TW202219275A - Production of glcnac containing bioproducts in a cell - Google Patents

Abstract

The present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of fermentation of metabolically engineered cells. The present invention describes a method for the production of a di- or oligosaccharide with an N-acetylglucosamine at the reducing end by a cell as well as the purification of said di- or oligosaccharide from the cultivation. Furthermore, the present invention provides a cell metabolically engineered for production of a di- or oligosaccharide with an N-acetylglucosamine at the reducing end.

Description

Production of GlcNAc-containing bioproducts in cells

The present invention belongs to the technical field of synthetic biology and metabolic engineering. More specifically, the present invention belongs to the technical field of fermentation of metabolically engineered cells. The present invention describes a method for producing disaccharides or oligosaccharides having N-acetylglucosamine units at the reducing end by cells and purifying the disaccharides or oligosaccharides from culture. Furthermore, the present invention provides a metabolically engineered cell for producing disaccharides or oligosaccharides having N-acetylglucosamine units at the reducing end.

Carbohydrates, often in sugar-conjugated forms of proteins and lipids, are involved in many important phenomena such as differentiation, development, and biorecognition processes associated with the development and progression of fertilization, embryogenesis, inflammation, metastasis, and host pathogen adhesion . Carbohydrates can also be present as unconjugated glycans in body fluids and breast milk, where they also regulate important developmental and immune processes (Bode, Early Hum. Dev. 1-4 (2015); Reily et al., Nat. Rev. Nephrol. 15, 346-366 (2019); Varki, Glycobiology 27, 3-49 (2017)).

Disaccharide Galβ1,3GlcNAc, also known as lacto-N-biose, LNB, N-acetyllactosamine type 1, or type 1 LacNAc, which is linked by β-1,3 to N- Galactose composition of acetylglucosamine (GlcNAc). GlcNAc is present at the reducing end of the disaccharide. LNB is known to be a precursor of several important blood group epitopes, such as Lewis A, Lewis B or sialylated Lewis A. The glycan-containing type 1 LacNAc also plays an important role in tumor metastasis and is therefore regarded as a tumor marker (Fischöder et al., Molecules 22, 1320 (2017)). They are also important components of mucin-type glycoproteins. The widespread disaccharide Galβ1,4GlcNAc, also known as N-acetyllactosamine, LacNAc, or LacNAc type 2, consists of galactose β-1,4 linked to N-acetylglucosamine (GlcNAc). Likewise, GlcNAc is present at the reducing end of the disaccharide. LacNAc is frequently overexpressed on the surface of cancer cells, where it binds to tumor-secreted galectins to promote cancer-related processes such as metastasis, adhesion, tumor survival, and immune escape (Romano and Oscarson, Org. Biomol. Chem. 17, 2265-2278 (2019)). LacNAc is also part of the Lewis X, Lewis Y and sialylated Lewis X epitopes. Dimeric and extended LacNAc structures of both type 1 (Galβ1,3GlcNAc) and type 2 (Galβ1,4GlcNAc) and poly-N-acetyllactosamine (poly-LacNAc) are commonly associated with inflammatory processes and cancer. The disaccharides LNB and LacNAc and their parts and Lewis-type epitopes are also present in human milk as part of human milk oligosaccharide (HMO) compositions (Prudden et al., PNAS USA 114, 6954-6959 (2017)). Parts of the Bifidobacterium species present in the colon are capable of specific consumption of HMOs, such as LNB or LNB containing carbohydrates. Some lactobacilli, such as L. casei , have been shown to metabolize the LacNAc disaccharide present in human milk during early lactation (Bidart et al., Sci. Rep. 8, 7152 (2018)). Thus, Lactobacillus and Bifidobacterium constitute up to 90% of the total gut flora of breastfed infants (Fischöder et al., Molecules 22, 1320 (2017)).

Since these disaccharides and oligosaccharides with GlcNAc at the reducing end are involved in the important process of plethora, many pharmaceutical and nutraceuticals are focusing on the development of novel N-acetyllactosamines (type 1 or type 2) A therapeutic or beneficial nutritional compound. Considerable effort has been devoted to developing synthetic processes for glycans such as the disaccharides LNB and LacNAc or glycans containing LNB or LacNAc at the reducing end.

Chemical synthesis methods are laborious and time-consuming, and they are difficult to scale up due to the large number of steps involved. Biocatalytic approaches offer many advantages, and thus there are numerous publications related to the production of LNB, LacNAc, and variants thereof. Bayón et al. (RSC Advances 3(30) (2013)) report the use of purified β-Gal-3 galactosidase from Bacillus circulans , self-supplemented with p-NP-Gal as supply LNB was produced in the biomass of the recipient and supplemented with GlcNAc as recipient. Patent application JP2017195793A Another galactosidase enzyme, recombinantly produced from and purified from a Bacillus species, for in vitro synthesis of galacto-oligosaccharides such as LNB via hydrolysis. Other reports utilize LNB phosphorylase from Bifidobacteria in an enzymatic reaction mixture starting from sucrose and supplemented with GlcNAc, additionally supplemented with sucrose phosphorylase, UDP-glucose-hexose-1-phosphate uridine UDP-glucose-hexose-1-phosphate uridylyltransferase and UDP-glucose 4-epimerase to produce LNB (Nishimoto and Kitaoka, Biosci. Biotechnol. Biochem. 71, 2101 -2104 (2007); Nishimoto, Biosci. Biotechnol. Biochem. 84, 17-24 (2020); US2010120096A; JP4264742 B2; JP2005341883 A2). Also, it is proposed to use an in vitro reaction mixture containing galactose, GlcNAc, galactokinase together with LNB phosphorylase (CN110527704 A). Another frequently reported mode of action is the use of coupled coupling for catalytic conversion. In such an example, several recombinant bacterial strains expressing enzymes involved in the carbohydrate catalytic pathway of the present invention were grown and lysed to obtain the necessary enzymes in the decomposition reaction mixture. The inventors of patent application JP2013201913 describe the treatment of Bifidobacterium breve MCC1320 or Bifidobacterium infantis in a mixture containing supplemental sucrose, GlcNAc, phosphate, UDP-glucose and/or UDP-galactose Bifidobacterium infantis strains and B. longum strains were coupled together to express LNB phosphorylase and sucrose phosphorylase, respectively, to make LNB. Endo and co-workers even report the coupled use of three bacterial strains, namely two recombinant E. coli strains for overexpressing galT, galK, galU, ppa and lgtB from Neisseria gonorrhoeae and for UTP production A strain of Corynebacterium ammoniagenes to produce LacNAc in a reaction supplemented with galactose and GlcNAc (Endo et al., Carb. Res. 316(1-4), 179-183 (1999)). The same group described a similar coupling system in which an E. coli strain expressed β1,4-galT from H. pylori , rather than NgLgtB, to participate in the enzymatic switch, supplemented with additional GlcNAc, to generate LacNAc (Endo et al., Glycobiology 10(8), 809-813 (2000)). The NmLgtB enzyme of N. meningitidis (Wakarchuk et al., Protein Engineering, Design and Selection 11(4), 295-302 (1998)) is also frequently selected for use with E. coli or Streptococcus thermophilus. ( Streptococcus thermophilus ) together as part of a fusion protein for the synthesis of LacNAc-like oligosaccharides in an additional GlcNAc reaction (Blixt et al., J. Org. Chem. 66(7), 2442-2448 (2001) ; Ruffing et al., Metab. Eng. 8(5), 465-473 (2006); Mao et al., Biotechnol. Prog. 22(2), 369-374 (2006)). Bacterial coupling, also known as 3'-fucosylated LacNAc, has also been described in the synthesis of Lewis X epitopes (Koizumi et al., J. Ind. Microbiol. Biotech. 25, 213-217 (2000)).

The above methods generally suffer from the following problems: relatively poor availability and/or stability of glycosyltransferases and/or glycosyl hydrolases, requirement for optimal stoichiometric balance, need for in situ regeneration of nucleotide-sugars, addition of multiple reactions Compounds, such as recipients including GlcNAc or oligosaccharides containing GlcNAc at their reducing end, and require the cultivation of different producing organisms, which respectively produce one or more enzymes or fusion enzymes necessary for catalytic conversion. The most important obstacle in these lists is the cellular synthesis of the primary acceptor including GlcNAc or oligosaccharides containing GlcNAc at its reducing end. In the examples mentioned, GlcNAc monosaccharides used to synthesize LNB, LacNAc or oligosaccharides with GlcNAc at their reducing end are externally supplemented to the relevant reaction or cell.

