fucose production can also be obtained by genomic knock-outs of the E. coli fucK and fucI genes and genomic knock-ins of constitutive transcriptional units containing a fucose permease like e.g., fucP from E. coli (UniProt ID P11551) and a bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase like e.g., fkp from Bacteroides fragilis (UniProt ID SUV40286.1). All mutant strains can be additionally modified with genomic knock-outs of the E. coli LacZ, LacY and LacA genes and with a genomic knock-in of a constitutive transcriptional unit for a lactose permease like e.g., the E. coli LacY (UniProt ID P02920). In a next step to produce fucosylated structures as used in the disclosure, the mutant GDP-fucose production strain was additionally modified with an expression plasmid comprising a constitutive transcriptional unit for an alpha-1,2-fucosyltransferase like e.g., HpFutC from H. pylori (GenBank: AAD29863.1), the polypeptide from Brachyspira pilosicoli with UniProt ID A0A2N5RQ26, the polypeptide from Dysgonomonas mossii with UniProt ID F8X274, the polypeptide from Dechlorosoma suillum with UniProt ID G8QLF4, the polypeptide from Desulfovibrio alaskensis with UniProt ID Q316B5 or the polypeptide from Polaribacter vadi with UniProt ID A0A1B8TNT0 and with a constitutive transcriptional unit for a selection marker like e.g., the E. coli thyA (UniProt ID P0A884). The constitutive transcriptional unit of the alpha-1,2-fucosyltransferase could also be presented to the mutant E. coli strain via genomic knock-ins.

Alternatively, and/or additionally, production of GDP-fucose and/or fucosylated structures can further be optimized in the mutant E. coli strains with genomic knock-ins of a constitutive transcriptional unit comprising a membrane transporter protein like e.g., MdfA from Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID P0AEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207) or iceT from Citrobacter youngae (UniProt ID D4B8A6).

In an example to produce lacto-N-triose (LNT-II, LN3, GlcNAc-b1,3-Gal-b1,4-Glc), the mutant strain was derived from E. coli K12 MG1655 and modified with knock-outs of the E. coli LacZ, LacY, LacA and nagB genes and with genomic knock-ins of constitutive transcriptional units for a lactose permease like e.g., LacY from E. coli (UniProt ID P02920) and a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g., LgtA from N. meningitidis (GenBank. AAM33849.1). For the production of lacto-N-tetraose (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,3-galactosyltransferase like e.g., WbgO from E. coli 055:H7 (UniProt ID D3QY14) that can be delivered to the strain either via genomic knock-in or from an expression plasmid. For the production of lacto-N-neotetraose (LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,4-galactosyltransferase like e.g., LgtB from Neisseria meningitidis (UniProt ID Q51116). Optionally, multiple copies of the lactose permease, the galactoside beta-1,3-N-acetylglucosaminyltransferase, the N-acetylglucosamine beta-1,3-galactosyltransferase and/or the N-acetylglucosamine beta-1,4-galactosyltransferase genes could be added to the mutant E. coli strains. The mutant strains can also be optionally modified for enhanced UDP-GlcNAc production with a genomic knock-in of a constitutive transcriptional unit for an L-glutamine-D-fructose-6-phosphate aminotransferase like e.g., the mutant glmS*54 from E. coli (differing from the wild-type E. coli glmS protein, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 2006, 88: 419-429).

In addition, LN3, LNT and/or LNnT production can further be optimized in the mutant E. coli strains with genomic knock-outs of the E. coli genes comprising any one or more of gaIT, ushA, ldhA and agp. The mutant E. coli strains can also optionally be adapted with a genomic knock-in of a constitutive transcriptional unit for an UDP-glucose-4-epimerase like e.g., galE from E. coli (UniProt ID P09147), a phosphoglucosamine mutase like e.g., glmM from E. coli (UniProt ID P31120) and an N-acetylglucosamine-1-phosphate uridylyltransferase/glucosamine-1-phosphate acetyltransferase like e.g., glmU from E. coli (UniProt ID P0ACC7). The mutant E. coli strains can also be adapted for UDP-GalNAc production with a genomic knock-in of a constitutive transcriptional unit for an 4-epimerase like e.g., WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) next to optional knock-ins for a phosphoglucosamine mutase like e.g., glmM from E. coli (UniProt ID P31120) and an N-acetylglucosamine-1-phosphate uridylyltransferase/glucosamine-1-phosphate acetyltransferase like e.g., glmU from E. coli (UniProt ID P0ACC7). Furthermore, the mutant strains can be modified with a constitutive transcriptional unit for an alpha-1,3-galactosyltransferase like e.g., SEQ ID NOs:3, 4, 5, 6, 7, 8, 9, 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. Alternatively and/or additionally, the mutant strains can be modified with a constitutive transcriptional unit for an alpha-1,3-N-acetylgalactosaminyl transferase like e.g., SEQ ID NOs:40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102.

Alternatively, and/or additionally, production of LN3, LNT, LNnT and oligosaccharides derived thereof can further be optimized in the mutant E. coli strains with a genomic knock-in of a constitutive transcriptional unit comprising a membrane transporter protein like e.g., MdfA from Cronobacter muytjensii (UniProt ID AOA2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID P0AEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207) or iceT from Citrobacter youngae (UniProt ID D4B8A6).

In one example for sialic acid production, the mutant strain was derived from E. coli K12 MG1655 comprising genomic knock-ins of constitutive transcriptional units containing one or more copies of a glucosamine 6-phosphate N-acetyltransferase like e.g., GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), an N-acetylglucosamine 2-epimerase like e.g., AGE from Bacteroides ovatus (UniProt ID A7LVG6) and an N-acetylneuraminate synthase like e.g., from Neisseria meningitidis (UniProt ID E0NCD4) or Campylobacter jejuni (UniProt ID Q93MP9).

Alternatively, and/or additionally, sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing an UDP-N-acetylglucosamine 2-epimerase like e.g., NeuC from C. jejuni (UniProt ID Q93MP8) and an N-acetylneuraminate synthase like e.g., from Neisseria meningitidis (UniProt ID E0NCD4) or Campylobacter jejuni (UniProt ID Q93MP9).

Alternatively and/or additionally, sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g., glmM from E. coli (UniProt ID P31120), an N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase like e.g., glmU from E. coli (UniProt ID P0ACC7), an UDP-N-acetylglucosamine 2-epimerase like e.g., NeuC from C. jejuni (UniProt ID Q93MP8) and an N-acetylneuraminate synthase like e.g., from Neisseria meningitidis (UniProt ID E0NCD4) or Campylobacter jejuni (UniProt ID Q93MP9).

Alternatively, and/or additionally, sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a bifunctional UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase like e.g., from Mus musculus (strain C57BL/6J) (UniProt ID Q91WG8), an N-acylneuraminate-9-phosphate synthetase like e.g., from Pseudomonas sp. UW4 (UniProt ID K9NPH9) and an N-acylneuraminate-9-phosphatase like e.g., from Candidatus Magnetomorum sp. HK-1 (UniProt ID KPA15328.1) or from Bacteroides thetaiotaomicron (UniProt ID Q8A712).

Alternatively, and/or additionally, sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g., glmM from E. coli (UniProt ID P31120), an N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase like e.g., glmU from E. coli (UniProt ID P0ACC7), a bifunctional UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase like e.g., from M. musculus (strain C57BL/6J) (UniProt ID Q91WG8), an N-acylneuraminate-9-phosphate synthetase like e.g., from Pseudomonas sp. UW4 (UniProt ID K9NPH9) and an N-acylneuraminate-9-phosphatase like e.g., from Candidatus Magnetomorum sp. HK-1 (UniProt ID KPA15328.1) or from Bacteroides thetaiotaomicron (UniProt ID Q8A712).

Sialic acid production can further be optimized in the mutant E. coli strain with genomic knock-outs of the E. coli genes comprising any one or more of nagA, nagB, nagC, nagD, nagE, nanA, nanE, nanK, manX, manY and manZ as described in WO 2018122225, and/or genomic knock-outs of the E. coli genes comprising any one or more of nanT, poxB, ldhA, adhE, aldB, pflA, pflC, ybiY, ackA and/or pta and with genomic knock-ins of constitutive transcriptional units comprising one or more copies of an L-glutamine-D-fructose-6-phosphate aminotransferase like e.g., the mutant glmS*54 from E. coli (differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006)), preferably a phosphatase like e.g., the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida, ScDOG1 from S. cerevisiae or BsAraL from Bacillus subtilis as described in WO 2018122225 and an acetyl-CoA synthetase like e.g., acs from E. coli (UniProt ID P27550).

For sialylated oligosaccharide production, the sialic acid production strains were further modified to express an N-acylneuraminate cytidylyltransferase like e.g., the NeuA enzyme from C. jejuni (UniProt ID Q93MP7), the NeuA enzyme from Haemophilus influenzae (GenBank NO. AGV11798.1) or the NeuA enzyme from Pasteurella multocida (GenBank NO. AMK07891.1) and to express one or more copies of a beta-galactoside alpha-2,3-sialyltransferase like e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity, NmeniST3 from N. meningitidis (GenBank NO. ARC07984.1) or PmultST2 from P. multocida subsp. multocida str. Pm70 (GenBank NO. AAK02592.1), a beta-galactoside alpha-2,6-sialyltransferase like e.g., PdST6 from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity or P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 having beta-galactoside alpha-2,6-sialyltransferase activity, and/or an alpha-2,8-sialyltransferase like e.g., from M. musculus (UniProt ID Q64689). Constitutive transcriptional units of the N-acylneuraminate cytidylyltransferase and the sialyltransferases can be delivered to the mutant strain either via genomic knock-in or via an expression plasmid. If the mutant strains producing sialic acid and CMP-sialic acid were intended to make sialylated lactose structures, the strains were additionally modified with genomic knock-outs of the E. coli LacZ, LacY and LacA genes and with a genomic knock-in of a constitutive transcriptional unit for a lactose permease like e.g., E. coli LacY (UniProt ID P02920).

Alternatively, and/or additionally, sialic acid and/or sialylated oligosaccharide production can further be optimized in the mutant E. coli strains with a genomic knock-in of a constitutive transcriptional unit comprising a membrane transporter protein like e.g., a sialic acid transporter like e.g., nanT from E. coli K-12 MG1655 (UniProt ID P41036), nanT from E. coli 06:H1 (UniProt ID Q8FD59), nanT from E. coli O157:H7 (UniProt ID Q8X9G8) or nanT from E. albertii (UniProt ID B1EFH1) or a porter like e.g., EntS from E. coli (UniProt ID P24077), EntS from Kluyvera ascorbata (UniProt ID A0A378GQ13) or EntS from Salmonella enterica subsp. arizonae (UniProt ID A0A6Y2K4E8), MdfA from Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID P0AEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207), iceT from Citrobacter youngae (UniProt ID D4B8A6), SetA from E. coli (UniProt ID P31675), SetB from E. coli (UniProt ID P33026) or SetC from E. coli (UniProt ID P31436) or an ABC transporter like e.g., oppF from E. coli (UniProt ID P77737), lmrA from Lactococcus lactis subsp. lactis bv. diacetylactis (UniProt ID AOA1V0NEL4), or Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4).

All mutant strains could also optionally be adapted for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g., CscB from E. coli W (UniProt ID E0IXR1), a fructose kinase like e.g., Frk originating from Z. mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g., BaSP from B. adolescentis (UniProt ID A0ZZH6).

Preferably but not necessarily, any one or more of the glycosyltransferases, the proteins involved in nucleotide-activated sugar synthesis and/or membrane transporter proteins were N- and/or C-terminally fused to a solubility enhancer tag like e.g., a SUMO-tag, an MBP-tag, His, FLAG®, Strep-II, Halo-tag, NusA, thioredoxin, GST and/or the Fh8-tag to enhance their solubility (Costa et al., Front. Microbiol. 2014, doi.org/10.3389/fmicb.2014.00063; Fox et al., Protein Sci. 2001, 10(3), 622-630; Jia and Jeaon, Open Biol. 2016, 6: 160196).

Optionally, the mutant E. coli strains were modified with a genomic knock-in of a constitutive transcriptional unit encoding a chaperone protein like e.g., DnaK, DnaJ, GrpE, or the GroEL/ES chaperonin system (Baneyx F., Palumbo J. L. (2003) Improving Heterologous Protein Folding via Molecular Chaperone and Foldase Co-Expression. In: Vaillancourt P. E. (eds) E. coli Gene Expression Protocols. Methods in Molecular Biology™, vol 205. Humana Press).

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

All constitutive promoters, UTRs and terminator sequences originated from the libraries described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360) and Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148). All genes were ordered synthetically at Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com) and the codon usage was adapted using the tools of the supplier. The SEQ ID NOs described in disclosure are summarized in Table 1.

All strains were stored in cryovials at −80° C. (overnight LB culture mixed in a 1:1 ratio with 70% glycerol).

