PRODUCTION OF GALACTOSYLATED DI- AND OLIGOSACCHARIDES

The disclosure is in the technical field of synthetic biology and metabolic engineering. Described is the use of a new type of galactosyltransferases for the production of a galactosylated di- or oligosaccharide. The disclosure also describes methods for the production of a galactosylated di- or oligosaccharide as well as the purification of the di- or oligosaccharide. Furthermore, the disclosure is in the field of cultivation or fermentation of metabolically engineered cells. The disclosure provides a cell metabolically engineered for production of a galactosylated di- or oligosaccharide.

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Description
CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International Patent Application PCT/EP2021/072270, filed Aug. 10, 2021, designating the United States of America and published as International Patent Publication WO 2022/034076 A2 on Feb. 17, 2022, which claimed the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial Nos. 21186202.4 and 21186203.2 filed Jul. 16, 2021; to European Patent Application Serial No. 21168997.1 filed Apr. 16, 2021; and to European Patent Application Serial Nos. 20190198.0, 20190200.4, 20190201.2, 20190202.0, 20190203.8, 20190204.6, 20190205.3, 20190206.1, 20190207.9, and 20190208.7, all filed on Aug. 10, 2020, the disclosure of each of which is hereby incorporated herein in its entirety by this reference.

TECHNICAL FIELD

The application is in the technical field of synthetic biology and metabolic engineering. Described is the use of a new type of galactosyltransferases for the production of a galactosylated di- or oligosaccharide. Also described are methods for producing a galactosylated di- or oligosaccharide as well as the purification of the di- or oligosaccharide. Furthermore, the disclosure is in the field of cultivation or fermentation of metabolically engineered cells. The disclosure provides a cell metabolically engineered for production of a galactosylated di- or oligosaccharide.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

Pursuant to 37 C.F.R. § 1.821, a Sequence Listing ASCII text file entitled “4006-P17268US_ST26.xml,” 102,741 bytes in size, generated Feb. 10, 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.

BACKGROUND

Disaccharides and 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)). There is large scientific and commercial interest in galactosylated di- and oligosaccharides due to the wide functional spectrum of these saccharides. Yet, the availability of galactosylated di- and oligosaccharides 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 using glycosyltransferases offer many advantages above chemical synthesis. Glycosyltransferases catalyze the transfer of a sugar moiety from an activated nucleotide-sugar donor onto saccharide or non-saccharide acceptors (Coutinho et al., J. Mol. Biol. 328 (2003) 307-317). These glycosyltransferases are the source for biotechnologists to synthesize oligosaccharides and are used both in (chemo)enzymatic approaches as well as in cell-based production systems. However, stereospecificity and regioselectivity of glycosyltransferases and of galactosyltransferases, as part of the glycosyltransferase family, are still a formidable challenge. In addition, chemo-enzymatic approaches need to regenerate in situ nucleotide-sugar donors. Cellular production of di- and oligosaccharides needs tight control of spatiotemporal availability of adequate levels of nucleotide-sugar donors in proximity of complementary glycosyltransferases.

BRIEF SUMMARY

Provided are tools and methods by means of which a galactosylated di- or oligosaccharide can be produced in an efficient, time and cost-effective way and if needed, continuous process.

Provided is the use of a new type of galactosyltransferase for the production of a galactosylated di- or oligosaccharide, methods and a cell for the production of a galactosylated di- or oligosaccharide, wherein the cell is genetically modified for the production of the galactosylated di- or oligosaccharide.

Surprisingly, it has now been found that it is possible to produce a galactosylated di- or oligosaccharide with a new type of galactosyltransferases, more specifically a new type of N-acetylglucosamine b-1,X-galactosyltransferases. The disclosure provides use of a new type of N-acetylglucosamine b-1,3-galactosyltransferases and N-acetylglucosamine b-1,4-galactosyltransferases that galactosylate acceptors like an N-acetylglucosamine and/or N-acetylgalactosamine as a monosaccharide and/or that galactosylate acceptors like an N-acetylglucosamine and/or N-acetylgalactosamine as part of a di- and/or oligosaccharide at the non-reducing end of the oligosaccharide. The disclosure provides the use of the N-acetylglucosamine b-1,X-galactosyltransferases to produce a galactosylated di- or oligosaccharide. The disclosure provides methods and a cell for the production of a galactosylated di- or oligosaccharide by using the galactosyltransferases. The methods comprise providing UDP-galactose and any one of the new galactosyltransferases, and contacting any one of the galactosyltransferases and UDP-galactose with one or more acceptor(s), under conditions where the galactosyltransferase catalyzes the galactosylation of the acceptor(s). One method comprises the steps of providing a cell that is capable of synthesizing UDP-galactose and any one or more of the acceptor(s) and that is capable of expressing any one of the galactosyltransferases capable of galactosylating the acceptors, and cultivation of the cell under conditions permissive for producing the galactosylated di- or oligosaccharide. Next, the disclosure also provides methods to separate the galactosylated di- or oligosaccharide. Furthermore, the disclosure provides a cell metabolically engineered for production of a galactosylated di- or oligosaccharide.

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 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 application, 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 element 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 application, 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.”

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 EP20190198, EP20190200 and EP20190207, is also incorporated by reference to the same extent as if the priority application was 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 phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. 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 that 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 that 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 terms “cell genetically modified for the production of a galactosylated di- or oligosaccharide” within the context of the disclosure refers to a cell of a microorganism that is genetically modified in the expression or activity of one or more enzyme(s) selected from the group comprising: glucosamine 6-phosphate N-acetyltransferase, phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase.

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 that originates from outside the cell under study and not a natural part of the cell or that 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 (e.g., the usage of siRNA, CrispR, CrispRi, riboswitches, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutating genes, knocking-out genes, transposon mutagenesis, etc.) that 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 (e.g., 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 that result in regulated expression or a repressible promoter that results in regulated expression. Overexpression or expression is obtained by means of common well-known technologies for a skilled person (e.g., 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 a “transcriptional unit” 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 application, unless explicitly stated otherwise, the expressions “capable of . . . <verb>” and “capable to . . . <verb>” may be 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 that 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 vivo activity as the original polypeptide as judged by any of a number of criteria, including but not limited to enzymatic activity, and that 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 proteins 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 in order 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, 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 that retains a usable, functional characteristic (e.g., activity) of the full-length polynucleotide molecule that 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%, more preferably at least 87%, even more preferably at least 90%, even more preferably at least 95%, most preferably at least 97%, of the full-length of the polynucleotide SEQ ID NO (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%, even more preferably no more than 10%, even more preferably no more than 5%, 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.

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

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 that 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 that 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%, more preferably at least 87%, even more preferably at least 90%, even more preferably at least 95%, most preferably at least 97% of the full-length of the polypeptide SEQ ID NO (or UniProt ID or GenBank NO.) and that 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 that 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 (or UniProt ID 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 polypeptide SEQ ID NO (or UniProt ID or GenBank NO.) and that 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 that can be routinely assessed by the skilled person.

Throughout the application, the sequence of a polypeptide can be represented by a SEQ ID NO or alternatively by a 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 that have substantially no effect on the polypeptide's activity. By “conservative substitutions” is intended substitution 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) or Conserved Domain Database (CDD) (ncbi.nlm.nih.gov/cdd) (Lu et al., Nucleic Acids Res. 48 (2020) D265-D268) designation. 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. The PFAM database (pfam.xfam.org/) used herein was Pfam version 33.1 released on Jun. 11, 2020. Protein 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) (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 a UniProt ID are referred herein using the respective GenBank Accession number (GenBank No.) as present in the NIH genetic sequence database (ncbi.nlm.nih.gov/genbank/) (Nucleic Acids Res. 2013, 41(D1), D36-D42) version of 5 May 2021.

In the disclosure, polypeptide sequence stretches are being used to refer to fragments of the galactosyltransferases used in the disclosure that are common to those galactosyltransferases. 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 (Xn) refers to a stretch of a protein sequence consisting of a number n of the amino acid residue X wherein each X is any amino acid possible and wherein n is 2, 3, 4 or more. The term (Xm) refers to a stretch of a protein sequence consisting of a number m of the amino acid residue X wherein each X is any amino acid possible and wherein m is 2, 3, 4 or more. The term (Xp) refers to a stretch of a protein sequence consisting of a number p of the amino acid residue X wherein each X is any amino acid possible and wherein p is 2, 3, 4 or more.

The term “[X, no A, G or S]” refers to any amino acid excluding the amino acid residues alanine (A), glycine (G) or serine (S). The term “[X, no F, H, W or Y]” refers to any amino acid excluding the amino acid residues phenylalanine (F), histidine (H), tryptophan (W) and tyrosine (Y). The term “[X, no V]” refers to any amino acid except for a valine (V).

The terms “nucleotide-sugar” or “activated sugar” as used herein refer to activated forms of monosaccharides. Examples of activated monosaccharides include but are not limited to UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-fucose (GDP-Fuc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy-L-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-glucuronate, UDP-galacturonate, GDP-rhamnose, or UDP-xylose. Nucleotide-sugars act as glycosyl donors in glycosylation reactions. Those reactions are catalyzed by a group of enzymes called glycosyltransferases.

The term “N-acetylglucosamine b-1,X-galactosyltransferase” as used in the disclosure refers to an N-acetylglucosamine b-1,3-galactosyltransferase or an N-acetylglucosamine b-1,4-galactosyltransferase that transfers a galactosyl residue from UDP-galactose to an acceptor in a beta-1,3 or beta-1,4 linkage, respectively.

The terms “N-acetylglucosamine b-1,3-galactosyltransferase,” “N-acetylglucosamine beta-1,3-galactosyltransferase,” “N-acetylglucosamine beta 1,3 galactosyltransferase,” “N-acetylglucosamine β-1,3-galactosyltransferase,” “N-acetylglucosamine β1,3 galactosyltransferase” as used in the disclosure, are used interchangeably and refer to a galactosyltransferase that catalyzes the transfer of galactose from the donor substrate UDP-galactose, to an acceptor in a beta-1,3 glycosidic linkage. A polynucleotide encoding an “N-acetylglucosamine b-1,3-galactosyltransferase” or any of the above terms, refers to a polynucleotide encoding such glycosyltransferase that catalyzes the transfer of galactose from the donor substrate UDP-galactose, to an acceptor in a beta-1,3 glycosidic linkage.

The terms “N-acetylglucosamine b-1,4-galactosyltransferase,” “N-acetylglucosamine beta-1,4-galactosyltransferase,” “N-acetylglucosamine beta 1,4 galactosyltransferase,” “N-acetylglucosamine β-1,4-galactosyltransferase,” “N-acetylglucosamine β1,4 galactosyltransferase” as used in the disclosure, are used interchangeably and refer to a galactosyltransferase that catalyzes the transfer of galactose from the donor substrate UDP-galactose, to an acceptor in a beta-1,4 glycosidic linkage. A polynucleotide encoding an “N-acetylglucosamine b-1,4-galactosyltransferase” or any of the above terms, refers to a polynucleotide encoding such glycosyltransferase that catalyzes the transfer of galactose from the donor substrate UDP-galactose, to an acceptor in a beta-1,4 glycosidic linkage.

The term “acceptor” as used herein refers to the monosaccharide N-acetylglucosamine (GlcNAc), the monosaccharide N-acetylgalactosamine (GalNAc), and/or an N-acetylglucosamine residue and/or an N-acetylgalactosamine residue as part of a di- and/or oligosaccharide at the non-reducing end of the di- and/or oligosaccharide, that is modified by any one of N-acetylglucosamine b-1,X-galactosyltransferases of the disclosure. Examples of the di- and/or oligosaccharides containing an N-acetylglucosamine and/or N-acetylgalactosamine at the non-reducing end include, but are not limited to, GlcNAc-b1,3-Glc, GlcNAc-b1,4-Glc, GalNAc-b1,3-Glc, GalNAc-b1,4-Glc, GlcNAc-b1,3-Gal-b1,4-Glc (lacto-N-triose, LN3), GalNAc-a1,3-Gal-b1,4-Glc (3′-GalNAcL), Gal-b1,4-GlcNAc-b1,6-(GlcNAc-b1,3)-Gal-b1,4-Glc, GalNAc-b1,3-Gal-b1,4-Glc (b3′-GalNAcL), GalNAc-b1,4-GlcNAc (LacdiNAc), GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, GalNAc-b1,4-Glc, Neu5Ac-a2,6-(GlcNAc-b1,3)-Gal-b1,4-Glc (6′-sialylated LN3), Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,6-(GlcNAc-b1,3)-Gal-b1,4-Glc, GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc, GalNAc-b1,4-(Neu5Ac-a2,3)-Gal-b1,4-Glc, GlcNAc-b1,6-(Gal-b1,3)-Gal-b1,4-Glc (Novo-LNT), lacto-N-pentaose (LNP), lacto-N-neopentaose, para lacto-N-pentaose, para lacto-N-neopentaose, lacto-N-novopentaose I, lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose.

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), Neu5Ac-a2,3-Gal, Neu5Ac-a2,6-Gal and fucopyranosyl-(1-4)-N-glycolylneuraminic acid (Fuc-(1-4)-Neu5Gc).

“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 disaccharides and 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 term “reducing end of a saccharide” as used in the disclosure, refers to the free anomeric carbon that is available in the saccharide to reduce another compound.

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, a sialic 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 “galactosylated disaccharide” as used in the disclosure comprises Gal-b1,3-GlcNAc, Gal-b1,4-GlcNAc, Gal-b1,3-GalNAc and Gal-b1,4-GalNAc wherein galactose is linked to an N-acetylglucosamine (GlcNAc) or to an N-acetylgalactosamine (GalNAc), respectively, in a beta-1,3-linkage or a beta-1,4-linkage, and wherein the N-acetylglucosamine or N-acetylgalactosamine is positioned at the reducing end of the disaccharide and galactose at the terminal non-reducing end of the disaccharide.

The terms “Gal-b1,3-GlcNAc,” “Gal-beta-1,3-GlcNAc,” “Gal-β1,3-GlcNAc,” “Galb1, 3GlcNAc,” “Galβ1,3GlcNAc,” “lacto-N-biose,” “LNB,” “LacNAc type I,” “type 1 LacNAc,” “LacNAc (I)” are used interchangeably and refer to a disaccharide wherein galactose is linked to an N-acetylglucosamine in a beta-1,3-linkage, and wherein N-acetylglucosamine is positioned at the reducing end of the disaccharide.

The terms “Gal-b1,4-GlcNAc,” “Gal-beta-1,4-GlcNAc,” “Gal-β1,4-GlcNAc,” “Galb1,4GlcNAc,” “Galβ1,4GlcNAc,” “N-acetyllactosamine,” “LacNAc,” “LacNAc type II,” “type 2 LacNAc,” “LacNAc (II)” are used interchangeably and refer to a disaccharide wherein galactose is linked to an N-acetylglucosamine in a beta-1,4-linkage, and wherein N-acetylglucosamine is positioned at the reducing end of the disaccharide.

The terms “Gal-b1,3-GalNAc,” “Gal-beta-1,3-GalNAc,” “Gal-β1,3-GlcNAc,” “Galb1,3GalNAc,” “Galβ1,3GalNAc” and “T-disaccharide” are used interchangeably and refer to a disaccharide wherein galactose is linked to an N-acetylgalactosamine in a beta-1,3-linkage, and wherein N-acetylgalactosamine is positioned at the reducing end of the disaccharide.

The terms “Gal-b1,4-GalNAc,” “Gal-beta-1,4-GalNAc,” “Gal-β1,4-GalNAc,” “Galb1,4GalNAc,” “Galβ1,4GalNAc” are used interchangeably and refer to a disaccharide wherein galactose is linked to an N-acetylgalactosamine in a beta-1,4-linkage, and wherein N-acetylgalactosamine is positioned at the reducing end of the disaccharide.

The term “galactosylated oligosaccharide” as used in the disclosure refers to an oligosaccharide built of three to twenty monosaccharide units, wherein a terminal non-reducing galactose is linked to an N-acetylglucosamine or an N-acetylgalactosamine of the oligosaccharide in a beta-1,3 or beta-1,4 linkage. 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,” “β-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 terms “glucosamine 6-phosphate N-acetyltransferase,” “glucosamine-phosphate N-acetyltransferase,” “GNA,” “GNA1,” “glucosamine-6P N-acetyltransferase,” “GlcN6P N-acetyltransferase” as used in the disclosure, are used interchangeably and refer to an enzyme that catalyzes the transfer of an acetyl group from acetyl-CoA to the primary amine in glucosamine-6-phosphate, generating N-acetyl-D-glucosamine-6-phosphate, the latter also known as GlcNAc-6P. A polynucleotide encoding a “glucosamine 6-phosphate N-acetyltransferase” or any of the above terms, refers to a polynucleotide encoding such an enzyme that catalyzes the transfer of an acetyl group from acetyl-CoA to the primary amine in glucosamine-6-phosphate, generating N-acetyl-D-glucosamine-6-phosphate.

The terms “fructose-6-phosphate aminotransferase,” “glutamine-fructose-6-phosphate-aminotransferase,” “glutamine-fructose-6-phosphate aminotransferase,” “L-glutamine D-fructose-6-phosphate aminotransferase,” “glmS,” “glms,” “glmS*54” as used in the disclosure, are used interchangeably and refer to an enzyme that catalyzes the conversion of fructose-6-phosphate into glucosamine-6-phosphate using glutamine as a nitrogen source. A polynucleotide encoding a “fructose-6-phosphate aminotransferase” or any of the above terms, refers to a polynucleotide encoding such an enzyme that catalyzes the conversion of fructose-6-phosphate into glucosamine-6-phosphate using glutamine as a nitrogen source.

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, and polypeptides, 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, proteins 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 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 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/IMM) 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 application, unless explicitly specified otherwise, a polypeptide (or DNA sequence) comprising/consisting/having an amino acid sequence (or nucleotide sequence) having at least 80% sequence identity to the full-length amino acid sequence (or nucleotide sequence) of a reference polypeptide (or nucleotide sequence), usually indicated with a SEQ ID NO or UniProt ID or GenBank NO., preferably has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, more preferably has at least 85%, even more preferably has at least 90%, most preferably has at least 95%, 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 “cultivation” refers to the culture medium wherein the cell is cultivated or fermented, the cell itself, and the galactosylated di- and/or 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.

“Mammalian milk oligosaccharides (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 (HMOs) are also known as human identical milk oligosaccharides that are chemically identical to the human milk oligosaccharides found in human breast milk but that 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.

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 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 17th June 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, and 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 vithe 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 that 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 vithese β-Barrel porins include but are not limited to nucleosides, raffinose, glucose, beta-glucosides, and 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 17th June 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 Jun. 17, 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 (tcdb.org).

“SET” or “Sugar Efflux Transporter” as used herein refers to membrane proteins of the SET family that 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 https://www.ebi.ac.uk/interpro/or a standalone version of InterProScan (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 that 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 “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-1-phosphate, galactose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, mannose-6-phosphate, mannose-1-phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-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, and GDP-fucose.

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

Description

According to a first aspect, the disclosure provides the use of an N-acetylglucosamine b-1,X-galactosyltransferase for the synthesis of a galactosylated disaccharide or oligosaccharide. Herein, N-acetylglucosamine b-1,X-galactosyltransferase galactosylates an N-acetylglucosamine and/or N-acetylgalactosamine as a monosaccharide, and/or an N-acetylglucosamine and/or N-acetylgalactosamine as part of a di- and/or oligosaccharide at the non-reducing end of the di- and/or oligosaccharide to produce the galactosylated disaccharide or oligosaccharide. Throughout the application, the feature “di- or oligosaccharide” is preferably replaced with “oligosaccharide,” the feature “di- and/or oligosaccharides” is preferably replaced with “oligosaccharides.” Throughout the application, the di- and/or oligosaccharide is preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO).

In the scope of the disclosure, the N-acetylglucosamine b-1,X-galactosyltransferase is used to transfer a galactose residue from the nucleotide-sugar donor UDP-galactose to an N-acetylglucosamine and/or N-acetylgalactosamine as a monosaccharide, and/or an N-acetylglucosamine and/or N-acetylgalactosamine as part of a di- and/or oligosaccharide at the non-reducing end of the di- and/or oligosaccharide resulting in the production of a galactosylated disaccharide or oligosaccharide as defined herein.

According to the disclosure, the N-acetylglucosamine b-1,X-galactosyltransferase is an N-acetylglucosamine b-1,3-galactosyltransferase or an N-acetylglucosarnine b-1,4-galactosyltransferase that transfers a galactose residue from UDP-galactose to an N-acetylglucosamine and/or N-acetylgalactosamine as a monosaccharide, and/or an N-acetylglucosamine and/or N-acetylgalactosamine as part of a di- and/or oligosaccharide at the non-reducing end of the di- and/or oligosaccharide in a beta-1,3 or beta-1,4 linkage, respectively, resulting in the production of a galactosylated disaccharide or oligosaccharide as defined herein.

A galactosylated disaccharide according to the disclosure is a saccharide of two monosaccharides consisting of a galactose residue at its non-reducing end, which is beta-1,3 or beta-1,4 linked to a GleNAc or a GalNAc residue at its reducing end, and a galactosylated oligosaccharide is a saccharide of three or more monosaccharides having a terminal galactose residue at its non-reducing end, which is beta-1,3 or beta-1,4 linked to a GlcNAc or a GalNAc residue.

According to the disclosure, the N-acetylglucosamine b-1,X-galactosyltransferase is an N-acetylglucosamine b-1,3-galactosyltransferase that has 1) PFAM domain PF00535 and that (i) comprises the sequence [AGPS]XXLN(Xn)RXDXD with SEQ ID NO: 1, wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17, or (ii) comprises the sequence PXXLN(Xn)RXDXD(Xm)[FWY]XX[HKR]XX[NQST] with SEQ ID NO: 2, wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17 and m is 100 to 115, or (iii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 3 or 4, or (iv) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 3 or 4 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide with SEQ ID NOs: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or (v) 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: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or (vi) is a functional fragment of any one of SEQ ID NOs: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or (vii) comprises 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: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity; or the N-acetylglucosamine b-1,X-galactosyltransferase is an N-acetylglucosamine b-1,3-galactosyltransferase that has 2) PFAM domain IPR002659 and that (i) comprises the sequence KT(Xn)[FY]XXKXDXD(Xm)[FHY]XXG(X, no A, G, S)(Xp)(X, no F, H, W, Y)[DE]D[ILV]XX[AG] with SEQ ID NO: 05, wherein X is any amino acid and wherein n is 13 to 16, m is 35 to 70 and p is 20 to 45, or (ii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 6, 7, 8 or 9, or (iii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 6, 7, 8 or 9 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide with SEQ ID NOs: 6, 7, 8 or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or (iv) 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: 6, 7, 8 or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or (v) is a functional fragment of any one of SEQ ID NOs: 6, 7, 8 or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or (vi) comprises 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: 6, 7, 8 or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity. In a preferred embodiment, the N-acetylglucosamine b-1,3-galactosyltransferase as disclosed herein is not beta1,3-galactosyltransferase (WbgO) from E. coli 055:H7 with UniProt ID D3QY14.