Bettler and co-workers describe the production of the hexasaccharide βGal(1,4)[βGlcNAc(1,4)]4GlcNAc constructed in a single cell without supplementation with GlcNAc ( _Bettler et al., Glycoconj. J . 16, 205-212 (1999)). This hexasaccharide has a GlcNAc unit at its reducing end and a terminal LacNAc moiety at its non-reducing end. To this end, in a recombinant E. coli cell, the NodC enzyme ( β1,4 -GlcNAc-oligosaccharide synthase) from bacterial Azorhizobium was combined with the LgtB enzyme (β(1) from Neisseria meningitidis. ,4)-galactosyltransferase (β(1,4)-galactosyltransferase)) co-expressed. In this example, however, the chitin structure (GlcNAc-GlcNAc) _n which is present in this hexasaccharide and has GlcNAc at its reducing end is created by the linkage of the UDP-GlcNAc moiety. Bettler and co-workers also described in a similar system the co-expression of NodC, LgtB and the bovine a1,3-galactosyltransferase GstA in recombinant E. coli cells to generate the xenograft antigen Galα1,3Galβ1 containing GlcNAc at its reducing end, ₄ [βGlcNAc(1,4)]4GlcNAc (Bettler et al., Biochem. Biophys. Res. Commun. 302(3), 620-624 (2003)). Likewise, the chitin structure and the LacNAc moiety present in the heptasaccharide were constructed from the use of the UDP-GlcNAc moiety instead of the inactivated GlcNAc. Deng and co-workers describe the successful production of GlcNAc via fermentation using recombinant E. coli cells (Deng et al., Metab. Eng. 7, 201-214 (2005); EP1576106). In the microbial system developed by Deng and co-workers, GlcNAc is extracellular in E. coli cells, more specifically, in the periplasm of E. coli, via a process that exports GlcNAc-6-phosphate produced when the latter is dephosphorylated. Thus, the obtained GlcNAc fraction can no longer be used for intracellular transformations, such as further saccharification, which is required for the manufacture of the saccharides of the present invention. Additionally, the process described by Deng and co-workers requires a two-stage feeding batch system that requires precise control to minimize host inhibition by phosphorylated amino sugars, which makes the process critical to the production of high-potency (Extracellular) GlcNAc has no commercial value.

The present invention overcomes the above problems as it provides a method and cell for producing the desired product with relative ease and, if desired, continuous production.

Surprisingly, it has now been found that GlcNAc can be produced by a single cell, and this GlcNAc monosaccharide can be further glycated by the same cell to produce a disaccharide or oligosaccharide with GlcNAc at its reducing end. The present invention provides a method for producing a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end by a cell. The method includes the steps of: providing cells capable of synthesizing nucleotide-sugars to synthesize GlcNAc to glycosylate the GlcNAc monosaccharide. The present invention also relates to a method for producing a disaccharide or oligosaccharide having GlcNAc at the reducing end by culturing the cell under conditions that allow production of the disaccharide or oligosaccharide. Furthermore, the present invention also provides a method for isolating the disaccharide or oligosaccharide from the culture. Furthermore, the present invention provides a metabolically engineered cell for producing disaccharides or oligosaccharides having N-acetylglucosamine units at the reducing end.

definition

The words used to describe the present invention and its various embodiments in this specification should not only be construed as the meanings of the commonly defined meanings, but also include structures and materials beyond the scope of the commonly defined meanings by special definitions in this specification and action. Thus, if an element can be understood within the context of this specification to contain more than one meaning, its use in the context of an invention claim must be understood as being generic to all possible meanings supported by the specification and the words themselves .

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

In the specification, embodiments of the present invention have been disclosed, and although specific terms are used, these terms are used in a descriptive sense only and are not intended to be limiting, and the scope of the present invention is set forth in the following claims. It should be understood that the illustrated embodiments are for illustrative purposes only and should not be construed as limiting the invention. Modifications, other embodiments, improvements, details and uses consistent with the letter and spirit of the invention herein and within the scope of the invention will be apparent to those of ordinary skill in the art, which are only limited by the invention The limitations of patent scope are interpreted in accordance with patent law, including the doctrine of equivalents. In the following claims for invention, the reference features used to designate the steps of the claim for invention are provided for convenience of description only and do not imply any particular order in which the steps are performed.

In this article and the scope of its patent application, the verb "to comprise" and its conjugations are used in their non-limiting sense to mean that the item following the word is included, but not specifically mentioned items are not excluded. In this application, the verb "comprise" may be replaced by "to consist" or "to consist essentially of" and vice versa. Furthermore, the verb "to consist" may be replaced by "to consist essentially of", which means that a composition as defined herein may include more ingredients than those specifically identified, the additional ingredients not changing Unique features of the present invention. Furthermore, reference to an element with the indefinite articles "a (a)" or "an (an)" does not preclude the presence of more than one element unless the context clearly requires the presence of one and only one element. Therefore, the indefinite article "a" or "an" usually means "at least one".

In this application, unless expressly stated otherwise, the articles "a" and "an" are preferably replaced by "at least two", more preferably "at least three" three)", more preferably "at least four", still more preferably "at least five", still more preferably "at least six" ", preferably with "at least two".

In this application, unless expressly stated otherwise, the features of "synthesize", "synthesized" and "synthesis" may be combined with "produce", "produced", respectively Used interchangeably with the "production" feature.

Unless otherwise stated, the various embodiments identified herein can be combined together. All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the extent that each individual publication, patent, or patent application is specifically and individually designated to be incorporated by reference Enter the same. The entire contents of the priority application, including EP20190198, EP20190200 and EP20190206, are 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 present invention, the term "polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA with single- and double-stranded regions or a mixture of single-, double-, and triple-stranded regions, and single- and double-stranded RNA and mixed single- and double-stranded regions RNA, including hybrid molecules of DNA and RNA, which may be single-stranded or more typically double- or triple-stranded regions, or a mixture of single- and double-stranded regions. Furthermore, "polynucleotide" as used herein refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The chains of these regions can be from the same molecule or from different molecules. These regions may encompass the entirety of one or more molecules, but are more typically regions for only some molecules. One of the molecules of the triple helix region is often an oligonucleotide. The term "polynucleotide" as used herein also includes the aforementioned DNA or RNA containing one or more modified bases. Thus, according to the present invention, DNA or RNA whose backbone has been modified for stability or other reasons is a "polynucleotide". In addition, DNA or RNA containing unusual bases (eg, inosine) or modified bases (eg, tritylated bases) should be understood to be covered by the term "polynucleotide" middle. It will be appreciated that a wide variety of modifications have been made to DNA and RNA for many useful purposes known to those of ordinary skill in the art. The term "polynucleotide" as used herein includes such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as chemical forms of DNA and RNA that are characteristic of viruses and cells, including, for example, simple and complex cells. The term "polynucleotide" also includes short polynucleotides often referred to as oligonucleotides.

"Polypeptide(s)" refers to any peptide or protein comprising two or more amino acids linked to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to both short chains (often called peptides, oligopeptides, and oligomers) and long chains (often called proteins). Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptide" includes polypeptides modified by processes such as processing and other post-translational modifications, as well as polypeptides modified by chemical modification techniques. Such modifications are well described in basic textbooks and more detailed monographs, as well as in the multi-volume research literature, and are known to those of ordinary skill in the art. The same type of modification may be present at several sites in a given polypeptide to the same degree or to different degrees. Furthermore, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Modifications include, for example, acetylation, acetylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moieties, nucleotide or nucleotide derivative Covalent attachment, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, pyroglutamine Acid formation, formylation, gamma-carboxylation, glycation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, isotope Prenylation, racemization, lipid linkage, sulfation, gamma-carboxylation, hydroxylation and ADP-ribosylation of glutamic acid residues, selenoylation, transfer RNA-mediated targeting Protein addition of amino acids (eg arginylation) and ubiquitination. Polypeptides can be branched or cyclic with or without branching. Cyclic, branched and branched cyclic polypeptides can be formed by post-translational natural processes, and can also be made by fully synthetic methods.