TABLE 1
Overview of SEQ ID NOs described in the disclosure
				Country of
				origin of
SEQ				digital
ID				sequence
NO	Name/identifier	Organism	Origin	information
01	motif	Not applicable	Synthetic	Not applicable
	YX[FHMQT]XAXX[ACG][ACG]
02	motif YXQXCXX[ACG][ACG]	Not applicable	Synthetic	Not applicable
03	WbnI	E. coli K-12 MG1655	Synthetic	USA
04	A0A379B3N6_9PAST	[Pasteurella] mairii	Synthetic	United
				Kingdom
05	A0A430GH65_ACIBZ	Acinetobacter bereziniae	Synthetic	USA
06	A0A1G6GGM3_9GAMM	Acinetobacter boissieri	Synthetic	Unknown
07	DOS616_ACICA	Acinetobacter calcoaceticus	Synthetic	Unknown
08	DOSG80_ACIJO	Acinetobacter johnsonii	Synthetic	Unknown
09	R5HLA7_9SPIR	Brachyspira sp. CAG:484	Synthetic	human
10	A0A327JA01_9BACT	Candidatus Melainabacteria	Synthetic	USA
11	A0A2D6M7J8_9ARCH	Candidatus Pacearchaeota	Synthetic	USA
12	A0A1F6ZBC2_9ARCH	Candidatus Pacearchaeota	Synthetic	USA
13	A0AIN6DQ13_9BACT	Chitinophaga niabensis	Synthetic	Unknown
14	A0A0L6XXM2_ECOLX	Escherichia coli	Synthetic	USA
15	A0A5F2KSQ8_ECOLX	Escherichia coli	Synthetic	USA
16	A0A377IY75_9PAST	Haemophilus pittmaniae	Synthetic	Denmark
17	F9Q7G3_9PAST	Haemophilus pittmaniae HK 85	Synthetic	Unknown
18	A0A268TKN7_9HELI	Helicobacter sp. 11S02629-2	Synthetic	The
				Netherlands
19	A0A268U1C6_9HELI	Helicobacter sp. 13S00401-1	Synthetic	The
				Netherlands
20	A0A2D8EN13_9RHOB	Hyphomonas sp.	Synthetic	Unknown
21	G4CKK7_9NEIS	Neisseria shayeganii 871	Synthetic	Unknown
22	A0A448K0X4_PASAE	Pasteurella aerogenes	Synthetic	Germany
23	A5WE44_PSYWF	Psychrobacter sp.	Synthetic	Unknown
		(strain PRwf-1)
24	A0A0Q9ZZ77_9GAMM	Psychrobacter sp. P11F6	Synthetic	Norway
25	U3GL52_SALER	Salmonella enterica	Synthetic	USA
26	A0A5VOIUT3_SALER	Salmonella enterica	Synthetic	USA
27	A0A3U0WT94_SALET	Salmonella enterica I	Synthetic	USA
28	A0A512AVE0_SALET	Salmonella enterica	Synthetic	United
		subsp. enterica serovar		Kingdom
		Ahuza
29	A0A5JIJHJ9_SALET	Salmonella enterica	Synthetic	United
		subsp. enterica serovar		Kingdom
		Kingabwa
30	A0A1I6Z135_SELRU	Selenomonas ruminantium	Synthetic	Japan
31	A0A0Q0W3X1_9DELT	Smithella sp. SDB	Synthetic	USA
32	A0A0E3HFV4_9CAUD	Synechococcus phage	Synthetic	Unknown
		ACG-2014f
33	A0A0E3F846_9CAUD	Synechococcus phage	Synthetic	Unknown
		ACG-2014f
34	A0A0E3HAU1_9CAUD	Synechococcus phage	Synthetic	Unknown
		ACG-2014f
35	A0A222YW15_9CAUD	Synechococcus phage	Synthetic	Unknown
		Bellamy
36	A0AID8KNT5_9CAUD	Synechococcus phage	Synthetic	Unknown
		S-CAM9
37	A0A0T9L978_YERKR	Yersinia kristensenii	Synthetic	Unknown
38	motif	Not applicable	Synthetic	Not applicable
	YX[ALIC]XGXX[ACG][ACG]
39	motif	Not applicable	Synthetic	Not applicable
	YX[AG]XAXX[ACG][ACG]
40	BgtA	Helicobacter mustelae	Synthetic	Unknown
41	R0BXE6_9FIRM	[Clostridium] bolteae	Synthetic	Unknown
		90A9
42	A0A2N8HMV8_9BACT	Akkermansia muciniphila	Synthetic	The
				Netherlands
43	A0A2N8HSR4_9BACT	Akkermansia muciniphila	Synthetic	The
				Netherlands
44	A0A5B5WWM4_9BACT	Akkermansia sp. BIOML-A61	Synthetic	USA
45	R7DUA1_9BACT	Akkermansia sp. CAG:344	Synthetic	Unknown
46	A0A139TN33_9BACT	Akkermansia sp. KLE1798	Synthetic	Unknown
47	A0A2D8CAM3_9BACT	Algoriphagus sp.	Synthetic	USA
48	A0A414EE60_BACOV	Bacteroides ovatus	Synthetic	Unknown
49	A0A395VXC9_BACOV	Bacteroides ovatus	Synthetic	Unknown
50	A0A5M5NEF9_BACOV	Bacteroides ovatus	Synthetic	Unknown
51	A0A5M5BDZ6_BACOV	Bacteroides ovatus	Synthetic	Unknown
52	D4WAD3_BACOV	Bacteroides ovatus SD	Synthetic	Unknown
		CMC 3f
53	W4UP70_9BACE	Bacteroides reticulotermitis	Synthetic	Unknown
		JCM 10512
54	A0A374VDT0_9BACE	Bacteroides sp. OM08-11	Synthetic	Unknown
55	A0A414GH63_9BACE	Bacteroides xylanisolvens	Synthetic	USA
56	A0A3A1YUS9_9PAST	Bisgaard taxon 44 str. 111	Synthetic	Germany
57	A0A3A1Y620_9PAST	Bisgaard Taxon 44 str. B96_3	Synthetic	Unknown
58	A0A3A1Y6E1_9PAST	Bisgaard taxon 44 str. B96_4	Synthetic	Belgium
59	A0A3A1YHR3_9PAST	Bisgaard taxon 44 str. EEAB3T1	Synthetic	Denmark
60	A0A1F6NMG0_9BACT	Candidatus Magasanikbacteria	Synthetic	USA
61	A0A3D4XY75_9BACT	Candidatus Nomurabacteria	Synthetic	Canada
62	A0A1F8JKB9_9BACT	Chlamydiae bacterium	Synthetic	USA
63	A0A1F8JQI5_9BACT	Chlamydiae bacterium	Synthetic	USA
64	R5SZE5_9CLOT	Clostridium hathewayi CAG:224	Synthetic	Unknown
65	A0A1F8V813_9COXI	Coxiella sp.	Synthetic	USA
66	M5PWK3_DESAF	Desulfocurvibacter	Synthetic	USA
		africanus PCS
67	M5PWJ9_DESAF	Desulfocurvibacter	Synthetic	USA
		africanus PCS
68	A0A1T4X4S8_9FIRM	Gemmiger formicilis	Synthetic	Unknown
69	A0A1T4WDI3_9FIRM	Gemmiger formicilis	Synthetic	Unknown
70	L1K4J1_GUITC	Guillardia theta	Synthetic	Unknown
71	LIJS30_GUITC	Guillardia theta	Synthetic	Unknown
72	A0A268TN00_9HELI	Helicobacter sp. 11S02629-2	Synthetic	The
				Netherlands
73	A0A268U494_9HELI	Helicobacter sp. 13S00401-1	Synthetic	The
				Netherlands
74	A0A113AV07_9FIRM	Lachnospiraceae bacterium	Synthetic	Unknown
75	A0A1M5FSV6_9GAMM	Marinomonas polaris	Synthetic	Unknown
76	A0A1H1WY84_9ACTN	Marmoricola scoriae	Synthetic	Unknown
77	A0A4Q0J8V7_9BACT	Muribaculaceae bacterium	Synthetic	Germany
78	A0A3S0CIL8_9NEIS	Neisseriaceae bacterium	Synthetic	USA
79	A0A1V1WBB2_9ACTN	Nocardioides sp. PD653	Synthetic	Japan
80	K5ZTG7_9BACT	Parabacteroides goldsteinii	Synthetic	USA
81	SOGK24_9BACT	Parabacteroides goldsteinii	Synthetic	USA
82	A0A0F5J8N9_9BACT	Parabacteroides goldsteinii	Synthetic	USA
83	A0A0F5IUL0_9BACT	Parabacteroides gordonii MS-1	Synthetic	Unknown
84	A0A0C1BYL5_9BACT	Parachlamydia acanthamoebae	Synthetic	Germany
85	F8KUR1_PARAV	Parachlamydia acanthamoebae	Synthetic	Germany
86	A0A2H9SLN5_9BACT	Parachlamydia sp.	Synthetic	Germany
87	A0A1Y3N080_PIRSE	Piromyces sp.	Synthetic	Unknown
88	A0A1Y3N1S0_PIRSE	Piromyces sp.	Synthetic	Unknown
89	A0A3C1Y7L0_9PORP	Porphyromonadaceae	Synthetic	Unknown
		bacterium
90	Q58M87_BPPRM	Prochlorococcus phage P-SSM2	Synthetic	Unknown
91	A0A3R5VYF4_9FIRM	Roseburia inulinivorans	Synthetic	United
				Kingdom
92	F2U7J8_SALR5	Salpingoeca rosetta	Synthetic	Unknown
93	A0A353BN36_9FIRM	Subdoligranulum sp.	Synthetic	human
94	A0A3D4DY51_9FIRM	Subdoligranulum sp.	Synthetic	human
95	A0A353BNA4_9FIRM	Subdoligranulum sp.	Synthetic	human
96	A0A1Q6SX55_9FIRM	Subdoligranulum sp.	Synthetic	human
97	A0A2V2FCK1_9FIRM	Subdoligranulum variabile	Synthetic	Denmark
98	A0A2V2FLV2_9FIRM	Subdoligranulum variabile	Synthetic	Denmark
99	D1PIY3_9FIRM	Subdoligranulum variabile	Synthetic	Denmark
100	D1PRD4_9FIRM	Subdoligranulum variabile	Synthetic	Denmark
101	D1B3X0_SULD5	Sulfurospirillum deleyianum	Synthetic	Germany
102	PDB: 4AYL_A	Bacteroides ovatus	Synthetic	USA

Cultivation Conditions

A preculture of 96-well microtiter plate experiments was started from a cryovial, in 150 μL LB and was incubated overnight at 37° C. on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96-well square microtiter plate, with 400 μL minimal medium by diluting 400×. These final 96-well culture plates were then incubated at 37° C. on an orbital shaker at 800 rpm for 72 h, or shorter, or longer. To measure sugar concentrations at the end of the cultivation experiment whole broth samples were taken from each well by boiling the culture broth for 15 min at 60° C. before spinning down the cells (=average of intra- and extracellular sugar concentrations).

A preculture for the bioreactor was started from an entire 1 mL cryovial of a certain strain, inoculated in 250 mL or 500 mL minimal medium in a 1 L or 2.5 L shake flask and incubated for 24 h at 37° C. on an orbital shaker at 200 rpm. A 5 L bioreactor was then inoculated (250 mL inoculum in 2 L batch medium); the process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). Culturing condition were set to 37° C., and maximal stirring; pressure gas flow rates were dependent on the strain and bioreactor. The pH was controlled at 6.8 using 0.5 M H₂SO₄ and 20% NH₄OH. The exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.

Optical Density

Cell density of the cultures was frequently monitored by measuring optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or with a Spark 10M microplate reader, Tecan, Switzerland).

Analytical Analysis

Standards such as but not limited to sucrose, lactose, lacto-N-triose II (LN3), lacto-N-tetraose (LNT) and LNFP-I were purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analyzed with in-house made standards.

The oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Evaporative Light Scattering Detector (ELSD) or a Refractive Index (RI) detection. A volume of 0.7 μL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1×100 mm; 130 Å; 1.7 μm) column with an Acquity UPLC BEH Amide VanGuard column, 130 Å, 2.1×5 mm. The column temperature was 50° C. The mobile phase consisted of a ¼ water and ¾ acetonitrile solution to which 0.2% triethylamine was added. The method was isocratic with a flow of 0.130 mL/min. The ELS detector had a drift tube temperature of 50° C. and the N2 gas pressure was 50 psi, the gain 200 and the data rate 10 pps. The temperature of the RI detector was set at 35° C.

The sugars were also analyzed on a Waters Acquity H-class UPLC with Refractive Index (RI) detection. A volume of 0.5 μL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1×100 mm; 130 Å; 1.7 μm). The column temperature was 50° C. The mobile phase consisted of a mixture of 72% acetonitrile and 28% ammonium acetate buffer (100 mM) to which 0.1% triethylamine was added. The method was isocratic with a flow of 0.260 mL/min. The temperature of the RI detector was set at 35° C.

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

For analysis of sugars at low concentrations (below 50 mg/L) a Dionex HPAEC system with pulsed amperometric detection (PAD) was used. A volume of 5 μL of sample was injected on a Dionex CarboPac PA200 column 4×250 mm with a Dionex CarboPac PA200 guard column 4×50 mm. The column temperature was set to 30° C. A gradient was used wherein eluent A was deionized water, wherein eluent B was 200 mM Sodium hydroxide and wherein eluent C was 500 mM Sodium acetate. The oligosaccharides were separated in 60 min while maintaining a constant ratio of 25% of eluent B using the following gradient: an initial isocratic step maintained for 10 min of 75% of eluent A, an initial increase from 0 to 4% of eluent C over 8 min, a second isocratic step maintained for 6 min of 71% of eluent A and 4% of eluent C, a second increase from 4 to 12% of eluent C over 2.6 min, a third isocratic step maintained for 3.4 min of 63% of eluent A and 12% of eluent C and a third increase from 12 to 48% of eluent C over 5 min. As a washing step 48% of eluent C was used for 3 min. For column equilibration, the initial condition of 75% of eluent A and 0% of eluent C was restored in 1 min and maintained for 11 min. The applied flow was 0.5 mL/min.

Example 2. Materials and Methods Saccharomyces cerevisiae

Media

Strains were grown on Synthetic Defined yeast medium with Complete Supplement Mixture (SD CSM) or CSM drop-out (SD CSM-Ura or SD CSM-His) containing 6.7 g/L Yeast Nitrogen Base without amino acids (YNB w/o AA, Difco), 20 g/L agar (Difco) (solid cultures), 22 g/L glucose monohydrate or 20 g/L lactose and 0.79 g/L CSM or 0.77 g/L CSM-Ura or 0.77 g/L CSM-His (MP Biomedicals).