Alternatively, the N-acetylglucosamine b-1,X-galactosyltransferase is an N-acetylglucosamine b-1,4-galactosyltransferase that has 1) PFAM domain PF01755 and that (i) comprises the sequence EXXCXXSHX[AFIL,TY]LW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 10, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or (ii) comprises the sequence EXXCXXSH[LR]VLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 11, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or (iii) comprises the sequence EXXCXXSH[VHI]SLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 12, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or (iv) comprises the sequence EXXCXXSHYMLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 13, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or (v) comprises the sequence EXXCXXSHXX(X, no V)Y(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 14, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or (vi) comprises a polypeptide sequence according to any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23, or (vii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (viii) 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: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (ix) is a functional fragment of any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (x) comprises 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: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity; or the N-acetylglucosamine b-1,X-galactosyltransferase is an N-acetylglucosamine b-1,4-galactosyltransferase that has 2) PFAM domain PF00535 and that (i) comprises the sequence R[KN]XXXXXXXGXXXX[FL](X, no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE] with SEQ ID NO: 24 wherein X is any amino acid and wherein n is 50 to 75 and m is 10 to 30, or (ii) comprises the sequence R[KN]XXXXXXXGXXXX[FL](X, no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE](Xp)[FWY]XX[HKR]XX[NQST] with SEQ ID NO: 25 wherein X is any amino acid and wherein n is 50 to 75, m is 10 to 30 and p is 20 to 25, or (iii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 26 or 27, or (iv) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 26 or 27 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (v) 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: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (vi) is a functional fragment of any one of SEQ ID NOs: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (vii) comprises 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: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity; or the N-acetylglucosamine b-1,X-galactosyltransferase is an N-acetylglucosamine b-1,4-galactosyltransferase that has 3) PFAM domain PF02709 and not PFAM domain PF00535 and that (i) comprises the sequence [FWY]XX[FWY](Xn)[FWY][GQ]X[DE]D with SEQ ID NO: 28 wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an IP or NL combination and wherein n is 21 to 26, or (ii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 29, 30, 31, 32, 33 or 34, or (iii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 29, 30, 31, 32, 33 or 34 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 29, 30, 31, 32, 33 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (iv) 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: 29, 30, 31, 32, 33 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (v) is a functional fragment of any one of SEQ ID NOs: 29, 30, 31, 32, 33 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (vi) comprises 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: 29, 30, 31, 32, 33 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity; or the N-acetylglucosamine b-1,X-galactosyltransferase is an N-acetylglucosamine b-1,4-galactosyltransferase that has 4) PFAM domain PF03808 and that (i) comprises the sequence [ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA with SEQ ID NO: 35, wherein X is any amino acid and wherein n is 20 to 25, or (ii) comprises the sequence [ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA(Xm) [HR]XG[FWY](Xp)GXGXXXQ[DE] with SEQ ID NO: 36, wherein X is any amino acid and wherein n is 20 to 25, m is 40 to 50 and p is 22 to 30, or (iii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 37, 38 or 39, or (iv) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 37, 38 or 39 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (v) 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: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (vi) is a functional fragment of any one of SEQ ID NOs: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (vii) comprises 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: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity. In a preferred embodiment, the N-acetylglucosamine b-1,4-galactosyltransferase as disclosed herein is not Neisseria meningitidis 1gtB (UniProt ID Q51116).

The PFAM domains used herein, PF00535, IPR002659, PF01755, PF02709, PF03808, are protein domains as annotated in the PFAM database version Pfam 33.1 as released on Jun. 11, 2020. PF00535 is found in the glycosyltransferase 2 (GT2) family that comprises enzymes that transfer sugar from UDP-glucose, UDP-N-acetyl-galactosamine, GDP-mannose or CDP-abequose, to a range of substrates including cellulose, dolichol phosphate and teichoic acids.

IPR002659 is found in the glycosyltransferase family 31 (GH31) that comprises enzymes with a number of known activities including N-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase (2.4.1.149), beta-1,3-galactosyltransferase (2.4.1), fucose-specific beta-1,3-N-acetylglucosaminyltransferase (2.4.1), globotriosylceramide beta-1,3-GalNAc transferase (2.4.1.79). PF01755 is found in the glycosyltransferase 25 (GT25) family. This is a family of glycosyltransferases involved in lipopolysaccharide (LPS) biosynthesis. These enzymes catalyze the transfer of various sugars onto the growing LPS chain during its biosynthesis. PF02709 refers to the Glyco_transf_7C family. This is the N-terminal domain of a family of galactosyltransferases from a wide range of Metazoa with three related galactosyltransferases activities, all three of which are possessed by one sequence in some cases: EC:2.4.1.90, N-acetyllactosamine synthase, EC:2.4.1.38, Beta-N-acetylglucosaminyl-glycopeptide beta-1,4-galactosyltransferase, and EC:2.4.1.22 Lactose synthase. PF03808 refers to the glycosyltransferase 26 (GT26) family that comprises enzymes with activities like β-N-acetyl mannosaminuronyltransferase (EC 2.4.1.-), β-N-acetyl-mannosaminyltransferase (EC 2.4.1.-), β-1,4-glucosyltransferase (EC 2.4.1.-) and β-1,4-galactosyltransferase (EC 2.4.1.-).

Proteins having the same PFAM domain and motifs as specified for each class of N-acetylglucosamine b-1,3-galactosyltransferase or N-acetylglucosamine b-1,4-galactosyltransferase can be searched via a RegEx analysis as exemplified in the disclosure. A RegEx, or Regular Expression, is a special sequence of characters that helps to match or find other strings or sets of strings, using a specialized syntax held in a pattern. Many programs are available to do RegEx search. One of them is the Python module “re,” which provides full support for regular expressions in Python. Detailed information, and known by the persons skilled in the art, is available from https://towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2, as released on 6 Apr. 2019.

The overall sequence identity may be 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.

At least 80% overall sequence identity to the full length of any one of the polypeptides with SEQ ID NOs: 3, 4, 6, 7, 8, 9, 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 or 39 should be understood as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to any one of the polypeptides with SEQ ID NOs: 3, 4, 6, 7, 8, 9, 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 or 39, respectively, as given herein. 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: 3, 4, 6, 7, 8, 9, 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 or 39 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 amino acid residues, of consecutive amino acid residues from any one of the polypeptides with SEQ ID NOs: 3, 4, 6, 7, 8, 9, 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 or 39, respectively, 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.

In a second aspect, provided is a method to synthesize a galactosylated disaccharide or oligosaccharide by use of an N-acetylglucosamine b-1,X-galactosyltransferase as described herein.

In a preferred embodiment, the synthesis comprises the steps of:

    • a. providing UDP-galactose and any one of the galactosyltransferase as defined herein, wherein the galactosyltransferase is capable of transferring a galactose residue from the UDP-galactose donor to one or more acceptor(s), and
    • b. contacting any one of the galactosyltransferase and UDP-galactose with one or more acceptor(s), under conditions where the galactosyltransferase catalyzes the transfer of a galactose residue from the UDP-galactose to the acceptor(s),
    • c. preferably, separating the galactosylated di- or oligosaccharide.

In the scope of the disclosure, the wording “conditions where the galactosyltransferase catalyzes the transfer of a galactose residue frorn the UDP-galactose to the acceptor(s)” 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.

In a preferred embodiment of disclosure, the monosaccharide N-acetylglucosamine (GlcNAc), the monosaccharide N-acetylgalactosamine (GalNAc), a non-reducing N-acetylglucosamine of a di- and/or oligosaccharide (i.e., an N-acetylglucosamine at the non-reducing end of a di- and/or oligosaccharide) and/or a non-reducing N-acetylgalactosamine of a di- and/or oligosaccharide (i.e., an N-acetylgalactosamine at the non-reducing end of a di- and/or oligosaccharide) are the acceptors of the N-acetylglucosamine b-1,X-galactosyltransferase. The di- and oligosaccharides having a non-reducing GlcNAc and/or GalNAc comprise di- and oligosaccharides, respectively, as defined herein.

Another preferred embodiment is a method as disclosed herein wherein any one of the galactosyltransferase is contacted with at least two different acceptors, preferably at least three different acceptors, more preferably at least four different acceptors, even more preferably at least five different acceptors, to synthesize a mixture of galactosylated disaccharides and/or oligosaccharides by use of a N-acetylglucosamine b-1,X-galactosyltransferase as described herein. According to the disclosure, the mixture comprises or consists of at least two different “di- or oligosaccharides,” preferably at least three different “di- or oligosaccharide,” more preferably at least four different “di- or oligosaccharide.” Preferably, the mixture comprises or consists of neutral di-/oligosaccharides. More preferably, the mixture comprises or consists of charged and/or neutral di- or oligosaccharides. In a preferred embodiment of the method and/or cell, the charged di- or oligosaccharides are sialylated di- or oligosaccharides. In a preferred embodiment of the method and/or cell, the neutral di- or oligosaccharides are fucosylated. In another preferred embodiment of the method and/or cell, the neutral di- or oligosaccharides are not fucosylated.

The N-acetylglucosamine, N-acetylgalactosamine and/or di- and/or oligosaccharide containing an N-acetylglucosamine and/or N-acetylgalactosamine at the non-reducing end, i.e., the preferred acceptors of the N-acetylglucosamine b-1,X-galactosyltransferase(s) as disclosed herein, are produced by methods comprising extraction from natural sources, biotechnological processes, physical processes, chemical processes, and combinations thereof.

In a further preferred embodiment of the method and/or cell, the galactosylated disaccharide or oligosaccharide synthesized as described herein is further glycosylated by providing a glycosyltransferase and a nucleotide-sugar, which is donor for the glycosyltransferase. The glycosyltransferase is preferably selected from the list comprising: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolyineuraminyltransferases, rhamnnosyltransferases, N-acetylrhamnnosyltransferases, UDP-4-amnino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyiltransferases.

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

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

In another more preferred embodiment of the method and/or cell, the galactosyltransferase is chosen from the list comprising beta-1,3-galactosyltransferase, N-acetylglucosamine beta-1,3-galactosyltransferase, beta-1,4-galactosyltransferase, N-acetylglucosamine beta-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase and alpha-1,4-galactosyltransferase.

In another more preferred embodiment of the method and/or cell, the glucosyltransferase is chosen from the list comprising alpha-glucosyltransferase, beta-1,2-glucosyltransferase, beta-1,3-glucosyltransferase and beta-1,4-glucosyltransferase.

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

In another more preferred embodiment of the method and/or cell, the N-acetylglucosaminyltransferase is chosen from the list comprising galactoside beta-1,3-N-acetylglucosaminyltransferase and beta-1,6-N-acetylglucosaminyltransferase.

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

The nucleotide-sugar is preferably selected from the list comprising GDP-fucose (GDP-Fuc), CMP-N-acetylneuraminic acid (CMP-Neu5Ac), UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-galactose (UDP-Gal), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy-L-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-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, UDP-glucuronate, UDP-galacturonate, GDP-rhamnose and UDP-xylose.

In a preferred embodiment of the method and/or cell, the galactosylated di- or oligosaccharide as described herein is additionally modified with one or more GleNAc moieties and the further glycosyltransferase is one or more N-acetylglucosaminyltransferase(s), preferably a galactoside beta-1,3-N-acetylglucosaminyltransferase and/or a beta-1,6-N-acetylglucosaminyltransferase and the nucleotide-activated sugar is UDP-GlcNAc.

In an alternative, and/or additional preferred embodiment of the method and/or cell, the galactosylated di- or oligosaccharide as described herein is additionally sialylated, the further glycosyltransferase is one or more sialyltransferase(s), preferably an alpha-2,3-sialyltransferase, an alpha-2,6-sialyltransferase and/or an alpha-2,8-sialyltransferase, and the nucleotide-activated sugar is CMP-Neu5Ac and/or CMP-Neu5Gc.

In an alternative, and/or additional preferred embodiment of the method and/or cell, the galactosylated di- or oligosaccharide as described herein is additionally fucosylated, the further glycosyltransferase is one or more fucosyltransferase(s), preferably an alpha-1,2-fucosyltransferase, an alpha-1,3-fucosyltransferase, an alpha-1,4-fucosyltransferase and/or an alpha-1,6-fucosyltransferase, and the nucleotide-activated sugar is GDP-fucose.

Preferably, at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, most preferably at least six, glycosyltransferases are provided to further glycosylate the galactosylated di- or oligosaccharide as synthesized herein.

According to the disclosure, the galactosylated disaccharide or oligosaccharide (or further glycosylated form thereof as described herein) can make use of a cell-free system, i.e., produced in a cell-free system. Alternatively, the galactosylated disaccharide or oligosaccharide (or further glycosylated form thereof as described herein) is produced by using a cell, preferably a single cell. Such cell can be a non-metabolically engineered cell or a metabolically engineered cell as disclosed herein.

In this context, it is a preferred embodiment that the cell is able to produce (i) any one of the galactosyltransferase as defined herein, and/or (ii) UDP-galactose, and/or (iii) one or more acceptor(s) as defined herein, wherein the cell is grown in a cultivation to obtain a sufficient number of cells for use in the method according to the disclosure at a desired scale. Upon growth of the cell to a desired cell density, the cell is processed for use in the methods of the disclosure. For example, the cell is generally permeabilized or otherwise disrupted to allow entry of the saccharide acceptor(s) into the cell (if not produced by the cell or if produced by another cell). The galactosyltransferase and/or UDP-galactose as produced by the cells can, however, in some situations, diffuse from the cells into the extracellular fluid or can be actively transported as described herein. Methods of permeabilizing cells so as to not significantly degrade enzymatic activity and nucleotide sugar stability are known to those of skill in the art. A cell can be subjected to concentration, drying, lyophilization, treatment with surfactants, ultrasonic treatment, mechanical disruption, enzymatic treatment, and the like. The skilled person will understand that one or more of (i) to (iii) can be produced by a same cell or by different cells. For example, one cell can provide the galactosyltransferase, while another cell can provide one or more acceptors. Any one of (i) to (iii) can also be provided as such, i.e., not produced by a cell. As such, the disclosure provides a method to synthesize a galactosylated disaccharide or oligosaccharide, comprising the steps of a. providing UDP-galactose and any one of the galactosyltransferase as defined herein, wherein the galactosyltransferase is capable of transferring a galactose residue from the UDP-galactose donor to one or more acceptor(s), b. contacting any one of the galactosyltransferase and UDP-galactose with one or more acceptor(s), under conditions where the galactosyltransferase catalyzes the transfer of a galactose residue from the UDP-galactose to the acceptor(s), and c. preferably, separating the galactosylated di- or oligosaccharide; wherein at least one of the UDP-galactose, galactosyltransferase and acceptor(s) is provided, preferably produced, by a cell, preferably a single cell.

In this context, it is another preferred embodiment that the galactosylated di- or oligosaccharide is produced by a cell, preferably a single cell. Such cell can be a non-metabolically engineered cell or a metabolically engineered cell as disclosed herein.

The N-acetylglucosamine, N-acetylgalactosamine and/or di- and/or oligosaccharide containing an N-acetylglucosamine and/or N-acetylgalactosamine at the non-reducing end being the acceptor(s) can be taken up by the cell and/or synthesized by the cell. In the context of the disclosure, it should be understood that the galactosylated di- or oligosaccharide according to the disclosure is preferably synthesized intracellularly. The skilled person will further understand that a fraction or substantially all of the synthesized galactosylated di- or oligosaccharide remains intracellularly and/or is excreted outside the cell either via passive or via active transport. As such, the disclosure provides a method to synthesize a galactosylated disaccharide or oligosaccharide, comprising the steps of: a. providing a cell, preferably a single cell, as described herein (it is referred to the second and third aspect of disclosure), b. optionally providing one or more acceptor(s) as described herein, c. cultivating and/or incubating the cell under conditions permissive for producing the galactosylated di- or oligosaccharide, and d. preferably, separating the galactosylated di- or oligosaccharide.

A third aspect of the disclosure relates to a cell that is metabolically engineered to synthesize a galactosylated disaccharide or oligosaccharide (or a further glycosylated form thereof as described herein) by use of an N-acetylglucosamine b-1,X-galactosyltransferase as disclosed herein. Throughout the application, 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 a galactosylated di- or oligosaccharide according to the disclosure. In the context of the disclosure, the galactosylated di- or oligosaccharide preferably does not occur in the wild type progenitor of the metabolically engineered cell.

Preferably, the cell as described herein (it is referred to the second and third aspect of disclosure).

    • expresses any one of the N-acetylglucosamine b-1,3-galactosyltransferases and/or N-acetylglucosamine b-1,4-galactosyltransferases, and
    • is capable of synthesizing UDP-galactose (UDP-Gal) as donor for the galactosyltransferases.

More preferably, the cell as described herein (it is referred to the second and third aspect of disclosure):

    • is capable of synthesizing one or more of the acceptor(s) as disclosed herein, and
    • expresses any one of the N-acetylglucosamine b-1,3-galactosyltransferases and/or N-acetylglucosamine b-1,4-galactosyltransferases, and
    • is capable of synthesizing UDP-galactose (UDP-Gal) as donor for the galactosyltransferases.

In another embodiment, the cell is capable of synthesizing one or more nucleotide-sugar donor(s) chosen from the list comprising: GDP-fucose (GDP-Fuc), CMP-N-acetylneuraminic acid (CMP-Neu5Ac), UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-galactose (UDP-Gal), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy-L-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-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose.

Alternatively or preferably, the cell is capable of expressing one or more glycosyltransferases selected from the list comprising: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases.

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

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

In another more preferred embodiment of the method and/or cell, the cell is capable of expressing a galactosyltransferase chosen from the list comprising beta-1,3-galactosyltransferase, N-acetylglucosamine beta-1,3-galactosyltransferase, beta-1,4-galactosyltransferase, N-acetylglucosamine beta-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase and alpha-1,4-galactosyltransferase.

In another more preferred embodiment of the method and/or cell, the cell is capable of expressing a glucosyltransferase chosen from the list comprising alpha-glucosyltransferase, beta-1,2-glucosyltransferase, beta-1,3-glucosyltransferase and beta-1,4-glucosyltransferase.

In another more preferred embodiment of the method and/or cell, the cell is capable of expressing a mannosyltransferase chosen from the list comprising alpha-1,2-mannosyltransferase, alpha-1,3-mannosyltransferase and alpha-1,6-manrnosyltransferase.

In another more preferred embodiment of the method and/or cell, the cell is capable of expressing an N-acetylglucosaminyltransferase chosen from the list comprising galactoside beta-1,3-N-acetylglucosaminyltransferase and beta-1,6-N-acetylglucosaminyltransferase.

In another more preferred embodiment of the method and/or cell, the cell is capable of expressing a N-acetylgalactosaminyltransferase, which is an alpha-1,3-N-acetylgalactosaminyltransferase.

The glycosyltransferase family is a very broad family of enzymes capable of catalyzing the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. A classification of glycosyltransferases using nucleotide diphospho-sugar, nucleotide monophospho-sugar and sugar phosphates and related proteins into distinct sequence-based families has been described (Campbell et al., Biochem. J. 326, 929-939 (1997)) and is available on the CAZy (CArbohydrate-Active EnZymes) website (cazy.org).

Alternatively or preferably, the cell described herein is a metabolically engineered cell Preferably, the metabolically engineered cell is modified with gene expression modules wherein the expression from any one of the expression modules is constitutive or is created by a natural inducer.

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).

According to a preferred embodiment, the cell is modified with one or more expression modules. 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 the galactosylated di- or oligosaccharide like, e.g., a glycosyltransferase, a polypeptide directly involved in the synthesis of a nucleotide-activated sugar or a membrane transporter protein as described herein; or the recombinant gene is linked to other pathways in the host cell that are not involved in the synthesis of the galactosylated di- or oligosaccharide. 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 that also expresses a heterologous protein.

According to a preferred embodiment of the disclosure, the expression of each of the expression modules is constitutive or created by a natural 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, 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.

According to a further embodiment, the recombinant polynucleotides are adapted to the codon usage of the respective cell or expression system.

In the method and cell described herein, 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 application, the feature “multiple” means at least two, preferably at least three, more preferably at least four, even more preferably at least five.

In a preferred embodiment of the method and/or cell, the cell used herein is genetically modified to produce a nucleotide-sugar selected from the group: 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-glucuronate, UDP-galacturonate, GDP-rhamnose, or UDP-xylose. In a further embodiment, the cell is genetically modified for the optimized production of any one of the nucleotide-sugars.

According to an embodiment of the method and/or cell, the cell is capable of synthesizing or producing UDP-galactose. Preferably, the cell is optimized for UDP-galactose production. In an optional embodiment, the cell is modified in the expression or activity of the UDP-glucose 4-epimerase GalE, which is capable of converting UDP-glucose into UDP-galactose.

In a further embodiment, the nucleotide-sugar synthesized by the cell is UDP-galactose and the glycosyltransferase an N-acetylglucosamine b-1,3-galactosyltransferase or an N-acetylglucosamine b-1,4-galactosyltransferase.

In another preferred embodiment of the method and/or cell, the cell used herein is capable of producing the nucleotide-sugar GDP-fucose. The 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 a 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.

In another additive and/or alternative embodiment of the method and/or cell, the cell used herein is capable of producing the nucleotide-sugar CMP-N-acetylneuraminic acid (CMP-sialic acid). The CMP-N-acetylneuraminic acid can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Preferably, the cell is modified to produce CMP-N-acetylneuraminic acid. More preferably, the cell is modified for enhanced CMP-N-acetylneuraminic acid production. The modification can be one or more chosen from the group comprising over-expression of a CMP-sialic acid synthetase encoding gene, over-expression of a sialate synthase encoding gene, and over-expression of an N-acetyl-D-glucosamine 2-epimerase encoding gene.

Synthesis of CMP-N-acetylneuraminic acid makes use of GlcNAc but GlcNAc in the cell as described herein can be used as acceptor for the N-acetylglucosamine b-1,-X galactosyltransferases. Production of CMP-N-acetylneuraminic acid in the cell may thus lower the GlcNAc available for the production of the saccharides of interest, i.e., a galactosylated di- or oligosaccharide. Production of both CMP-N-acetylneuraminic acid and GlcNAc needs to be optimized to ensure high levels of both CMP-N-acetylneuraminic acid and GlcNAc. Such optimization may include efficient balancing and fine-tuning of the expression levels of polypeptides involved in the synthesis of both CMP-N-acetylneuraminic acid and GlcNAc.

In another additive or alternative embodiment of the method and/or cell, the cell as described herein is modified in the expression or activity of any glycosyltransferase expressed in the cell, preferably any of the glycosyltransferases as described herein.

In a preferred embodiment of the method and/or cell, the cell is genetically modified in the expression or activity of an enzyme selected from the group: a glucosamine 6-phosphate N-acetyltransferase, a phosphatase, a glycosyltransferase, an L-glutamine-D-fructose-6-phosphate aminotransferase, or a UDP-glucose-4-epimerase. According to the disclosure, the as such enlisted enzymes comprising glucosamine 6-phosphate N-acetyltransferase, a phosphatase, a glycosyltransferase, an L-glutamine-D-fructose-6-phosphate aminotransferase, or a UDP-glucose 4-epimerase are either endogenous proteins with a modified expression or activity, preferably the endogenous proteins are overexpressed; or the enzymes of the as such enlisted group are heterologous proteins, which can be heterologously expressed by the cell. The heterologously expressed proteins will then be introduced and expressed, preferably overexpressed. In another embodiment, the endogenous proteins can have a modified expression in the cell that also expresses a heterologous protein. Heterologous expression can either be from the host's genome or from a vector introduced in the cell as described herein.

In another preferred embodiment, the cell described herein expresses at least one glucosamine 6-phosphate N-acetyltransferase and a phosphatase to synthesize N-acetylglucosamine. In this preferred embodiment, the glucosamine 6-phosphate N-acetyltransferase is an enzyme capable of converting glucosamine-6-phosphate to N-acetylglucosamine-6-phosphate in the cell and the phosphatase is capable of dephosphorylating N-acetylglucosamine-6-phosphate to produce N-acetylglucosamine in the cell. In a more preferred embodiment, this phosphatase is a HAD-like phosphatase. Phosphatases from the HAD superfamily and the HAD-like family are described in the art. Examples from these families can be found in the enzymes expressed from genes yqaB, inhX, yniC, ybiV, yidA, ybjI, yigL or coffrom Escherichia coli or one or more of, 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 WO18122225. One phosphatase that catalyzes this reaction is identified in Blastocladiella emersonii. Phosphatases are generally non-specific and the activity is generally not related to the family or structure. Other examples can thus be found in all phosphatase families. Specific phosphatases are easily identified and screened by well-known methods as described by Fahs et a1. (ACS Chem. Biol. 11(11), 2944-2961 (2016)). In a preferred embodiment, the phosphatase is encoded by a heterologous nucleic acid. In other words, the phosphatase is preferably heterologously expressed in the cell. In another preferred embodiment, the glucosamine 6-phosphate N-acetyltransferase is encoded by a heterologous nucleic acid. In other words, the glucosamine 6-phosphate N-acetyltransferase is preferably heterologously expressed in the cell.