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

"Isolated" means "by the hand of man" that has changed its natural state, that is, if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that is naturally present in an organism is not "isolated," but the same polynucleotide or polypeptide is "isolated," as the term is used herein, that is isolated from coexisting material in its natural state. Likewise, a "synthetic" sequence as used herein refers to any sequence produced synthetically, rather than directly isolated from a natural source. The term "Synthesized" as used herein refers to any synthetically produced sequence, rather than directly isolated from a natural source.

The terms "recombinant" or "transgenic" or "metabolically engineered" or "genetically modified" are used interchangeably herein with reference to a cell or host cell and mean Bacterial cells replicate a heterologous nucleic acid, or express a peptide or protein encoded by a heterologous nucleic acid (i.e., sequences that are "foreign to the cell" or "foreign to this location or environment in the cell" sequence). Such cells are described as transformed with at least one heterologous or exogenous gene, or described as transformed by the introduction of at least one heterologous or exogenous gene. Metabolically engineered or recombinant or transgenic cells may contain genes that were not present in the original (non-recombinant) form of the cell. Recombinant cells may also contain genes found in native forms of cells, wherein the genes have been modified and reintroduced into the cells by artificial means. These terms also encompass cells that contain nucleic acid endogenous to the cell, which has been modified, or whose expression or activity has been modified without removing the nucleic acid from the cell; such modifications include substitution of genes, substitution of promoters, Site-specific mutations; and related art-obtained modifications. Thus, a "recombinant polypeptide" is a polypeptide produced by a recombinant cell. As used herein, a "heterologous sequence" or "heterologous nucleic acid" refers to an origin other than a particular cell (eg, from a different species), or if the same origin, from its original form or its position in the genome is modified. Thus, the source of the heterologous nucleic acid operably linked to the promoter is different from the source of the derived promoter, or if the same source, modified from its original form or position in the genome. Heterologous sequences can be stably introduced into the genome of a host microbial cell by, for example, transfection, transformation, conjugation, or transduction, where applicable techniques will depend on the cell and the sequence to be introduced. Various techniques are known to those of ordinary skill in the art, and are described, for example, 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 present invention refers to a genetically modified cell or microorganism.

The term "cellular genetic modification for the production of disaccharides or oligosaccharides having N-acetylglucosamine (GlcNAc) units at the reducing end" used within the context of the present invention means that one or more selected from the group consisting of Microbial cells genetically modified in terms of the expression or activity of enzymes: N-acetylglucosamine-6-phosphate transferase (glucosamine 6-phosphate N-acetyltransferase), phosphatase (phosphatase), glycosyltransferase, L- Glutamate-D-fructose-6-phosphate aminotransferase (L-glutamine-D-fructose-6-phosphate aminotransferase) and UDP-glucose 4-epimerase.

In the context of the present context, the term "endogenous" refers to any polynucleotide, polypeptide or protein sequence that is a natural part of the cell and is present in its natural location in the cell's chromosome, and that acts on The control of its performance was not altered compared to the natural control mechanism of its performance. The term "exogenous" refers to any polynucleotide, polypeptide, or protein sequence that is derived from outside the cell under study and is not a native part of the cell, or is not naturally present in the cell's chromosomes or plastids. Location.

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 derived from or derived from a source other than the host species. In contrast, "homologous" polynucleotides, genes, nucleic acids, polypeptides or enzymes as used herein refers to polynucleotides, genes, nucleic acids, polypeptides or enzymes derived from a species of host organism. When referring to gene regulatory sequences or auxiliary nucleic acid sequences for maintaining or manipulating gene sequences (e.g., promoters, 5' untranslated regions, 3' untranslated regions, poly A appendages, intron sequences, splice sites, ribosome binding sites, internal ribosomal entry sequences, regions of homology in the genome, recombination sites, etc.), "heterologous" means a regulatory sequence or ancillary sequence that is associated with a regulatory or auxiliary sequence in a construct, genome, chromosome or episome. Genes whose helper nucleic acid sequences are juxtaposed are not naturally associated. Thus, as used herein, a "heterologous promoter" refers to a promoter operably linked to a gene that is not operably linked in its native state (ie, in the genome of a non-engineered organism), even if The promoter can 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 enzyme refers to a change in the activity of the protein or enzyme compared to the wild-type (ie, native) activity of the protein or enzyme. The modified activity may be an abolished, impaired, reduced or delayed activity of the protein or enzyme compared to the wild-type activity of the protein or enzyme, but may also be compared to the wild-type activity of the protein or enzyme, An accelerated or enhanced activity of the protein or enzyme. The modification activity of a protein or enzyme is obtained by modifying the expression of the protein or enzyme or by expressing a modified, ie mutated, form of the protein or enzyme. The modification activity of the enzyme is further related to the change in the significant Michaelis constant Km and/or the significant maximum velocity (Vmax) of the enzyme.

The term "modified expression" of a gene refers to a change in expression compared to the wild type of the gene at any stage during the production of the encoded protein. The modified expression is a lower or higher expression compared to the wild type, where the term "higher expression" refers to an endogenous gene that is also defined as "over-expression (over-expression) of that gene. Overexpression)", or "overexpression" in the case of a heterologous gene not present in the wild-type strain. Lower expression or reduced expression is achieved by techniques commonly known to those of ordinary skill in the art (eg, using siRNA, CrispR, CrispRi, riboswitches, recombination, homologous recombination, ssDNA mutagenesis). ), RNAi, miRNA, asRNA, mutated genes, knockout genes, transposon mutagenesis..), this technique is used to alter a gene to make it less-able (ie, comparable to a functional wild-type gene) In contrast, statistically significantly "less capable") or not at all (such as a knockout gene) to produce a functional end product. The term "riboswitch" as used herein is defined as a portion of a message RNA that folds into a complex structure to prevent expression by interfering with translation. Binding of effector molecules induces conformational changes that allow post-transcriptionally regulated expression. In addition to altering the gene of interest in the manner described above to achieve lower performance, lower performance can also be achieved by altering transcription units, promoters, untranslated regions, ribosome binding sites, Shine Dalgarno sequences, or transcription terminators . For example, induction of regulated expression can be achieved by mutating one or more base pairs in the promoter sequence or by completely changing the promoter sequence to a constitutive promoter with lower expression strength compared to wild type Inducible promoters or repressible promoters that result in regulated performance to obtain lower or reduced performance. Overexpression or expression is a technique commonly known to those of ordinary skill in the art (eg, use of artificial transcription factors, de novo design of promoter sequences, ribosome engineering, introduction on euchromatin) or re-introduction of expression modules, the use of high-copy number plastids), in which the gene is part of an "expression cassette", which is about any sequence, in which there is a promoter sequence, a non-translated region sequence ( Contains ribosome binding sequences, Shine Dalgarno or Kozak sequences), coding sequences and optional transcription terminators, and results in the expression of a functionally active protein. The performance is persistent or modulating.

The term "constitutive expression" is defined as the absence under some growth conditions of transcription factors other than subunits of RNA polymerase (such as σ ⁷⁰ , σ ⁵⁴ , or related bacterial σ factors and those associated with the RNA polymerase core enzyme. Expression of the co-associated yeast mitochondrial RNA polymerase-specific factor MTF1). Non-limiting examples of such transcription factors are CRP, LacI, ArcA, Cra, IclR in E. coli, or Aft2p, Crz1p, Skn7 in Saccharomyces cerevisiae , or B. subtilis DeoR, GntR, Fur. These transcription factors bind to a specific sequence that may prevent or promote expression under some growth conditions. RNA polymerase is a catalytic machine that synthesizes RNA from a DNA template. RNA polymerase binds to specific sequences to initiate transcription, such as via sigma factor in prokaryotic hosts or via MTF1 in yeast. Persistent performance provides a constant level of performance that does not require induction or inhibition.

The term "regulated expression" is defined as an expression regulated by transcription factors other than subunits of RNA polymerase (eg, bacterial sigma factors) under some growth conditions. Examples of such transcription factors are described above. Common performance modulations are obtained by means of induction or inhibition, such as but not limited to IPTG, arabinose, rhamnose, fucose, allo-lactose or pH shift or temperature shift or carbon depletion Either acceptor or produced product or chemical inhibition.