Strains

S. cerevisiae BY4742 created by Brachmann et al. (Yeast (1998) 14:115-32) was used, available in the Euroscarf culture collection. All mutant strains were created by homologous recombination or plasmid transformation using the method of Gietz (Yeast 11:355-360, 1995).

Plasmids

In an example to produce GDP-fucose, a yeast expression plasmid like e.g., p2a_2μ_Fuc (Chan 2013, Plasmid 70, 2-17) was used for expression of foreign genes in S. cerevisiae. This plasmid contained an ampicillin resistance gene and a bacterial origin of replication to allow for selection and maintenance in E. coli and the 2μ yeast ori and the Ura3 selection marker for selection and maintenance in yeast. This plasmid further contained constitutive transcriptional units for a lactose permease like e.g., LAC12 from Kluyveromyces lactis (UniProt ID P07921), a GDP-mannose 4,6-dehydratase like e.g., gmd from E. coli (UniProt ID P0AC88) and a GDP-L-fucose synthase like e.g., fcl from E. coli (UniProt ID P32055). The yeast expression plasmid p2a_2μ_Fuc2 was used as an alternative expression plasmid of the p2a_2μ_Fuc plasmid comprising next to the ampicillin resistance gene, the bacterial ori, the 2μ yeast ori and the Ura3 selection marker constitutive transcriptional units for a lactose permease like e.g., LAC12 from K. lactis (UniProt ID P07921), a fucose permease like e.g., fucP from E. coli (UniProt ID P11551) and a bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase like e.g., fkp from B. fragilis (UniProt ID SUV40286.1). To further produce fucosylated oligosaccharides, the p2a_2μ_Fuc and its variant the p2a_2μ_Fuc2, additionally contained a constitutive transcriptional unit for an alpha-1,2-fucosyltransferase like e.g., HpFutC from H. pylori (GenBank: AAD29863.1).

In an example to produce UDP-galactose, a yeast expression plasmid was derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the HIS3 selection marker and a constitutive transcriptional unit for an UDP-glucose-4-epimerase like e.g., galE from E. coli (UniProt ID P09147). To produce LN3 and LNT, this plasmid was further modified with constitutive transcriptional units for a lactose permease like e.g., LAC12 from K. lactis (UniProt ID P07921), a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g., lgtA from N. meningitidis (GenBank: AAM33849.1) and an N-acetylglucosamine beta-1,3-galactosyltransferase like e.g., WbgO from E. coli 055:H7 (UniProt ID D3QY14). To produce UDP-GalNAc, the plasmid was additionally modified with constitutive transcriptional units for an 4-epimerase like e.g., WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) next to optional knock-ins for a phosphoglucosamine mutase like e.g., glmM from E. coli (UniProt ID P31120) and an N-acetylglucosamine-1-phosphate uridylyltransferase/glucosamine-1-phosphate acetyltransferase like e.g., glmU from E. coli (UniProt ID P0ACC7). Furthermore, the mutant strains can be modified with a constitutive transcriptional unit for an alpha-1,3-galactosyltransferase like e.g., SEQ ID NOs:3, 4, 5, 6, 7, 8, 9, 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. Alternatively and/or additionally, the mutant strains can be modified with a constitutive transcriptional unit for an alpha-1,3-N-acetylgalactosaminyl transferase like e.g., SEQ ID NOs:40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102.

In one example to produce sialic acid and CMP-sialic acid, a yeast expression plasmid can be derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the TRP1 selection marker and constitutive transcriptional units for one or more copies of an L-glutamine-D-fructose-6-phosphate aminotransferase like e.g., the mutant glmS*54 from E. coli (differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006)), a phosphatase like e.g., the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida, ScDOG1 from S. cerevisiae or BsAraL from Bacillus subtilis as described in WO 2018122225, an N-acetylglucosamine 2-epimerase like e.g., AGE from B. ovatus (UniProt ID A7LVG6), an N-acetylneuraminate synthase like e.g., from Neisseria meningitidis (UniProt ID E0NCD4) or Campylobacter jejuni (UniProt ID Q93MP9), and an N-acylneuraminate cytidylyltransferase like e.g., NeuA from C. jejuni (UniProt ID Q93MP7), NeuA from Haemophilus influenzae (GenBank No. AGV11798.1) or NeuA from Pasteurella multocida (GenBank No. AMK07891.1). Optionally, a constitutive transcriptional unit comprising one or more copies for a glucosamine 6-phosphate N-acetyltransferase like e.g., GNA1 from S. cerevisiae (UniProt ID P43577) was/were added as well. To produce sialylated oligosaccharides, the plasmid further comprised constitutive transcriptional units for a lactose permease like e.g., LAC12 from Kluyveromyces lactis (UniProt ID P07921), and one or more copies of a beta-galactoside alpha-2,3-sialyltransferase like e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity, NmeniST3 from N. meningitidis (GenBank No. ARC07984.1) or PmultST2 from P. multocida subsp. multocida str. Pm70 (GenBank No. AAK02592.1), a beta-galactoside alpha-2,6-sialyltransferase like e.g., PdST6 from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity or P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 having beta-galactoside alpha-2,6-sialyltransferase activity, and/or an alpha-2,8-sialyltransferase like e.g., from M. musculus (UniProt ID Q64689).

Preferably but not necessarily, any one or more of the glycosyltransferases, the proteins involved in nucleotide-activated sugar synthesis and/or membrane transporter proteins were N- and/or C-terminally fused to a SUMOstar tag (e.g., obtained from pYSUMOstar, Life Sensors, Malvern, Pa.) to enhance their solubility.

Optionally, the mutant yeast strains were modified with a genomic knock-in of a constitutive transcriptional unit encoding a chaperone protein like e.g., Hsp31, Hsp32, Hsp33, Sno4, Kar2, 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).

Plasmids were maintained in the host E. coli DH5alpha (F⁻, phi80dlacZdeltaM15, delta(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk⁻, mk⁺), phoA, supE44, lambda, thi-1, gyrA96, relA1) bought from Invitrogen.

Heterologous and Homologous Expression

Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9, IDT or Twist Bioscience. Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.

Cultivations Conditions

In general, yeast strains were initially grown on SD CSM plates to obtain single colonies. These plates were grown for 2-3 days at 30° C. Starting from a single colony, a preculture was grown over night in 5 mL at 30° C., shaking at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL media. These shake flasks were incubated at 30° C. with an orbital shaking of 200 rpm.

Gene Expression Promoters

Genes were expressed using synthetic constitutive promoters, as described by Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012).

Example 3. Production of an Oligosaccharide Mixture Comprising 2′FL, DiFL, LN3, LNT, LNFP-I and Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 is further adapted for LN3 and LNT production by genomic knock-ins of a constitutive transcriptional unit for the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (GenBank: AAM33849.1) and for the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt ID D3QY14). To produce LNFP-I, the novel strain is further modified with an expression plasmid containing a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1). In a final step to produce Gal-LNFP-I, the mutant strain is modified with a second compatible expression plasmid containing a constitutive transcriptional unit for the alpha-1,3-galactosyltransferase WbnI from E. coli with SEQ ID NO:03. The novel strain is evaluated in a growth experiment for the production of 2′FL, DiFL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L lactose. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 4. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified E. coli Host in Fed-Batch Fermentations

A mutant E. coli strain as described in Example 3 is further evaluated in a fed-batch fermentation process. Fed-batch fermentations at bioreactor scale are performed as described in Example 1. In these examples, sucrose is used as a carbon source and lactose is added in the batch medium as a precursor. Regular broth samples are taken, and the production of oligosaccharides is measured using UPLC as described in Example 1.

Example 5. Production of an Oligosaccharide Mixture Comprising 2′FL, DiFL, LN3, LNT, LNFP-I and GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and for LNFP-I production and growth on sucrose as described in Example 3 is further transformed with a second compatible expression plasmid containing constitutive transcriptional units for the alpha-1,3-N-acetylgalactosaminyltransferase BgtA from H. mustelae with SEQ ID NO:40 and the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) for UDP-GalNAc production. The novel strain is evaluated in a growth experiment for the production of 2′FL, DiFL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L lactose. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 6. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified E. coli Host in Fed-Batch Fermentations

A mutant E. coli strain as described in Example 5 is further evaluated in a fed-batch fermentation process. Fed-batch fermentations at bioreactor scale are performed as described in Example 1. In these examples, sucrose is used as a carbon source and lactose is added in the batch medium as a precursor. Regular broth samples are taken, and the production of oligosaccharides is measured using UPLC as described in Example 1.

Example 7. Production of an Oligosaccharide Mixture Comprising 2′FL, DiFL, LN3, LNT, LNFP-I and Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified S. cerevisiae Host

An S. cerevisiae strain is adapted for production of GDP-fucose and LNT and for expression of an a-1,2-fucosyltransferase and an alpha-1,3-galactosyltransferase as described in Example 2 with a first yeast expression plasmid comprising constitutive transcriptional units for the lactose permease LAC12 from K. lactis (UniProt ID P07921), the GDP-mannose 4,6-dehydratase gmd from E. coli (UniProt ID P0AC88), the GDP-L-fucose synthase fcl from E. coli (UniProt ID P32055) and the a1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) and with a second yeast expression plasmid comprising constitutive transcriptional units for the UDP-glucose 4-epimerase galE from E. coli (UniProt ID P09147), the galactoside beta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis (GenBank: AAM33849.1), the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt ID D3QY14) and the alpha-1,3-galactosyltransferase WbnI from E. coli with SEQ ID NO:03. The novel strain is evaluated in a growth experiment for the production of 2′FL, DiFL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) according to the culture conditions provided in Example 2, in which the SD CSM-Ura-His drop-out medium comprises lactose as precursor. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 8. Production of an Oligosaccharide Mixture Comprising 2′FL, DiFL, LN3, LNT, LNFP-I and GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified S. cerevisiae Host

An S. cerevisiae strain is adapted for production of GDP-fucose and LNT and for expression of an a-1,2-fucosyltransferase and an alpha-1,3-N-acetylgalactosaminyltransferase as described in Example 2 with a first yeast expression plasmid comprising constitutive transcriptional units for the lactose permease LAC12 from K. lactis (UniProt ID P07921), the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) for the production of UDP-GalNAc, the GDP-mannose 4,6-dehydratase gmd from E. coli (UniProt ID P0AC88), the GDP-L-fucose synthase fcl from E. coli (UniProt ID P32055) and the a1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) and with a second yeast expression plasmid comprising constitutive transcriptional units for the UDP-glucose 4-epimerase galE from E. coli (UniProt ID P09147), the galactoside beta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis (GenBank: AAM33849.1), the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt ID D3QY14) and the alpha-1,3-N-acetylgalactosaminyltransferase BgtA from H. mustelae with SEQ ID NO:40. The novel strain is evaluated in a growth experiment for the production of 2′FL, DiFL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) according to the culture conditions provided in Example 2, in which the SD CSM-Ura-His drop-out medium comprises lactose as precursor. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 9. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified E. coli Host Expressing the Alpha-1,2-Fucosyltransferase from Brachyspira pilosicoli

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 was further adapted for LN3 and LNT production by genomic knock-ins of constitutive transcriptional units for the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (GenBank: AAM33849.1) and the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt ID D3QY14). The strain was also adapted for UDP-GalNAc production with a genomic knock-in of a constitutive transcriptional unit for the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66). To produce GalNAc-LNFP-I, the novel strain was further modified with a first expression plasmid comprising a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase from Brachyspira pilosicoli with UniProt ID AOA2N5RQ26 and a second compatible expression plasmid comprising a constitutive transcriptional unit for one alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising BgtA from Helicobacter mustelae with SEQ ID NO:40, the polypeptide from Bacteroides ovatus with SEQ ID NO:49, the polypeptide from Lachnospiraceae bacterium with SEQ ID NO:74, the polypeptide from Roseburia inulinivorans with SEQ ID NO:91 and the polypeptide from Bacteroides ovatus with SEQ ID NO:102. The novel strains were evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contained 30 g/L sucrose and 20 g/L lactose. Each strain was grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth was harvested, and the sugars were analyzed on UPLC. The experiment demonstrated all strains produced GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples, as summarized in Table 2.

TABLE 2
Production of GalNAc-a1,3-LNFP-I in whole broth samples of
mutant E. coli strains expressing the alpha-1,2-fucosyltransferase
from B. pilosicoli with UniProt ID A0A2N5RQ26 and one
alpha-1,3-N-acetylgalactosaminyltransferase, when evaluated in a
growth experiment according the cultivation conditions as described
in Example 1, in which the culture medium contained sucrose as
carbon source and lactose as precursor.
                                 Production of
Alpha-1,3-N-acetylgalactosaminyltransferase GalNAc-a1,3-LNFP-I
expressed in the mutant E. coli strain (g/L)
SEQ ID NO: 40                    0.71 ± 0.07
SEQ ID NO: 49                    0.36 ± 0.36
SEQ ID NO: 74                    1.30 ± 0.14
SEQ ID NO: 91                    1.22 ± 0.13
SEQ ID NO: 102                   0.92 ± 0.08

Example 10. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified E. coli Host Expressing the Alpha-1,2-Fucosyltransferase from Dysgonomonas mossii

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 was further adapted for LN3 and LNT production by genomic knock-ins of constitutive transcriptional units for the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (GenBank: AAM33849.1) and the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt ID D3QY14). The strain was also adapted for UDP-GalNAc production with a genomic knock-in of a constitutive transcriptional unit for the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66). To produce GalNAc-LNFP-I, the novel strain was further modified with a first expression plasmid comprising a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase from Dysgonomonas mossii with UniProt ID F8X274 and a second compatible expression plasmid comprising a constitutive transcriptional unit for one alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising BgtA from Helicobacter mustelae with SEQ ID NO:40, the polypeptide from Bacteroides ovatus with SEQ ID NO:49 and the polypeptide from Bacteroides ovatus with SEQ ID NO:102. The novel strains were evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contained 30 g/L sucrose and 20 g/L lactose. Each strain was grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth was harvested, and the sugars were analyzed on UPLC. The experiment demonstrated all strains produced GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples, as summarized in Table 3.