In a further preferred embodiment, the glucosamine 6-phosphate N-acetyltransferase is (i) a polypeptide sequence with UniProt ID P43577, or (ii) is a functional homologue, variant or derivative of the polypeptide with UniProt ID P43577 having at least 80% overall sequence identity to the full-length of the polypeptide with UniProt ID P43577 and having glucosamine 6-phosphate N-acetyltransferase activity, or (iii) is a functional fragment of the polypeptide with UniProt ID P43577 and having glucosamine 6-phosphate N-acetyltransferase activity, or (iv) comprises a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of the polypeptide with UniProt ID P43577 and having glucosamine 6-phosphate N-acetyltransferase activity. In another preferred embodiment the L-glutamine-D-fructose-6-phosphate aminotransferase is (i) a polypeptide sequence with UniProt ID P17169, or (ii) is a functional homologue, variant or derivative of the polypeptide with UniProt ID P17169 having at least 80% overall sequence identity to the full-length of the polypeptide with UniProt ID P17169 and having L-glutamine D-fructose-6-phosphate aminotransferase activity, or (iii) is a functional fragment of the polypeptide with UniProt ID P17169 and having L-glutamine-D-fructose-6-phosphate aminotransferase activity, or (iv) comprises a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of the polypeptide with UniProt ID P17169 and having L-glutamine-D-fructose-6-phosphate aminotransferase activity. In an alternative preferred embodiment, the L-glutamine-D-fructose-6-phosphate aminotransferase (i) is a glmS*54 polypeptide sequence differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and a G472S mutation as described by Deng et a1. (Biochimie 88, 419-29 (2006) and having L-glutamine-D-fructose-6-phosphate aminotransferase activity, or (ii) is a functional homologue, variant or derivative of the mutant glmS*54 polypeptide differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and a G472S mutation having at least 80% overall sequence identity to the full-length of the mutant glmS*54 polypeptide differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and a G472S mutation and having L-glutamine-D-fructose-6-phosphate aminotransferase activity, or (iii) is a functional fragment of the mutant glmS*54 polypeptide differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and a G472S mutation and having L-glutamine-D-fructose-6-phosphate aminotransferase activity, or (iv) comprises a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of the mutant glmS*54 polypeptide differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and a G472S mutation and having L-glutamine-D-fructose-6-phosphate aminotransferase activity.

In another preferred embodiment of the method and/or cell, the cell is unable to convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or unable to convert glucosamine-6-phosphate to fructose-6-phosphate. In a cell N-acetylglucosamine-6-phosphate can be converted to glucosamine-6-phosphate by activity of an N-acetylglucosamine-6-phosphate deacetylase like nagA and glucosamine-6-phosphate can be converted to fructose-6-phosphate by activity of a glucosamine-6-phosphate deaminase like nagB. Such N-acetylglucosamine-6-phosphate deacetylase and/or glucosamine-6-phosphate deaminase can obtain reduced expression or reduced activity or can be inactivated by mutagenesis or by partial or full deletion of the corresponding polynucleotides encoding for the coding sequences or by mutagenesis of the promoter sequences controlling the expression of the corresponding encoding polynucleotides by methods well-known in the art.

In a further preferred embodiment of the method and/or cell as described herein, the cell is modified for enhanced UDP-galactose production and the modification is chosen from the group comprising: knock-out of a 5′-nucleotidase/UDP-sugar hydrolase encoding gene or knock-out of a galactose-1-phosphate uridylyltransferase encoding gene.

In a further preferred embodiment of the method and/or cell, the cell 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 cell 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 preferred 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 another preferred embodiment of the method and/or cell, 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, 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 that also expresses a heterologous membrane transporter protein or polypeptide having transport activity.

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

In another preferred embodiment of the method and/or cell, the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of the galactosylated disaccharide or oligosaccharide. In an alternative and or additional preferred embodiment of the method and/or cell, the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of a mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one of the galactosylated disaccharide or oligosaccharide. In an alternative and or additional preferred embodiment of the method and/or cell, the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of a mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one of the galactosylated oligosaccharide.

In an alternative and/or additional preferred embodiment of the method and/or cell, 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 the galactosylated disaccharide or oligosaccharide. In an alternative and/or additional preferred embodiment of the method and/or cell, 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 the mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one of the galactosylated disaccharide or oligosaccharide. In an alternative and/or additional preferred embodiment of the method and/or cell, 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 the mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one of the galactosylated oligosaccharide.

In an alternative and/or additional preferred embodiment of the method and/or cell, 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 the galactosylated disaccharide or oligosaccharide. In an alternative and/or additional preferred embodiment of the method and/or cell, 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 the mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one of the galactosylated disaccharide or oligosaccharide. In an alternative and/or additional preferred embodiment of the method and/or cell, 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 the mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one of the galactosylated oligosaccharide.

In another preferred embodiment of the method and/or cell, the membrane transporter protein or polypeptide having transport activity provides improved production of the galactosylated disaccharide or oligosaccharide. In another preferred embodiment of the method and/or cell, the membrane transporter protein or polypeptide having transport activity provides improved production of the mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one of the galactosylated disaccharide or oligosaccharide. In another preferred embodiment of the method and/or cell, the membrane transporter protein or polypeptide having transport activity provides improved production of the mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one of the galactosylated oligosaccharide.

In an alternative and/or additional preferred embodiment of the method and/or cell, the membrane transporter protein or polypeptide having transport activity provides enabled efflux of the galactosylated disaccharide or oligosaccharide. In an alternative and/or additional preferred embodiment of the method and/or cell, the membrane transporter protein or polypeptide having transport activity provides enabled efflux of the mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one of the galactosylated disaccharide or oligosaccharide. In an alternative and/or additional preferred embodiment of the method and/or cell, the membrane transporter protein or polypeptide having transport activity provides enabled efflux of the mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one of the galactosylated oligosaccharide.

In an alternative and/or additional preferred embodiment of the method and/or cell, the membrane transporter protein or polypeptide having transport activity provides enhanced efflux of the galactosylated disaccharide or oligosaccharide. In an alternative and/or additional preferred embodiment of the method and/or cell, the membrane transporter protein or polypeptide having transport activity provides enhanced efflux of the mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one of the galactosylated disaccharide or oligosaccharide. In an alternative and/or additional preferred embodiment of the method and/or cell, the membrane transporter protein or polypeptide having transport activity provides enhanced efflux of the mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one of the galactosylated oligosaccharide.

In another preferred embodiment of the method and/or cell, the cell expresses a polypeptide selected from the group comprising a lactose transporter like, e.g., the LacY or lac12 permease, a glucose transporter, a galactose transporter, a fucose transporter, a transporter for a nucleotide-activated sugar like, for example, a transporter for UDP-Gal, UDP-GlcNAc, GDP-Fuc or CMP-sialic acid. As such, the transporter internalizes a to the medium added precursor and/or acceptor for the synthesis of a galactosylated disaccharide or oligosaccharide, a mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one of the galactosylated disaccharide or oligosaccharide or a mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one of the galactosylated oligosaccharide.

In a more preferred embodiment of the method and/or cell, 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 POAEY8), Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), Citrobacter youngae (UniProt ID D4BC23) and Yokenella regensburgei (UniProt ID G9Z5F4). In another more preferred embodiment of the method and/or cell, 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 more preferred embodiment of the method and/or cell, 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 more preferred embodiment of the method and/or cell, 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 A0A1V0NEL4) and Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4). In an even more preferred embodiment of the method and/or cell, the cell expresses a 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, GDP-Fuc and/or CMP-sialic acid.

In a preferred embodiment of the method and/or cell, the cell uses lactose in a glycosylation reaction to produce an oligosaccharide. Lactose can be produced by the cell (for example, by the cell's metabolism and/or upon metabolically engineering the cell for this purpose as known to the skilled person), preferably intracellularly, or can be added to the cell, which can import the lactose through passive or active transport. Lactose production by a cell can be obtained by expression of an N-acetylglucosamine beta-1,4-galactosyltransferase and a 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 an N-acetylglucosamine beta-1,4-galactosyltransferase, over-expression of a UDP-glucose 4-epimerase.

In a preferred embodiment of the method and/or cell of disclosure, a cell using lactose as acceptor in a glycosylation reaction 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. It is preferred the cell constitutively expresses the lactose permease. Lactose can be added at the start of the cultivation or it can be added as soon as enough biomass has been formed during the growth phase of the cultivation, i.e., the oligosaccharide production phase that is initiated by the addition of lactose to the cultivation is decoupled from the growth phase. In a preferred embodiment, lactose is added at the start and/or during the cultivation, i.e., the growth phase and production phase are not decoupled.

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.

According to another embodiment of the method and/or cell, the cell is capable of producing phosphoenolpyruvate (PEP). According to another embodiment of the method and/or cell, the cell comprises a pathway for production of a galactosylated disaccharide or oligosaccharide comprising a pathway for production of PEP. In a preferred embodiment of the method and/or cell, the cell is modified for enhanced production and/or supply of PEP compared to a non-modified progenitor.

In another preferred embodiment, the cell comprises a pathway for production of a galactosylated disaccharide or oligosaccharide comprising any one or more modifications for enhanced production and/or supply of PEP compared to a non-modified progenitor.

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 II 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 fructose-1,6 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 (PTSsugar) of E. coli K-12. PtsI is one of two (PtsI and PtsH) sugar non-specific protein constituents of the PTSsugar that, 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 PTSsugar. 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 PTSsugar. Crr or EIIAGIc 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, the cell comprises a modification for reduced production of acetate compared to a non-modified progenitor. 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, 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 that 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 80% 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, 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, 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, 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 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, pyruvate decarboxylase compared to a non-modified progenitor.

According to another preferred embodiment of the method and/or cell, 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 a galactosylated disaccharide or oligosaccharide.

According to another preferred embodiment of the method and/or cell, the cell is using a precursor for the production of a galactosylated disaccharide or oligosaccharide, preferably the precursor being fed to the cell from the cultivation medium. According to a more preferred aspect of the method and/or cell, the cell is using at least two precursors for the production of the galactosylated disaccharide or oligosaccharide, preferably the precursors being fed to the cell from the cultivation medium. According to another preferred aspect of the method and/or cell, the cell is producing at least one precursor, preferably at least two precursors, for the production of the galactosylated disaccharide or oligosaccharide. In a preferred embodiment of the method and/or cell, the precursor that is used by the cell for the production of a galactosylated disaccharide or oligosaccharide is completely converted into the galactosylated disaccharide or oligosaccharide.

According to another preferred embodiment of the method and/or cell, the cell produces 90 g/L or more of a galactosylated disaccharide or oligosaccharide in the whole broth and/or supernatant. In a more preferred embodiment, the galactosylated disaccharide or oligosaccharide produced in the whole broth and/or supernatant has a purity of at least 80% measured on the total amount of the galactosylated disaccharide or oligosaccharide and its precursor produced by the cell in the whole broth and/or supernatant, respectively.

In a preferred embodiment of the method and/or cell, the cell is capable of catabolizing a carbon source selected from the list comprising: glucose, fructose, mannose, galactose, lactose, sucrose, maltose, malto-oligosaccharides, trehalose, starch, cellulose, hemi-cellulose, corn-steep liquor, high-fructose syrup, glycerol, acetate, citrate, lactate, and pyruvate.

In an alternative embodiment of the method and/or cell, the cell as described herein is capable of growing 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 “complex medium” is meant a medium for which the exact constitution is not determined. With the term main is meant the most important carbon source for the galactosylated di- or oligosaccharide, biomass formation, carbon dioxide and/or by-products formation (e.g., 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.

Another embodiment of the disclosure provides for a method and a cell wherein a galactosylated di- or oligosaccharide is produced in and/or by a fungal, yeast, bacterial, insect, animal, plant or protozoan cell as described herein. The cell is chosen from the list comprising a bacterium, a yeast, a protozoan or a fungus, or, refers to a plant or animal 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—that 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), 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.

In a preferred embodiment of the method and/or cell, 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 compared to a non-modified progenitor.

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-1-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 α-1,2-glucosyltransferase, UDP-D-glucose:(glucosyl)LPS α-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, wbbl, 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.

According to another embodiment of the method of the disclosure, the conditions permissive to produce the galactosylated disaccharide or oligosaccharide comprise the use of a culture medium comprising at least one precursor and/or acceptor for the production of the galactosylated disaccharide or oligosaccharide. 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 galactosylated disaccharide or oligosaccharide comprise adding to the culture medium at least one precursor and/or acceptor feed for the production of the galactosylated disaccharide or oligosaccharide.

According to an alternative embodiment of the method of the disclosure, the conditions permissive to produce the galactosylated disaccharide or oligosaccharide comprise the use of a culture medium to cultivate a cell of disclosure for the production of a galactosylated disaccharide or oligosaccharide wherein the culture medium lacks any precursor and/or acceptor for the production of the galactosylated disaccharide or oligosaccharide 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 galactosylated disaccharide or oligosaccharide.

In a preferred embodiment, the method for the production of a galactosylated disaccharide or oligosaccharide 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 (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 a galactosylated disaccharide or oligosaccharide 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 broth.

In another and/or additional preferred embodiment, the method for the production of a galactosylated disaccharide or oligosaccharide 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 min., 10 min., 30 min., 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 min., 10 min., 30 min., 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, or 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 a galactosylated disaccharide or oligosaccharide 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 broth.

In a further embodiment of the methods described herein the host 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 precursor is added to the cultivation 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, followed by a second phase wherein only a carbon-based substrate, preferably glucose or sucrose, is added to the cultivation.

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, followed by a second phase wherein a carbon-based substrate, preferably glucose or sucrose, and a precursor are added to the cultivation.

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

According to the disclosure, the method as described herein preferably comprises a step of separating the galactosylated di- or oligosaccharide. The term “separating” means harvesting, collecting, or retrieving the galactosylated di- or oligosaccharide from the enzyme reaction or the cell and/or the medium of its growth.

The galactosylated di- or oligosaccharide can be separated in a conventional manner from the enzyme mixture or the aqueous culture medium, in which the cell was grown. In case the saccharide is still present in the cells producing the saccharide, conventional manners to free or to extract the saccharide 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, etc. The enzyme reaction mixture, the culture medium and/or cell extract together and separately can then be further used for separating the saccharide. This preferably involves clarifying the saccharide-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 saccharide-containing mixture can be clarified in a conventional manner. Preferably, the saccharide-containing mixture is clarified by centrifugation, flocculation, decantation and/or filtration. Another step of separating the saccharide from the saccharide-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 saccharide-containing mixture, preferably after it has been clarified. In this step, proteins and related impurities can be removed from the saccharide-containing mixture in a conventional manner. Preferably, proteins, salts, by-products, color, endotoxins and other related impurities are removed from the saccharide-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 (e.g., but not limited to cation exchange, anion exchange, mixed bed ion exchange, inside-out ligand attachment), hydrophobic interaction chromatography and/or gel filtration (i.e., size exclusion chromatography), 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 saccharide remains in the saccharide-containing mixture.

In a further preferred embodiment, the methods as described herein also provide for a further purification of the galactosylated di- or oligosaccharide. A further purification of the saccharide 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 saccharide.

In an exemplary embodiment, the separation and purification 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 saccharide 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, and
    • c) collecting the retentate enriched in the saccharide in the form of a salt from the cation of the electrolyte and preferably spray drying the retentate.

In an alternative exemplary embodiment, the separation and purification of the produced galactosylated di- or oligosaccharide 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 to about 500 Dalton, and the other membrane has a molecular weight cut-off of between about 600 to about 800 Dalton, and preferably spray drying.

In an alternative exemplary embodiment, the separation and purification of the produced galactosylated di- or oligosaccharide 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, and preferably spray drying.

In an alternative exemplary embodiment, the separation and purification of the produced galactosylated di- or oligosaccharide is made in the following way. The cultivation comprising the produced oligosaccharide, 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, and
    • iv) nanofiltration step and/or electrodialysis step,
    • wherein a purified solution comprising the produced saccharide 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 the produced galactosylated di- or oligosaccharide 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. Optionally the process further comprises a step of drying 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 a specific embodiment, the disclosure provides the produced galactosylated di- or oligosaccharide, 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.

For identification of the produced galactosylated di- or oligosaccharide 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 saccharide sequence, a partial depolymerization is carried out using an acid or enzymes to determine the structures. To identify the anomeric configuration, the saccharide 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.

Provided is the use of the N-acetylglucosamine b-1,X-galactosyltransferases as described herein for the synthesis of a galactosylated disaccharide or oligosaccharide. In a preferred embodiment, described is use of the N-acetylglucosamine b-1,X-galactosyltransferases as described herein for the synthesis of a mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide. In another preferred embodiment, the disclosure provides the use of the N-acetylglucosamine b-1,X-galactosyltransferases as described herein for the synthesis of a mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide.

Also provided for is the use of a metabolically engineered cell as described herein for the production of a galactosylated disaccharide or oligosaccharide. In a preferred embodiment, the disclosure provides the use of a metabolically engineered cell as described herein for the production of a mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide. In another preferred embodiment, the disclosure also provides for the use of a metabolically engineered cell as described herein for the production of a mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide. In another preferred embodiment, a metabolically engineered cell as described herein is used for the production of a galactosylated disaccharide or oligosaccharide. In another preferred embodiment, a metabolically engineered cell as described herein is used for the production of a mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide. In another preferred embodiment, a metabolically engineered cell as described herein is used for the production of a mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide.

Also provided for is the use of a method as described herein for producing a galactosylated disaccharide or oligosaccharide. In a preferred embodiment, the disclosure provides the use of a method as described herein for the production of a mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide. In another preferred embodiment, the disclosure also provides for the use of a method as described herein for the production of a mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide.

Products comprising the galactosylated oligosaccharides (or further glycosylated form thereof) produced in the disclosure

In some embodiments, the galactosylated oligosaccharide (or further glycosylated form thereof) 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 galactosylated oligosaccharide (or further glycosylated form thereof) 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 galactosylated oligosaccharide 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 (e.g., HMOs) and plant polysaccharides (e.g., 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, a galactosylated 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 (e.g., lactose), monosaccharides (e.g., glucose and galactose), thickeners (e.g., gum Arabic), acidity regulators (e.g., trisodium citrate), water, skimmed milk, and flavorings.

In some embodiments, the galactosylated oligosaccharide (or further glycosylated form thereof) 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 roughly mimic human breast milk. In some embodiments, a galactosylated oligosaccharide (or further glycosylated form thereof) produced and/or purified by a process herein is included in infant formula to provide nutritional benefits similar to those provided by the oligosaccharides in human breast milk. In some embodiments, the galactosylated oligosaccharide is mixed with one or more ingredients of the infant formula. Examples of infant formula ingredients include nonfat 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 (e.g., vitamins A, Bb, Bi2, C and D), minerals (e.g., potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate) and possibly human milk oligosaccharides (HMOs). 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 galactosylated oligosaccharide (or further glycosylated form thereof) in the infant formula is approximately the same concentration as the oligosaccharide's concentration generally present in human breast milk. In some embodiments, the concentration of the galactosylated oligosaccharide in the infant formula is approximately the same concentration as the concentration of that oligosaccharide generally present in human breast milk.

In some embodiments, the galactosylated oligosaccharide (or further glycosylated form thereof) is incorporated into a feed preparation, wherein the feed is chosen from the list comprising pet food, animal milk replacer, veterinary product, post weaning feed, and creep feed.

In some embodiments, a food, a feed, a dietary supplement, a pharmaceutical ingredient and/or a medicine comprises at least one immunomodulatory ingredient.

“An immunomodulatory” ingredient is a substance that modifies the immune response or the functioning of the immune system. An “immunomodulatory” ingredient can modify the immune response by an increase (immunostimulators) or a decrease (immunosuppressives) of the production of serum antibodies. Immunostimulators are used, among others, to enhance the immune response against infectious diseases, tumors, primary or secondary immunodeficiency, and alterations in antibody transfer. Immunosuppressive ingredients are used to reduce the immune response against transplanted organs and to treat autoimmune diseases such as pemphigus, lupus, or allergies. In some embodiments, the immunomodulatory ingredient has anti-inflammatory activity. In some embodiments, a food, a feed, a dietary supplement, a pharmaceutical ingredient and/or a medicine provides multiple immunomodulators, including the galactosylated oligosaccharide (or further glycosylated form thereof) produced and/or purified by a process disclosed in this specification, to adapt the immune system for proper functioning. Immunity varies strongly in distinguishable life stages. Different food components can affect specific immune reactions, depending on the characteristics of deviating metabolic processes, and consumers and patients. A food supplemented with an immunomodulatory ingredient is also called a functional food. A functional food is a food product with specific health benefits for specific groups of consumers. Examples of immunomodulatory ingredients present in functional food as well as in feed and dietary supplements comprise other immunomodulatory molecules, such as the galactosylated oligosaccharides as specified in this specification and fatty acids (PUFAs), fish oil, amino acids (e.g., arginine and glutamine), lectins (e.g., selectins), vitamins (e.g., vitamins A, B6, C, E, thiamine, folate) and minerals (e.g., zinc). Such galactosylated oligosaccharides may include LNB, LacNAc, poly-LacNAc, novo-LNP I, Gal-b1,4-GlcNAc-b1,6-(GlcNAc-b1,3)-Gal-b1,4-Glc, 3-FLN (Gal-b1,4-(Fuc-a1,3)-GlcNAc, SLNPa (Gal-b1,4-GlcNAc-b1,6-(Neu5Ac-a2,3-Gal-b1,3)-Gal-b1,4-Glc), LNT, iso-LNT, novo LNT, Gal-Novo-LNP I, Gal-Novo-LNP II, LNnT, LNnH, DGal-LNnH, Gal-LNFP III, DF DGal-LNnH, DF DGal-LNnT, TF DGal-LNnH a, TF DGal-LNnH b, FS Gal-LNnH, galilipentasaccharide, para-LNnH. In addition, examples of immunomodulatory ingredients present in pharmaceutical ingredients and medicines comprise other immunomodulatory molecules, such as a galactosylated oligosaccharide as specified in this specification and interleukins, lipopolysaccharides, glucan, interferon gamma and specific antibodies. Such galactosylated oligosaccharides present in pharmaceutical mixtures and/or medicines may include LNB, LacNAc, poly-LacNAc, novo-LNP I, Gal-b1,4-GlcNAc-b1,6-(GlcNAc-b1,3)-Gal-b1,4-Glc, 3-FLN (Gal-b1,4-(Fuc-a1,3)-GlcNAc, SLNPa (Gal-b1,4-GlcNAc-b1,6-(Neu5Ac-a2,3-Gal-b1,3)-Gal-b1,4-Glc), LNT, iso-LNT, novo LNT, Gal-Novo-LNP I, Gal-Novo-LNP II, LNnT, LNnH, DGal-LNnH, Gal-LNFP III, DF DGal-LNnH, DF DGal-LNnT, TF DGal-LNnH a, TF DGal-LNnH b, FS Gal-LNnH, galilipentasaccharide, para-LNnH.

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.