The term "expression by a natural inducer" is defined as a facultative or regulatory expression of a gene that occurs only under certain natural conditions in the host (for example, the organism is giving birth, or during lactation) as a response to environmental changes (e.g., including but not limited to hormones, heat, cold, pH shifts, light, oxidative or osmotic stress/signals) or depending on the developmental stage or cell cycle position of the host cell ( including but not limited to apoptosis and autophagy).

The term "inducible expression upon chemical treatment" is defined as an episodic or regulatory expression of a gene that is only expressed upon treatment with a chemical inducer or inhibitor, wherein the inducer and inhibitor Compounds include, but are not limited to, alcohol (eg, ethanol, methanol), carbohydrates (eg, glucose, galactose, glycerol, lactose, arabinose, rhamnose, fucose, allolactose), metal ions (eg, aluminum, copper, zinc ), nitrogen, phosphate, IPTG, acetate, formate, xylene.

The term "control sequences" refers to sequences recognized by cellular transcription and translation systems that permit transcription and translation of polynucleotide sequences into polypeptides. Thus, this DNA sequence is necessary for the expression of the operably associated coding sequence in a particular cell or organism. This control sequence can be, but is not limited to, a promoter sequence, a ribosome binding sequence, a Shine Dalgarno sequence, a Kozak sequence, a transcription terminator sequence. For example, control sequences suitable for use in prokaryotes include promoters, optional operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers. The DNA of the presequence or secretory leader may be operably associated with the DNA of the polypeptide if expressed as a preprotein involved in the secretion of the polypeptide; operably associated with the coding sequence if the promoter or enhancer affects the transcription of the coding sequence; or if A ribosome binding site affects the transcription of the coding sequence and is operably associated with the coding sequence; or if the ribosome binding site facilitates translation, it is operably associated with the coding sequence. The control sequence can be further transcribed or translated into the polynucleotide via an inducible promoter or via induction or inhibition of transcription or translation of the polynucleotide with an external chemical such as, but not limited to, IPTG, arabinose, lactose, allolactose, rhamnose or fucose. controlled by the genetic circuit of the polypeptide.

In general, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader, contiguous and in the reading phase. However, enhancers are not necessarily continuous.

The term "wild type" refers to a genetic or phenotypic condition commonly found 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) a difference between wild-type (ie, native) Expression and/or overexpression of a variant protein with higher activity compared to the protein.

The term "mammary cell(s)" as used herein generally refers to mammary epithelial cells, mammary-epithelial luminal cell(s) or mammalian epithelial alveolar cells ( s)) or any combination thereof. The term "mammary-like cell(s)" as used herein generally refers to cells that have a similar (or substantially similar) phenotype/genotype to natural breast cells, but which are derived from non-mammary cells source. Such mammary gland-like cells can be engineered to remove at least one unwanted genetic component and/or contain at least one predetermined genetic structure typical of mammary gland cells. Non-limiting examples of mammary-like cells can include mammary-like epithelial cells, mammary-like epithelial luminal cells, non-mammary cells exhibiting one or more characteristics of cells of mammary cell lineages, or any combination thereof. Further non-limiting examples of mammary gland-like cells may include cells having a phenotype similar (or substantially similar) to native breast cells, or more specifically a phenotype similar (or substantially similar) to native mammary epithelial cells cells. Cells that have a phenotype similar (or substantially similar) to, or exhibit at least one characteristic of, native breast cells or mammary epithelial cells may include cells that naturally exhibit or are engineered to express at least one milk component (eg, derived from breast or non-mammary cell lines).

The term "non-mammary cell(s)" as used herein may generally include any cell of a non-mammary lineage. In the context of the present invention, a non-mammary gland cell can be any mammalian cell that can be engineered to express at least one milk component. Non-limiting examples of such non-mammary cells include hepatocytes, blood cells, kidney cells, cord blood cells, epithelial cells, epidermal cells, muscle cells, fibroblasts, interstitial cells, or any combination thereof. In some cases, molecular biology and genome editing technologies can be engineered to simultaneously eliminate, silence or attenuate countless genes.

In this application, unless expressly stated otherwise, "capable of...<verb>" and "capable of...<verb>" (capable to...<verb>)" are preferred Substitute 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, that is, "capable of expressing" is preferably replaced with "capable of expressing".

As used herein, the term "Variant(s)" 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 the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. In general, differences are constrained so that the sequences of the reference polypeptide and the variant are closely similar overall and are identical in many regions. The 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 an amino acid residue encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring variant, such as a counterpart gene variant, or it may be a variant that is not known to be naturally occurring. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those of ordinary skill in the art.

As used herein, the term "derivative" of a polypeptide refers to deletions, additions, or substitutions of amino acid residues that may include in the amino acid sequence of a polypeptide, but which result in a silent change such that A functionally equivalent polypeptide is produced. Substitution of amino acids can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the relevant residues. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; planar neutral amino acids Includes glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and Histamine; negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In the context of the present invention, a derived polypeptide as used herein refers to a polypeptide capable of exhibiting substantially similar in vitro and/or in vivo activities as the original polypeptide, as judged by any of a number of criteria, including but not limited to enzymes activity, and various modifications can be made during or after translation. In addition, non-classical amino acids or chemical amino acid analogs can be introduced into the original polypeptide sequence as substitutes or additions.

In some embodiments, the present invention contemplates making functional variants by modifying the structure of the enzymes used in the present invention. Variants can be created by amino acid substitutions, deletions, additions, or combinations thereof. For example, it is reasonable to expect isoleucine or valine to replace leucine alone, glutamic acid to replace aspartic acid, serine acid to replace threonine alone, or a structurally related amino acid to replace Similar substitutions (eg, conservative mutations) to amino acids do not have a significant effect on the biological activity of the resulting molecule. Conservative substitutions are those that take place within a family of amino acids related to their side chains. Whether a change in the amino acid sequence of a polypeptide of the invention results in a functional homologue can be readily determined by assessing the ability of the variant polypeptide to generate a response in cells in a manner similar to the wild-type polypeptide.

The term "functional homolog" as used herein describes those molecules that share sequence similarity (in other words, homology) and also share at least one functional characteristic, such as biochemical activity (Altenhoff et al. , PLoS Comput. Biol. 8 (2012) e1002514). Functional homologues generally yield similar, but not necessarily the same, degree for the same characteristics. Functionally homologous proteins have the same characteristics, wherein the quantitative measure produced by one homolog is at least 10% of the other; more typically at least 20% of the quantitative measure produced by the original molecule, at about 30% and between about 40%; for example, between about 50% and about 60%; between about 70% and about 80%; or between about 90% and about 95%; between about 98% and about 100% %, or more than 100%. Thus, when the molecule has enzymatic activity, the functional homologue will have the aforementioned percentage of enzymatic activity compared to the original enzyme. When the molecule is a DNA binding molecule (eg, a polypeptide), then the homologue will have the above-described percentage of binding affinity as determined by the weight of the binding molecule compared to the original molecule.

Functional homologs and reference polypeptides can be naturally occurring polypeptides, and sequence similarity can result from convergent or divergent evolutionary events. Functional homologs are sometimes referred to as xenologs, where "ortholog" refers to a homologous gene or protein that is functionally equivalent to a reference gene or protein in another species.

Orthologous genes refer to homologous genes in different species, which originate from a single gene in the last common ancestor through vertical inheritance, and the genes and their main functions are conserved. Homologous genes refer to genes inherited from a common ancestor in two species.

The term "ortholog" when referring to an amino acid or nucleotide/nucleic acid sequence of a particular species refers to the same amino acid or nucleotide/nucleic acid sequence from a different species. It will be understood that two sequences are heterologous to each other when they are derived from a common ancestral sequence via linear kinship and/or are closely related in their sequence and biological function. Heterologs usually have a high degree of sequence identity, but do not necessarily (and often do not) have 100% sequence identity.