TABLE 3
Production of GalNAc-a1,3-LNFP-I in whole broth samples of
mutant E. coli strains expressing the alpha-1,2-fucosyltransferase
from D. mossii with UniProt ID F8X274 and one
alpha-1,3-N-acetylgalactosaminyltransferase, when evaluated
in a growth experiment according the cultivation conditions as
described in Example 1, in which the culture medium contained
sucrose as carbon source and lactose as precursor.
                                 Production of
Alpha-1,3-N-acetylgalactosaminyltransferase GalNAc-a1,3-LNFP-I
expressed in the mutant E. coli strain (g/L)
SEQ ID NO: 40                    1.07 ± 0.12
SEQ ID NO: 49                    0.80 ± 0.03
SEQ ID NO: 102                   0.92 ± 0.04

Example 11. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified E. coli Host Expressing the Alpha-1,2-Fucosyltransferase from Dechlorosoma suillum

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 was further adapted for LN3 and LNT production by genomic knock-ins of constitutive transcriptional units for the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (GenBank: AAM33849.1) and the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt ID D3QY14). The strain was also adapted for UDP-GalNAc production with a genomic knock-in of a constitutive transcriptional unit for the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66). To produce GalNAc-LNFP-I, the novel strain was further modified with a first expression plasmid comprising a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase from Dechlorosoma suillum with UniProt ID G8QLF4 and a second compatible expression plasmid comprising a constitutive transcriptional unit for one alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising BgtA from Helicobacter mustelae with SEQ ID NO:40 and the polypeptide from Bacteroides ovatus with SEQ ID NO:102. The novel strains were evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contained 30 g/L sucrose and 20 g/L lactose. Each strain was grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth was harvested, and the sugars were analyzed on UPLC. The experiment demonstrated all strains produced GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples, as summarized in Table 4.

TABLE 4
Production of GalNAc-a1,3-LNFP-I in whole broth samples of
mutant E. coli strains expressing the alpha-1,2-fucosyltransferase
from D. suillum with UniProt ID G8QLF4 and one
alpha-1,3-N-acetylgalactosaminyltransferase, when evaluated
in a growth experiment according the cultivation conditions as
described in Example 1, in which the culture medium contained
sucrose as carbon source and lactose as precursor.
                                 Production of
Alpha-1,3-N-acetylgalactosaminyltransferase GalNAc-a1,3-LNFP-I
expressed in the mutant E. coli strain (g/L)
SEQ ID NO: 40                    0.23 ± 0.01
SEQ ID NO: 102                   0.30 ± 0.02

Example 12. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified E. coli Host Expressing the Alpha-1,2-Fucosyltransferase from Desulfovibrio alaskensis

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 was further adapted for LN3 and LNT production by genomic knock-ins of constitutive transcriptional units for the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (GenBank: AAM33849.1) and the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt ID D3QY14). The strain was also adapted for UDP-GalNAc production with a genomic knock-in of a constitutive transcriptional unit for the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66). To produce GalNAc-LNFP-I, the novel strain was further modified with a first expression plasmid comprising a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase from Desulfovibrio alaskensis with UniProt ID Q316B5 and a second compatible expression plasmid comprising a constitutive transcriptional unit for one alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising BgtA from Helicobacter mustelae with SEQ ID NO:40 and the polypeptide from Bacteroides ovatus with SEQ ID NO:102. The novel strains were evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contained 30 g/L sucrose and 20 g/L lactose. Each strain was grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth was harvested, and the sugars were analyzed on UPLC. The experiment demonstrated all strains produced GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples, as summarized in Table 5.

TABLE 5
Production of GalNAc-a1,3-LNFP-I in whole broth samples of
mutant E. coli strains expressing the alpha-1,2-fucosyltransferase
from D. alaskensis with UniProt ID Q316B5 and one
alpha-1,3-N-acetylgalactosaminyltransferase, when evaluated
in a growth experiment according the cultivation conditions as
described in Example 1, in which the culture medium contained
sucrose as carbon source and lactose as precursor.
                                 Production of
Alpha-1,3-N-acetylgalactosaminyltransferase GalNAc-a1,3-LNFP-I
expressed in the mutant E. coli strain (g/L)
SEQ ID NO: 40                    0.02 ± 0.01
SEQ ID NO: 102                   0.40 ± 0.15

Example 13. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 is further adapted for LN3 and LNT production by genomic knock-ins of constitutive transcriptional units for the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (GenBank: AAM33849.1) and the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt ID D3QY14). To produce LNFP-I, the novel strain is further modified with an expression plasmid containing a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1). In a final step to produce Gal-LNFP-I, the mutant strain is modified with a second compatible expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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 novel strain is evaluated in a growth experiment for the production of Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L lactose. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 14. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified E. coli Host

An E. coli strain modified for production of GDP-fucose and LNFP-I and growth on sucrose as described in Example 3 is further modified with a constitutive transcriptional unit for the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) for UDP-GalNAc production, and transformed with a second compatible expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98; 99, 100, 101 and 102. The novel strains are evaluated in a growth experiment for the production of 2′FL, DiFL, LN3, LNT, LNFP-I, Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc and GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L lactose. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 15. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc and Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 is further transformed with a first expression plasmid containing a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) and a second compatible expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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 novel strain is evaluated in a growth experiment for the production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc and Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L lactose. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 16. Production of an Oligosaccharide Mixture Comprising Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc and 3′-SL with a Modified E. coli Host

An E. coli strain modified as described in Example 15 is further modified with a genomic knock-out of the nagA and nagB genes and genomic knock-ins of constitutive expression units comprising the genes encoding the L-glutamine-D-fructose-6-phosphate aminotransferase (glmS*54) from E. coli (differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation), the phosphoglucosamine mutase (glmM) from E. coli (UniProt ID P31120), the N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase (glmU) from E. coli (UniProt ID P0ACC7), the UDP-N-acetylglucosamine 2-epimerase (NeuC) from C. jejuni (UniProt ID Q93MP8), the N-acetylneuraminate synthase (NeuB) from N. meningitidis (UniProt ID E0NCD4), the sialic acid transporter (nanT) from E. coli ((UniProt ID P41036), the N-acylneuraminate cytidylyltransferase from C. jejuni (UniProt ID Q93MP7) and the beta-galactoside alpha-2,3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3). The novel strain is evaluated in a growth experiment for the production of a mixture comprising Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc and 3′-sialyllactose (3′-SL) according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L lactose. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 17. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 is further modified with a constitutive transcriptional unit for the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) for UDP-GalNAc production, and transformed with a first expression plasmid containing a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) and a second compatible expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98; 99, 100, 101 and 102. The novel strain is evaluated in a growth experiment for the production of the alpha-tetrasaccharide (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc) according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L lactose. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 18. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 is further transformed with a first expression plasmid containing a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) and a second compatible expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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 novel strain is evaluated in a growth experiment for the production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L lacto-N-biose (LNB, Gal-b1,3-GlcNAc). The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 19. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 is further transformed with a genomic knock-out of the nagA and nagB genes and with genomic knock-ins of constitutive transcriptional units for the mutant L-glutamine-D-fructose-6-phosphate aminotransferase glmS*54 from E. coli (differing from the wild-type E. coli glmS protein, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation), the glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), the phosphatase BsAraL from Bacillus subtilis (UniProt ID P94526) and the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt iD D3QY14) for the production of lacto-N-biose (LNB, Gal-b1,3-GlcNAc). In a final step, the novel strain is further transformed with an expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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 novel strain is evaluated in a growth experiment for the production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 20. Production of an Oligosaccharide Mixture of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc, Sialylated LNB and 6′-SL with a Modified E. coli Host

The modified E. coli strain as described in Example 19 is further modified with a genomic knock-in of the N-acetylglucosamine 2-epimerase (AGE) from B. ovatus (UniProt ID A7LVG6) and the N-acetylneuraminate synthase (NeuB) from N. meningitidis (UniProt ID E0NCD4) and transformed with an expression plasmid comprising constitutive expression units comprising the N-acylneuraminate cytidylyltransferase from C. jejuni (UniProt ID Q93MP7) and the alpha-2,6-sialyltransferase PdbST from P. damselae (UniProt ID 066375). The novel strain is evaluated in a growth experiment for the production of LNB, Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc, sialylated LNB and 6′-sialyllactose (6′-SL) according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L lactose. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 21. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 is further modified with a constitutive transcriptional unit for the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) for UDP-GalNAc production, and transformed with a first expression plasmid containing a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) and a second compatible expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98; 99, 100, 101 and 102. The novel strain is evaluated in a growth experiment for the production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L lacto-N-biose (LNB, Gal-b1,3-GlcNAc). The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 22. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 is further modified with a constitutive transcriptional unit for the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) for UDP-GalNAc production, and transformed with a genomic knock-out of the nagA and nagB genes and with genomic knock-ins of constitutive transcriptional units for the mutant L-glutamine-D-fructose-6-phosphate aminotransferase glmS*54 from E. coli (differing from the wild-type E. coli glmS protein, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation), the glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), the phosphatase BsAraL from Bacillus subtilis (UniProt ID P94526) and the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt iD D3QY14) for the production of lacto-N-biose (LNB, Gal-b1,3-GlcNAc). In a final step, the novel strain is further transformed with an expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98; 99, 100, 101 and 102. The novel strain is evaluated in a growth experiment for the production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 23. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 is further transformed with a first expression plasmid containing a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) and a second compatible expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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 novel strain is evaluated in a growth experiment for the production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L N-acetyllactosamine (LacNAc, Gal-b1,4-GlcNAc). The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 24. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 is further transformed with a genomic knock-out of the nagA and nagB genes and with genomic knock-ins of constitutive transcriptional units for the mutant L-glutamine D-fructose-6-phosphate aminotransferase glmS*54 from E. coli (differing from the wild-type E. coli glmS protein, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation), the glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), the phosphatase BsAraL from Bacillus subtilis (UniProt ID P94526) and the N-acetylglucosamine beta-1,4-galactosyltransferase LgtB from N. meningitidis (UniProt ID Q51116) for the production of N-acetyllactosamine (LacNAc, Gal-b1,4-GlcNAc). In a final step, the novel strain is further transformed with an expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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 novel strain is evaluated in a growth experiment for the production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 25. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 is further modified with a constitutive transcriptional unit for the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) for UDP-GalNAc production, and transformed with a first expression plasmid containing a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) and a second compatible expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98; 99, 100, 101 and 102. The novel strain is evaluated in a growth experiment for the production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L N-acetyllactosamine (LacNAc, Gal-b1,4-GlcNAc). The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 26. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 is further modified with a constitutive transcriptional unit for the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) for UDP-GalNAc production, and transformed with a genomic knock-out of the nagA and nagB genes and with genomic knock-ins of constitutive transcriptional units for the mutant L-glutamine-D-fructose-6-phosphate aminotransferase glmS*54 from E. coli (differing from the wild-type E. coli glmS protein, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation), the glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), the phosphatase BsAraL from Bacillus subtilis (UniProt ID P94526) and the N-acetylglucosamine beta-1,4-galactosyltransferase LgtB from N. meningitidis (UniProt ID Q51116) for the production of N-acetyllactosamine (LacNAc, Gal-b1,4-GlcNAc). In a final step, the novel strain is further transformed with an expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98; 99, 100, 101 and 102. The novel strain is evaluated in a growth experiment for the production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 27. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc with a Modified E. coli Host

An E. coli strain modified for GDP-fucose production and growth on sucrose as described in Example 1 is further transformed with a genomic knock-in of a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) together with a first expression plasmid containing a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) and a second compatible expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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 novel strain is evaluated in a growth experiment for the production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L lactose. The strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 28. Material and Methods Bacillus subtilis

Media

Two different media are used, namely a rich Luria Broth (LB) and a minimal medium for shake flask (MMsf). The minimal medium uses a trace element mix.

Trace element mix consisted of 0.735 g/L CaCl₂)·2H₂O, 0.1 g/L MnCl₂·2H₂O, 0.033 g/L CuCl₂·2H₂O, 0.06 g/L COCl₂·6H₂O, 0.17 g/L ZnCl₂, 0.0311 g/L H₃BO₄, 0.4 g/L Na₂EDTA·2H₂O and 0.06 g/L Na₂MoO₄. The Fe-citrate solution contained 0.135 g/L FeCl₃·6H₂O, 1 g/L Na-citrate (Hoch 1973 PMC1212887).

The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). Luria Broth agar (LBA) plates consisted of the LB media, with 12 g/L agar (Difco, Erembodegem, Belgium) added.

The minimal medium for the shake flasks (MMsf) experiments contained 2.00 g/L (NH₄)₂SO₄, 7.5 g/L KH₂PO₄, 17.5 g/L K₂HPO₄, 1.25 g/L Na-citrate, 0.25 g/L MgSO₄·7H₂O, 0.05 g/L tryptophan, from 10 up to 30 g/L glucose or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose when specified in the examples, 10 ml/L trace element mix and 10 ml/L Fe-citrate solution. The medium was set to a pH of 7 with 1M KOH. Depending on the experiment lactose, LNB or LacNAc could be added as a precursor.

Complex medium, e.g., LB, was sterilized by autoclaving (121° C., 21′) and minimal medium by filtration (0.22 μm Sartorius). When necessary, the medium was made selective by adding an antibiotic (e.g., zeocin (20 mg/L)).

Strains, Plasmids and Mutations

Bacillus subtilis 168, available at Bacillus Genetic Stock Center (Ohio, USA).