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, the examples and the attached drawings. 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 the following specific embodiments:

    • 1. Use of an N-acetylglucosamine b-1,X-galactosyltransferase for the synthesis of a galactosylated disaccharide or oligosaccharide, wherein the N-acetylglucosamine b-1,X-galactosyltransferase galactosylates
      • an N-acetylglucosamine and/or N-acetylgalactosamine as a monosaccharide, and/or
      • an N-acetylglucosamine and/or N-acetylgalactosamine as part of a di- and/or oligosaccharide at the non-reducing end of the di- and/or oligosaccharide,
      • characterized in that the N-acetylglucosamine b-1,X-galactosyltransferase is:
      • A. an N-acetylglucosamine b-1,3-galactosyltransferase that has
        • a. PFAM domain PF00535 and
          • i) comprises the sequence [AGPS]XXLN(Xn)RXDXD with SEQ ID NO: 1, wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17, or
          • ii) comprises the sequence PXXLN(Xn)RXDXD(Xm) [FWY]XX[HKR]XX[NQST] with SEQ ID NO: 2, wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17 and m is 100 to 115, or
          • iii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 3 or 4, or
          • iv) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 3 or 4 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide with SEQ ID NOs: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
          • v) comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acid residues from any one of SEQ ID NOs: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
        • b. PFAM domain 1PR002659 and
          • i) comprises the sequence KT(Xn)[FY]XXKXDXD (Xm)[FHY]XXG(X, no A, G, S)(Xp)(X, no F, H, W, Y)[DE]D[ILV]XX [AG] with SEQ ID NO: 05, wherein X is any amino acid and wherein n is 13 to 16, m is 35 to 70 and p is 20 to 45, or
          • ii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 6, 7, 8 or 9, or
          • iii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 6, 7, 8 or 9 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide with SEQ ID NOs: 6, 7, 8 or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
          • iv) 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: 6, 7, 8 or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
      • B. an N-acetylglucosamine b-1,4-galactosyltransferase that has
        • a. PFAM domain PF01755 and
          • i) comprises the sequence EXXCXXSHX[AFILTY]LW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 10, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75,
          • ii) comprises the sequence EXXCXXSH[LR]VLW(Xn) EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 11, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75,
          • iii) comprises the sequence EXXCXXSH[VHI]SLW (Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 12, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75,
          • iv) comprises the sequence EXXCXXSHYMLW(Xn) EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 13, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75,
          • v) comprises the sequence EXXCXXSHXX(X, no V)Y(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 14, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75,
          • vi) comprises a polypeptide sequence according to any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23,
          • vii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • viii) 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: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
        • b. PFAM domain PF00535 and
          • i) comprises the sequence R[KN]XXXXXXXGXXXX [FL](X, no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE] with SEQ ID NO: 24 wherein X is any amino acid and wherein n is 50 to 75 and m is 10 to 30, or
          • ii) comprises the sequence R[KN]XXXXXXXGXXXX [FL](X, no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE](Xp)[FWY]XX[HKR]XX[NQST] with SEQ ID NO: 25 wherein X is any amino acid and wherein n is 50 to 75, m is 10 to 30 and p is 20 to 25, or
          • iii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 26 or 27, or
          • iv) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 26 or 27 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • v) 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: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
        • c. PFAM domain PF02709 and not PFAM domain PF00535, and
          • i) comprises the sequence [FWY]XX[FWY](Xn) [FWY][GQ]X[DE]D with SEQ ID NO: 28 wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an IP or NL combination and wherein n is 21 to 26, or
          • ii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 29, 30, 31, 32, 33 or 34, or
          • iii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 29, 30, 31, 32, 33 or 34 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 29, 30, 31, 32, 33 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • iv) 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: 29, 30, 31, 32, 33 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
        • d. PFAM domain PF03808 and
          • i) comprises the sequence [ST][FHY]XN(Xn)DGXXX XXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA with SEQ ID NO: 35, wherein X is any amino acid and wherein n is 20 to 25, or
          • ii) comprises the sequence [ST][FHY]XN(Xn)DGXXX XXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA(Xm)[HR]XG[FW Y](Xp)GXGXXXQ[DE] with SEQ ID NO: 36, wherein X is any amino acid and wherein n is 20 to 25, m is 40 to 50 and p is 22 to 30, or
          • iii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 37, 38 or 39, or
          • iv) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 37, 38 or 39 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • v) 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: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity.
    • 2. A method to synthesize a galactosylated disaccharide or oligosaccharide by use of an N-acetylglucosamine b-1,X-galactosyltransferase according to embodiment 1.
    • 3. Method according to embodiment 2, wherein the synthesis comprises the steps of:
      • a. providing UDP-galactose and any one of the galactosyltransferase, wherein the galactosyltransferase is capable of transferring a galactose residue from the UDP-galactose donor to one or more acceptor(s),
      • b. contacting any one of the galactosyltransferase and UDP-galactose with one or more acceptor(s), under conditions where the galactosyltransferase catalyzes the transfer of a galactose residue from the UDP-galactose to the acceptor(s), and
      • c. preferably, separating the galactosylated di- or oligosaccharide.
    • 4. Method according to embodiment 3, wherein the acceptor(s) is/are an N-acetylglucosamine and/or an N-acetylgalactosamine as a monosaccharide, and/or a di- and/or oligosaccharide having an N-acetylglucosamine and/or N-acetylgalactosamine at its non-reducing end.
    • 5. Method according to any one of embodiments 2 to 4, wherein the galactosylated disaccharide or oligosaccharide is produced in a cell-free system.
    • 6. Method according to any one of embodiments 2 to 4, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell.
    • 7. Method according to embodiment 6, wherein the cell
      • is capable of synthesizing one or more of the acceptor(s), and
      • expresses any one of the N-acetylglucosamine b-1,3-galactosyltransferases and/or N-acetylglucosamine b-1,4-galactosyltransferases, and
      • is capable of synthesizing UDP-galactose (UDP-Gal) as donor for the galactosyltransferases.
    • 8. Method according to any one of embodiments 6 or 7, wherein the cell is further capable of synthesizing one or more nucleotide-sugar donor(s) chosen from the list comprising: GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose.
    • 9. Method according to any one of embodiments 6 to 8, wherein the cell is further capable of expressing one or more glycosyltransferases selected from the list comprising: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolyineuraminyltransferases, rhamnosyltransferases.
    • 10. Method according to any one of embodiments 6 to 9, wherein the cell is a metabolically engineered cell.
    • 11. Method according to any one of embodiments 6 to 10, wherein the cell is modified in the expression or activity of an enzyme selected from the group comprising: glucosamine 6-phosphate N-acetyltransferase, phosphatase, glycosyltransferase, L-glutanine-D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase.
    • 12. Method according to any one of embodiments 6 to 11, wherein the cell is unable to convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or unable to convert glucosamine-6-phosphate to fructose-6-phosphate.
    • 13. Method according to any one of embodiments 6 to 12, wherein the cell is modified for enhanced UDP-galactose production and wherein the modification is chosen from the group comprising: knock-out of a 5′-nucleotidase/UDP-sugar hydrolase encoding gene or knock-out of a galactose-1-phosphate uridylyltransferase encoding gene.
    • 14. Method according to any one of embodiments 6 to 13, wherein the cell is capable of catabolizing a carbon source selected from the list comprising glucose, fructose, mannose, galactose, lactose, sucrose, maltose, malto-oligosaccharides, trehalose, starch, cellulose, hemi-cellulose, corn-steep liquor, high-fructose syrup, glycerol, acetate, citrate, lactate and pyruvate.
    • 15. The method according to any one of embodiments 3 to 14, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography.
    • 16. The method according to any one of embodiments 3 to 15, further comprising purification of the galactosylated di- or oligosaccharide.
    • 17. The method according to embodiment 16, wherein the purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying or lyophilization.
    • 18. A cell metabolically engineered to synthesize a galactosylated disaccharide or oligosaccharide by use of an N-acetylglucosamine b-1,X-galactosyltransferase according to embodiment 1.
    • 19. Cell according to embodiment 18, wherein the cell
      • expresses any one of the N-acetylglucosamine b-1,3-galactosyltransferases and/or N-acetylglucosamine b-1,4-galactosyltransferases,
      • is capable of synthesizing UDP-galactose (UDP-Gal) as donor for the galactosyltransferases, and
      • is capable of synthesizing one or more acceptor(s) for the galactosyltransferases, wherein the acceptor(s) is/are an N-acetylglucosamine as a monosaccharide, and/or a di- or oligosaccharide having an N-acetylglucosamine and/or N-acetylgalactosamine at its non-reducing end.
    • 20. Cell according to any one of embodiments 18 or 19, wherein the cell is further capable of synthesizing one or more nucleotide-sugar donor(s) chosen from the list comprising: GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose.
    • 21. Cell according to any one of embodiments 18 to 20, wherein the cell is further capable of expressing one or more glycosyltransferases selected from the list comprising: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases.
    • 22. Cell according to any one of embodiments 18 to 21, wherein the cell is modified in the expression or activity of an enzyme selected from the group comprising: glucosamine 6-phosphate N-acetyltransferase, phosphatase, glycosyltransferase, L-glutamine D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase.
    • 23. Cell according to any one of embodiments 18 to 22, wherein the cell is unable to convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or unable to convert glucosamine-6-phosphate to fructose-6-phosphate.
    • 24. Cell according to any one of embodiments 18 to 23, wherein the cell is modified for enhanced UDP-galactose production and wherein the modification is chosen from the group comprising: knock-out of a 5′-nucleotidase/UDP-sugar hydrolase encoding gene or knock-out of a galactose-1-phosphate uridylyltransferase encoding gene.
    • 25. Cell according to any one of embodiments 18 to 24, wherein the cell is capable of catabolizing a carbon source selected from the list comprising glucose, fructose, mannose, galactose, lactose, sucrose, maltose, malto-oligosaccharides, trehalose, starch, cellulose, hemi-cellulose, corn-steep liquor, high-fructose syrup, glycerol, acetate, citrate, lactate and pyruvate.
    • 26. The cell according to any one of embodiments 18 to 25 or method according to any one of embodiments 3 to 17, wherein the cell is selected from the group consisting of microorganism, plant, or animal cells, preferably the microorganism is a bacterium, fungus or a yeast, preferably the plant is a rice, cotton, rapeseed, soy, maize or corn plant, preferably the animal is an insect, fish, bird or non-human mammal, preferably the animal cell is a mammalian cell line.
    • 27. The cell according to any one of embodiments 18 to 26 or method according to any one of embodiments 3 to 17 and 26, 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 1K coil MG1655.
    • 28. The cell according to any one of embodiments 18 to 26 or method according to any one of embodiments 3 to 17 and 26, wherein the cell is a yeast cell.

Moreover, the disclosure relates to the following preferred specific embodiments:

    • 1. Use of an N-acetylglucosamine b-1,X-galactosyltransferase for the synthesis of a galactosylated disaccharide or oligosaccharide, wherein the N-acetylglucosamine b-1,X-galactosyltransferase galactosylates
      • an N-acetylglucosamine and/or N-acetylgalactosamine as a monosaccharide, and/or
      • an N-acetylglucosamine and/or N-acetylgalactosamine as part of a di- and/or oligosaccharide at the non-reducing end of the di- and/or oligosaccharide,
      • characterized in that the N-acetylglucosamine b-1,X-galactosyltransferase is:
      • A. an N-acetylglucosamine b-1,3-galactosyltransferase that has
        • a. PFAM domain PF00535 and
          • i) comprises the sequence [AGPS]XXLN(Xn)RXDXD with SEQ ID NO: 1, wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17, or
          • ii) comprises the sequence PXXLN(Xn)RXDXD(Xm) [FWY]XX[HKR]X[NQST] with SEQ ID NO: 2, wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17 and m is 100 to 115, or
          • iii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 3 or 4, or
          • iv) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 3 or 4 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide with SEQ ID NOs: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
          • v) 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: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
          • vi) is a functional fragment of any one of SEQ ID NOs: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
          • vii) comprises 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: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
        • b. PFAM domain 1PR002659 and
          • i) comprises the sequence KT(Xn)[FY]XXKXDXD (Xm)[FHY]XXG(X, no A, G, S)(Xp)(X, no F, H, W, Y)[DE]D[ILV]XX[AG] with SEQ ID NO: 05, wherein X is any amino acid and wherein n is 13 to 16, m is 35 to 70 and p is 20 to 45, or
          • ii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 6, 7, 8 or 9, or
          • iii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 6, 7, 8 or 9 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide with SEQ ID NOs: 6, 7, 8 or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
          • iv) 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: 6, 7, 8 or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
          • v) is a functional fragment of any one of SEQ ID NOs: 6, 7, 8 or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
          • vi) comprises 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: 6, 7, 8 or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
      • B. an N-acetylglucosamine b-1,4-galactosyltransferase that has
        • a. PFAM domain PF01755 and
          • i) comprises the sequence EXXCXXSHX[AFILTY]LW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 10, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or
          • ii) comprises the sequence EXXCXXSH[LR]VLW(Xn) EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 11, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or
          • iii) comprises the sequence EXXCXXSH[VHI]SLW (Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 12, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or
          • iv) comprises the sequence EXXCXXSHYMLW(Xn) EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 13, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or
          • v) comprises the sequence EXXCXXSHXX(X, no V)Y (Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 14, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or
          • vi) comprises a polypeptide sequence according to any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23, or
          • vii is a functional homologue, variant or derivative of any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • viii) 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: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • ix) is a functional fragment of any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • x) comprises 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: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
        • b. PFAM domain PF00535 and
          • i) comprises the sequence R[KN]XXXXXXXGXXX X[FL](X, no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE] with SEQ ID NO: 24 wherein X is any amino acid and wherein n is 50 to 75 and m is 10 to 30, or
          • ii) comprises the sequence R[KN]XXXXXXXGXXX X[FL](X, no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE](Xp)[FWY]XX[HKR]XX[NQST] with SEQ ID NO: 25 wherein X is any amino acid and wherein n is 50 to 75, m is 10 to 30 and p is 20 to 25, or
          • iii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 26 or 27, or
          • iv) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 26 or 27 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • v) 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: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • vi) is a functional fragment of any one of SEQ ID NOs: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • vii) comprises 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: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
        • c. PFAM domain PF02709 and not PFAM domain PF00535, and
          • i) comprises the sequence [FWY]XX[FWY](Xn) [FWY][GQ]X[DE]D with SEQ ID NO: 28 wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an IP or NL combination and wherein n is 21 to 26, or
          • ii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 29, 30, 31, 32, 33 or 34, or
          • iii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 29, 30, 31, 32, 33 or 34 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 29, 30, 31, 32, 33 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • iv) 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: 29, 30, 31, 32, 33 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • v) is a functional fragment of any one of SEQ ID NOs: 29, 30, 31, 32, 33 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • vi) comprises 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: 29, 30, 31, 32, 33 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
        • d. PFAM domain PF03808 and
          • i) comprises the sequence [ST][FHY]XN(Xn)DGXX XXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA with SEQ ID NO: 35, wherein X is any amino acid and wherein n is 20 to 25, or
          • ii) comprises the sequence [ST][FHY]XN(Xn)DGXXX XXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA(Xm)[HR]XG[FW Y](Xp)GXGXXXQ[DE] with SEQ ID NO: 36, wherein X is any amino acid and wherein n is 20 to 25, m is 40 to 50 and p is 22 to 30, or
          • iii) comprises a polypeptide sequence according to any one of SEQ ID NOs: 37, 38 or 39, or
          • iv) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 37, 38 or 39 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • v) 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: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • vi) functional fragment of any one of SEQ ID NOs: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
          • ii) comprises 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: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity.
    • 2. A method to synthesize a galactosylated disaccharide or oligosaccharide by use of an N-acetylglucosamine b-1,X-galactosyltransferase according to preferred embodiment 1.
    • 3. Method according to preferred embodiment 2, wherein the synthesis comprises the steps of:
      • a. providing UDP-galactose and any one of the galactosyltransferase, wherein the galactosyltransferase is capable of transferring a galactose residue from the UDP-galactose donor to one or more acceptor(s), and
      • b. contacting any one of the galactosyltransferase and UDP-galactose with one or more acceptor(s), under conditions where the galactosyltransferase catalyzes the transfer of a galactose residue from the UDP-galactose to the acceptor(s),
      • c. preferably, separating the galactosylated di- or oligosaccharide.
    • 4. Method according to preferred embodiment 3, wherein the acceptor(s) is/are an N-acetylglucosamine and/or an N-acetylgalactosamine as a monosaccharide, and/or a di- and/or oligosaccharide having an N-acetylglucosamine and/or N-acetylgalactosamine at its non-reducing end.
    • 5. Method according to any one of preferred embodiments 2 to 4, wherein the galactosylated disaccharide or oligosaccharide is produced in a cell-free system,
    • 6. Method according to any one of preferred embodiments 2 to 4, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell.
    • 7. Method according to preferred embodiment 6, wherein the cell
      • is capable of synthesizing one or more of the acceptor(s), and
      • expresses any one of the N-acetylglucosamine b-1,3-galactosyltransferases and/or N-acetylglucosamine b-1,4-galactosyltransferases, and
      • is capable of synthesizing UDP-galactose (UDP-Gal) as donor for the galactosyltransferases.
    • 8. Method according to any one of specific embodiments 6 or 7, wherein the cell is further capable of synthesizing one or more nucleotide-sugar donor(s) chosen from the list comprising: GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy-L-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, C1P—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-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose.
    • 9. Method according to any one of preferred embodiments 6 to 8, wherein the cell is further capable of expressing one or more glycosyltransferases selected from the list comprising: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases,
      • preferably, the fucosyltransferase is chosen from the list comprising alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase,
      • preferably, the sialyltransferase is chosen from the list comprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase,
      • preferably, the galactosyltransferase is chosen from the list comprising beta-1,3-galactosyltransferase, N-acetylglucosamine beta-1,3-galactosyltransferase, beta-1,4-galactosyltransferase, N-acetylglucosamine beta-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase and alpha-1,4-galactosyltransferase,
      • preferably, the glucosyltransferase is chosen from the list comprising alpha-glucosyltransferase, beta-1,2-glucosyltransferase, beta-1,3-glucosyltransferase and beta-1,4-glucosyltransferase,
      • preferably, the mannosyltransferase is chosen from the list comprising alpha-1,2-mannosyltransferase, alpha-1,3-mannosyltransferase and alpha-1,6-mannosyltransferase,
      • preferably, the N-acetylglucosaminyltransferase is chosen from the list comprising galactoside beta-1,3-N-acetylglucosaminyltransferase and beta-1,6-N-acetylglucosaminyltransferase,
      • preferably, the N-acetylgalactosaminyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase.
    • 10. Method according to any one of preferred embodiments 6 to 9, wherein the cell is a metabolically engineered cell.
    • 11. The method according to preferred embodiment 10, wherein the cell is modified with one or more gene expression modules, characterized in that the expression from any of the expression modules is either constitutive or is created by a natural inducer.
    • 12. The method according to any one of preferred embodiment 10 or 11, wherein the cell comprises multiple copies of the same coding DNA sequence encoding for one protein.
    • 13. Method according to any one of preferred embodiments 6 to 12, wherein the cell is modified in the expression or activity of an enzyme selected from the group comprising: glucosamine 6-phosphate N-acetyltransferase, phosphatase, glycosyltransferase, L-glutanine-D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase.
    • 14. Method according to any one of preferred embodiments 6 to 13, wherein the cell is unable to convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or unable to convert glucosamine-6-phosphate to fructose-6-phosphate.
    • 15. Method according to any one of preferred embodiments 6 to 14, wherein the cell is modified for enhanced UDP-galactose production and wherein the modification is chosen from the group comprising: knock-out of a 5′-nucleotidase/UDP-sugar hydrolase encoding gene or knock-out of a galactose-1-phosphate uridylyltransferase encoding gene.
    • 16. Method according to any one of preferred embodiments 6 to 15, wherein the cell is using one or more precursor(s) for the production of the galactosylated disaccharide or oligosaccharide, the precursor(s) being fed to the cell from the cultivation medium.
    • 17. Method according to any one of preferred embodiments 6 to 16, wherein the cell is producing one or more precursor(s) for the production of the galactosylated disaccharide or oligosaccharide.
    • 18. Method according to any one of preferred embodiments 16 or 17, wherein the precursor for the production of the galactosylated disaccharide or oligosaccharide is completely converted into the galactosylated disaccharide or oligosaccharide.
    • 19. Method according to any one of preferred embodiments 6 to 18, wherein the cell produces the galactosylated disaccharide or oligosaccharide intracellularly and wherein a fraction or substantially all of the produced galactosylated disaccharide or oligosaccharide remains intracellularly and/or is excreted outside the cell via passive or active transport.
    • 20. Method according to any one of preferred embodiments 6 to 19, 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,
      • preferably, the cell is modified in the expression or activity of the membrane transporter protein or polypeptide having transport activity.
    • 21. Method according to preferred embodiment 20, wherein the membrane transporter protein or polypeptide having transport activity is chosen from the list comprising porters, P—P-bond-hydrolysis-driven transporters, β-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.
    • 22. Method according to any one of preferred embodiments 20 or 21, wherein the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of the galactosylated disaccharide or oligosaccharide and/or of one or more precursor(s) and/or acceptor(s) to be used in the production of the galactosylated disaccharide or oligosaccharide.
    • 23. Method according to any one of preferred embodiments 20 to 22, wherein the membrane transporter protein or polypeptide having transport activity provides improved production and/or enabled and/or enhanced efflux of the galactosylated disaccharide or oligosaccharide.
    • 24. Method according to any one of the preferred embodiments 6 to 23, wherein the cell comprises a modification for reduced production of acetate compared to a non-modified progenitor.
    • 25. Method according to preferred embodiment 24, 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 isomnerase, aerobic respiration control protein, transcriptional repressor IclR, 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, pyruvate decarboxylase compared to a non-modified progenitor.
    • 26. Method according to any one of the preferred embodiments 6 to 25, wherein the cell is capable of producing phosphoenolpyruvate (PEP).
    • 27. Method according to any one of the preferred embodiments 6 to 26, wherein the cell is modified for enhanced production and/or supply of phosphoenolpyruvate (PEP) compared to a non-modified progenitor.
    • 28. Method according to any one of the preferred embodiments 6 to 27, wherein the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides that is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the production of the galactosylated disaccharide or oligosaccharide.
    • 29. Method according to any one of the preferred embodiments 6 to 28, 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).
    • 30. Method according to any one of the preferred embodiments 6 to 29, wherein the cell produces 90 g/L or more of the galactosylated disaccharide or oligosaccharide in the whole broth and/or supernatant and/or wherein the galactosylated disaccharide or oligosaccharide in the whole broth and/or supernatant has a purity of at least 80% measured on the total amount of the galactosylated disaccharide or oligosaccharide and its precursor(s) in the whole broth and/or supernatant, respectively.
    • 31. Method according to any one of the preferred embodiments 6 to 30, wherein the cell is stably cultured in a medium.
    • 32. Method according to any one of the preferred embodiments 6 to 31, wherein the conditions comprise:
      • use of a culture medium comprising at least one precursor and/or acceptor for the production of the galactosylated disaccharide or oligosaccharide, and/or
      • adding to the culture medium at least one precursor and/or acceptor feed for the production of the galactosylated disaccharide or oligosaccharide.
    • 33. Method according to any one of the preferred embodiments 6 to 32, 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 the galactosylated disaccharide or oligosaccharide 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.
    • 34. Method according to any one of the preferred embodiments 3 to 32, 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 two-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 two-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 the galactosylated disaccharide or oligosaccharide 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.
    • 35. Method according to preferred embodiment 34, 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.
    • 36. Method according to any one of preferred embodiment 34 or 35, 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.
    • 37. Method according to any one of preferred embodiments 6 to 36, wherein the cells are cultivated for at least about 60, 80, 100, or about 120 hours or in a continuous manner.
    • 38. Method according to any one of preferred embodiments 6 to 37, wherein the culture medium contains at least one precursor selected from the group comprising lactose, galactose, fucose and sialic acid.
    • 39. Method according to any one of preferred embodiments 6 to 38, 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, followed by a second phase wherein only a carbon-based substrate, preferably glucose or sucrose, is added to the culture medium.
    • 40. Method according to any one of preferred embodiments 6 to 38, 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, followed by a second phase wherein a carbon-based substrate, preferably glucose or sucrose, and a precursor are added to the culture medium.
    • 41. Method according to any one of preferred embodiments 6 to 40, wherein the cell is capable of catabolizing a carbon source selected from the list comprising glucose, fructose, mannose, galactose, lactose, sucrose, maltose, malto-oligosaccharides, trehalose, starch, cellulose, hemi-cellulose, corn-steep liquor, molasses, high-fructose syrup, glycerol, acetate, citrate, lactate and pyruvate.
    • 42. Method according to any one of preferred embodiments 1 to 5, wherein the method produces a mixture of charged and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide.
    • 43. Method according to any one of preferred embodiments 1 to 5, wherein the method produces a mixture of charged and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide.
    • 44. Method according to any one of preferred embodiments 6 to 41, wherein the cell produces a mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide.
    • 45. Method according to any one of preferred embodiments 6 to 41, wherein the cell produces a mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide.
    • 46. Method according to any one of preferred embodiments 3 to 45, 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.
    • 47. Method according to any one of preferred embodiments 3 to 46, further comprising purification of the galactosylated di- or oligosaccharide.
    • 48. Method according to preferred embodiment 47, 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.
    • 49. A cell metabolically engineered to synthesize a galactosylated disaccharide or oligosaccharide by use of an N-acetylglucosamine b-1,X-galactosyltransferase according to embodiment 1.
    • 50. The cell of preferred embodiment 49, wherein the cell
      • expresses any one of the N-acetylglucosamine b-1,3-galactosyltransferases and/or N-acetylglucosamine b-1,4-galactosyltransferases, and
      • is capable of synthesizing UDP-galactose (UDP-Gal) as donor for the galactosyltransferases, and
      • is capable of synthesizing one or more acceptor(s) for the galactosyltransferases, wherein the acceptor(s) is/are an N-acetylglucosamine as a monosaccharide, and/or a di- or oligosaccharide having an N-acetylglucosamine and/or N-acetylgalactosamine at its non-reducing end.
    • 51. Cell according to any one of preferred embodiment 49 or 50, wherein the cell is modified with one or more gene expression modules, characterized in that the expression from any of the expression modules is either constitutive or is created by a natural inducer.
    • 52. Cell according to any one of preferred embodiment 49 to 51, wherein the cell comprises multiple copies of the same coding DNA sequence encoding for one protein.
    • 53. The cell of any one of preferred embodiments 49 to 52, wherein the cell is further capable of synthesizing one or more nucleotide-sugar donor(s) chosen from the list comprising: GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy-L-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-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose.
    • 54. The cell of any one of preferred embodiments 49 to 53, wherein the cell is further capable of expressing one or more glycosyltransferases selected from the list comprising: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolyineurarninyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases,
      • preferably, the fucosyltransferase is chosen from the list comprising alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase,
      • preferably, the sialyltransferase is chosen from the list comprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase,
      • preferably, the galactosyltransferase is chosen from the list comprising beta-1,3-galactosyltransferase, N-acetylglucosamine beta-1,3-galactosyltransferase, beta-1,4-galactosyltransferase, N-acetylglucosamine beta-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase and alpha-1,4-galactosyltransferase,
      • preferably, the glucosyltransferase is chosen from the list comprising alpha-glucosyltransferase, beta-1,2-glucosyltransferase, beta-1,3-glucosyltransferase and beta-1,4-glucosyltransferase,
      • preferably, the mannosyltransferase is chosen from the list comprising alpha-1,2-mannosyltransferase, alpha-1,3-mannosyltransferase and alpha-1,6-mannosyltransferase,
      • preferably, the N-acetylglucosaminyltransferase is chosen from the list comprising galactoside beta-1,3-N-acetylglucosaminyltransferase and beta-1,6-N-acetylglucosaminyltransferase,
      • preferably, the N-acetylgalactosaminyltransferase is an alpha-1,3-N-acetylgalactosaminyltransferase,
      • preferably, the cell is modified in the expression or activity of the further glycosyltransferase.
    • 55. The cell of any one of preferred embodiments 49 to 54, wherein the cell is modified in the expression or activity of an enzyme selected from the group comprising: glucosamine 6-phosphate N-acetyltransferase, phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase.
    • 56. The cell of any one of preferred embodiments 49 to 55, wherein the cell is unable to convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or unable to convert glucosamine-6-phosphate to fructose-6-phosphate.
    • 57. The cell of any one of preferred embodiments 49 to 56, wherein the cell is modified for enhanced UDP-galactose production and wherein the modification is chosen from the group comprising: knock-out of a 5′-nucleotidase/UDP-sugar hydrolase encoding gene or knock-out of a galactose-1-phosphate uridylyltransferase encoding gene.
    • 58. The cell of any one of preferred embodiments 49 to 57, wherein the cell is using one or more precursor(s) for the production of the galactosylated disaccharide or oligosaccharide, the precursor(s) being fed to the cell from the cultivation medium.
    • 59. The cell of any one of preferred embodiments 49 to 58, wherein the cell is producing one or more precursor(s) for the production of the galactosylated disaccharide or oligosaccharide.
    • 60. The cell of any one of preferred embodiments 58 or 59, wherein the precursor for the production of the galactosylated disaccharide or oligosaccharide is completely converted into the galactosylated disaccharide or oligosaccharide.
    • 61. The cell of any one of preferred embodiments 49 to 60, wherein the cell produces the galactosylated disaccharide or oligosaccharide intracellularly and wherein a fraction or substantially all of the produced galactosylated disaccharide or oligosaccharide remains intracellularly and/or is excreted outside the cell via passive or active transport.
    • 62. The cell of any one of preferred embodiments 49 to 61, 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, preferably, the cell is modified in the expression or activity of the membrane transporter protein or polypeptide having transport activity.
    • 63. The cell of preferred embodiment 62, wherein the membrane transporter protein or polypeptide having transport activity is chosen from the list comprising porters, P—P-bond-hydrolysis-driven transporters, β-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, and preferably, the P—P-bond-hydrolysis-driven transporters comprise ABC transporters and siderophore exporters.
    • 64. The cell of any one of preferred embodiments 62 or 63, wherein the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of the galactosylated disaccharide or oligosaccharide and/or of one or more precursor(s) and/or acceptor(s) to be used in the production of the galactosylated disaccharide or oligosaccharide.
    • 65. The cell of any one of preferred embodiments 62 to 64, wherein the membrane transporter protein or polypeptide having transport activity provides improved production and/or enabled and/or enhanced efflux of the galactosylated disaccharide or oligosaccharide.
    • 66. The cell of any one of preferred embodiments 49 to 65, wherein the cell comprises a modification for reduced production of acetate compared to a non-modified progenitor.
    • 67. The cell of preferred embodiment 66, 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 0-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 IBC component ptsG, glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIAGlc, beta-glucoside specific PT'S 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 compared to a non-modified progenitor.
    • 68. The cell of any one of preferred embodiments 49 to 67, wherein the cell is capable of producing phosphoenolpyruvate (PEP).
    • 69. The cell of any one of preferred embodiments 49 to 68, wherein the cell is modified for enhanced production and/or supply of phosphoenolpyruvate (PEP) compared to a non-modified progenitor.
    • 70. The cell of any one of preferred embodiments 49 to 69, wherein the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides that is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the production of the galactosylated disaccharide or oligosaccharide.
    • 71. The cell of any one of preferred embodiments 49 to 70, 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).
    • 72. The cell of any one of preferred embodiments 49 to 71, wherein the cell is capable of catabolizing a carbon source selected from the list comprising glucose, fructose, mannose, galactose, lactose, sucrose, maltose, malto-oligosaccharides, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, glycerol, acetate, citrate, lactate and pyruvate.
    • 73. The cell of any one of preferred embodiments 49 to 72 or method according to any one of preferred embodiments 6 to 48, 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.
    • 74. The cell of preferred embodiment 73 or method according to preferred embodiment 73, 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 compared to a non-modified progenitor.
    • 75. The cell of any one of preferred embodiments 49 to 74, wherein the cell produces a mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide.
    • 76. The cell of any one of preferred embodiments 49 to 74, wherein the cell produces a mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide.
    • 77. Use of the cell of any one of preferred embodiments 49 to 74, or a method according to any one of preferred embodiments 1 to 41, 73 to 74 for the production of a galactosylated disaccharide or oligosaccharide.
    • 78. Use of the cell of any one of preferred embodiments 49 to 75, or a method according to any one of preferred embodiments 1 to 42, 73 to 74 for the production of a mixture of charged, preferably sialylated, and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide.
    • 79. Use of the cell of any one of preferred embodiments 49 to 76, or a method according to any one of preferred embodiments 1 to 43, 73 to 74 for the production of a mixture of charged, preferably sialylated, and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide.