Paralogous genes are homologous genes that result from gene duplication events. Homologous genes usually belong to the same species, but they are not required. Homologs can be divided into in-paralogs (pairs of homologs that occur after a species evolution event) and out-paralogs (same species that occur before a species evolution event) homologous pair). Exologs between species refer to a pair of homologs that existed between two organisms due to duplication before the evolution of the species. Within a species, a ectolog is a pair of homologs that exist within the same organism, but whose duplication occurs after the evolution of the species. Homologs usually have the same or similar function.

Functional homologs can be determined by analyzing nucleotide and polypeptide sequence alignments. For example, queries against databases of nucleotide or polypeptide sequences can identify homologs of polypeptides of interest, such as biomass-regulating polypeptides, glycosyltransferases, proteins involved in the synthesis of nucleotide-active sugars, or membrane transporters. Sequence analysis can be BLAST, Reciprocal BLAST, or PSI-BLAST analysis on non-redundant databases using bio-modulating polypeptides, glycosyltransferases, proteins involved in nucleotide-active sugar synthesis, or membrane transporters, respectively. The amino acid sequence served as a reference sequence. In some cases, the amino acid sequence is deduced from the nucleotide sequence. Typically, those polypeptides in the database with greater than 40% sequence identity are candidates for further evaluation as suitable for further evaluation as biomass-regulating polypeptides, glycosyltransferases, proteins involved in the synthesis of nucleotide-active sugars, or membrane transporters, respectively. The similarity of amino acid sequences 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. One acidic amino acid, or replacing one basic amino acid with another basic amino acid, etc. Preferably, conservative substitutions refer to combinations such as alanine for glycine and vice versa; methionine for valine, isoleucine and leucine and vice versa; Amino acid replaces aspartic acid and vice versa; glutamine replaces asparagine and vice versa; threonine replaces serine and vice versa; arginine replaces lysine and vice versa of course; substitution of cysteine by methionine, and vice versa; and substitution of amphetamine and tyrosine by tryptophan, and vice versa. If desired, these candidates can be manually inspected to narrow down the number of candidates for further evaluation. Manual inspection can be performed by selecting candidates for domains (eg, conserved functional domains) present in the polypeptide that appear to have the ability to modulate production.

"Fragment", in relation to a polynucleotide, refers to a colony or any portion of a polynucleotide molecule, particularly a portion of a polynucleotide, that retains the functional characteristics of a full-length polynucleotide molecule. Useful fragments include oligonucleotides and polynucleotides, which are useful in hybridization or amplification techniques or the regulation of replication, transcription or translation. "Polynucleotide fragment" refers to any subsequence of a polynucleotide SEQ ID NO (or Genbank NO.), typically including or consisting of at least about 9, 10, 11 of the polynucleotide SEQ ID NO (or Genbank NO.) , 12 contiguous nucleotides, eg, at least about 30 nucleotides or at least about 50 nucleotides of any of the polynucleotide sequences provided herein. Exemplary fragments may additionally or alternatively include fragments that include, consist essentially of, or consist of a region encoding a conserved family domain of a polypeptide. Exemplary fragments may additionally or alternatively include fragments that include conserved domains of polypeptides. Therefore, a fragment of a polynucleotide SEQ ID NO (or Genbank NO.) preferably represents a nucleotide sequence comprising or consisting of the polynucleotide SEQ ID NO (or Genbank NO.), wherein the deleted contiguous nucleosides No more than 200, 150, 100, 50, or 25 acids, preferably no more than 50 consecutive nucleotides deleted, and retain useful functional characteristics (eg, activity) of the full-length polynucleotide molecule, which can be obtained from the art Those with ordinary knowledge in the field are assessed by routine experimentation. In addition, a fragment of a polynucleotide SEQ ID NO (or Genbank NO.) is preferably represented as comprising or consisting of a certain amount of contiguous nucleotides from the polynucleotide SEQ ID NO (or Genbank NO.) The composed nucleotide sequence, wherein the amount of the continuous nucleotide is at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0%, 82.0% of the full length of the polynucleotide SEQ ID NO. (or Genbank NO.) , 83.0 %, 84.0 %, 85.0 %, 86.0 %, 87.0 %, 88.0 %, 89.0 %, 90.0 %, 91.0 %, 92.0 %, 93.0 %, 94.0 %, 95.0 %, 95.5 %, 96.0 %, 96.5 % , 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 100%, preferably at least 80.0%, more preferably at least 87.0%, still more preferably at least 90%, still more preferably at least 95.0%, Optimally at least 97.0% and retain the functional characteristics (eg, activity) of the full-length polynucleotide molecule available. Accordingly, a fragment of a polynucleotide of SEQ ID NO (or Genbank NO.) preferably represents a nucleotide sequence comprising or consisting of the polynucleotide of SEQ ID NO (or Genbank NO.) in which a number of contiguous nuclei are missing nucleotides, and the number does not exceed 50.0%, 40.0%, 30.0% of the full length of the polynucleotide SEQ ID NO (or Genbank NO.), preferably not more than the full length of the polynucleotide SEQ ID NO (or Genbank NO.) of the , 0.5%, better not more than 15.0%, better not more than 10.0%, better not more than 5.0%, best not more than 2.5%, and wherein the fragment retains the available functional characteristics of the full-length polynucleotide molecule (eg, activity), this characteristic can be routinely assessed by one of ordinary skill in the art.

In the present application, the sequence of the polynucleotide can be represented by SEQ ID NO or GenBank NO. Accordingly, the terms "polynucleotide SEQ ID NO" and "polynucleotide GenBank NO." are used interchangeably unless expressly stated otherwise. Fragments may additionally or alternatively comprise subsequences of polypeptides and protein molecules, or subsequences of polypeptides. In some cases, a fragment or domain is a subsequence of a polypeptide that performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar degree, as the intact polypeptide. A "subsequence of the polypeptide" as defined herein refers to a sequence of contiguous amino acid residues derived from a polypeptide. For example, polypeptide fragments can include identifiable structural motifs or functional domains, such as DNA binding sites or binding domains, activation domains, or protein-protein interaction domains that bind to DNA promoter regions and can initiate transcription. Fragments can vary in size from as few as 3 amino acid residues to the full length of the complete polypeptide, eg, at least about 20 amino acid residues in length, eg, at least about 30 amino acid residues in length. Therefore, a fragment of a polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) preferably represents a polypeptide sequence comprising or consisting of the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.), wherein no lack of more than 80, 60 , 50, 40, 30, 20 or 15 contiguous amino acid residues, preferably not lacking more than 40 contiguous amino acid residues, and which are in substantially the same manner, preferably similar, to the intact polypeptide or to a greater extent, perform at least one biological function of the intact polypeptide, which can be routinely assessed by one of ordinary skill in the art. In addition, a fragment of a polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) preferably represents a polypeptide comprising or consisting of a number of consecutive amino acid residues from the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) sequence, wherein the number of consecutive amino acid residues is at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0%, 82.0%, 83.0%, of the full length of the polypeptide SEQ ID NO (or UniProt ID or Genbank NO), 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 100%, preferably at least 80.0%, more preferably at least 87.0%, still more preferably at least 90.0%, still more preferably at least 95.0%, most preferably at least 97.0% %, and it performs at least one biological function of the intact polypeptide in substantially the same manner, preferably in a similar or greater manner, as the intact polypeptide, which can be routinely assessed by those of ordinary skill in the art. Thus, a fragment of a polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) preferably represents a polypeptide sequence comprising or consisting of the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.), wherein a number of contiguous amines are lacking amino acid residues, and not 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 not more than the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) ) 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 % of the full length , 0.5%, better not more than 15.0%, better not more than 10.0%, better not more than 5.0%, best not more than 2.5%, and in substantially the same way as the intact polypeptide, preferably similar or to a greater extent, perform at least one biological function of the intact polypeptide, which can be routinely assessed by one of ordinary skill in the art.

In the present application, the sequence of the polypeptide can be represented by SEQ ID NO, or by UniProt ID or GenBank No. Accordingly, the terms "polypeptide SEQ ID NO" and "polypeptide Uniprot ID" and "polypeptide GenBank No." are used interchangeably unless expressly stated otherwise.