Plasmids for gene deletion via Cre/lox are constructed as described by Yan et al. (Appl. & Environm. Microbial., September 2008, p5556-5562). Gene disruption is done via homologous recombination with linear DNA and transformation via electroporation as described by Xue et al. (J. Microb. Meth. 34 (1999) 183-191). The method of gene knockouts is described by Liu et al. (Metab. Engine. 24 (2014) 61-69). This method uses 1000 bp homologies up- and downstream of the target gene.

Integrative vectors as described by Popp et al. (Sci. Rep., 2017, 7, 15158) are used as expression vector and could be further used for genomic integrations if necessary. A suitable promoter for expression can be derived from the part repository (iGem): sequence id: Bba_K143012, Bba_K823000, Bba_K823002 or Bba_K823003. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

In an example for the production of lactose-based oligosaccharides, Bacillus subtilis mutant strains are created to contain a gene coding for a lactose importer (such as e.g., the E. coli lacY with UniProt ID P02920). For 2′FL, 3FL and diFL production, an alpha-1,2- and/or alpha-1,3-fucosyltransferase expression construct is additionally added to the strains.

In an example for the production of lacto-N-triose (LNT-II, LN3, GlcNAc-b1,3-Gal-b1,4-Glc), the B. subtilis strain is modified with a genomic knock-in of constitutive expression units comprising a lactose importer (such as e.g., the E. coli lacY with UniProt ID P02920) and a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g., LgtA from N. meningitidis (GenBank: AAM33849.1). For LNT production, the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,3-galactosyltransferase like e.g., WbgO from E. coli 055:H7 (UniProt ID D3QY14). For the production of lacto-N-neotetraose (LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,4-galactosyltransferase like e.g., LgtB from Neisseria meningitidis (UniProt ID Q51116). Both the N-acetylglucosamine beta-1,3-galactosyltransferase and the N-acetylglucosamine beta-1,4-galactosyltransferase can be delivered to the strain either via genomic knock-in or from an expression plasmid. For the production of LNFP-I and other fucosylated derivatives of LNT and/or LNnT, the LNT and LNnT producing strains can further be modified with an alpha-1,2-fucosyltransferase and/or an alpha-1,3-fucosyltransferase expression construct.

Furthermore, the mutant strains can be modified with a constitutive transcriptional unit for an alpha-1,3-galactosyltransferase like e.g., SEQ ID NOs:3, 4, 5, 6, 7, 8, 9, 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. Alternatively and/or additionally, the mutant strains can be modified with constitutive transcriptional units for an alpha-1,3-N-acetylgalactosaminyl transferase like e.g., SEQ ID NOs:40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102.

For sialic acid production, a mutant B. subtilis strain is created by overexpressing the native fructose-6-P-aminotransferase (UniProt ID P0CI73) to enhance the intracellular glucosamine-6-phosphate pool. Further on, the enzymatic activities of the genes nagA, nagB and gamA are disrupted by genetic knockouts and a glucosamine-6-P-aminotransferase from S. cerevisiae (UniProt ID P43577), an N-acetylglucosamine-2-epimerase from B. ovatus (UniProt ID A7LVG6) and an N-acetylneuraminate synthase from C. jejuni (UniProt ID Q93MP9) are overexpressed on the genome. To allow sialylated oligosaccharide production, the sialic acid producing strain is further modified with expression constructs comprising the N-acylneuraminate cytidylyltransferase NeuA from C. jejuni (UniProt ID Q93MP7) and one or more copies of a beta-galactoside alpha-2,3-sialyltransferase like e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity, NmeniST3 from N. meningitidis (GenBank No. ARC07984.1) or PmultST2 from P. multocida subsp. multocida str. Pm70 (GenBank No. AAK02592.1), a beta-galactoside alpha-2,6-sialyltransferase like e.g., PdST6 from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity or P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 having beta-galactoside alpha-2,6-sialyltransferase activity, and/or an alpha-2,8-sialyltransferase like e.g., from M. musculus (UniProt ID Q64689).

Heterologous and Homologous Expression

Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.

Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.

Cultivation Conditions

A preculture of 96-well microtiter plate experiments was started from a cryovial or a single colony from an LB plate, in 150 μL LB and was incubated overnight at 37° C. on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96-well square microtiter plate, with 400 μL MMsf medium by diluting 400×. Each strain was grown in multiple wells of the 96-well plate as biological replicates. These final 96-well culture plates were then incubated at 37° C. on an orbital shaker at 800 rpm for 72 h, or shorter, or longer. At the end of the cultivation experiment samples were taken from each well to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 15 min at 90° C. or for 60 min at 60° C. before spinning down the cells (=whole broth concentration, intra- and extracellular sugar concentrations, as defined herein).

Also, a dilution of the cultures was made to measure the optical density at 600 nm. The cell performance index or CPI was determined by dividing the oligosaccharide concentrations by the biomass, in relative percentages compared to a reference strain. The biomass is empirically determined to be approximately ⅓rd of the optical density measured at 600 nm.

Example 29. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified B. subtilis Host

A B. subtilis strain is first modified for LN3 production and growth on sucrose by genomic knock-out of the nagB, glmS and gamA genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding the lactose permease (LacY) from E. coli (UniProt ID P02920), the native fructose-6-P-aminotransferase (UniProt ID P0CI73), the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (GenBank: AAM33849.1), the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6). In a next step, the mutant strain is further modified with a genomic knock-in of a constitutive transcriptional unit comprising the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt ID D3QY14) to produce LNT. In a subsequent step, the LNT producing strain is transformed with an expression plasmid comprising constitutive transcriptional units for an alpha-1,2-fucosyltransferase chosen from the list comprising HpFutC from H. pylori (GenBank: AAD29863.1), the polypeptide from Brachyspira pilosicoli (UniProt ID AOA2N5RQ26), the polypeptide from Dysgonomonas mossii (UniProt ID F8X274), the polypeptide from Dechlorosoma suillum (UniProt ID G8QLF4), the polypeptide from Desulfovibrio alaskensis (UniProt ID Q316B5) and the polypeptide from Polaribacter vadi (UniProt ID A0A1B8TNT0) and an alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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 novel strain is evaluated for the production of an oligosaccharide mixture comprising LN3, LNFP-I and Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 28. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 30. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified B. subtilis Host

In this example, a B. subtilis strain is modified for LNT production and growth on sucrose as described in Example 29. In a next step, the mutant LNT producing strain is further modified with a constitutive transcriptional unit for the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) for UDP-GalNAc production, and transformed with an expression plasmid comprising constitutive transcriptional units for an alpha-1,2-fucosyltransferase chosen from the list comprising HpFutC from H. pylori (GenBank: AAD29863.1), the polypeptide from Brachyspira pilosicoli (UniProt ID AOA2N5RQ26), the polypeptide from Dysgonomonas mossii (UniProt ID F8X274), the polypeptide from Dechlorosoma suillum (UniProt ID G8QLF4), the polypeptide from Desulfovibrio alaskensis (UniProt ID Q316B5) and the polypeptide from Polaribacter vadi (UniProt ID AOA1B8TNT0) and an alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98; 99, 100, 101 and 102. The novel strain is evaluated for the production of GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 28. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 31. Production of an Oligosaccharide Mixture Comprising LNT, Sialylated LN3, LSTa and GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified B. subtilis Host

The mutant B. subtilis strain producing GalNAc-a1,3-LNFP-I as described in Example 30 is further modified with a genomic knock-out of the nagA gene and a second compatible expression plasmid comprising the TRP1 selection marker and constitutive transcriptional units for two copies of the mutant L-glutamine-D-fructose-6-phosphate aminotransferase (glmS*54) from E. coli (differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006)), a phosphatase like e.g., a phosphatase chosen from the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from P. putida, ScDOG1 from S. cerevisiae or BsAraL from B. subtilis as described in WO 2018122225, the N-acetylglucosamine 2-epimerase (AGE) from B. ovatus (UniProt ID A7LVG6), the N-acetylneuraminate synthase (NeuB) from N. meningitidis (UniProt ID E0NCD4), the N-acylneuraminate cytidylyltransferase NeuA from Haemophilus influenzae (GenBank No. AGV11798.1) and three copies of the PmultST3 polypeptide from P. multocida (UniProt ID Q9CLP3). The novel strain is evaluated for the production of a mixture comprising LN3, sialylated LN3, LNT, LNFP-I, 2′-FL, GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), 3′-SL and LSTa (Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 28. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 32. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc with a Modified B. subtilis Host

A B. subtilis strain is modified for 2′-FL production as described in Example 28 by genomic knock-ins of constitutive transcriptional units for the lactose permease (LacY) from E. coli (UniProt ID P02920) and the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank. AAD29863.1). In a next step, the mutant strain is transformed with an expression plasmid containing a constitutive transcriptional unit for an alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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 novel strain is evaluated for the production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 28. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 33. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc with a Modified B. subtilis Host

A B. subtilis strain is transformed as described in Example 28 with an expression plasmid comprising constitutive transcriptional units for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1), the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) for UDP-GalNAc production and an alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98; 99, 100, 101 and 102. The novel strain is evaluated for the production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc in a growth experiment on MMsf medium comprising LacNAc as precursor according to the culture conditions provided in Example 28. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 34. Material and Methods Corynebacterium glutamicum

Media

Two different media are used, namely a rich tryptone-yeast extract (TY) medium and a minimal medium for shake flask (MMsf). The minimal medium uses a 1000×stock trace element mix.

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

The minimal medium for the shake flasks (MMsf) experiments contained 20 g/L (NH₄)₂SO₄, 5 g/L urea, 1 g/L KH₂PO₄, 1 g/L K₂HIPO₄, 0.25 g/L MgSO₄·7H₂O, 42 g/L MOPS, from 10 up to 30 g/L glucose or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose when specified in the examples and 1 ml/L trace element mix. Depending on the experiment lactose, LNB or LacNAc could be added as a precursor.

The TY medium consisted of 1.6% tryptone (Difco, Erembodegem, Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). TY agar (TYA) plates consisted of the TY media, with 12 g/L agar (Difco, Erembodegem, Belgium) added.

Complex medium, e.g., TY, was sterilized by autoclaving (121° C., 21′) and minimal medium by filtration (0.22 μm Sartorius). When necessary, the medium was made selective by adding an antibiotic (e.g., kanamycin, ampicillin).

Strains and Mutations

Corynebacterium glutamicum ATCC 13032, available at the American Type Culture Collection.

Integrative plasmid vectors based on the Cre/loxP technique as described by Suzuki et al. (Appl. Microbiol. Biotechnol., 2005 Apr., 67(2):225-33) and temperature-sensitive shuttle vectors as described by Okibe et al. (Journal of Microbiological Methods 85, 2011, 155-163) are constructed for gene deletions, mutations and insertions. Suitable promoters for (heterologous) gene expression can be derived from Yim et al. (Biotechnol. Bioeng., 2013 November, 110(11):2959-69). Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

In an example for the production of lactose-based oligosaccharides, C. glutamicum mutant strains are created to contain a gene coding for a lactose importer (such as e.g., the E. coli lacY with UniProt ID P02920). For 2′FL, 3FL and diFL production, an alpha-1,2- and/or alpha-1,3-fucosyltransferase expression construct is additionally added to the strains.

In an example for the production of lacto-N-triose (LNT-II, LN3, GlcNAc-b1,3-Gal-b1,4-Glc), the C. glutamicum strain is modified with a genomic knock-in of constitutive expression units comprising a lactose importer (such as e.g., the E. coli lacY with UniProt ID P02920) and a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g., LgtA from N. meningitidis (GenBank: AAM33849.1). For LNT production, the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,3-galactosyltransferase like e.g., WbgO from E. coli 055:H7 (UniProt ID D3QY14). For the production of lacto-N-neotetraose (LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,4-galactosyltransferase like e.g., LgtB from Neisseria meningitidis (UniProt ID Q51116). Both the N-acetylglucosamine beta-1,3-galactosyltransferase and the N-acetylglucosamine beta-1,4-galactosyltransferase can be delivered to the strain either via genomic knock-in or from an expression plasmid. For the production of LNFP-I, the LNT producing strain can further be modified with an alpha-1,2-fucosyltransferase expression construct.

Furthermore, the mutant strains can be modified with a constitutive transcriptional unit for an alpha-1,3-galactosyltransferase like e.g., SEQ ID NOs:3, 4, 5, 6, 7, 8, 9, 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. Alternatively and/or additionally, the mutant strains can be modified with a constitutive transcriptional unit for an alpha-1,3-N-acetylgalactosaminyl transferase like e.g., SEQ ID NOs:40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102.

For sialic acid production, a mutant C. glutamicum strain is created by overexpressing the native fructose-6-P-aminotransferase (UniProt ID Q8NND3) to enhance the intracellular glucosamine-6-phosphate pool. Further on, the enzymatic activities of the genes nagA, nagB and gamA are disrupted by genetic knockouts and a glucosamine-6-P-aminotransferase from S. cerevisiae (UniProt ID P43577), an N-acetylglucosamine-2-epimerase from B. ovatus (UniProt ID A7LVG6) and an N-acetylneuraminate synthase from C. jejuni (UniProt ID Q93MP9) are overexpressed on the genome. To allow sialylated oligosaccharide production, the sialic acid producing strain is further modified with expression constructs comprising an N-acylneuraminate cytidylyltransferase like e.g., the NeuA enzyme from C. jejuni (UniProt ID Q93MP7), the NeuA enzyme from H. influenzae (GenBank No. AGV11798.1) or the NeuA enzyme from P. multocida (GenBank No. AMK07891.1), and one or more copies of a beta-galactoside alpha-2,3-sialyltransferase like e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity, NmeniST3 from N. meningitidis (GenBank No. ARC07984.1) or PmultST2 from P. multocida subsp. multocida str. Pm70 (GenBank No. AAK02592.1), a beta-galactoside alpha-2,6-sialyltransferase like e.g., PdST6 from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity or P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 having beta-galactoside alpha-2,6-sialyltransferase activity, and/or an alpha-2,8-sialyltransferase like e.g., from M. musculus (UniProt ID Q64689).