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

DETAILED DESCRIPTION Examples Example 1: Materials and Methods Escherichia coli

Media: The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, BE), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, BE). The 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 medium was set to a pH of 7 with 1 M 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 and/or 20 g/L lactose, were additionally added to the minimal medium for fermentations 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, BE in 2007).

Plasmids were maintained in the host E. coli DH5alpha (F−, phi80dlacZΔM15, Δ(lacZYA-argF) U169, deoR, recAl, endAl, hsdR17(rk−, mk+), phoA, supE44, lambda−, thi-1, gyrA96, relAl) bought from Invitrogen.

Strains and Mutaions: 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 hour 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 Dpnl, 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 and fucosylated oligosaccharide 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 expression constructs containing a sucrose transporter like, e.g., CscB originating from E. coli W (UniProt ID EOIXR1), a fructose kinase like, e.g., frk originating from Zymomonas mobilis (ZmFrk) (UniProt ID Q03417), a sucrose phosphorylase like, e.g., BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6), additionally comprising expression plasmids with constitutive expression constructs for an alpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBank No. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProt ID 030511) and with a constitutive expression construct for the E. coli thyA (UniProt ID P0A884) as selective marker. The constitutive expression constructs of the fucosyltransferase genes can also be present in the mutant E. coli strain via genomic knock-ins. GDP-fucose production can further be optimized in the mutant E. coli strain by genomic knock-outs of the E. coli genes comprising glgC, agp, pfkA, pfkB, pgi, arcA, iclR, pgi and lon as described in WO2016075243 and WO2012007481. GDP-fucose production can additionally be optimized comprising genomic knock-ins of constitutive expression constructs for a mannose-6-phosphate isomerases 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 together with genomic knock-ins of constitutive expression constructs containing fucose permease like, e.g., fucP from E. coli (UniProt ID P11551) and a bifunctional enzyme with fucose kinase/fucose-1-phosphate guanylyltransferase activity like, e.g., fkp from Bacteroides fragilis (UniProt ID SUV40286.1). If the mutant strain producing GDP-fucose was intended to make fucosylated lactose structures, the strain was additionally modified with genomic knock-outs of the E. coli LacZ, LacY and LacA genes and with a genomic knock-in of a constitutive expression construct for a lactose permease like, e.g., the E. coli LacY (UniProt ID P02920).

Alternatively, and/or additionally, GDP-fucose and/or fucosylated oligosaccharide production can further be optimized in the mutant E. coli strains with genomic knock-ins of constitutive transcriptional units 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 POAEY8), 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 a 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), a 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, idhA, 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 a G472S mutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), preferably one phosphatase like any one or more of, 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 O66375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID O66375 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 expression plasmids. 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). All mutant strains producing sialic acid, CMP-sialic acid and/or sialylated oligosaccharides could 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).

Alternatively, and/or additionally, sialic acid and/or sialylated oligosaccharide production can further be optimized in the mutant E. coli strains with genomic knock-ins of constitutive transcriptional units 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 O6: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), 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 POAEY8), 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 A0A1V0NEL4), or Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4).

In an example for enhanced UDP-galactose production, the E. coli K12 MG1655 strain was modified with genomic knock-outs of any one or more of the E. coli ushA, galT, ldhA and agp genes and with a genomic knock-in of a constitutive expression construct for the UDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147).

In an example for enhanced UDP-GlcNAc production, the E. coli K12 MG1655 strain was modified 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 a G472S mutation as described by Deng et a1. (Biochimie 2006, 88: 419-429).

In an example to produce lacto-N-triose (LN3, GlcNAc-b1,3-Gal-b1,4-Glc), the mutant strain was derived from E. coli K12 MG1655 and modified with a knock-out 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., the E. coli LacY (UniProt ID P02920) and a galactoside beta-1,3-N-acetylglucosaminyltransferase like, e.g., lgtA (UniProt ID Q9JXQ6) from N. meningitidis.

In an example for production of LN3 derived oligosaccharides like lacto-N-tetraose (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), the mutant LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N-acetylglucosamine beta-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9.

In an example for production of LN3 derived oligosaccharides like lacto-N-neotetraose (LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), the mutant LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N-acetylglucosamine beta-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39.

In an example for production of lacto-N-biose (LNB, Gal-b1,3-GlcNAc) and LNB-derived oligosaccharides the strains were modified with genomic knock-ins or expression plasmids comprising constitutive transcriptional units for one or more copies of a glucosamine 6-phosphate N-acetyltransferase like, e.g., GNA1 from S. cerevisiae (UniProt ID P43577) and an N-acetylglucosamine beta-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9.

In an example for production of N-acetyllactosamine (LacNAc, Gal-b1,4-GlcNAc) and LacNAc-derived oligosaccharides the strains were modified with genomic knock-ins or expression plasmids comprising constitutive transcriptional units for one or more copies of a glucosamine 6-phosphate N-acetyltransferase like, e.g., GNA1 from S. cerevisiae (UniProt ID P43577) and an N-acetylglucosamine beta-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39.

The mutant LNB, LacNAc, LN3, LNT and LNnT producing E. coli strains can 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 EOIXR1), 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).

Alternatively, and/or additionally, production of LN3, LNT, LNnT, LNB, LacNAc and oligosaccharides derived thereof 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 POAEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207) or iceT from Citrobacter youngae (UniProt ID D4B8A6).

Preferably, but not necessarily, any one or more of the glycosyltransferases, the proteins involved in nucleotide-activated sugar synthesis and/or membrane transporter protein 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, https://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, wbbl, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA and yaiP.

All constitutive promoters and UTRs originated from the libraries described by De Mey et a1. (BMC Biotechnology, 2007), Dunn et a1. (Nucleic Acids Res. 1980, 8(10), 2119-2132), Kim and Lee (FEBS letters 1997, 407(3), 353-356) and Mutalik et a1. (Nat. Methods 2013, No. 10, 354-360).

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

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.

All strains are 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 SEQ Country of origin ID of digital NO Organism Origin sequence information 01 N.A. Synthetic Artificial sequence 02 N.A. Synthetic Artificial sequence 03 Corynebacterium glutamicum Synthetic Japan 04 Photobacterium leiognathi Synthetic USA 05 N.A. Synthetic Artificial sequence 06 A. thaliana Synthetic USA 07 Trypanosoma brucei Synthetic Scotland 08 Mus musculus Synthetic USA 09 Homo sapiens Synthetic Unknown 10 N.A. Synthetic Artificial sequence 11 N.A. Synthetic Artificial sequence 12 N.A. Synthetic Artificial sequence 13 N.A. Synthetic Artificial sequence 14 N.A. Synthetic Artificial sequence 15 Basilea psittacipulmonis Synthetic Switzerland 16 Neisseria arctica Synthetic USA 17 Glaesserella parasuis Synthetic USA 18 Actinobacillus seminis Synthetic Australia 19 Alysiella filiformis Synthetic Australia 20 Conchiformibius steedae Synthetic United Kingdom 21 Acinetobacter haemolyticus Synthetic South-Korea 22 Campylobacter pylori Synthetic Switzerland 23 Histophilus somni Synthetic Canada 24 N.A. Synthetic Artificial sequence 25 N.A. Synthetic Artificial sequence 26 Streptococcus pneumoniae Synthetic USA 27 Hafnia alvei Synthetic Unknown 28 N.A. Synthetic Artificial sequence 29 Mycolicibacterium flavescens Synthetic USA 30 Sphingomonas sp. Synthetic United Kingdom 31 Parachlamydiaceae bacterium Synthetic Japan HS-T3 32 Coxiella sp. DG_40 Synthetic USA 33 Corallococcus exercitus Synthetic United Kingdom 34 Hypericibacter adhaerens Synthetic Germany 35 N.A. Synthetic Artificial sequence 36 N.A. Synthetic Artificial sequence 37 Bacteroides vulgatus Synthetic United Kingdom 38 Prevotella copri Synthetic USA 39 Pseudomonas fluorescens Synthetic USA 90F12-2

Cultivation Conditions: A pre-culture 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 MMsf 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 hours, 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 pre-culture for the bioreactor was started from an entire 1 mL cryovial of a certain strain, inoculated in 250 mL or 500 mL of MMsf medium in a 1 L or 2.5 L shake flask and incubated for 24 hours 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 conditions 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% NH40H. 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, BE or with a Spark 10 M microplate reader, Tecan, CH).

Analytical Analysis: Standards such as, but not limited to, sucrose, glucose, N-acetylglucosamine, N-acetyllactosamine, lacto-N-biose, fucosylated N-acetyllactosamine (2′FLAcNAc, 3-FlacNAc), fucosylated lacto-N-biose (2′FLNB, 4-FLNB), sialylated N-acetyllactosamine (3′SLacNAc, 6′SLacNAc were purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analyzed with in-house made standards.

N-acetylglucosamine and N-acetyllactosamine were analyzed on a Dionex HPAEC system with pulsed amperometric detection (PAD). A volume of 5 μL of sample was injected on a Dionex CarboPac PA1 column 4×250 mm with a Dionex CarboPac PA1 guard column 4×50 mm. The column temperature was 20° C. The separation was performed using 3 eluents: A) deionized water B) 200 mM Sodium hydroxide and C) 500 mM Sodium acetate. The elution profile was applied as follow: 0-10 min. 50% A and 50% B; 10-18 min. 50-44% A and 50% B; 18-28 min. 44% A and 50% B; 28-32 min. 44-30.8% A and 50% B; 32-39 min. 30.8% A and 50% B; 39-40 min. 30.8-2% A and 50% B; 40-43 min. 2% A and 50% B; 43-44 min. 2-50% A and 50% B; 44-50 min. 50% A and 50% B. Flow rate was 1.0 mL/minute.

N-acetylglucosamine, N-acetyllactosamine, lacto-N-biose, fucosylated N-acetyllactosamine and fucosylated LNB 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 consists of a 1/4 water and 3/4 acetonitrile solution were 0.2% Triethylamine was added. The method was isocratic with a flow of 0.130 mL/minute. The ELS detector had a drift tube temperature of 50° C. and N2 gas pressure was 50 psi, gain 200 and data rate is 10 pps. The temperature of the RI detector was set at 35° C.

Sialylated N-acetyllactosamine and sialylated lacto-N-biose were analyzed with 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 with 1.7 μm particle size) with a mobile phase containing 70 mL acetonitrile, 26 mL 150 mM ammonium acetate in ultrapure water and 4 mL methanol with 0.05% pyrrolidine added. The method was isocratic with a flow rate of 0.150 mL/minute. The temperature of the column was set at 50° C.

Sugars at low concentrations (below 50 mg/L) were analyzed on a Dionex HPAEC system with pulsed amperometric detection (PAD). 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 minute and maintained for 11 min. The applied flow was 0.5 mL/minute.

Normalization of the data: For all types of cultivation conditions, data obtained from the mutant strains was normalized against data obtained in identical cultivation conditions with reference strains.

Example 2: Production of GlcNAc in a Modified E. coli Host

In this example, a wild-type E. coli K-12 MG1655 was modified with a knock-out of the homologous E. coli N-acetylglucosamine-6-phosphate deacetylase (nagA) gene and the E. coli glucosamine-6-phosphate deaminase (nagB) gene and then transformed with an expression plasmid comprising a constitutive transcriptional unit for the glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577). The thus obtained mutant E. coli strain produced GlcNAc in whole broth samples when evaluated in a growth experiment, according to the culture conditions in Example 1, in which the culture medium contained glycerol.

Example 3: Production of GlcNAc in a Modified E. coli Host

The mutant E. coli strain modified to produce GlcNAc as described in Example 2 was further transformed with a second compatible expression plasmid comprising a constitutive transcriptional unit for the mutated variant of the L-glutamine-D-fructose-6-phosphate aminotransferase (glmS*54) from E. coli differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation as described by Deng et a1. (Biochimie 2006: 88, 419-429). The novel strain produced GlcNAc in whole broth samples when evaluated in a growth experiment, according to the culture conditions in Example 1, in which the culture medium contained glycerol.

Example 4: Production of G/cNAc in a Modified E. coli Host

A wild-type E. coli K-12 MG1655 was modified with a knock-out of the E. coli nagA and the nagB genes and a genomic knock-in of a constitutive transcriptional unit for GNA1 from S. cerevisiae (UniProt ID P43577). The thus obtained mutant E. coli strain produces GlcNAc in whole broth samples when evaluated in a growth experiment, according to the culture conditions in Example 1, in which the culture medium contains glycerol.

Example 5: Production of GlcNAc in a Modified E. coli Host

The mutant E. coli strain modified to produce GlcNAc as described in Example 4 was further transformed with an expression plasmid comprising a constitutive transcriptional unit for glmS*54 from E. coli differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation as described by Deng et a1. (Biochimie 2006: 88, 419-429). The novel strain produced GlcNAc in whole broth samples when evaluated in a growth experiment, according to the culture conditions in Example 1, in which the culture medium contained glycerol.

Example 6: Production of GlcNAc in a Modified E. coli Host

The mutant E. coli strain modified to produce GlcNAc as described in Example 4 was further transformed with a genomic knock-in comprising a constitutive transcriptional unit for glmS*54 from E. coli differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation as described by Deng et a1. (Biochimie 2006: 88, 419-429). The novel strain produced GlcNAc in whole broth samples when evaluated in a growth experiment, according to the culture conditions in Example 1, in which the culture medium contained glycerol.

Example 7: Production of G/cNAc in a Modified E. coli Host

The mutant E. coli strain modified to produce GlcNAc as described in Example 2 was further modified with a genomic knock-in comprising a constitutive transcriptional unit for glmS*54 from E. coli differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation as described by Deng et a1. (Biochimie 2006: 88, 419-429). The novel strain produced GlcNAc in whole broth samples when evaluated in a growth experiment, according to the culture conditions in Example 1, in which the culture medium contained glycerol.

Example 8: Production of LacNAc or LNB in a Modified E. coli Host

The mutant GlcNAc-producing E. coli K-12 MG1655 strains having a nagAB KO and expressing GNA1 (UniProt ID P43577) with or without additional expression of the mutant glmS*54 differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation as described by Deng et a1. (Biochimie 2006: 88, 419-429) as described in Examples 2 and 4 to 7 are in a next example transformed with a plasmid containing a constitutive transcriptional unit to express either an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 or an N-acetylglucosamine β1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8, and 9.

Each of the novel strains expressing an N-acetylglucosamine β1,4-galactosyltransferase are evaluated for production of GlcNAc and LacNAc in whole broth samples in a growth experiment according to the culture conditions in Example 1 in which the culture medium contains glycerol.