Preferably, a fragment of a polypeptide is a functional fragment having at least one property or activity of the polypeptide from which it is derived, preferably to a similar or greater extent. For example, functional fragments can include functional or conserved domains of polypeptides. It is understood that polypeptides or fragments thereof may have conservative amino acid substitutions that have no substantial effect on the activity of the polypeptide. The so-called conservative substitution refers to the substitution of one hydrophobic amino acid for another hydrophobic amino acid, or the substitution of one polar amino acid for another polar amino acid, or the substitution of one acidic amino acid for another acidic amino acid, Or replace another basic amino acid with one basic amino acid, etc. Preferably, conservative substitution refers to a combination, such as substitution of alanine for glycine, and vice versa; substitution of valine, isoleucine, and leucine by methionine, and vice versa; substitution of glutamine Acid replaces aspartic acid and vice versa; glutamine replaces asparagine and vice versa; threonine replaces serine and vice versa; arginine replaces lysine and vice versa ; substitution of cysteine by methionine, and vice versa; and substitution of amphetamine and tyrosine by tryptophan, and vice versa. For example, a domain can be identified by Pfam (El-Gebali et al., Nucleic Acids Res. 47 (2019) D427-D432) or the Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih. gov/cdd) (Lu et al., Nucleic Acids Res. 48 (2020) D265-D268) specified for characterization. The content of each repository is fixed with each release and may not be changed. When the content of a specific repository is changed, this specific repository receives the new release version and has a new release date. All published versions of each repository with their corresponding publication dates and specific content annotated at those specific publication dates are available and known to those of ordinary skill in the art. The PFAM repository (https://pfam.xfam.org/) used in this article is Pfam 33.1 version released on June 11, 2020. Protein sequence information and functional information can be provided by comprehensive sources of protein sequence and annotation data, such as the Universal Protein Resource (UniProt) (www.uniprot.org) (Nucleic Acids Res. 2021, 49(D1), D480-D489). UniProt includes a specialized and rich protein database known as the UniProt Knowledge Base (UniProtKB), as well as the UniProt Reference Cluster (UniRef) and the UniProt Archive (UniParc). The UniProt ID (UniProt ID) is unique to each protein present in the database. The UniProt ID used in this article is the UniProt ID from the May 5, 2021 UniProt Repository release. Proteins that do not have UniProt IDs use their respective GenBank accession numbers (GenBank No.) available in the NIH Gene Sequence Database on May 5, 2021 (https://www.ncbi.nlm.nih .gov/genbank/) (Nucleic Acids Res. 2013, 41(D1), D36-D42) edition.

In the present invention, polypeptide sequence extension is used to refer to N-acetylglucosamine b-1,3-galactosyltransferase and N-acetylglucosamine b-1,4-galactosyltransferase used in the present invention Fragments of enzymes that are common to those N-acetylglucosamine b-1,3-galactosyltransferases and N-acetylglucosamine b-1,4-galactosyltransferases. Such polypeptide extensions are written in the form of amino acid sequences in one-letter codes. If the amino acid at a particular position of such a polypeptide extension can be several amino acids, that particular position will be the amino acid code X. Unless otherwise described herein, the letter "X" refers to possibly any amino acid. The term (Xn) refers to an extension of a protein sequence consisting of a number n of amino acid residues X, wherein each said X is any amino acid possible, and wherein n is 2, 3, 4, or more. The term (Xm) refers to an extension of a protein sequence consisting of a number m of amino acid residues X, where each said X is any amino acid possible, where m is 2, 3, 4 or more. The term (Xp) refers to an extension of a protein sequence consisting of a number p of amino acid residues X, where each said X is any amino acid possible, where 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 Amino acid (Y).

The term "nucleotide-sugar" or "activated sugar" as used herein refers to the activated form of a monosaccharide. 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-Acetyl Amino-2,6-dideoxy--L-arabino-4-hexulose (UDP-2-acetamido-2,6-dideoxy--L-arabino-4-hexulose), UDP-2-acetamide Alkyl-2,6-dideoxy--L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamide yl-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine (dTDP-N-acetylfucosamine), UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylamino-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-N-acetyl-L-pneumosamine) (UDP -L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose (UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N- UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinosamine (UDP-L-QuiNAc or UDP-2- Acetylamino-2,6-dideoxy-L-glucose), GDP-L-isorhamnose (GDP-L-quinovose), CMP-N-acetylneuraminic acid (CMP-N-acetylneuraminic) acid) (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N ₃ , CMP-Neu4,5Ac ₂ , CMP-Neu5 ,7Ac ₂ , CMP-Neu5,9Ac ₂ , CMP-Neu5,7(8,9)Ac ₂ , UDP-glucuronate, UDP-galacturonate, GDP -Rhamnose or UDP-xylose. Nucleotide-sugars serve as glycoside donors in glycation reactions. These reactions are catalyzed by a group of enzymes called glycosyltransferases.

Terms used in the present invention "N-acetylglucosamine b-1,3-galactosyltransferase (N-acetylglucosamine b-1,3-galactosyltransferase)", "N-acetylglucosamine beta-1,3- N-acetylglucosamine beta-1,3-galactosyltransferase, "N-acetylglucosamine beta 1,3 galactosyltransferase", "N-acetylglucosamine beta 1,3 galactosyltransferase" N-acetylglucosamine β-1,3-galactosyltransferase, N-acetylglucosamine β-1,3-galactosyltransferase 1,3 galactosyltransferase)" is used interchangeably and refers to a galactosyltransferase that catalyzes the transfer of galactose from a donor UDP-galactose to a recipient N-acetylglucosamine via a β-1,3 glycosidic bond . A polynucleotide encoding "N-acetylglucosamine b-1,3-galactosyltransferase" or any of the above terms refers to a polynucleotide encoding a glycosyltransferase that catalyzes the conversion of Galactose from the donor UDP-galactose is transferred to the recipient N-acetylglucosamine with a β-1,3 glycosidic linkage.

Terms used in the present invention "N-acetylglucosamine b-1,4-galactosyltransferase (N-acetylglucosamine b-1,4-galactosyltransferase)", "N-acetylglucosamine beta-1,4- N-acetylglucosamine beta-1,4-galactosyltransferase, N-acetylglucosamine beta 1,4 galactosyltransferase, N-acetylglucosamine beta 1,4 galactosyltransferase N-acetylglucosamine β-1,4-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase 1,4 galactosyltransferase)" is used interchangeably and refers to a galactosyltransferase that catalyzes the transfer of galactose from a donor UDP-galactose to a recipient N-acetylglucosamine via a β-1,4 glycosidic bond . A polynucleotide encoding "N-acetylglucosamine b-1,4-galactosyltransferase" or any of the above terms refers to a polynucleotide encoding a glycosyltransferase that catalyzes the conversion of Galactose from the donor UDP-galactose is transferred to the recipient N-acetylglucosamine with a β-1,4 glycosidic linkage.

Terms used in the present invention "N-acetylglucosamine-6-phosphate transferase (glucosamine 6-phosphate N-acetyltransferase)", "glucosamine-phosphate N-acetyltransferase (glucosamine-phosphate N-acetyltransferase)" , "GNA", "GNA1", "glucosamine-6P N-acetyltransferase", "GlcN6P N-acetyltransferase" are used interchangeably and refer to the The acetyl group of acetyl-CoA (acetyl-CoA) is transferred to the primary amine in glucosamine-6-phosphate, resulting in N-acetyl-D-glucosamine-6-phosphate (also known as GlcNAc-6P) the enzyme. A polynucleotide encoding "N-acetylglucosamine-6-phosphotransferase" or any of the above terms refers to a polynucleotide encoding an enzyme that catalyzes the transfer of an acetyl group from acetyl-CoA to Primary amine in glucosamine-6-phosphate, yielding N-acetyl-D-glucosamine-6-phosphate.

The terms used in the present invention "fructose-6-phosphate aminotransferase", "glutamine--fructose-6-phosphate-aminotransferase" )", "glutamine--fructose-6-phosphate aminotransferase", "L-glutamine-D-fructose-6-phosphate aminotransferase (L- glutamine—D-fructose-6-phosphate aminotransferase)”, “glmS”, “glms”, and “glmS*54” are used interchangeably and refer to the catalysis of the conversion of fructose-6-phosphate by glutamine as a nitrogen source The enzyme that converts to glucosamine-6-phosphate. A polynucleotide encoding "fructose-6-phosphate transaminase" or any of the above terms refers to a polynucleotide encoding an enzyme that catalyzes the conversion of fructose-6-phosphate using glutamine as a nitrogen source into glucosamine-6-phosphate.