Heterologous and Homologous Expression

Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.

Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.

Cultivation Conditions

A preculture of 96-well microtiter plate experiments was started from a cryovial or a single colony from a TY plate, in 150 μL TY and was incubated overnight at 37° C. on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96-well square microtiter plate, with 400 μL MMsf medium by diluting 400×. Each strain was grown in multiple wells of the 96-well plate as biological replicates. These final 96-well culture plates were then incubated at 37° C. on an orbital shaker at 800 rpm for 72 h, or shorter, or longer. At the end of the cultivation experiment samples were taken from each well to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 15 min at 60° C. before spinning down the cells (=whole broth concentration, intra- and extracellular sugar concentrations, as defined herein).

Also, a dilution of the cultures was made to measure the optical density at 600 nm. The cell performance index or CPI was determined by dividing the oligosaccharide concentrations, e.g., sialyllactose concentrations, measured in the whole broth by the biomass, in relative percentages compared to the reference strain. The biomass is empirically determined to be approximately ⅓rd of the optical density measured at 600 nm.

Example 35. Production of an Oligosaccharide Mixture Comprising LN3, LNT, LNFP-I, 2′-FL and Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified C. glutamicum Host

A C. glutamicum strain is first modified for LN3 production and growth on sucrose by genomic knock-out of the ldh, cgl2645 and nagB genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding the lactose permease (LacY) from E. coli (UniProt ID P02920), native fructose-6-P-aminotransferase (UniProt ID Q8NND3), the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (GenBank: AAM33849.1), the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6). In a next step, the mutant strain is further modified with a genomic knock-in of a constitutive transcriptional unit comprising the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt ID D3QY14) to produce LNT. In a subsequent step, the LNT producing strain is transformed with an expression plasmid comprising constitutive transcriptional units for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) and an alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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 novel strain is evaluated for the production of an oligosaccharide mixture comprising LN3, LNT, LNFP-I, 2′-FL and Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 34. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 36. Production of an Oligosaccharide Mixture Comprising LN3, Sialylated LN3, LNT, 3′-SL, LSTa, 2′-FL and Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified C. glutamicum Host

The C. glutamicum strain modified as described in Example 34 is further modified with a genomic knock-out of the gamA and nagA genes together with genomic knock-ins of constitutive transcriptional units comprising native fructose-6-P-aminotransferase (UniProt ID Q8NND3), a glucosamine-6-P-aminotransferase from S. cerevisiae (UniProt ID P43577), an N-acetylglucosamine-2-epimerase from B. ovatus (UniProt ID A7LVG6) and an N-acetylneuraminate synthase from C. jejuni (UniProt ID Q93MP9). In a next step, the mutant strain is transformed with a compatible expression plasmid comprising constitutive transcriptional units comprising the gene encoding the NeuA enzyme from C. jejuni (UniProt ID Q93MP7) and the gene encoding the beta-galactoside alpha-2,3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3). The novel strain is evaluated for the production of an oligosaccharide mixture comprising LN3, sialylated LN3, LNT, LSTa, LNFP-I, 2′-FL, 3′-SL and Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 34. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 37. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a Modified C. glutamicum Host

A wild-type C. glutamicum strain is first modified with genomic knockouts of the C. glutamicum genes ldh, cgl2645, nagB and glmS, together with genomic knock-ins of constitutive transcriptional units comprising genes encoding the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6), the native fructose-6-P-aminotransferase (UniProt ID Q8NND3), the mutant L-glutamine-D-fructose-6-phosphate aminotransferase glmS*54 from E. coli (differing from the wild-type E. coli glmS protein, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation), the glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), the phosphatase BsAraL from Bacillus subtilis (UniProt ID P94526) and the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt ID D3QY14) to produce LNB. In a next step, the mutant strain is transformed with an expression plasmid comprising constitutive transcriptional units for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) and an alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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 novel strain is evaluated for the production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc in a growth experiment on MMsf medium according to the culture conditions provided in Example 34. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 38. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a Modified C. glutamicum Host

A wild-type C. glutamicum strain is first modified with genomic knockouts of the C. glutamicum genes ldh, cgl2645, nagB and glmS, together with genomic knock-ins of constitutive transcriptional units comprising genes encoding the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6), the 4-epimerase WbpP from P. aeruginosa (UniProt ID Q8KN66), the native fructose-6-P-aminotransferase (UniProt ID Q8NND3), the mutant L-glutamine-D-fructose-6-phosphate aminotransferase glmS*54 from E. coli (differing from the wild-type E. coli glmS protein, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation), the glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), the phosphatase BsAraL from Bacillus subtilis (UniProt ID P94526) and the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt ID D3QY14) to produce LNB. In a next step, the mutant strain is transformed with an expression plasmid comprising constitutive transcriptional units for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) and an alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98; 99, 100, 101 and 102. The novel strain is evaluated for the production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc in a growth experiment on MMsf medium according to the culture conditions provided in Example 34. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 39. Materials and Methods Chlamydomonas reinhardtii

Media

C. reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7.0). The TAP medium uses a 1000×stock Hutner's trace element mix. Hutner's trace element mix consisted of 50 g/L Na₂EDTA·H₂O (Titriplex III), 22 g/L ZnSO₄·7H₂O, 11.4 g/L H₃BO₃, 5 g/L MnCl₂·4H₂O, 5 g/L FeSO₄·7H₂O, 1.6 g/L COCl₂·6H₂O, 1.6 g/L CuSO₄·5H₂O and 1.1 g/L (NH₄)₆MoO₃.

The TAP medium contained 2.42 g/L Tris (tris(hydroxymethyl)aminomethane), 25 mg/L salt stock solution, 0.108 g/L K₂HIPO₄, 0.054 g/L KH₂PO₄ and 1.0 mL/L glacial acetic acid. The salt stock solution consisted of 15 g/L NH₄Cl, 4 g/L MgSO₄·7H₂O and 2 g/L CaCl₂)·2H₂O. As precursor for saccharide synthesis, precursors like e.g., galactose, glucose, fructose, fucose, GlcNAc, LNB and/or LacNAc could be added. Medium was sterilized by autoclaving (121° C., 21′). For stock cultures on agar slants TAP medium was used containing 1% agar (of purified high strength, 1000 g/cm²).

Strains, Plasmids and Mutations

C. reinhardtii wild-type strains 21 gr (CC-1690, wild-type, mt+), 6145C (CC-1691, wild-type, mt−), CC-125 (137c, wild-type, mt+), CC-124 (137c, wild-type, mt−) as available from Chlamydomonas Resource Center (www.chlamycollection.org), University of Minnesota, U.S.A.

Expression plasmids originated from pSI103, as available from Chlamydomonas Resource Center. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation. Suitable promoters for (heterologous) gene expression can be derived from e.g., Scranton et al. (Algal Res. 2016, 15: 135-142). Targeted gene modification (like gene knock-out or gene replacement) can be carried using the Crispr-Cas technology as described e.g., by Jiang et al. (Eukaryotic Cell 2014, 13(11): 1465-1469).

Transformation via electroporation was performed as described by Wang et al. (Biosci. Rep. 2019, 39: BSR2018210). Cells were grown in liquid TAP medium under constant aeration and continuous light with a light intensity of 8000 Lx until the cell density reached 1.0-2.0×10⁷ cells/mL. Then, the cells were inoculated into fresh liquid TAP medium in a concentration of 1.0×10⁶ cells/mL and grown under continuous light for 18-20 h until the cell density reached 4.0×10⁶ cells/mL. Next, cells were collected by centrifugation at 1250 g for 5 min at room temperature, washed and resuspended with pre-chilled liquid TAP medium containing 60 mM sorbitol (Sigma, U.S.A.), and iced for 10 min. Then, 250 μL of cell suspension (corresponding to 5.0×10⁷ cells) were placed into a pre-chilled 0.4 cm electroporation cuvette with 100 ng plasmid DNA (400 ng/mL). Electroporation was performed with 6 pulses of 500 V each having a pulse length of 4 ms and pulse interval time of 100 ms using a BTX ECM830 electroporation apparatus (1575 Ω, 50 FD). After electroporation, the cuvette was immediately placed on ice for 10 min. Finally, the cell suspension was transferred into a 50 ml conical centrifuge tube containing 10 mL of fresh liquid TAP medium with 60 mM sorbitol for overnight recovery at dim light by slowly shaking. After overnight recovery, cells were recollected and plated with starch embedding method onto selective 1.5% (w/v) agar-TAP plates containing ampicillin (100 mg/L) or chloramphenicol (100 mg/L). Plates were then incubated at 23+/−0.5° C. under continuous illumination with a light intensity of 8000 Lx. Cells were analyzed 5-7 days later.

In an example for production of UDP-galactose, C. reinhardtii cells are modified with transcriptional units comprising the genes encoding the galactokinase from Arabidopsis thaliana (KIN, UniProt ID Q9SEE5) and the UDP-sugar pyrophosphorylase (USP) from A. thaliana (UniProt ID Q9C5I1).

In an example for production of UDP-N-acetylgalactosamine, C. reinhardtii cells are modified with a transcriptional unit comprising the UDP-N-acetylglucosamine 4-epimerase wbpP from Pseudomonas aeruginosa serotype 06 (UniProt ID Q8KN66).

In an example for production of LNB, C. reinhardtii cells modified for UDP-galactose production are further modified with an expression plasmid comprising a transcriptional unit for the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (UniProt ID D3QY14). In an example for production of LacNAc, C. reinhardtii cells modified for UDP-galactose production are further modified with an expression plasmid comprising a transcriptional unit for the N-acetylglucosamine beta-1,4-galactosyltransferase LgtB from Neisseria meningitidis (UniProt ID Q51116).

Additionally, the mutant C. reinhardtii cells can be modified with an expression plasmid comprising transcriptional units for an alpha-1,2-fucosyltransferase, an alpha-1,3-fucosyltransferase, an alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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/or an alpha-1,3-N-acetylgalactosaminyltransferase like e.g., SEQ ID NOs:40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102.

In an example for CMP-sialic acid synthesis, C. reinhardtii cells are modified with constitutive transcriptional units for an UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase like e.g., GNE from Homo sapiens (UniProt ID Q9Y223) or a mutant form of the human GNE polypeptide comprising the R263L mutation, an N-acylneuraminate-9-phosphate synthetase like e.g., NANS from Homo sapiens (UniProt ID Q9NR45) and an N-acylneuraminate cytidylyltransferase like e.g., CMAS from Homo sapiens (UniProt ID Q8NFW8). In an example for production of sialylated oligosaccharides, C. reinhardtii cells are modified with a CMP-sialic acid transporter like e.g., CST from Mus musculus (UniProt ID Q61420), and a Golgi-localized sialyltransferase chosen from species like e.g., Homo sapiens, Mus musculus, Rattus norvegicus.

Heterologous and Homologous Expression

Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.

Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.

Cultivation Conditions

Cells of C. reinhardtii were cultured in selective TAP-agar plates at 23+/−0.5° C. under 14/10 h light/dark cycles with a light intensity of 8000 Lx. Cells were analyzed after 5 to 7 days of cultivation.

For high-density cultures, cells could be cultivated in closed systems like e.g., vertical or horizontal tube photobioreactors, stirred tank photobioreactors or flat panel photobioreactors as described by Chen et al. (Bioresour. Technol. 2011, 102: 71-81) and Johnson et al. (Biotechnol. Prog. 2018, 34: 811-827).

Example 40. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc in Mutant C. reinhardtii Cells

C. reinhardtii cells are engineered as described in Example 39 for production of UDP-Gal with genomic knock-ins of constitutive transcriptional units comprising the Arabidopsis thaliana genes encoding the galactokinase (KIN, UniProt ID Q9SEE5) and the UDP-sugar pyrophosphorylase (USP) (UniProt ID Q9C5I1). In a next step, the mutant cells are transformed with an expression plasmid comprising transcriptional units comprising the N-acetylglucosamine beta-1,4-galactosyltransferase LgtB from Neisseria meningitidis (UniProt ID Q51116), the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1) and an alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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 novel strains are evaluated in a cultivation experiment on TAP-agar plates comprising galactose and GlcNAc as precursors according to the culture conditions provided in Example 39. After 5 days of incubation, the cells are harvested, and the production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc is analyzed on UPLC.

Example 41. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc in Mutant C. reinhardtii Cells

C. reinhardtii cells are engineered as described in Example 39 for production of UDP-Gal with genomic knock-ins of constitutive transcriptional units comprising the Arabidopsis thaliana genes encoding the galactokinase (KIN, UniProt ID Q9SEE5) and the UDP-sugar pyrophosphorylase (USP) (UniProt ID Q9C5I1). In a next step, the mutant cells are transformed with an expression plasmid comprising transcriptional units comprising the N-acetylglucosamine beta-1,4-galactosyltransferase LgtB from Neisseria meningitidis (UniProt ID Q51116), the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank: AAD29863.1), the 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) for UDP-GalNAc production, and an alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising SEQ ID NOs:40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 and 102. The novel strains are evaluated in a cultivation experiment on TAP-agar plates comprising galactose and GlcNAc as precursors according to the culture conditions provided in Example 39. After 5 days of incubation, the cells are harvested, and the production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc is analyzed on UPLC.

Example 42. Materials and Methods Animal Cells

Isolation of Mesenchymal Stem Cells from Adipose Tissue of Different Mammals

Fresh adipose tissue is obtained from slaughterhouses (e.g., cattle, pigs, sheep, chicken, ducks, catfish, snake, frogs) or liposuction (e.g., in case of humans, after informed consent) and kept in phosphate buffer saline supplemented with antibiotics. Enzymatic digestion of the adipose tissue is performed followed by centrifugation to isolate mesenchymal stem cells. The isolated mesenchymal stem cells are transferred to cell culture flasks and grown under standard growth conditions, e.g., 370 C, 5% CO₂. The initial culture medium includes DMEM-F12, RPMI, and Alpha-MEM medium (supplemented with 15% fetal bovine serum), and 1% antibiotics. The culture medium is subsequently replaced with 10% FBS (fetal bovine serum)-supplemented media after the first passage. For example, Ahmad and Shakoori (2013, Stem Cell Regen Med. 9(2): 29-36), which is incorporated herein by reference in its entirety for all purposes, describes certain variation(s) of the method(s) described herein in this example.