Each of the novel strains expressing an N-acetylglucosamine β1,3-are evaluated for production of GlcNAc and LNB in whole broth samples in a growth experiment according to the culture conditions in Example 1 in which the culture medium contains glycerol.

Example 9: Production of GlcNAc and LacNAc in Modified E. coli Hosts

An E. coli K-12 MG1655 mutant strain optimized for GDP-fucose production as described in Example 1, is further modified with a knock-out of the E. coli nagA and nagB genes and with a genomic knock-in of a constitutive expression construct of an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39. In a next step, cells of the mutant strain are transformed with an expression vector comprising constitutive transcriptional units of the mutant glmS*54 from E. coli (differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation) and GNA1 from S. cerevisiae (UniProt ID P43577). The novel strain is evaluated for production of GlcNAc and LacNAc in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose. The strain is grown for 72 hours in multiple wells of a 96-well plate; afterwards the culture broth is harvested and the GlcNAc and LacNAc are analyzed on UPLC.

Example 10: Production of GlcNAc and LacNAc in Modified E. coli Hosts

In a next experiment, an E. coli K-12 MG1655 strain producing sialic acid, as described in Example 1 and containing knock-outs of the E. coli nagA and nagB genes and genomic knock-ins of constitutive expression constructs containing glmS*54, differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation as described by Deng et a1. (Biochimie 2006: 88, 419-429), GNA1 (UniProt ID P43577), the N-acetylglucosamine 2-epimerase (AGE) from Bacteroides ovatus (UniProt ID A7LVG6) and the N-acetylneuraminate synthase from N. meningitidis (UniProt ID E0NCD4), is further modified with a knock-in of an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39.

Also, an E. coli K-12 MG1655 strain optimized for enhanced UDP-galactose production with genomic knock-outs of the E. coli ushA and galT genes and with a genomic knock-in of a constitutive expression construct for the UDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147) as described in Example 1, was additionally mutated by a knock-out of the E. coli nagB gene and with a genomic knock-in of a constitutive expression construct of an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39. The novel strains are evaluated for production of GlcNAc and LacNAc in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose. Each strain is grown for 72 hours in multiple wells of a 96-well plate; afterwards the culture broth is harvested and the GlcNAc and LacNAc are analyzed on UPLC.

Example 11: Production of Galactosylated Oligosaccharides in a Modified E. coli Host

An E. coli K-12 MG1655 strain optimized for enhanced UDP-galactose production and capable of producing GlcNAc and LacNAc as described in Example 10, can additionally be transformed with an expression vector containing a constitutive expression construct for an N-acetylglucosamine β1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9. The novel strain is evaluation for production of Gal-b14-(Galb13)-GlcNAc, containing two galactose moieties linked beta-1,3 and beta-1,4 to GlcNAc, in addition to GlcNAc, LacNAc and LNB when evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose.

Example 12: Production of Modified LacNAc in a E. coli Host

An E. coli K-12 MG1655 strain optimized for GDP-fucose production and capable of produce GlcNAc and LacNAc as described in Example 9, can additionally be transformed with an expression plasmid containing a constitutive expression construct for the b1,3-N-acetyl-hexosaminyl-transferase LgtA from N. meningitidis (UniProt ID Q9JXQ6). By subsequent action of the mutant glmS*54 (differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation), the homologous EcGlmM and EcGlmU and the heterologous LgtA (UniProt ID Q9JXQ6), the thus created strain is capable of intracellularly converting fructose-6-phosphate into UDP-GlcNAc, and of using this UDP-GlcNAc to intracellularly modify LacNAc leading to production of GlcNAc-b1,3-Gal-b1,4-GlcNAc in whole broth samples when evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose. The novel strain is also capable of producing poly-LacNAc structures, i.e., (Gal-b1,4-GlcNAc)n which are built of repeated N-acetyllactosamine that are beta1,3-linked to each other by alternate activity of the N-acetylglucosamine β1,4-galactosyltransferase and LgtA expressed in the strain.

An E. coli K-12 MG1655 strain optimized for UDP-galactose production and capable of producing GlcNAc and LacNAc as described in Example 10, is modified to constitutively express the UDP-GlcNAc epimerase wbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) and the glycosyltransferase lgtD from Haemophilus influenzae (UniProt ID A0A2X4DBP3). By subsequent action of the mutant E. coli glmS*54, the homologous E. coli glmM and glmU and the P. aeruginosa wbpP, the cell is capable of intracellularly converting fructose-6-phosphate into UDP-GalNAc vithe intermediate compounds glucosamine-6-phosphate, glucosamine-1-phosphate and UDP-GlcNAc. By subsequent action of the newly expressed LgtD enzyme, the novel strain is capable of modifying the intracellularly produced LacNAc with GalNAc, producing GalNAc-b1,3-Gal-b1,4-GlcNAc in whole broth samples when evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains 30 g/L sucrose.

Example 13: Production of LNB in a Modified E. coli Host

In a next experiment, an E. coli K-12 MG1655 strain optimized for GDP-fucose production was modified with genomic knock-ins of constitutive expression constructs for glmS*54 (differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation) and GNA1 from S. cerevisiae (UniProt ID P43577) to intracellularly produce GlcNAc. In a next step, the mutant strain was further modified for constitutive expression of the N-acetylglucosamine β1,3-galactosyltransferase from C. glutamicum with SEQ ID NO: 3, either from plasmid or from a genomic knock-in. The novel strains were evaluated in a growth experiment according to the culture conditions provided in Example 1. Table 2 shows the production of LNB (g/L) in whole broth samples from each of the mutant strains, taken after 72 hours of cultivation in minimal medium with 30 g/L sucrose. The data demonstrates that both novel strains produced LNB in whole broth samples, independent of how the N-acetylglucosamine β1,3-galactosyltransferase gene was presented to the strain.

TABLE 2 Production of LNB (g/L) in whole broth samples taken from mutant E. coli strains after 72 hours of cultivation in minimal medium comprising 30 g/L sucrose Expression of a transcriptional unit for the N- acetylglucosamine β1,3-galactosyltransferase with LNB (g/L) SEQ ID NO: 3 (± sd) From genomic knock-in 0.63 (±0.12) From an expression plasmid 2.81 (±0.11)

Example 14: Production of Galactosylated LNB in a Modified E. coli Host

An E. coli K-12 MG1655 strain optimized for enhanced production of UDP-galactose as described in Example 1 was additionally mutated by a knock-out of the E. coli nagB gene and was further modified for constitutive expression of the N-acetylglucosamine β1,3-galactosyltransferase from C. glutamicum with SEQ ID NO: 3, either from plasmid or from a genomic knock-in. Both novel strains produce LNB in whole broth samples when evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose. When the novel LNB production strains are additionally transformed with expression constructs for galactosyltransferases each of the novel strains produces GlcNAc and LNB together with galactosylated LNB forms in whole broth samples, when evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose.

Example 15: Fermentative Production of LacNAc with a Mutant E. coli Host

A mutant E. coli K-12 MG1655 strain optimized for GDP-fucose production as described in Example 1 and modified to produce GlcNAc and LacNAc by genomic knock-ins of constitutive transcriptional units for glmS*54 (differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation) and GNA1 (UniProt ID P43577), was evaluated in a fed-batch fermentation at 5 L bioreactor scale according to the conditions provided in Example 1. In this example, sucrose was used as a carbon source. Regular samples were taken and the production of LacNAc was measured, as described in Example 1.

Example 16: Production of LacNAc or LNB in a Modified E. coli Host when Grown on Other Carbon Sources than Sucrose

Mutant E. coli strains modified for the production of GlcNAc and LacNAc as described in Examples 9 and 10 or mutant E. coli strains modified for the production of GlcNAc and LNB as described in Example 13 and 14 are capable of producing GlcNAc and LacNAc or GlcNAc and LNB, respectively, when evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains glycerol. The mutant strains are also capable of producing GlcNAc and LacNAc or GlcNAc and LNB, respectively, when evaluated in fed-batch fermentations at bioreactor scale, as described in Example 1, using any one or more of but not limited to following carbon sources: glycerol, glucose, fructose, lactose, arabinose, maltotriose, sorbitol, xylose, rhamnose and mannose.

Example 17: Materials and Methods in Saccharomyces cerevisiae

Media: Strains are grown on Synthetic Defined yeast medium with Complete Supplement Mixture (SD CSM) or CSM drop-out (SD CSM-Ura) 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 (MP Biomedicals).

Strains: Saccharomyces cerevisiae BY4742 created by Brachmann et a1. (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: Yeast expression plasmid p2a_2μ (Chan 2013, Plasmid 70, 2-17) was used for expression of foreign genes in Saccharomyces cerevisiae. This plasmid contained an ampicillin resistance gene and a bacterial origin of replication to allow for selection and maintenance in E. coli. The plasmid further contained the 2p yeast on and the Ura3 selection marker for selection and maintenance in yeast. In one example, the yeast expression plasmid p2a_2μ can be modified to obtain the mutant fructose-6-phosphate aminotransferase glmS*54 from E. coli (differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation) as described in WO18122225, the glucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae (UniProt ID P43577), a phosphatase like any one or more of, 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 WO18122225. The modified plasmids can further be modified to obtain an N-acetylglucosamine b-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and/or an N-acetylglucosamine b-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39.

In one example to produce GDP-fucose, a yeast expression plasmid like p2a_2μ_Fuc (Chan 2013, Plasmid 70, 2-17) is further modified with constitutive transcriptional units for a lactose permease like, e.g., LAC12 from K. 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 can be 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 on 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 enzyme with fucose kinase/fucose-1-phosphate guanylyltransferase activity like, e.g., fkp from Bacteroides 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 No. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProt ID 030511).

In one example to produce UDP-galactose, a yeast expression plasmid can be 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 a UDP-glucose-4-epimerase like, e.g., galE from E. coli (UniProt ID P09147). This plasmid can be further modified with constitutive transcriptional units for a lactose permease like, e.g., LAC12 from K. lactis (UniProt ID P07921) and a galactoside beta-1,3-N-acetylglucosaminyltransferase activity like, e.g., lgtA from N. meningitidis (UniProt ID Q9JXQ6) to produce LN3. To further produce LN3-derived oligosaccharides like LNT, the mutant LN3 producing strains were further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9. To further produce LN3-derived oligosaccharides like LNnT, the mutant LN3 producing strains were further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39.

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 a G472S mutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), one phosphatase like any one or more of, 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 WO18122225, 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), and 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). 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, the glycosyltransferase proteins and/or the proteins involved in nucleotide-activated sugar synthesis were N-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 knock-ins 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, recAl, endAl, 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 pre-culture 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 pre-culture, 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 18: Production of GlcNAc and LacNAc; or GlcNAc and LNB in S. cerevisiae

Another example provides use of a eukaryotic organism, in the form of S. cerevisiae, for performing the disclosure. Using the strains, plasmids and methods as described in Example 17, a mutant S. cerevisiae strain is created that produces GlcNAc and LacNAc. These modifications comprise the addition of constitutive expression units for the mutant fructose-6-phosphate aminotransferase glmS*54 of E. coli (differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and a G472S mutation), the glucosamine 6-phosphate N-acetyltransferase GNA1 of S. cerevisiae (UniProt ID P43577), one phosphatase chosen from the list comprising any one or more of the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida, ScDOG1 from S. cerevisiae and BsAraL from Bacillus subtilis as described in WO18122225 and an N-acetylglucosamine b-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39. The mutant S. cerevisiae strain is capable of growing on glucose or glycerol as carbon source, converting the carbon source into fructose-6-phosphate, which is then converted to GlcNAc and subsequently LacNAc, by activity of the novel expressed fructose-6-phosphate aminotransferase, glucosamine 6-phosphate N-acetyltransferase, the phosphatase and N-acetylglucosamine b-1,4-galactosyltransferase.

Pre-culture of the strain is made in 5 mL of the synthetic defined medium SD-CSM containing 22 g/L glucose and grown at 30° C. as described in Example 17. This pre-culture is then inoculated in 25 mL medium in a shake flask with 10 g/L glucose as sole carbon source and grown at 30° C. Regular samples are taken and the production of GlcNAc and LacNAc is measured as described in Example 1.

A similar yeast strain containing an N-acetylglucosamine b-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 instead of the N-acetylglucosamine b-1,4-galactosyltransferase is capable of producing GlcNAc and LNB in a similar cultivation experiment.

Example 19: Enzymatic Production of Galactosylated Di- or Oligosaccharides

Another example provides the use of N-acetylglucosamine b-1,3-galactosyltransferases and N-acetylglucosamine b-1,4-galactosyltransferases of the disclosure to synthesize galactosylated di- or oligosaccharides. These enzymes are produced in a cell-free expression system such as but not limited to the PURExpress system (NEB), or in a host organism such as but not limited to Escherichia coli or Saccharomyces cerevisiae, after which the above-listed enzymes can be isolated and optionally further purified.

An N-acetylglucosamine b-1,3-galactosyltransferase or an N-acetylglucosamine b-1,4-galactosyltransferase, selected from the above enzyme extracts or purified enzymes are added to a reaction mixture together UDP-galactose and a buffering component such as Tris-HCl or HEPES and GlcNAc, GalNAc or a di- or oligosaccharide containing a non-reducing (terminal) GlcNAc or GalNAc as acceptor. The reaction mixture is then incubated at a certain temperature (for example, 37° C.) for a certain amount of time (for example, 24 hours), during which the acceptor will be galactosylated on GlcNAc or GalNAc. The resulting galactosylated di- or oligosaccharide is then separated from the reaction mixture by methods known in the art. Further purification of the galactosylated di- or oligosaccharide can be performed if preferred. At the end of the reaction or after separation and/or purification, the production of the galactosylated di- or oligosaccharide is measured as described in Example 1.

Example 20: RegEx Search for N-Acetylglucosamine b-1,3-Galactosyltransferase Genes Having PFAM Domain PF00535

A RegEx analysis was performed for the N-acetylglucosamine b-1,3-galactosyltransferase genes having PFAM domain PF00535 to find members comprising the sequence [AGPS]XXLN(Xn)RXDXD with SEQ ID NO: 1, wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17, or members comprising the sequence PXXLN(Xn)RXDXD(Xm)[FWY]XX[HKR]XX[NQST] with SEQ ID NO: 2, wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17 and m is 100 to 115. To this end, all N-acetylglucosamine b-1,3-galactosyltransferase genes having PFAM domain PF00535 as annotated in the Pfam database version Pfam 33.1 (as released on Jun. 11, 2020) were downloaded from the UniProt database (as released on Jul. 3, 2020) and analyzed for the presence of the motifs according the method as available on: towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (as released on Apr. 6, 2019). Corresponding members from the RegEx search comprised A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3.

Example 21: RegEx Searches for Other N-Acetylglucosamine b-1,3-Galactosyltransferase or N-Acetylglucosamine b-1,4-Galactosyltransferase Genes

A similar RegEx analysis as exemplified in Example 20 can be performed for the N-acetylglucosamine b-1,3-galactosyltransferase genes having PFAM domain IPR002659 to find members comprising the sequence KT(Xn)[FY]XXKXDXD(Xm)[FHY]XXG(X, no A, G, S)(Xp)(X, no F, H, W, Y)[DE]D[ILV]XX[AG] with SEQ ID NO: 05, wherein X is any amino acid and wherein n is 13 to 16, m is 35 to 70 and p is 20 to 45. To this end, all N-acetylglucosamine b-1,3-galactosyltransferase genes having PFAM domain IPR002659 as annotated in the Pfam database version Pfam 33.1 (as released on Jun. 11, 2020) were downloaded from the UniProt database (as released on Jul. 3, 2020) and analyzed for the presence of the motifs according the method as available on: towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (as released on 6 Apr. 2019).

Similarly, a RegEx analysis can be performed for the N-acetylglucosamine b-1,4-galactosyltransferase genes having PFAM domain PF01755 to find members comprising the sequence EXXCXXSHX[AFILTY]LW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 10, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or comprising the sequence EXXCXXSH[LR]VLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 11, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or comprising the sequence EXXCXXSH[VHI]SLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 12, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or comprising the sequence EXXCXXSHYMLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 13, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or comprising the sequence EXXCXXSHXX(X, no V)Y(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 14, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75. To this end, all N-acetylglucosamine b-1,4-galactosyltransferase genes having PFAM domain PF01755 as annotated in the Pfam database version Pfam 33.1 (as released on Jun. 11, 2020) were downloaded from the UniProt database (as released on Jul. 3, 2020) and analyzed for the presence of the motifs according the method as available on: towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (as released on Apr. 6, 2019).

A RegEx analysis can also be performed for the N-acetylglucosamine b-1,4-galactosyltransferase genes having PFAM domain PF00535 to find members comprising the sequence R[KN]XXXXXXXGXXXX[FL](X, no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE] with SEQ ID NO: 24 wherein X is any amino acid and wherein n is 50 to 75 and m is 10 to 30, or comprising the sequence R[KN]XXXXXXXGXXXX[FL](X, no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE](Xp)[FWY]XX[HKR]XX[NQST] with SEQ ID NO: 25 wherein X is any amino acid and wherein n is 50 to 75, m is 10 to 30 and p is 20 to 25. To this end, all N-acetylglucosamine b-1,4-galactosyltransferase genes having PFAM domain PF00535 as annotated in the Pfam database version Pfam 33.1 (as released on Jun. 11, 2020) were downloaded from the UniProt database (as released on Jul. 3, 2020) and analyzed for the presence of the motifs according the method as available on: towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (as released on Apr. 6, 2019).

A RegEx analysis can also be performed for the N-acetylglucosamine b-1,4-galactosyltransferase genes having PFAM domain PF02709 and not having PFAM domain PF00535 to find members comprising the sequence [FWY]XX[FWY](Xn)[FWY][GQ]X[DE]D with SEQ ID NO: 28 wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an IP or NL combination and wherein n is 21 to 26. To this end, all N-acetylglucosamine b-1,4-galactosyltransferase genes having PFAM domain PF02709 and not having PFAM domain PF00535 as annotated in the Pfam database version Pfam 33.1 (as released on Jun. 11, 2020) were downloaded from the UniProt database (as released on Jul. 3, 2020) and analyzed for the presence of the motifs according the method as available on: towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (as released on Apr. 6, 2019).

Finally, a RegEx analysis can be performed for the N-acetylglucosamine b-1,4-galactosyltransferase genes having PFAM PF03808 to find members comprising the sequence [ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA with SEQ ID NO: 35, wherein X is any amino acid and wherein n is 20 to 25, or comprising the sequence [ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA(Xm)[HR]XG[F WY](Xp)GXGXXXQ[DE] with SEQ ID NO: 36, wherein X is any amino acid and wherein n is 20 to 25, m is 40 to 50 and p is 22 to 30. To this end, all N-acetylglucosamine b-1,4-galactosyltransferase genes having PFAM domain PF03808 as annotated in the Pfam database version Pfam 33.1 (as released on Jun. 11, 2020) were downloaded from the UniProt database (as released on Jul. 3, 2020) and analyzed for the presence of the motifs according the method as available on: towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (as released on Apr. 6, 2019).

Example 22: Production of an Oligosaccharide Mixture Comprising 6′-SL, LacNAc, Sialylated LacNAc, LN3, Sialylated LN3, LNnT and LSTc with a Modified E. coli Host

An E. coli K-12 strain MG1655 is modified for sialic acid production as described in Example 1 comprising knock-outs of the E. coli nagA, nagB, nanA, nanT, nanE, nanK, LacZ, LacY and LacA genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding the lactose permease (LacY) from E. coli (UniProt ID P02920), the sialic acid transporter (nanT) from E. coli (UniProt ID P41036), 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 a G472S mutation), the glucosamine 6-phosphate N-acetyltransferase (GNA1) from S. cerevisiae (UniProt ID P43577), the N-acetylglucosamine 2-epimerase (AGE) from B. ovatus (UniProt ID A7LVG6), the N-acetylneuraminate synthase (NeuB) from C. jejuni (UniProt ID Q93MP9), 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 thus obtained mutant E. coli strain producing sialic acid is further modified with a genomic knock-in of constitutive transcriptional units to express the N-acylneuraminate cytidylyltransferase enzyme NeuA from C. jejuni (UniProt ID Q93MP7) and the alpha-2,6-sialyltransferase PdbST from P. damselae (UniProt ID 066375) to produce 6′-sialyllactose. In a next step, the mutant strain is further modified with genomic knock-ins comprising constitutive transcriptional units for the galactoside beta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis (UniProt ID Q9JXQ6) and an N-acetylglucosamine beta-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 to produce a mixture of oligosaccharides comprising 6′-SL, LacNAc, sialylated LacNAc, LN3, sialylated LN3, LNnT and LSTc (Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc). The novel strains are evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose and lactose. The strains are grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 23: Production of an Oligosaccharide Mixture LN3, Sialylated LN3, LNT, LNB, Sialylated LNB, 3′-SL and LSTa with a Modified E. coli Host

An E. coli strain modified to produce sialic acid (Neu5Ac) as described in Example 22 is further modified with a genomic knock-in of constitutive transcriptional units to express the N-acylneuraminate cytidylyltransferase enzyme NeuA from C. jejuni (UniProt ID Q93MP7) and the alpha-2,3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) to produce 3′-siayllactose. In a next step, the mutant strain is further modified with genomic knock-ins comprising constitutive transcriptional units for the galactoside beta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis (UniProt ID Q9JXQ6) and an N-acetylglucosamine beta-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 to produce a mixture of oligosaccharides comprising LN3,3′-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc), LNT, LNB, sialylated LNB, 3′-SL and LSTa (Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc). The novel strains are evaluated in a growth experiment per the culture conditions of Example 1, in which the culture medium contains sucrose and lactose. The strains are grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 24: Production of Fucosylated LNB Forms in Modified E. coli Hosts

An E. coli K-12 MG1655 mutant strain optimized for GDP-fucose production and growth on sucrose as described in Example 1, is further modified with a knock-out of the E. coli nagA and nagB genes and with a genomic knock-in of a constitutive expression construct of an N-acetylglucosamine β1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9. In a next step, cells of the mutant strain are transformed with an expression vector comprising constitutive transcriptional units of the mutant glmS*54 from E. coli (differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation) and GNA1 from S. cerevisiae (UniProt ID P43577). In a further step, the novel strain is transformed with a second compatible expression vector comprising a constitutive transcriptional unit for the a1,2-fucosyltransferase HpFutC (GenBank NO. AAD29863.1) and/or the a1,3-fucosyltransferase HpFucT (UniProt ID 030511). The novel strains are evaluated for production of GlcNAc, LNB, and fucosylated LNB forms (2′FLNB, 4′FLNB and/or difucosylated LNB) in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose.

Example 25: Production of Fucosylated LNB and Lactose Forms in Modified E. coli Hosts

The mutant E. coli strains described in Example 24 can further be modified comprising genomic knock-out of the E. coli genes galT, ushA, ldhA, LacZ, LacY and LacA and a genomic knock-in of a constitutive transcriptional unit for the lactose permease LacY from E. coli (UniProt ID P02920).

When the novel mutant strains are cultivated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose and lactose, the strains can be evaluated for the production of GlcNAc, LNB and fucosylated LNB and lactose forms like 2′-FLNB, 4-FLNB, 2′FL, 3-FL and/or DiFL.