The term "bioproduct" as used herein refers to a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end, which is biosynthesized, ie, by microbial synthesis, cellular synthesis.

The term "disaccharide" as used herein refers to a saccharide consisting of two monosaccharide units. The term "Oligosaccharide" as used herein, as generally understood in the art, refers to a saccharide polymer containing a small amount (usually three to twenty) of a monosaccharide, ie, a monosaccharide. Monosaccharides as used herein are reducing sugars. Disaccharides and oligosaccharides can be reducing or non-reducing sugars and have reducing and non-reducing ends. A reducing sugar is any sugar that is capable of reducing another compound and is itself oxidized (ie, the carbonyl group of the sugar is oxidized to a carboxyl group). The term "reducing end of a saccharide" as used in the present invention refers to a free isomeric carbon in a sugar that can be used to reduce another compound.

As used herein, the term "monosaccharide" refers to a sugar that cannot be broken down by hydrolysis into simpler sugars, either aldose or ketose, containing one or more hydroxyl groups per molecule. Monosaccharides are sugars that contain only one type of monosaccharide. Examples of monosaccharides include hexose, D-Glucopyranose, D-galactofuranose, D-galactopyranose, L-galactofuranose Sugar, D-Mannopyranose, D-Allopyranose, D-Gulopyranose, D-idopyranose , D-talopyranose (D-talopyranose), D-ribofuranose (D-ribofuranose), D-ribopyranose (D-ribopyranose), D-arabinofuranose (D-arabinofuranose), D- D-arabinopyranose, L-arabinofuranose, L-arabinopyranose, D-xylopyranose, D-lyxopyranose, D- D-erythrofuranose, D-threofuranose, heptose, L-glycero-D-manno-Heptopyranose, LDmanHep), D-glycero-D-manno-heptopyranose (D-glycero-D-manno-Heptopyranose, DDmanHep), 6-Deoxy-L-altropyranose (6-Deoxy-L-altropyranose) , 6-deoxy--D-gulpyranose, 6-deoxy-D-talopyranose, 6-deoxy-D-galactopyranose, 6-deoxy-L-galactose 6-Deoxy-D-mannanose, 6-deoxy-L-mannanose, 6-deoxy-D-glucopyranose, 2-deoxy-D-arabino-hexose (2-Deoxy-D-arabino-hexose), 2-Deoxy-D-erythro-pentose (2-Deoxy-D-erythro-pentose), 2,6-dideoxy-D-arabino-hexose Ranose (2,6-Dideoxy-D-arabino-hexopyranose), 3,6-dideoxy-D-arabino-hexopyranose, 3,6-dideoxy-L-arabino-hexopyranose, 3,6-Dideoxy-D-xylose-hexopyranose (3,6-Dideoxy-D-xylo-hexopyranose), 3,6-dideoxy-D-ribose-hexopyranose (3,6-Dideoxy-D-ribose-hexopyranose) 6-Dideoxy-D-ribo-hexopyranose), 2,6-dideoxy-D-ribose-hexopyranose, 3,6-dideoxy-L-xylose- Hexanose, 2-amino-2-deoxy-D-glucopyranose, 2-amino-2-deoxy-D-galactopyranose, 2-amino-2-deoxy-D -Mannphenanose, 2-amino-2-deoxy-D-allofanose, 2-amino-2-deoxy-L-aldropyranose, 2-amino-2-des Oxy-D-gulpyranose, 2-amino-2-deoxy-L-iduropyranose, 2-amino-2-deoxy-D-talopyranose, 2-acetonitrile Amino-2-deoxy-D-glucopyranose, 2-acetamido-2-deoxy-D-galactopyranose, 2-acetamido-2-deoxy-D-pyranose Mannose, 2-acetamido-2-deoxy-D-allopyranosyl, 2-acetamido-2-deoxy-L-aldropyranose, 2-acetamido- 2-Deoxy-D-gulpyranose, 2-acetamido-2-deoxy-L-iduranose, 2-acetamido-2-deoxy-D-talopeptide Ranose, 2-Acetamino-2,6-dideoxy-D-galactopyranose, 2-Acetamino-2,6-dideoxy-L-galactopyranose, 2 -Acetamido-2,6-dideoxy-L-mannanose, 2-acetamido-2,6-dideoxy-D-glucopyranose, 2-acetamido-2 ,6-dideoxy-L-aldropyranose, 2-acetamido-2,6-dideoxy-D-talopyranose, D-glucopyranuronic acid (D-glucopyranuronic acid) acid), D-Galactopyranuronic acid, D-Mannopyranuronic acid, D-Allopyranuronic acid, L -L-Altropyranuronic acid, D-Gulopyranuronic acid, L-Gulopyranuronic acid, L-Iduronic acid (L-Idopyranuronic acid), D-Talopyranuronic acid (D-Talopyranuronic acid), sialic acid, 5-amino-3,5-dideoxy-D-glycerol-D-half Lactose-nonan-2-ketonic acid (5-Amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid), 5-acetamido-3,5-dideoxy -D-glycerol-D-galactose-nonan-2-ketonic acid, 5-ethanolamido-3,5-dideoxy-D-glycerol-D-galactose-nonan-2-ketonic acid ( 5-Glycolylamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid), erythritol ), Arabinitol, Xylitol, Ribitol, Glucitol, Galactitol, Mannitol, D-ribose-hex-2-one D-ribo-Hex-2-ulopyranose, D-arabino-Hex-2-ulofuranose (D-fructofuranose), D-arabino-hex-2-ketopyranose (D-arabino-Hex-2-ulopyranose), L-xylo-hex-2-ketopyranose (L-xylo-Hex-2-ulopyranose), D - Xylose-Hex-2-ketopyranose (D-lyxo-Hex-2-ulopyranose), D-threo-Pent-2-ketopyranose (D-threo-Pent-2-ulopyranose), D- D-altro-Hept-2-ulopyranose, 3-C-(Hydroxymethyl)-D-erythrofuranose (3-C-(Hydroxymethyl)-D -erythofuranose), 2,4,6-Trideoxy-2,4-diamino-D-glucopyranose (2,4,6-Trideoxy-2,4-diamino-D-glucopyranose), 6-Deoxy Oxy-3-O-methyl-D-glucose, 3-O-methyl-D-rhamnose, 2,6-dideoxy-3-methyl-D-ribose-hexose, 2-amino -3-O-[(R)-1-Carboxyethyl]-2-deoxy-D-glucopyranose, 2-acetamido-3-O-[(R)-carboxyethyl]-2 -Deoxy-D-glucopyranose, 2-ethanolamido-3-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose, 3-deoxy-D -Xylose-hept-2-ketopyranoic acid (3-Deoxy-D-lyxo-hept-2-ulopyranosaric acid), 3-deoxy-D-manno-oct-2-ketopyranoic acid (3 -Deoxy-D-manno-oct-2-ulopyranosonic acid), 3-Deoxy-D-glycero-D-galactose-non-2-ketopyranosic acid (3-Deoxy-D-glycero-D-galacto -non-2-ulopyranosonic acid), 5,7-diamino-3,5,7,9-tetradeoxy-L-glycerol-L-manno-non-2-ketopyranoic acid (5,7 -Diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulopy ranosonic acid), 5,7-diamino-3,5,7,9-tetradeoxy-L-glycerol o-L-altrose-nonan-2-ketopyranoic 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-glycerol-D -Galactose-nonan-2-ketopyranosonic 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-glycerol-D-tarot-non-2-ketopyranoic 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-acetylglucosamine N-glycolyl neuraminic acid, N-glycolyl neuraminic acid, N-acetylgalactosamine, galactosamine, fucose, rhamnose, glucuronic acid, gluconic acid, fructose and polyols.

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

The term "disaccharide having an N-acetylglucosamine unit at the reducing end" includes, but is not limited to, the products Gal-b1,3-GlcNAc or Gal-b1,4-GlcNAc, wherein galactose is bound by a β-1,3-bond, respectively It is linked to N-acetylglucosamine by means of a knot or β-1,4-bond, and wherein N-acetylglucosamine is located at the reducing end of the disaccharide.