Isolation of Mesenchymal Stem Cells from Milk

This example illustrates isolation of mesenchymal stem cells from milk collected under aseptic conditions from human or any other mammal(s) such as described herein. An equal volume of phosphate buffer saline is added to diluted milk, followed by centrifugation for 20 min. The cell pellet is washed thrice with phosphate buffer saline and cells are seeded in cell culture flasks in DMEM-F12, RPMI, and Alpha-MEM medium supplemented with 10% fetal bovine serum and 1% antibiotics under standard culture conditions. For example, Hassiotou et al. (2012, Stem Cells. 30(10): 2164-2174), which is incorporated herein by reference in its entirety for all purposes, describes certain variation(s) of the method(s) described herein in this example.

Differentiation of Stem Cells Using 2D and 3D Culture Systems

The isolated mesenchymal cells can be differentiated into mammary-like epithelial and luminal cells in 2D and 3D culture systems. See, for example, Huynh et al. 1991. Exp Cell Res. 197(2): 191-199; Gibson et al. 1991, In Vitro Cell Dev Biol Anim. 27(7): 585-594; Blatchfordetal. 1999; Animal Cell Technology: Basic & Applied Aspects, Springer, Dordrecht. 141-145; Williams et al. 2009, Breast Cancer Res 11(3): 26-43; and Arevalo et al. 2015, Am J Physiol Cell Physiol. 310(5): C348-C356; each of which is incorporated herein by reference in their entireties for all purposes.

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

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

Method of Making Mammary-Like Cells

Mammalian cells are brought to induced pluripotency by reprogramming with viral vectors encoding for Oct4, Sox2, Klf4, and c-Myc. The resultant reprogrammed cells are then cultured in Mammocult media (available from Stem Cell Technologies), or mammary cell enrichment media (DMEM, 3% FBS, estrogen, progesterone, heparin, hydrocortisone, insulin, EGF) to make them mammary-like, from which expression of select milk components can be induced. Alternatively, epigenetic remodeling are performed using remodelling systems such as CRISPR/Cas9, to activate select genes of interest, such as casein, a-lactalbumin to be constitutively on, to allow for the expression of their respective proteins, and/or to down-regulate and/or knock-out select endogenous genes as described e.g., in WO 2021067641, which is incorporated herein by reference in its entirety for all purposes.

Cultivation

Completed growth media includes high glucose DMEM/F12, 10% FBS, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, and 5 pg/ml hydrocortisone. Completed lactation media includes high glucose DMEM/F12, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, 5 pg/ml hydrocortisone, and 1 pg/ml prolactin (5 ug/ml in Hyunh 1991). Cells are seeded at a density of 20,000 cells/cm² onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media. Upon exposure to the lactation media, the cells start to differentiate and stop growing. Within about a week, the cells start secreting lactation product(s) such as milk lipids, lactose, casein and whey into the media. A desired concentration of the lactation media can be achieved by concentration or dilution by ultrafiltration. A desired salt balance of the lactation media can be achieved by dialysis, for example, to remove unwanted metabolic products from the media. Hormones and other growth factors used can be selectively extracted by resin purification, for example, the use of nickel resins to remove His-tagged growth factors, to further reduce the levels of contaminants in the lactated product.

Example 43. Making of Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in a Non-Mammary Adult Stem Cell

Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 42 are modified via CRISPR-CAS to over-express the GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), the human galactoside alpha-1,2-fucosyltransferase FUT1 (UniProt ID P19526), and a codon-optimized alpha-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: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. Cells are seeded at a density of 20,000 cells/cm² onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media for about 7 days. After cultivation as described in Example 42, cells are subjected to UPLC to analyze for production of Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc).

Example 44. Making of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc in a Non-Mammary Adult Stem Cell

Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 42 are modified via CRISPR-CAS to over-express the GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), the human galactoside alpha-1,2-fucosyltransferase FUT1 (UniProt ID P19526), and a codon-optimized alpha-1,3-N-acetylgalactosaminyltransferase chosen from the list comprising SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98; 99, 100, 101 and 102. Cells are seeded at a density of 20,000 cells/cm2 onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media for about 7 days. After cultivation as described in Example 42, cells are subjected to UPLC to analyze for production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc.

Example 45. Making of an Oligosaccharide Mixture Comprising Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, 2′-FL and 3′-SL in a Non-Mammary Adult Stem Cell

Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 42 are modified via CRISPR-CAS to over-express the GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), the human galactoside alpha-1,2-fucosyltransferase FUT1 (UniProt ID P19526), a codon-optimized alpha-1,3-N-acetylgalactosyltransferase chosen from the list comprising SEQ ID NOs: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 N-acylneuraminate cytidylyltransferase from Mus musculus (UniProt ID Q99KK2) and the CMP-N-acetylneuraminate-beta-1,4-galactoside alpha-2,3-sialyltransferase ST3GAL3 from Homo sapiens (UniProt ID Q11203). Cells are seeded at a density of 20,000 cells/cm2 onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media for about 7 days. After cultivation as described in Example 42, cells are subjected to UPLC to analyze for production of 2′-FL, 3-SL and Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc.

Example 46. Evaluation of the Expression of a Membrane Transporter Protein on the Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc in a Modified E. coli Host

The modified E. coli hosts described in Examples 9, 10, 11 and 12 are further modified with by a genomic knock-in of a constitutive transcriptional unit for one heterologous membrane transporter protein chosen from the list comprising MdfA from Cronobacter muytjensii (UniProt ID AOA2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID P0AEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207) and iceT from Citrobacter youngae (UniProt ID D4B8A6). The novel strains, each expressing one of the heterologous membrane transporter proteins, are evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L lactose. Each strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth (i.e., extracellular and intracellular fractions) together with the extracellular and intracellular fractions separately, is harvested and the sugars are analyzed on UPLC.

Example 47. Evaluation of the Expression of a Membrane Transporter in a Modified E. coli Host

The modified E. coli hosts described in Examples 16 and 20 are further modified with by a genomic knock-ins of a constitutive transcriptional unit for one membrane transporter protein chosen from the list comprising MdfA from Cronobacter muytjensii (UniProt ID AOA2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID P0AEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207), iceT from Citrobacter youngae (UniProt ID D4B8A6), nanT from E. coli 06:H1 (UniProt ID Q8FD59), nanT from E. coli O157:H7 (UniProt ID Q8X9G8), nanT from E. albertii (UniProt ID B1EFH1), EntS from E. coli (UniProt ID P24077), EntS from Kluyvera ascorbata (UniProt ID A0A378GQ13) EntS from Salmonella enterica subsp. arizonae (UniProt ID A0A6Y2K4E8), SetA from E. coli (UniProt ID P31675), SetB from E. coli (UniProt ID P33026), SetC from E. coli (UniProt ID P31436), oppF from E. coli (UniProt ID P77737), lmrA from Lactococcus lactis subsp. lactis bv. diacetylactis (UniProt ID A0A1V0NEL4) and Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4). The novel strains, each expressing one of the membrane transporter proteins, are evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose and 20 g/L lactose. Each strain is grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth (i.e., extracellular and intracellular fractions) together with the extracellular and intracellular fractions separately, is harvested and the sugars are analyzed on UPLC.

Claims

1. A method to produce an alpha-1,3 glycosylated form of fucose-alpha-1,2-galactose-R (Fuc-a1,2-Gal-R) by a cell, wherein the alpha-1,3 glycosylation occurs at a terminal “fucose-a1,2-galactose”-group of fucose-alpha-1,2-galactose R (Fuc-a1,2-Gal-R), wherein the method comprises the steps of: i. providing a cell capable to synthesize Fuc-a1,2-Gal-R, expressing an alpha-1,3-glycosyltransferase and capable to synthesize a nucleotide-sugar which is donor for the alpha-1,3-glycosyltransferase, andii. cultivating the cell under conditions permissive to synthesize the Fuc-a1,2-Gal-R, to express the alpha-1,3-glycosyltransferase, to synthesize the nucleotide-sugar and to synthesize the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, andiii. optionally separating the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R from the cultivation.

2. Method according to claim 1, wherein the galactose (Gal) residue within the Fuc-a1,2-Gal-R is bound to R via a beta-1,3 or a beta-1,4 glycosidic linkage.

3. Method according to claim 1, wherein the R comprises a monosaccharide, a disaccharide, an oligosaccharide, a peptide, a protein, a glycopeptide, a glycoprotein, a lipid or a glycolipid.

4. Method according to claim 1, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-R, Fuc-a1,2-Gal-b1,3-GlcNAc-R, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,4-R, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R, lacto-N-fucopentaose I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), Fuc-a1,2-Gal-b1,4-R, Fuc-a1,2-Gal-b1,4-Glc or Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc.

5.-7. (canceled)

8. Method according to claim 1, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is: a structure of the histo blood group antigen (HBGA) system,an alpha-1,3 glycosylated form of lacto-N-fucopentaose I (LNFP-I) which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (Gal-a1,3-LNFP-1), the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase and the nucleotide-sugar is UDP-galactose (UDP-Gal),an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-Glc which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, optionally an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc, the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase and the nucleotide-sugar is UDP-galactose (UDP-Gal),an alpha-1,3 glycosylated form of lacto-N-fucopentaose I (LNFP-I) which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (GalNAc-a1,3-LNFP-I), the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase and the nucleotide-sugar is UDP-N-acetylgalactosamine (UDP-GalNAc), oran alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-Glc which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc (alpha-tetrasaccharide or A-tetrasaccharide), optionally an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc, the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase and the nucleotide-sugar is UDP-N-acetylgalactosamine (UDP-GalNAc).

9. Method according to claim 1, wherein the alpha-1,3-glycosyltransferase is i) an alpha-1,3-galactosyltransferase which is a glycosyltransferase with the ability to transfer a galactose (Gal) residue from UDP-galactose (UDP-Gal) to the terminal “fucose-a1,2-galactose” group of:a. fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R),b. LNFP-I, orc. Fuc-a1,2-Gal-b1,4-Glc, optionally the glucose residue in the Fuc-a1,2-Gal-b1,4-Glc is fucosylated;orii) an alpha-1,3-N-acetylgalactosaminyltransferase which is a glycosyltransferase with the ability to transfer an N-acetylgalactosamine (GalNAc) residue from UDP-N-acetylgalactosamine (UDP-GalNAc) to the terminal “fucose-a1,2-galactose” group of:a. fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R),b. LNFP-I, orc. Fuc-a1,2-Gal-b1,4-Glc, optionally the glucose residue in the Fuc-a1,2-Gal-b1,4-Glc is fucosylated.

10.-13. (canceled)

14. Method according to claim 9, wherein the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain, and a. comprises the motif YX[FHMQT]XAXX[ACG][ACG] with SEQ ID NO: 01 wherein X can be any amino acid residue, orb. comprises the motif YXQXCXX[ACG][ACG] with SEQ ID NO: 02 wherein X can be any amino acid residue, orc. comprises a polypeptide sequence according to any one of SEQ ID NOs: 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, ord. is a functional homolog, variant or derivative of any one of SEQ ID NOs: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 having at least 80% overall sequence identity to the full length of any one of the a-1,3-galactosyltransferase polypeptide with SEQ ID NOs: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 having a-1,3-galactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R), ore. is a functional fragment comprising an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs: 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 having a-1,3-galactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R).

15.-19. (canceled)

20. Method according to claim 9, wherein the alpha-1,3-N-acetylgalactosaminyltransferase has a PFAM PF03414 domain, and a. comprises the motif YX[ACIL]XGXX[ACG][ACG] with SEQ ID NO: 38 wherein X can be any amino acid residue, orb. comprises the motif YX[AG]XAXX[ACG][ACG] with SEQ ID NO: 39 wherein X can be any amino acid residue, orc. comprises a polypeptide sequence according to any one of SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102, ord. is a functional homolog, variant or derivative of any one of SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 having at least 80% overall sequence identity to the full length of any one of the a-1,3-N-acetylgalactosyltransferase polypeptide with SEQ ID NOs:40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and having a-1,3-N-acetylgalactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R), ore. is a functional fragment comprising an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs:40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and having a-1,3-N-acetylgalactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R).

21. Method according to claim 4, wherein the LNFP-I is synthesized in the cell by the transfer of fucose from GDP-fucose to a terminal galactose residue of lacto-N-tetraose (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) by action of a glycosyltransferase which is: a. an alpha-1,2-fucosyltransferase selected from the group consisting of polypeptides from Brachyspira pilosicoli with UniProt ID AOA2N5RQ26, Dysgonomonas mossii with UniProt ID F8X274, Dechlorosoma suillum with UniProt ID G8QLF4, Desulfovibrio alaskensis with UniProt ID Q316B5 and Polaribacter vadi with UniProt ID AOA1B8TNT0, orb. a functional fragment of any one of the polypeptide from B. pilosicoli (UniProt ID AOA2N5RQ26), the polypeptide from D. mossii (UniProt ID F8X274), the polypeptide from D. suillum (UniProt ID G8QLF4), the polypeptide from D. alaskensis (UniProt ID Q316B5) and the polypeptide from P. vadi (UniProt ID AOA1B8TNT0) having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of LNT, orc. a functional homolog, variant or derivative of any one of the polypeptides from B. pilosicoli with UniProt ID AOA2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, D. alaskensis with UniProt ID Q316B5 and P. vadi with UniProt ID AOA1B8TNT0 having at least 80% overall sequence identity to the full length of any one of the polypeptides from B. pilosicoli with UniProt ID AOA2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, D. alaskensis with UniProt ID Q316B5 and P. vadi with UniProt ID AOA1B8TNT0, respectively, and having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of lacto-N-tetraose (LNT), ord. a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any one of the polypeptide from B. pilosicoli (UniProt ID AOA2N5RQ26), the polypeptide from D. mossii (UniProt ID F8X274), the polypeptide from D. suillum (UniProt ID G8QLF4), the polypeptide from D. alaskensis (UniProt ID Q316B5) or the polypeptide from P. vadi (UniProt ID AOA1B8TNT0) and having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of LNT.