Example 26: Production of a Neutral Oligosaccharide Mixture Comprising Fucosylated Structures in Modified E. coli Hosts

An E. coli K-12 MG1655 mutant strain optimized for GDP-fucose production and growth on sucrose as described in Example 1, is further modified with a knock-out of the E. coli nagA and nagB genes and with a genomic knock-in of a constitutive expression construct of the galactoside beta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis (GenBank: AAM33849.1) and an N-acetylglucosamine β1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9. In a next step, cells of the mutant strain are transformed with an expression vector comprising constitutive transcriptional units of the mutant glmS*54 from E. coli (differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation) and GNA1 from S. cerevisiae (UniProt ID P43577). In a further step, the novel strain is transformed with a second compatible expression vector comprising a constitutive transcriptional unit for the a1,2-fucosyltransferase HpFutC (GenBank NO. AAD29863.1).

The novel mutant strain is evaluated for the production of a neutral oligosaccharide mixture comprising LNB, fucosylated LNB, 2′FL, DiFL, LN3 (lacto-N-triose), LNT (lacto-N-tetraose) and LNFP-I (Lacto-N-fucopentaose I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose and lactose.

Example 27: Production of a Neutral Oligosaccharide Mixture Comprising Fucosylated Structures in Modified E. coli Hosts

An E. coli K-12 MG1655 mutant strain optimized for GDP-fucose production and growth on sucrose as described in Example 1, is further modified with a knock-out of the E. coli nagA and nagB genes and with a genomic knock-in of a constitutive expression construct of the galactoside beta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis (GenBank: AAM33849.1) and an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39. In a next step, cells of the mutant strain are transformed with an expression vector comprising constitutive transcriptional units of the mutant glmS*54 from E. coli (differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation) and GNA1 from S. cerevisiae (UniProt ID P43577). In a further step, the novel strain is transformed with a second compatible expression vector comprising a constitutive transcriptional unit for the a1,3-fucosyltransferase HpFucT (UniProt ID 030511).

The novel mutant strain is evaluated for the production of a neutral oligosaccharide mixture comprising LacNAc, fucosylated LacNAc, 3-FL, LN3 (lacto-N-triose), LNnT (lacto-N-tetraose) and LNFP-III (Lacto-N-fucopentaose III, Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose and lactose.

Example 28: Production of LacNAc and 3-FLN in Modified E. coli Hosts

An E. coli K-12 MG1655 mutant strain optimized for GDP-fucose production as described in Example 1, is further modified with a knock-out of the E. coli nagA and nagB genes and with a genomic knock-in of a constitutive expression construct of an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39. In a next step, cells of the mutant strain are transformed with an expression vector comprising constitutive transcriptional units of the mutant glmS*54 from E. coli (differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation) and GNA1 from S. cerevisiae (UniProt ID P43577). In a further step, the novel strain is transformed with a second compatible expression vector comprising a constitutive transcriptional unit for the a1,3-fucosyltransferase HpFucT (UniProt ID 030511). The novel strains are evaluated for production of GlcNAc, LacNAc and 3-FLN (Gal-b1,4-(Fuc-a1,3)-GlcNAc) in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose.

Example 29: Production of the T-Disaccharide (Gal-b1,3-GalNAc) in a Modified E. Coli Host

An E. coli K-12 MG1655 strain is optimized for enhanced UDP-galactose production as described in Example 1 with genomic knock-outs of the E. coli ushA and galT genes and with a genomic knock-in of a constitutive expression construct for the UDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147). In a next step the strain is additionally mutated by a knock-out of the E. coli nagB gene and with a genomic knock-in of a constitutive expression construct of an N-acetylglucosamine β1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9. The novel strain is evaluated for the production of the T-disaccharide (Gal-b1,3-GalNAc) when cultivated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains glucose and GalNAc.

Example 30: Production of Gal-b1,4-GalNAc in a Modified E. coli Host

An E. coli K-12 MG1655 strain is optimized for enhanced UDP-galactose production as described in Example 1 with genomic knock-outs of the E. coli ushA and galT genes and with a genomic knock-in of a constitutive expression construct for the UDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147). In a next step, the strain is additionally mutated by a knock-out of the E. coli nagB gene and with a genomic knock-in of a constitutive expression construct of an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39. In a final step, the mutant strain is adapted for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing the sucrose transporter CscB from E. coli W (UniProt ID E0IXR1), the fructose kinase Frk originating from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B. adolescentis (UniProt ID A0ZZH6). The strain is evaluated for the production of Gal-b1,4-GalNAc when cultivated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose and GalNAc.

Example 31: Production of LN3 and LNT in a Modified E. coli Host

An E. coli K-12 strain MG1655 is modified as described in Example 1 comprising knock-outs of the E. coli nagB, galT, ushA, agp, ldhA, LacZ, LacY and LacA genes and genomic knock-ins of constitutive transcriptional units comprising the genes encoding the lactose permease (LacY) from E. coli (UniProt ID P02920), the sucrose transporter (CscB) from E. coli W (UniProt ID EOIXR1), 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 E. coli strain is modified for LN3 production with a genomic knock-in of a constitutive transcriptional unit for the galactoside beta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis (GenBank: AAM33849.1). In a further step, the mutant strain is modified for LNT production with a genomic knock-in of a constitutive transcriptional unit for an N-acetylglucosamine β1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8, 9, and the polypeptides with UniProt ID A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3. The novel strain is evaluated for production of LN3 and LNT 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. The strain is grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 32: Production of LN3 and LNnT in a Modified E. coli Host

An E. coli K-12 strain MG1655 is modified as described in Example 1 comprising knock-outs of the E. coli nagB, galT, ushA, agp, ldhA, LacZ, LacY and LacA genes and genomic knock-ins of constitutive transcriptional units comprising the genes encoding the lactose permease (LacY) from E. coli (UniProt ID P02920), 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 E. coli strain is modified for LN3 production with a genomic knock-in of a constitutive transcriptional unit for the galactoside beta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis (GenBank: AAM33849.1). In a further step, the mutant strain is modified for LNT production with a genomic knock-in of a constitutive transcriptional unit for an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38, 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides identified as described by Example 21. The novel strain is evaluated for production of oligosaccharides comprising LN3, LNnT, lacto-N-neohexaose (LNnH) and para-lacto-N-neohexaose (pLNH) 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. The strain is grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 33: 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 H3B04, 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, BE), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, BE). Luria Broth agar (LBA) plates consisted of the LB media, with 12 g/L agar (Difco, Erembodegem, BE) 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 sialic acid or lactose could be added as a precursor.

Complex medium, e.g., LB, 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., zeocin (20 mg/L)).

Strains, plasmids and mutations: B. subtilis 168, available at Bacillus Genetic Stock Center (Ohio, USA).

Plasmids for gene deletion via Cre/lox are constructed as described by Yan et a1. (Appl. & Environm. Microbial., September 2008, p 5556-5562). Gene disruption is done via homologous recombination with linear DNA and transformation via electroporation as described by Xue et a1. (J. Microb. Meth. 34 (1999) 183-191). The method of gene knockouts is described by Liu et a1. (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 a1. (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 LNB, Bacillus subtilis mutant strains are modified with genomic knock-ins comprising constitutive transcriptional units for 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 a G472S mutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae (UniProt ID P43577), one phosphatase chosen from the list comprising any one or more of the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida, DOG1 from S. cerevisiae or AraL from Bacillus subtilis as described in WO18122225 and an N-acetylglucosamine beta-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E311B28 and A0A538TXM3.

In an example for the production of LacNAc, Bacillus subtilis mutant strains are modified with genomic knock-ins comprising constitutive transcriptional units for 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 a G472S mutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae (UniProt ID P43577), one phosphatase chosen from the list comprising any one or more of the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida, DOG1 from S. cerevisiae or AraL from Bacillus subtilis as described in WO18122225 and an N-acetylglucosamine beta-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides identified as described in Example 21. To further fucosylate the LNB or LacNAc, the LNB or LacNAc producing strains are further modified with a constitutive transcriptional unit for an alpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBank No. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProt ID 030511).

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 transcriptional 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 chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3, that can be delivered to the strain either via genomic knock-in or from an expression plasmid. For LNnT production, the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides identified as described in Example 21. To further fucosylate the LN3, LNT or LNnT, the mutant strains are further modified with a constitutive transcriptional unit for an alpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBank No. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProt ID 030511).

In an example 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 an N-acylneuraminate cytidylyltransferase like, e.g., the NeuA enzyme from C. jejuni (UniProt ID Q93MP7), the NeuA enzyme from H. influenzae (GenBank No. AGV 11798.1) 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). In an example for the production of lactose-based sialylated oligosaccharides, the Bacillus subtilis mutant strains are further modified with a constitutive transcriptional unit for a lactose importer (such as, e.g., the E. coli lacY with UniProt ID P02920).

For growth on sucrose, the mutant strains can additionally be modified with genomic knock-ins of constitutive transcriptional units comprising 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).

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 pre-culture 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 hours, 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 1/3rd of the optical density measured at 600 nm.

Example 34: Production of 2′FLNB with a Modified B. subtilis Strain

A B. subtilis strain is first modified for LNB 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 native fructose-6-P-aminotransferase (UniProt ID P0CI73), 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 a G472S mutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae (UniProt ID P43577), the phosphatase AraL from B. subtilis (UniProt ID P94526), an N-acetylglucosamine beta-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3, the sucrose transporter (CscB) from E. coli W (UniProt ID EOIXR1), 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 LNB producing strain is transformed with an expression plasmid comprising a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBank No. AAD29863.1).

The novel strain is evaluated for the production 2′FLNB in a growth experiment on MMsf medium lacking a precursor according to the culture conditions provided in Example 33. After 72 hours of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 35: Production of an Oligosaccharide Mixture Comprising 6′-SL, LacNAc, Sialylated LacNAc, LN3, Sialylated LN3, LNnT and LSTc with a Modified B. subtilis Strain

In a first step, a B. subtilis strain is modified for sialic acid production with genetic knockouts of the nagA, nagB and gamA genes and with genomic knock-ins of constitutive transcriptional units comprising genes encoding the native fructose-6-P-aminotransferase (UniProt ID P0CI73), 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 further modified with genomic knock-ins of constitutive transcriptional units comprising genes encoding the N-acylneuraminate cytidylyltransferase NeuA from C. jejuni (UniProt ID Q93MP7), and the alpha-2,6-sialyltransferase PdbST from P. damselae (UniProt ID 066375) to produce 6′-sialyllactose. In a next step, the mutant strain is further modified with genomic knock-ins comprising constitutive transcriptional units for the galactoside beta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis (UniProt ID Q9JXQ6) and an N-acetylglucosamine beta-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides identified as described in Example 21. The novel strain is evaluated for production of a mixture of oligosaccharides comprising 6′-SL, LacNAc, sialylated LacNAc, LN3, sialylated LN3, LNnT and LSTc (Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experiment on MMsf medium containing lactose as precursor according to the culture conditions provided in Example 33. After 72 hours of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 36: Production of Gal-b1,4-GalNAc with a Modified B. subtilis Strain

A B. subtilis strain is modified with a genomic knock-in of a constitutive expression construct for the UDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147), an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides identified as described in Example 21, the sucrose transporter CscB from E. coli W (UniProt ID EOIXR1), the fructose kinase Frk originating from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B. adolescentis (UniProt ID A0ZZH6). The novel strain is evaluated for the production of Gal-b1,4-GalNAc when cultivated in a growth experiment according to the culture conditions provided in Example 33, in which the culture medium contains sucrose and GalNAc.

Example 37: Production of Gal-b1,3-GalNAc with a Modified B. subtilis Strain

A B. subtilis strain is modified with a genomic knock-in of a constitutive expression construct for the UDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147), an N-acetylglucosamine β1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3, the sucrose transporter CscB from E. coli W (UniProt ID EOIXR1), the fructose kinase Frk originating from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B. adolescentis (UniProt ID A0ZZH6). The novel strain is evaluated for the production of Gal-b1,3-GalNAc when cultivated in a growth experiment according to the culture conditions provided in Example 33, in which the culture medium contains sucrose and GalNAc.

Example 38: 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 K2HPO4, 0.25 g/L MgSO4.7H2O, 42 g/L MOPS, from 10 up to 30 g/L glucose or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose when specified in the examples and 1 ml/L trace element mix. Depending on the experiment lactose and/or sialic acid could be added as precursor(s).

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

Complex medium, e.g., TY, 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., 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 a1. (Appl. Microbiol. Biotechnol., 2005 April, 67(2):225-33) and temperature-sensitive shuttle vectors as described by Okibe et a1. (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 a1. (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 LNB, the C. glutamicum strain is modified with a genomic knock-in of constitutive expression units comprising 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 a G472S mutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae (UniProt ID P43577), one phosphatase chosen from the list comprising any one or more of the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from P. putida, ScDOG1 from S. cerevisiae or BsAraL from B. subtilis as described in WO18122225 and an N-acetylglucosamine beta-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3.

In an example for the production of LacNAc, the C. glutamicum strain is modified with a genomic knock-in of constitutive expression units comprising 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 a G472S mutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae (UniProt ID P43577), one phosphatase chosen from the list comprising any one or more of the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida, ScDOG1 from S. cerevisiae or BsAraL from Bacillus subtilis as described in WO18122225 and an N-acetylglucosamine beta-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides identified as described in Example 21. To further fucosylate the LNB or LacNAc, the LNB or LacNAc producing strains are further modified with a constitutive transcriptional unit for an alpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBank No. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProt ID 030511).

In an example for the production of lactose-based oligosaccharides, a mutant C. glutamicum strain is 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-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBank No. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProt ID 030511) is additionally added to the strain.

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 transcriptional 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 chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3H1B28 and A0A538TXM3, that can be delivered to the strain either via genomic knock-in or from an expression plasmid. For LNnT production, the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides identified as described in Example 21. To further fucosylate the LN3, LNT or LNnT, the mutant strains are further modified with a constitutive transcriptional unit for an alpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBank No. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProt ID 030511).

In an example 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 C. glutamicum genes ldh, cg12645, nagB, gamA and nagA 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). In an example for the production of lactose-based sialylated oligosaccharides, the C. glutamicum mutant strains are further modified with a constitutive transcriptional unit for a lactose importer (such as, e.g., the E. coli lacY with UniProt ID P02920).

For growth on sucrose, the mutant strains can additionally be modified with genomic knock-ins of constitutive transcriptional units comprising 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).

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 pre-culture 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 hours, 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 measured in the whole broth by the biomass, in relative percentages compared to the reference strain. The biomass is empirically determined to be approximately 1/3rd of the optical density measured at 600 nm.

Example 39: Production of 2′FLNB with a Modified C. glutamicum Strain

A C. glutamicum strain is first modified for LNB production and growth on sucrose by genomic knock-out of the ldh, cg12645 and nagB genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding 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 a G472S mutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae (UniProt ID P43577), the phosphatase AraL from B. subtilis (UniProt ID P94526), the N-acetylglucosamine beta-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3, the sucrose transporter (CscB) from E. coli W (UniProt ID EOIXR1), 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 LNB producing strain is transformed with an expression plasmid comprising a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank No. AAD29863.1). The novel strain is evaluated for the production 2′FLNB in a growth experiment on MMsf medium lacking a precursor according to the culture conditions provided in Example 38. After 72 hours of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 40: Production of a Mixture Comprising Sialylated LacNAc with a Modified C. glutamicum Strain

A C. glutamicum strain is first modified for LacNAc production and growth on sucrose by genomic knock-out of the ldh, cgl2645, nagB, nagA and gamA genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding the native fructose-6-P-aminotransferase (UniProt ID Q8NND3), 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 a G472S mutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae (UniProt ID P43577), the phosphatase AraL from B. subtilis (UniProt ID P94526), the N-acetylglucosamine beta-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides identified as described in Example 21, the sucrose transporter (CscB) from E. coli W (UniProt ID EOIXR1), 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 for sialic acid synthesis, the mutant strain was further modified with genomic knock-ins of constitutive transcriptional units comprising genes encoding the N-acetylglucosamine-2-epimerase from B. ovatus (UniProt ID A7LVG6), and the N-acetylneuraminate synthase from C. jejuni (UniProt ID Q93MP9). In a next step, the novel strain is transformed with an expression plasmid comprising constitutive transcriptional units comprising the gene encoding the NeuA enzyme from C. jejuni (UniProt ID Q93MP7) combined with the gene encoding either the beta-galactoside alpha-2,3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) or the beta-galactoside alpha-2,6-sialyltransferase PdST6 from P. damselae (UniProt ID 066375). The novel strains are evaluated for production of LacNAc, sialic acid and sialylated LacNAc in a growth experiment on MMsf medium lacking a precursor according to the culture conditions provided in Example 38. When adding lactose as precursor to the MMsf medium, the mutant strains are also evaluated for additional production of 3′-SL or 6′-SL, depending on the alpha-sialyltransferase expressed. After 72 hours of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 41: Production of Gal-b1,4-GalNAc with a Modified C. glutamicum Strain

A C. glutamicum strain is modified with a genomic knock-in of a constitutive expression construct for the UDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147), an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides identified as described in Example 21, the sucrose transporter CscB from E. coli W (UniProt ID EOIXR1), the fructose kinase Frk originating from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B. adolescentis (UniProt ID A0ZZH6). The novel strain is evaluated for the production of Gal-b1,4-GalNAc when cultivated in a growth experiment according to the culture conditions provided in Example 38, in which the culture medium contains sucrose and GalNAc.

Example 42: Production of Gal-b1,3-GalNAc with a Modified C. glutamicum Strain

A C. glutamicum strain is modified with a genomic knock-in of a constitutive expression construct for the UDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147), an N-acetylglucosamine β1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3, the sucrose transporter CscB from E. coli W (UniProt ID E0IXR1), the fructose kinase Frk originating from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B. adolescentis (UniProt ID A0ZZH6). The novel strain is evaluated for the production of Gal-b1,3-GalNAc when cultivated in a growth experiment according to the culture conditions provided in Example 38, in which the culture medium contains sucrose and GalNAc.

Example 43: 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 K2HPO4, 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 and/or fucose could be added. Medium was sterilized by autoclaving (121° C., 21 min.). 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 (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 a1. (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 a1. (Eukaryotic Cell 2014, 13(11): 1465-1469).

Transformation via electroporation was performed as described by Wang et a1. (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 hours 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 (1575Ω, 50 μFD). 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 gene encoding the galactokinase from Arabidopsis thaliana (KIN, UniProt ID Q9SEE5) and the gene encoding the UDP-sugar pyrophosphorylase (USP) from A. thaliana (UniProt ID Q9C5I1).

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 chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3. Additionally, the mutant C. reinhardtii cells can be modified with an expression plasmid comprising a transcriptional unit for an alpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBank No. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProt ID 030511).

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 chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides identified as described in Example 21. Additionally, the mutant C. reinhardtii cells can be modified with an expression plasmid comprising a transcriptional unit for an alpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBank No. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProt ID 030511).

In an example for CMP-sialic acid synthesis, C. reinhardtii cells are modified with constitutive transcriptional units for a 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-localised sialyltransferase chosen from species like, e.g., Homo sapiens, Mus musculus, and 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-hour 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 a1. (Bioresour. Technol. 2011, 102: 71-81) and Johnson et a1. (Biotechnol. Prog. 2018, 34: 811-827).

Example 44: Production of LNB and 2′FLNB in Modified C. reinhardtii Cells

C. reinhardtii cells are engineered as described in Example 43 for production of UDP-Gal with genomic knock-ins of constitutive transcriptional units comprising the galactokinase from A. thaliana (KIN, UniProt ID Q9SEE5) and the UDP-sugar pyrophosphorylase (USP) from A. thaliana (UniProt ID Q9C5I1). In a next step, the mutant cells are transformed with an expression plasmid comprising transcriptional units comprising an N-acetylglucosamine beta-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3 and the alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank No. AAD29863.1). The novel strain is evaluated in a cultivation experiment on TAP-agar plates comprising galactose as precursor according to the culture conditions provided in Example 43. After 5 days of incubation, the cells are harvested, and evaluated for the production of LNB and 2′FLNB via analysis on UPLC.

Example 45: Production of LacNAc and 3′-Fucosylated LacNAc (3-FLN) in Modified C. reinhardtii Cells

C. reinhardtii cells are engineered as described in Example 43 for production of UDP-Gal with genomic knock-ins of constitutive transcriptional units comprising the galactokinase from A. thaliana (KIN, UniProt ID Q9SEE5) and the UDP-sugar pyrophosphorylase (USP) from A. thaliana (UniProt ID Q9C5I1). In a next step, the mutant cells are transformed with an expression plasmid comprising transcriptional units comprising an N-acetylglucosamine beta-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides and the alpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProt ID 030511). The novel strain is evaluated in a cultivation experiment on TAP-agar plates comprising galactose as precursor according to the culture conditions provided in Example 43. After 5 days of incubation, the cells are harvested, and cells are evaluated for the production of LacNAc and 3′-fucosylated LacNAc (3-FLN, Gal-b1,4-(Fuc-a1,3)-GlcNAc) via analysis on UPLC.

Example 46: Production of Gal-b1,4-GalNAc with a Modified C. reinhardtii Strain

C. reinhardtii cells are modified with a genomic knock-in of a constitutive expression construct for the UDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147), and an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides identified as described in Example 21. The novel strain is evaluated in a cultivation experiment on TAP-agar plates comprising GalNAc as precursor according to the culture conditions provided in Example 43. After 5 days of incubation, the cells are harvested, and cells are evaluated for the production of Gal-b1,4-GalNAc via analysis on UPLC.

Example 47: Production of Gal-b1,3-GalNAc with a Modified C. reinhardtii Strain

C. reinhardtii cells are modified with a genomic knock-in of a constitutive expression construct for the UDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147) and an N-acetylglucosamine β1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3. The novel strain is evaluated in a cultivation experiment on TAP-agar plates comprising GalNAc as precursor according to the culture conditions provided in Example 43. After 5 days of incubation, the cells are harvested, and cells are evaluated for the production of Gal-b1,3-GalNAc via analysis on UPLC.

Example 48: 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., 37° 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 a1. (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; Blatchford et al. 1999; Animal Cell Technology: Basic & Applied Aspects, Springer, Dordrecht. 141-145; Williams et al. 2009, Breast Cancer Res. 11(3): 26-43; and Arevalo et al. 2015, Am. J. Physiol. Cell Physiol. 310(5): C348-C356; each of which is incorporated herein by reference in 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 hours. 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 hours, 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 hours. 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 hours, 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 remodeling 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 49: Evaluation of 2′FL, LNFP-I and 2′FLNB Production in a Non-Mammary Adult Stem Cell

Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 48 are modified via CRISPR-CAS to over-express a codon-optimized N-acetylglucosamine beta-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3, the GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), and a codon-optimized alpha-1,2-fucosyltransferase from H. pylori (GenBank No. AAD29863.1). 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 48, cells are subjected to UPLC to analyze for production of 2′FL, LNFP-I and 2′FLNB.

Example 50: Evaluation of LacNAc, Sialylated LacNAc and Sialyl-Lewis×Production in a Non-Mammary Adult Stem Cell

Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 48 are modified via CRISPR-CAS to over-express a beta-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides identified as described in Example 21, the GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), the galactoside alpha-1,3-fucosyltransferase FUT3 from H. sapiens (UniProt ID P21217), 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). All genes introduced in the cells are codon-optimized to the host. 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 48, cells are subjected to UPLC to analyze for production of LacNAc, sialylated LacNAc and sialyl-Lewis x.

Example 51: Evaluation of Production of Gal-b1,4-GalNAc in a Non-Mammary Adult Stem Cell

Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 48 are modified via CRISPR-CAS to over-express the UDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147), and an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides identified as described in Example 21. Both genes introduced in the cells are codon-optimized to the host. 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 48, cells are subjected to UPLC to analyze for production of Gal-b1,4-GalNAc.

Example 52: Evaluation of Production of Gal-b1,3-GalNAc in a Non-Mammary Adult Stem Cell

Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 48 are modified via CRISPR-CAS to over-express the UDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147), and an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3. Both genes introduced in the cells are codon-optimized to the host. 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 48, cells are subjected to UPLC to analyze for production of Gal-b1,3-GalNAc.

Example 53: Production of LNB in a Modified E. coli Host

An E. coli K-12 MG1655 strain optimized for GDP-fucose production was modified with genomic knock-ins of constitutive expression constructs for glmS*54 (differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation) and GNA1 from S. cerevisiae (UniProt ID P43577) to intracellularly produce GlcNAc. In a next step, the mutant strain was further modified for constitutive expression of the N-acetylglucosamine β1,3-galactosyltransferase from Photobacterium leiognathi with SEQ ID NO: 4 from plasmid. The novel strain was evaluated in a growth experiment according to the culture conditions provided in Example 1. The strain demonstrated that the strain produced 0.37 (±0.18) g/L LNB in whole broth samples, taken after 72 hours of cultivation in minimal medium with 30 g/L sucrose. The cell performance index (CPI) was determined by dividing the LNB concentration measured in the whole broth by the biomass. The biomass was empirically determined to be approximately ⅓rd of the optical density measured at 600 nm. The CPI related to LNB production in whole broth samples for the novel strain was 0.10±0.05.

Example 54: Production of LacNAc in Modified E. coli Hosts

An E. coli K-12 MG1655 strain optimized for GDP-fucose production was modified with genomic knock-ins of constitutive expression constructs for glmS*54 (differing from the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation) and GNA1 from S. cerevisiae (UniProt ID P43577) to intracellularly produce GlcNAc. In a next step, the mutant strain was further modified for constitutive expression of an N-acetylglucosamine β1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs: 17, 20, 22, 27 and 37 from plasmid. The novel strains were evaluated in a growth experiment according to the culture conditions provided in Example 1. Table 3 shows the production of LacNAc (g/L) in whole broth samples from each of the mutant strains, taken after 72 hours of cultivation in minimal medium with 30 g/L sucrose, together with the cell performance index (CPI) calculated for the samples. The CPI was determined by dividing the LacNAc concentrations, measured in the whole broth by the biomass. The biomass was empirically determined to be approximately ⅓rd of the optical density measured at 600 nm. The data demonstrated that all novel strains expressing either SEQ ID NOs: 17, 20, 22, 27 or 37 produced LacNAc.

TABLE 3 Production of LacNAc (g/L) and cell performance index (CPI) data related to the LacNAc production in whole broth samples taken from mutant E. coli strains after 72 hours of cultivation in minimal medium comprising 30 g/L sucrose SEQ ID NO of the β1,4-galactosyltransferase expressed from a transcriptional unit from an LacNAc CPI_ expression plasmid (g/L) (± sd) LacNAc (± sd) 17  1.89 ± 0.10 0.50 ± 0.01 20  9.32 ± 4.45 2.55 ± 1.24 22  3.40 ± 0.39 0.93 ± 0.05 27  9.33 ± 0.20 2.51 ± 0.13 37 10.50 ± 0.48 2.94 ± 0.11

Example 55: Production of LNT in a Modified E. coli Host

An E. coli K-12 strain MG1655 was modified as described in Example 1 comprising knock-outs of the E. coli nagB, galT, ushA, agp, ldhA, LacZ, LacY and LacA genes and genomic knock-ins of constitutive transcriptional units comprising the genes encoding the lactose permease (LacY) from E. coli (UniProt ID P02920), the sucrose transporter (CscB) from E. coli W (UniProt ID EOIXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B. adolescentis (UniProt ID 35 A0ZZH6). In a next step, the mutant E. coli strain was modified for LN3 production with a genomic knock-in of a constitutive transcriptional unit for the galactoside beta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis (GenBank: AAM33849.1). In a further step, the mutant strain was modified for LNT production with a constitutive transcriptional unit for an N-acetylglucosamine β1,3-galactosyltransferase expressed from plasmid chosen from the list comprising SEQ ID NOs: 3 and 4. The novel strains were evaluated in a growth experiment according to the culture conditions provided in Example 1. Table 4 shows the production of LNT in whole broth samples from each of the mutant strains, taken after 72 hours of cultivation in minimal medium with 30 g/L sucrose, together with the cell performance index (CPI) calculated for the samples. The CPI was determined by dividing the LNT concentrations, measured in the whole broth by the biomass. The biomass was empirically determined to be approximately ⅓rd of the optical density measured at 600 nm. The experiment demonstrated that the strains expressing a N-acetylglucosamine β1,3-galactosyltransferase with SEQ ID NOs: 3 or 4 were able to produce LNT.

TABLE 4 Production of LNT (g/L) and cell performance index (CPI) data related to the LNT production in whole broth samples taken from mutant E. coli strains after 72 hours of cultivation in minimal medium comprising 30 g/L sucrose 20 g/L lactose SEQ ID NO of the β1,3-galactosyltransferase expressed from a transcriptional unit from an LNT CPI_ expression plasmid (g/L) (± sd) LNT ± sd 03 5.25 ± 1.50 1.54 ± 0.42 04 0.83 ± 0.26 0.27 ± 0.06

Example 56: Production of LNnT in a Modified E. coli Host

An E. coli K-12 strain MG1655 was modified as described in Example 1 comprising knock-outs of the E. coli nagB, galT, ushA, agp, ldhA, LacZ, LacY and LacA genes and genomic knock-ins of constitutive transcriptional units comprising the genes encoding the lactose permease (LacY) from E. coli (UniProt ID P02920), the sucrose transporter (CscB) from E. coli W (UniProt ID EOIXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B. adolescentis (UniProt ID 20 A0ZZH6). In a next step, the mutant E. coli strain was modified for LN3 production with a genomic knock-in of a constitutive transcriptional unit for the galactoside beta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis (GenBank: AAM33849.1). In a further step, the mutant strain was modified for LNnT production with a constitutive transcriptional unit for an N-acetylglucosamine β1,4-galactosyltransferase expressed from plasmid chosen from the list comprising SEQ ID NOs: 15, 17, 18, 19, 20, 22, 32 and 33. The novel strains were evaluated in a growth experiment according to the culture conditions provided in Example 1. Table 5 shows the production of LNnT in whole broth samples from each of the mutant strains, taken after 72 hours of cultivation in minimal medium with 30 g/L sucrose, together with the cell performance index (CPI) calculated for the samples. The CPI was determined by dividing the LNnT concentrations, measured in the whole broth by the biomass. The biomass was empirically determined to be approximately ⅓rd of the optical density measured at 600 nm. The experiment demonstrated that the strains expressing a N-acetylglucosamine β1,4-galactosyltransferase with SEQ ID NOs: 15, 17, 18, 19, 20, 22, 32 or 33 were able to produce LNnT.

TABLE 5 Production of LNnT (g/L) and cell performance index (CPI) data related to the LNnT production in whole broth samples taken from mutant E. coli strains after 72 hours of cultivation in minimal medium comprising 30 g/L sucrose 20 g/L lactose SEQ ID NO of the β1,4-galactosyltransferase expressed from a transcriptional unit from an LNnT CPI_ expression plasmid (g/L) (± sd) LNnT ± sd 15 2.82 ± 0.82 1.57 ± 0.49 17 0.59 ± 0.02 0.41 ± 0.01 18 1.64 ± 0.05 0.73 ± 0.06 19 0.58 ± 0.04 0.31 ± 0.02 20 0.88 ± 0.16 0.94 ± 0.19 22 1.11 ± 0.13 1.00 ± 0.02 33 0.25 ± 0.03 0.24 ± 0.07

Claims

1. An N-acetylglucosamine b-1,X-galactosyltransferase that galactosylates wherein the N-acetylglucosamine b-1,X-galactosyltransferase is: A. an N-acetylglucosamine b-1,3-galactosyltransferase which has B. an N-acetylglucosamine b-1,4-galactosyltransferase which has

an N-acetylglucosamine and/or N-acetylgalactosamine as a monosaccharide, and/or
an N-acetylglucosamine and/or N-acetylgalactosamine as part of a di- and/or oligosaccharide at the non-reducing end of the di- and/or oligosaccharide, and
a. PFAM domain PF00535 and i) comprises the sequence [AGPS]XXLN(Xn)RXDXD with SEQ ID NO: 1, wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17 ii) comprises the sequence PXXLN(Xn)RXDXD(Xm)[FWY]XX[HKR]XX[NQST] with SEQ ID NO: 2, wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17 and m is 100 to 115, iii) comprises a polypeptide sequence of SEQ ID NO: 3 or 4, or iv) is a functional homologue, variant or derivative of SEQ ID NO: 3 or 4 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide with SEQ ID NOs: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, v) comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acid residues from SEQ ID NO: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, vi) is a functional fragment of SEQ ID NO: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or vii) comprises a polypeptide comprising a peptide having at least 80% sequence identity to the full-length peptide of SEQ ID NO: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
b. PFAM domain IPR002659 and i) comprises the sequence KT(Xn)[FY]XXKXDXD(Xm)[FHY]XXG(X, no A, G, S)(Xp)(X, no F, H, W, Y)[DE]D[ILV]XX[AG] with SEQ ID NO: 5, wherein X is any amino acid and wherein n is 13 to 16, m is 35 to 70 and p is 20 to 45, ii) comprises the polypeptide sequence of any one of SEQ ID NOs: 6, 7, 8 or 9, iii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 6, 7, 8 or 9 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide with SEQ ID NOs: 6, 7, 8 or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, iv) 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: 6, 7, 8, or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, v) is a functional fragment of any one of SEQ ID NOs: 6, 7, 8, or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or vi) comprises a polypeptide comprising a peptide having at least 80% sequence identity to the full-length peptide of any one of SEQ ID NOs: 6, 7, 8, or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or
a. PFAM domain PF01755 and i) comprises the sequence EXXCXXSHX[AFILTY]LW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 10, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or ii) comprises the sequence EXXCXXSH[LR]VLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 11, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, iii) comprises the sequence EXXCXXSH[VHI]SLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 12, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, iv) comprises the sequence EXXCXXSHYMLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 13, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, v) comprises the sequence EXXCXXSHXX(X, no V)Y(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 14, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, vi) comprises the polypeptide sequence of any one of SEQ ID NOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23, vii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, viii) 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: 15, 18, 22, 20, 17, 19, 16, 21 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, ix) is a functional fragment of any one of SEQ ID NOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or x) comprises a polypeptide comprising a peptide having at least 80% sequence identity to the full-length peptide of any one of SEQ ID NOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
b. PFAM domain PF00535 and i) comprises the sequence R[KN]XXXXXXXGXXXX[FL](X, no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE] with SEQ ID NO: 24 wherein X is any amino acid and wherein n is 50 to 75 and m is 10 to 30, ii) comprises the sequence R[KN]XXXXXXXGXXXX[FL](X, no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE](Xp)[FWY]XX[HKR]XX[NQST] with SEQ ID NO: 25 wherein X is any amino acid and wherein n is 50 to 75, m is 10 to 30 and p is 20 to 25, iii) comprises a polypeptide sequence of SEQ ID NO: 26 or 27, iv) is a functional homologue, variant or derivative of SEQ ID NO: 26 or 27 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NO: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, v) 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 SEQ ID NO: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, vi) is a functional fragment of SEQ ID NO: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or vii) comprises a polypeptide comprising a peptide having at least 80% sequence identity to the full-length peptide of SEQ ID NO: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
c. PFAM domain PF02709 and not PFAM domain PF00535, and i) comprises the sequence [FWY]XX[FWY](Xn)[FWY][GQ]X[DE]D with SEQ ID NO: 28 wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an I1P or NL combination and wherein n is 21 to 26, ii) comprises the polypeptide sequence of any one of SEQ ID NOs: 33, 29, 30, 31, 32 or 34, or iii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 33, 29, 30, 31, 32 or 34 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 33, 29, 30, 31, 32 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, iv) 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: 33, 29, 30, 31, 32 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, v) is a functional fragment of any one of SEQ ID NOs: 33, 29, 30, 31, 32 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or vi) comprises a polypeptide comprising a peptide having at least 80% sequence identity to the full-length peptide of any one of SEQ ID NOs: 33, 29, 30, 31, 32 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or
d. PFAM domain PF03808 and i) comprises the sequence [ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA with SEQ ID NO: 35, wherein X is any amino acid and wherein n is 20 to 25, ii) comprises the sequence [ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA (Xm)[HR]XG[FWY](Xp)GXGXXXQ[DE] with SEQ ID NO: 36, wherein X is any amino acid and wherein n is 20 to 25, m is 40 to 50 and p is 22 to 30, iii) comprises the polypeptide sequence of any one of SEQ ID NOs: 37, 38 or 39, iv) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 37, 38 or 39 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, v) 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: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, vi) functional fragment of any one of SEQ ID NOs: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or vii) comprises a polypeptide comprising a peptide having at least 80% sequence identity to the full-length peptide of any one of SEQ ID NOs: 37, 38, or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity.

2. A method of synthesizing a galactosylated disaccharide or oligosaccharide, the method comprising:

utilizing the N-acetylglucosamine b-1,X-galactosyltransferase of claim 1 in a method of synthesizing a galactosylated disaccharide or oligosaccharide.

3. The method according to claim 2, wherein the synthesis comprises:

a. providing UDP-galactose and any one of the galactosyltransferase, wherein the galactosyltransferase is capable of transferring a galactose residue from the UDP-galactose donor to one or more acceptor(s), and
b. contacting any one of the galactosyltransferase and UDP-galactose with one or more acceptor(s), under conditions where the galactosyltransferase catalyses the transfer of a galactose residue from the UDP-galactose to the acceptor(s), and
c. optionally, separating the galactosylated di- or oligosaccharide.

4. The method according to claim 3, wherein the acceptor(s) is/are an N-acetylglucosamine and/or an N-acetylgalactosamine as a monosaccharide, and/or a di- and/or oligosaccharide having an N-acetylglucosamine and/or N-acetylgalactosamine at its non-reducing end.

5. The method according to claim 2, wherein the galactosylated disaccharide or oligosaccharide is produced in a cell-free system or is produced by a cell.

6. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell

is capable of synthesizing one or more of the acceptor(s),
expresses any one of the N-acetylglucosamine b-1,3-galactosyltransferases and/or N-acetylglucosamine b-1,4-galactosyltransferases, and
is capable of synthesizing UDP-galactose (UDP-Gal) as donor for the galactosyltransferases.

7. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is capable of synthesizing one or more nucleotide-sugar donor(s) selected from the group consisting of GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy-L-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-pneumosamnine (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-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, UDP-glucuronate, UDP-galacturonate, DP-rhamnose, and UDP-xylose.

8. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is capable of expressing one or more glycosyltransferases selected from the group consisting of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases.

9. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is a metabolically engineered cell.

10. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is modified in the expression or activity of an enzyme selected from the group consisting of glucosamine 6-phosphate N-acetyltransferase, phosphatase, glycosyltransferase, L-glutamine D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase.

11. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is unable to convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or unable to convert glucosamine-6-phosphate to fructose-6-phosphate.

12. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is modified for enhanced UDP-galactose production and wherein the modification is selected from the group consisting of knock-out of a 5′-nucleotidase/UDP-sugar hydrolase encoding gene or knock-out of a galactose-1-phosphate uridylyltransferase encoding gene.

13. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell uses at least one precursor for producing the galactosylated disaccharide or oligosaccharide, the precursor(s) being fed to the cell from the cultivation medium, and/or wherein the cell produces at least one precursor for producing galactosylated disaccharide or oligosaccharide.

14. The method according to claim 13, wherein the precursor for producing galactosylated disaccharide or oligosaccharide is completely converted into the galactosylated disaccharide or oligosaccharide.

15. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell produces the galactosylated disaccharide or oligosaccharide intracellularly and wherein a fraction or substantially all of the produced galactosylated disaccharide or oligosaccharide remains intracellularly and/or is excreted outside the cell via passive or active transport.

16. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell expresses a membrane transporter protein or a polypeptide having transport activity hereby transporting compounds across the outer membrane of the cell wall.

17. The method according to claim 16, wherein the membrane transporter protein or polypeptide having transport activity:

controls the flow over the outer membrane of the cell wall of the galactosylated disaccharide or oligosaccharide and/or of at least one precursor and/or acceptor(s) to be used in the production of the galactosylated disaccharide or oligosaccharide, and/or
provides improved production and/or enabled and/or enhanced efflux of the galactosylated disaccharide or oligosaccharide.

18. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell comprises a modification for reduced production of acetate compared to a non-modified progenitor, optionally 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 0-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 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, pyruvate decarboxylase compared to a non-modified progenitor.

19. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is capable of producing phosphoenolpyruvate (PEP), optionally the cell is modified for enhanced production and/or supply of PEP compared to a non-modified progenitor.

20. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and 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 producing galactosylated disaccharide or oligosaccharide.

21. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and 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).

22. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell produces 90 g/L or more of the galactosylated disaccharide or oligosaccharide in the whole broth and/or supernatant and/or wherein the galactosylated disaccharide or oligosaccharide in the whole broth and/or supernatant has a purity of at least 80% measured on the total amount of the galactosylated disaccharide or oligosaccharide and its precursor(s) in the whole broth and/or supernatant, respectively.

23. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and wherein the conditions comprise:

use of a culture medium comprising at least one precursor and/or acceptor for producing galactosylated disaccharide or oligosaccharide, and/or
adding to the culture medium at least one precursor and/or acceptor feed for producing galactosylated disaccharide or oligosaccharide.

24. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell, wherein the culture medium contains at least one precursor selected from the group consisting of lactose, galactose, fucose, and sialic acid.

25. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell, wherein a first phase of exponential cell growth is provided by adding a carbon-based substrate to the culture medium comprising a precursor, followed by a second phase wherein:

only a carbon-based substrate is added to the culture medium, or
a carbon-based substrate and a precursor are added to the culture medium.

26. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is capable of catabolizing a carbon source selected from the group consisting of glucose, fructose, mannose, galactose, lactose, sucrose, maltose, malto-oligosaccharides, trehalose, starch, cellulose, hemi-cellulose, corn-steep liquor, molasses, high-fructose syrup, glycerol, acetate, citrate, lactate, and pyruvate.

27. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell.

28. The method according to claim 27, 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 compared to a non-modified progenitor.

29. The method according to claim 2, wherein the method produces a mixture of charged and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide.

30. The method according to claim 2, wherein the method produces a mixture of charged and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide.

31. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell produces a mixture of charged and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide.

32. The method according to claim 5, wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell produces a mixture of charged and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide.

33. The method according to claim 3, 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, and ligand exchange chromatography.

34. The method according to claim 3, further comprising purification of the galactosylated di- or oligosaccharide, optionally 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, and vacuum roller drying.

35. A cell metabolically engineered to synthesize a galactosylated disaccharide or oligosaccharide by utilization of the N-acetylglucosamine b-1,X-galactosyltransferase of claim 1.

36. The cell of claim 35, wherein the cell

expresses any one of the N-acetylglucosamine b-1,3-galactosyltransferases and/or N-acetylglucosamine b-1,4-galactosyltransferases,
is capable of synthesizing UDP-galactose (UDP-Gal) as donor for the galactosyltransferases, and
is capable of synthesizing one or more acceptor(s) for the galactosyltransferases, wherein the acceptor(s) is/are an N-acetylglucosamine as a monosaccharide, and/or a di- or oligosaccharide having an N-acetylglucosamine and/or N-acetylgalactosamine at its non-reducing end.

37. The cell of claim 35, wherein the cell is further capable of synthesizing one or more nucleotide-sugar donor(s) selected from the group consisting of GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy-L-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-26-dideoxy-L-glucose), GDP-L-quinovose, 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-glucuronate, UDP-galacturonate, GDP-rhamnose, and UDP-xylose.

38. The cell of claim 35, wherein the cell is further capable of expressing at least one glycosyltransferase selected from the group consisting of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases.

39. The cell of claim 35, wherein the cell is modified in the expression or activity of an enzyme selected from the group consisting of glucosamine 6-phosphate N-acetyltransferase, phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase.

40. The cell of claim 35, wherein the cell is unable to convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or unable to convert glucosamine-6-phosphate to fructose-6-phosphate.

41. The cell of claim 35, wherein the cell is modified for enhanced UDP-galactose production and wherein the modification is selected from the group consisting of knock-out of an 5′-nucleotidase/UDP-sugar hydrolase encoding gene, and knock-out of a galactose-1-phosphate uridylyltransferase encoding gene.

42. The cell of claim 35, wherein the cell uses at least one precursor for producing galactosylated disaccharide or oligosaccharide, the precursor(s) being fed to the cell from the cultivation medium and/or wherein the cell is producing at least one precursor for producing galactosylated disaccharide or oligosaccharide.

43. The cell of claim 42, wherein the precursor for producing galactosylated disaccharide or oligosaccharide is completely converted into the galactosylated disaccharide or oligosaccharide.

44. The cell of claim 35, wherein the cell produces the galactosylated disaccharide or oligosaccharide intracellularly and wherein a fraction or substantially all of the produced galactosylated disaccharide or oligosaccharide remains intracellularly and/or is excreted outside the cell via passive or active transport.

45. The cell of claim 35, 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.

46. The cell of claim 45, wherein the membrane transporter protein or polypeptide having transport activity:

controls the flow over the outer membrane of the cell wall of the galactosylated disaccharide or oligosaccharide and/or of at least one precursor and/or acceptor(s) to be used in the production of the galactosylated disaccharide or oligosaccharide, and/or
provides improved production and/or enabled and/or enhanced efflux of the galactosylated disaccharide or oligosaccharide.

47. The cell of claim 35, wherein the cell comprises a modification for reduced production of acetate compared to a non-modified progenitor, optionally 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 0-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 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, and pyruvate decarboxylase compared to a non-modified progenitor.

48. The cell of claim 35, wherein the cell is capable of producing phosphoenolpyruvate (PEP), optionally the cell is modified for enhanced production and/or supply of PEP compared to a non-modified progenitor.

49. The cell of claim 35, 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 producing galactosylated disaccharide or oligosaccharide.

50. The cell of claim 35, 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).

51. The cell of claim 35, wherein the cell is capable of catabolizing a carbon source selected from the group consisting of glucose, fructose, mannose, galactose, lactose, sucrose, maltose, malto-oligosaccharides, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, glycerol, acetate, citrate, lactate, and pyruvate.

52. The cell of claim 35, wherein the cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell.

53. The cell of claim 52, 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 compared to a non-modified progenitor.

54. The cell of claim 35, wherein the cell produces a mixture of charged and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide.

55. The cell of claim 35, wherein the cell produces a mixture of charged and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide.

56. A method of producing a galactosylated disaccharide or oligosaccharide, the method comprising:

cultivating the cell of claim 35 so as to produce a galactosylated disaccharide or oligosaccharide.

57. A method of producing a mixture of charged and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide, the method comprising:

cultivating the cell of claim 35 so as to produce a mixture of charged and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide.

58. A method of producing a mixture of charged and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide, the method comprising:

cultivating the cell of claim 35 so as to produce a mixture of charged and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide.
Patent History
Publication number: 20230416796
Type: Application
Filed: Feb 10, 2023
Publication Date: Dec 28, 2023
Inventors: Sofie Aesaert (Zwijnaarde), Joeri Beauprez (Zwijnaarde), Pieter Coussement (Zwijnaarde), Thomas Decoene (Zwijnaarde), Nausicaä Lannoo (Zwijnaarde), Gert Peters (Zwijnaarde), Kristof Vandewalle (Zwijnaarde), Annelies Vercauteren (Zwijnaarde)
Application Number: 18/167,687
Classifications
International Classification: C12P 19/18 (20060101); C12N 9/10 (20060101);