22. Method according to claim 1, wherein said cell is modified in the expression or activity of a glycosyltransferase.

23. Method according to claim 1, wherein the cell has a cell wall and expresses a membrane transporter protein or a polypeptide having transport activity, wherein the membrane transporter protein or the polypeptide having transport activity: transports compounds across an outer membrane of the cell wall,is a porter, a P-P-bond-hydrolysis-driven transporter, a β-barrel porin, an auxiliary transport protein, a putative transport protein or a phosphotransfer-driven group translocator,controls the flow over the outer membrane of the cell wall of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R and/or of one or more precursor(s) and/or acceptor(s) to be used in the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, and/orprovides improved production and/or enabled and/or enhanced efflux of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.

24.-26. (canceled)

27. Method according to claim 1, wherein the cell is a metabolically engineered cell.

28.-29. (canceled)

30. Method according to claim 27, wherein the cell: comprises a modification for reduced production of acetate,a lower or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the proteins comprising beta-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose undecaprenyl-phosphate glucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridylyltransferase, glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IcIR, Ion protease, glucose-specific translocating phosphotransferase enzyme IIBC component ptsG, glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIAGlc, beta-glucoside-specific PTS enzyme II, fructose-specific PTS multiphosphoryl transfer protein FruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, or pyruvate decarboxylase,is capable to produce phosphoenolpyruvate (PEP), and/oris modified for enhanced production and/or supply of phosphoenolpyruvate (PEP).

31.-33. (canceled)

34. Method according to claim 1, wherein the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.

35. Method according to claim 1, wherein the cell resists a phenomenon of lactose killing when grown in an environment in which lactose is combined with one or more other carbon source(s).

36.-37. (canceled)

38. Method according to claim 1, wherein the conditions comprise: (i) use of a culture medium comprising at least one precursor and/or acceptor for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, and/or(ii) adding to the culture medium at least one precursor and/or acceptor feed for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.

39.-43. (canceled)

44. Method according to claim 1, wherein the cell is cultivated in culture medium, comprising a carbon source comprising a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract, and/orcontaining at least one precursor comprising lactose, galactose, fucose, sialic acid, GlcNAc, GalNAc, lacto-N-biose (LNB), or N-acetyllactosamine (LacNAc).

45. (canceled)

46. Method according to claim 44, wherein a first phase of exponential cell growth is provided by adding a carbon-based substrate to the culture medium comprising the at least one precursor, followed by a second phase wherein: only a carbon-based substrate is added to the culture medium, ora carbon-based substrate and a precursor are added to the culture medium.

47. (canceled)

48. Method according to claim 1, wherein the cell produces: a mixture of charged and/or neutral, di- and oligosaccharides comprising an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R ora mixture of charged and/or neutral oligosaccharides comprising an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.

49. (canceled)

50. A cell metabolically engineered for producing an alpha-1,3 glycosylated form of fucose-alpha-1,2-galactose-R (Fuc-a1,2-Gal-R), wherein the alpha-1,3 glycosylation occurs at a terminal “fucose-a1,2-galactose”-group of fucose-alpha-1,2-galactose R (Fuc-a1,2-Gal-R), and wherein the cell synthesizes Fuc-a1,2-Gal-R, andexpresses an alpha-1,3-glycosyltransferase, andis capable to produce a nucleotide-sugar, wherein the nucleotide-sugar is donor for the alpha-1,3-glycosyltransferase.

51. Cell according to claim 50, wherein the galactose (Gal) residue within the Fuc-a1,2-Gal-R is bound to R via a beta-1,3 or a beta-1,4 glycosidic linkage.

52. Cell according to claim 50, wherein the R comprises a monosaccharide, a disaccharide, an oligosaccharide, a peptide, a protein, a glycopeptide, a glycoprotein, a lipid or a glycolipid.

53. Cell according to claim 50, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-R, Fuc-a1,2-Gal-b1,3-GlcNAc-R, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R, lacto-N-fucopentaose I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), Fuc-a1,2-Gal-b1,4-R, Fuc-a1,2-Gal-b1,4-Glc, or Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc.

54. Cell according to claim 53, wherein the N-acetylglucosamine (GIcNAc) residue within the Fuc-a1,2-Gal-b1,3-GlcNAc-R is bound to R via a beta-1,3 or a beta-1,4 glycosidic linkage.

55.-56. (canceled)

57. Cell according to claim 50, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is: a structure of the histo blood group antigen (HBGA) system,an alpha-1,3 glycosylated form of lacto-N-fucopentaose I (LNFP-I) which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (Gal-a1,3-LNFP-I), the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase and the nucleotide-sugar is UDP-galactose (UDP-Gal),an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-Glc which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, optionally an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc, the alpha-1,3-glycosyltransferase is an alpha-1,3-galactosyltransferase and the nucleotide-sugar is UDP-galactose (UDP-Gal),an alpha-1,3 glycosylated form of lacto-N-fucopentaose I (LNFP-I) which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (GalNAc-a1,3-LNFP-I), the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase and the nucleotide-sugar is UDP-N-acetylgalactosamine (UDP-GalNAc), oran alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-Glc which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc (alpha-tetrasaccharide or A-tetrasaccharide), optionally an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc, the alpha-1,3-glycosyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase and the nucleotide-sugar is UDP-N-acetylgalactosamine (UDP-GalNAc).

58. Cell according to claim 50, wherein said alpha-1,3-glycosyltransferase is: i) an alpha-1,3-galactosyltransferase which is a glycosyltransferase with the ability to transfer a galactose (Gal) residue from UDP-galactose (UDP-Gal) to the terminal “fucose-a1,2-galactose” group of:a. fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R),b. LNFP-I, orc. Fuc-a1,2-Gal-b1,4-Glc, optionally the glucose residue in the Fuc-a1,2-Gal-b1,4-Glc is fucosylated;orii) an alpha-1,3-N-acetylgalactosaminyltransferase which is a glycosyltransferase with the ability to transfer an N-acetylgalactosamine (GalNAc) residue from UDP-N-acetylgalactosamine (UDP-GalNAc) to the terminal “fucose-a1,2-galactose” group of:a. fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R),b. LNFP-I, orc. Fuc-a1,2-Gal-b1,4-Glc, optionally the glucose residue in the Fuc-a1,2-Gal-b1,4-Glc is fucosylated.

59.-62. (canceled)

63. Cell according to claim 58, wherein the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain, and a. comprises the motif YX[FHMQT]XAXX[ACG][ACG] with SEQ ID NO: 01 wherein X can be any amino acid residue, orb. comprises the motif YXQXCXX[ACG][ACG] with SEQ ID NO 02 wherein X can be any amino acid residue, orc. comprises a polypeptide sequence according to any one of SEQ ID NOs: 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, ord. is a functional homolog, variant or derivative of any one of SEQ. ID NOs: 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 having at least 80% overall sequence identity to the full length of any one of the a-1,3-galactosyltransferase polypeptide with SEQ ID NOs: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 having a-1,3-galactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R), ore. is a functional fragment comprising an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs: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 having a-1,3-galactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R).

64.-68. (canceled)

69. Cell according to claim 58, wherein the alpha-1,3-N-acetylgalactosaminyltransferase has a PFAM PF03414 domain, and a. comprises the motif YX[ACIL]XGXX[ACG][ACG] with SEQ ID NO: 38 wherein X can be any amino acid residue, orb. comprises the motif YX[AG]XAXX[ACG][ACG] with SEQ ID NO: 39 wherein X can be any amino acid residue, orc. comprises a polypeptide sequence according to any one of SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102, ord. is a functional homolog, variant or derivative of any one of SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 having at least 80% overall sequence identity to the full-length of any one of the a-1,3-N-acetylgalactosyltransferase polypeptide with SEQ ID NOs:40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and having a-1,3-N-acetylgalactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R), ore. is a functional fragment comprising an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs:40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and having a-1,3-N-acetylgalactosyltransferase activity on the terminal “fucose-a1,2-galactose” group of fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R).

70. Cell according to claim 53, wherein the LNFP-I is synthesized in the cell by the transfer of fucose from GDP-fucose to a terminal galactose residue of lacto-N-tetraose (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) by action of a glycosyltransferase which is: a. an alpha-1,2-fucosyltransferase selected from the group consisting of polypeptides from Brachyspira pilosicoli with UniProt ID AOA2N5RQ26, Dysgonomonas mossii with UniProt ID F8X274, Dechlorosoma suillum with UniProt ID G8QLF4, Desulfovibrio alaskensis with UniProt ID Q316B5 and Polaribacter vadi with UniProt ID AOA1B8TNT0, orb. a functional fragment of any one of the polypeptide from B. pilosicoli (UniProt ID AOA2N5RQ.26), the polypeptide from D. mossii (UniProt ID F8X274), the polypeptide from D. suillum (UniProt ID G8QLF4), the polypeptide from D. alaskensis (UniProt ID Q.316B5) and the polypeptide from P. vadi (UniProt ID AOA1B8TNT0) having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of LNT, orc. a functional homolog, variant or derivative of any one of the polypeptides from B. pilosicoli with UniProt ID AOA2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, D. alaskensis with UniProt ID Q316B5 and P. vadi with UniProt ID AOA1B8TNT0 having at least 80% overall sequence identity to the full length of any one of the polypeptides from B. pilosicoli with UniProt ID AOA2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, D. alaskensis with UniProt ID Q316B5 and P. vadi with UniProt ID AOA1B8TNT0, respectively, and having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of lacto-N-tetraose (LNT), ord. a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any one of the polypeptide from B. pilosicoli (UniProt ID AOA2N5RQ26), the polypeptide from D. mossii (UniProt ID F8X274), the polypeptide from D. suillum (UniProt ID G8QLF4), the polypeptide from D. alaskensis (UniProt ID Q316B5) or the polypeptide from P. vadi (UniProt ID AOA1B8TNT0) and having alpha-1,2-fucosyltransferase activity on the terminal galactose residue of LNT.

71. Cell according to claim 50, wherein the cell is modified in the expression or activity of a glycosyltransferase.

72. Cell according to claim 50, wherein the cell has a cell wall and expresses a membrane transporter protein or a polypeptide having transport activity, wherein the membrane transporter protein or the polypeptide having transport activity: transports compounds across an outer membrane of the cell wall,is a porter, a P-P-bond-hydrolysis-driven transporter, a β-barrel porin, an auxiliary transport protein, a putative transport protein or a phosphotransfer-driven group translocator,controls the flow over the outer membrane of the cell wall of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R and/or of one or more precursor(s) and/or acceptor(s) to be used in the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, and/orprovides improved production and/or enabled and/or enhanced efflux of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.

73.-77. (canceled)

78. Cell according to claim 50, wherein the cell: comprises a modification for reduced production of acetate,comprises a lower or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the proteins comprising beta-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose undecaprenyl-phosphate glucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridylyltransferase, glucose-I-phosphate adenylyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IcIR, Ion protease, glucose-specific translocating phosphotransferase enzyme IIBC component ptsG, glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIAGlc, beta-glucoside-specific PTS enzyme II, fructose-specific PTS multiphosphoryl transfer protein FruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, or pyruvate decarboxylase,is capable to produce phosphoenolpyruvate (PEP), and/oris modified for enhanced production and/or supply of phosphoenolpyruvate (PEP).

79.-81. (canceled)

82. Cell according to claim 50, wherein the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.

83. Cell according to claim 50, wherein the cell resists a phenomenon of lactose killing when grown in an environment in which lactose is combined with one or more other carbon source(s).

84. (canceled)

85. Cell according to claim 50, wherein the cell produces: a mixture of charged and/or neutral, di- and oligosaccharides comprising an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, ora mixture of charged and/or neutral oligosaccharides comprising an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.

86. (canceled)

87. Method according to claim 1, wherein the cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell.

88. Method according to claim 87, wherein the bacterium is: an Escherichia coli strain, and/ora viable Gram-negative bacterium that comprises a reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans (OPG), Glucosyl glycerol, glycan, and/or trehalose.

89. (canceled)

90. Method according to claim 87, wherein the yeast belongs to a genus selected from the group consisting of Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces or Debaromyces.

91. Method according to claim 1, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography.

92. Method according to claim 1 further comprising purification of any one of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R from the cell.

93. Method according to claim 92, wherein the purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying or vacuum roller drying.

94. A method to produce an alpha-1,3 glycosylated form of fucose-alpha-1,2-galactose-R (Fuc-a1,2-Gal-R) by a cell, wherein the alpha-1,3 glycosylation occurs at a terminal “fucose-a1,2-galactose”-group of fucose-alpha-1,2-galactose R (Fuc-a1,2-Gal-R), wherein the method comprises the steps of: i. cultivating a cell according to claim 50 under conditions permissive to synthesize the Fuc-a1,2-Gal-R, to express the alpha-1,3-glycosyltransferase, to synthesize the nucleotide-sugar and to synthesize the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, andii. optionally separating the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R from the cultivation.

95. The cell of claim 50, wherein the cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell.

96. The cell of claim 95, wherein the bacterium: is an Escherichia coli strain, and/oris a viable Gram-negative bacterium that comprises a reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose.

97. The cell of claim 95, wherein the cell is a yeast belonging to a genus selected from the group comprising Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces or Debaromyces.