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 in which galactose is β-1,3-linked to N-acetylglucosamine is linked and wherein N-acetylglucosamine is located at the reducing end of the disaccharide.

Terms "Gal-b1,4-GlcNAc", "Gal-beta-1,4-GlcNAc", "Gal-β1,4-GlcNAc", "Galb1,4GlcNAc", "Galβ1,4GlcNAc", "N-acetylene" Lactosamine, "LacNAc", "LacNAc type II", "type 2 LacNAc", "LacNAc (ii)" are used interchangeably and refer to a disaccharide in which galactose is β-1,4-bonded Linked to N-acetylglucosamine, and wherein N-acetylglucosamine is located at the reducing end of the disaccharide.

該寡醣由以下以α或β糖苷鍵鍵結的單醣組成，其中B為N-乙醯葡萄糖胺，且其中A、V、W、X、Y及/或Z不存在，或為半乳糖、葡萄糖、岩藻糖、甘露糖、木糖、葡萄糖醛酸、半乳糖醛酸、艾杜糖醛酸、N-乙醯基神經胺酸、N-乙醇醯神經胺酸、葡萄糖胺、N-乙醯半乳糖胺、N-乙醯甘露糖胺、N-乙醯葡萄糖胺及/或由通式2定義的寡醣結構：

其中B是N-乙醯葡萄糖胺，且其中A、V、W、X、Y及/或Z不存在，或為半乳糖、葡萄糖、岩藻糖、甘露糖、木糖、葡萄糖醛酸、半乳糖醛酸、艾杜糖醛酸、N-乙醯基神經胺酸、N-乙醇醯神經胺酸、葡萄糖胺、N-乙醯半乳糖胺、N-乙醯甘露糖胺或N-乙醯葡萄糖胺；且其中，在通式1和通式2中，m是3且可選地v是4，或者m是4且可選地v是3，並且其中p是3及/或4，且其中w是6，並且其中如果n＞1且p是3，x是3且X不是單醣，並且其中如果n＞1且p為4，y是4且Y不是單醣，並且其中z是6，且其中n從1至10。 The term "oligosaccharide having an N-acetylglucosamine unit at the reducing end" used in the present invention refers to an oligosaccharide consisting of three to twenty monosaccharide units, wherein N-acetylglucosamine is present in the oligosaccharide. restore end. Oligosaccharides used in the present invention may be linear in structure or may include branches. The linkage between two sugar units (eg, glycosidic linkage, galactosidic linkage, glucosidic linkage, etc.) can be expressed as, for example, 1,4, 1->4 or (1-4), in This is used interchangeably. 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 The meaning of , that the β-glycosidic bond connects carbon 1 of galactose (Gal) to carbon 4 of glucose (Glc). Each monosaccharide can be in a cyclic form (eg, the piperanose form of furanose). The linkages between individual monosaccharide units may include α1->2, α1->3, α1->4, α1->6, α2->1, α2->3, α2->4, α2->6 , β1->2, β1->3, β1->4, β1->6, β2->1, β2->3, β2->4 and β2->6. Oligosaccharides may contain both alpha- and beta-glycosidic linkages or may contain beta-glycosidic linkages only. The oligosaccharides used in the present invention can be defined according to general formula 1:

The oligosaccharide consists of the following monosaccharides linked by alpha or beta glycosidic linkages, wherein B is N-acetylglucosamine and wherein A, V, W, X, Y and/or Z are absent, or galactose , glucose, fucose, mannose, xylose, glucuronic acid, galacturonic acid, iduronic acid, N-acetylneuraminic acid, N-glycolylneuraminic acid, glucosamine, N- Acetylgalactosamine, N-acetylmannosamine, N-acetylglucosamine and/or oligosaccharide structures defined by general formula 2:

wherein B is N-acetylglucosamine, and wherein A, V, W, X, Y, and/or Z are absent, or galactose, glucose, fucose, mannose, xylose, glucuronic acid, semi- Lacturonic acid, iduronic acid, N-acetylneuraminic acid, N-glycolylneuraminic acid, glucosamine, N-acetylgalactosamine, N-acetylmannosamine or N-acetyl Glucosamine; and wherein, in Formula 1 and Formula 2, m is 3 and optionally v is 4, or m is 4 and optionally v is 3, and wherein p is 3 and/or 4, and where w is 6, and where if n > 1 and p is 3, x is 3 and X is not a monosaccharide, and where if n > 1 and p is 4, y is 4 and Y is not a monosaccharide, and where z is 6 , and where n ranges from 1 to 10.

"mammalian milk oligosaccharide" (MMO) as used herein refers to oligosaccharides such as but not limited to lacto-N-triose II, 3-fucose 3-fucosyllactose, 2'-fucosyllactose, 6-fucosyllactose, 2',3-difucosyllactose, 2',2-difucosyllactose, 3 ,4-disialyllactose, 6'-sialyllactose, 3'-sialyllactose, 3,6-disialylactose, 6,6'-disialylactose, 8 ,3-disialyllactoose, 3,6-disialyllacto-N-tetraose (3,6-disialyllacto-N-tetraose), lactodifucotetraose, lactodifucotetraose (lactodifucotetraose) lacto-N-tetraose), lacto-N-neotetraose (lacto-N-neotetraose), lacto-N-fucopentaose II (lacto-N-fucopentaose II), lacto-N-fucopentaose I, Lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentose VI, sialyllacto-N-tetraose c (sialyllacto-N-tetraose c), sialic acid Lacto-N-tetrasaccharide b, sialic acid lacto-N-tetrasaccharide a, lacto-N-difucohexaose I (lacto-N-difucohexaose I), lacto-N-difucohexaose II, lacto-N-difucohexaose I N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, monofucosyl monosialolacto-N-tetra Sugar c (monofucosylmonosialyllacto-N-tetraose c), monofucosyl para-lacto-N-hexaose (monofucosyl para-lacto-N-hexaose), monofucosyl lacto-N-hexaose III, isomerite fucosylated lacto-N-hexaose III (isomeric fucosylated lacto-N-hexaose III), isofucosylated lacto-N-hexaose I, sialo-N-hexaose, sialo- N-Neohexaose II, Difucose-p-lacto-N-hexaose, Difucoselacto-N-hexaose, Difucoselacto-N-hexaose a, Difucoselacto-N - hexasaccharide c, fucosylated chitosan, fucosylated oligosaccharides, neutral oligosaccharides and/or sialylated oligosaccharides. Mammalian milk oligosaccharides (MMOs) include oligosaccharides present in milk at any stage of the lactation period, including human (ie, human milk oligosaccharides or HMOs) and colostrum of mammals including, but not limited to, bovine ( Bos Taurus ), sheep ( Ovis aries ), goats ( Capra aegagrus hircus ), bactrian camels ( Camelus bactrianus ), horses ( Equus ferus caballus ), pigs ( Sus scropha ), dogs ( Canis lupus familiaris ), Japanese brown bears ( Ursus arctos ) yesoensis ), polar bear ( Ursus maritimus ), Japanese black bear ( Ursus thibetanus japonicus ), striped skunk ( Mephitis mephitis ), hooded seal ( Cystophora cristata ), Asian elephant ( Elephas maximus ), African elephant ( Loxodonta africana ), giant anteater ( Myrmecophaga tridactyla ), bottlenose dolphin ( Tursiops truncates ), northern minke whale ( Balaenoptera acutorostrata ), eugene kangaroo ( Macropus eugenii ), red kangaroo ( Macropus rufus ), brush-tailed possum ( Trichosurus Vulpecula ), koala ( Phascolarctos cinereus ), eastern quoll ( Dasyurus viverrinus ), platypus ( Ornithorhynchus anatinus ). Human milk oligosaccharides (HMOs), also known as human identical milk oligosaccharides, are chemically identical to human milk oligosaccharides in human milk, but are produced by biotechnology (e.g., using cell-free oligosaccharides). systems or cells and organisms including bacterial, fungal, yeast, plant, animal or protozoan cells, preferably genetically engineered cells and organisms). Doujin milk oligosaccharides are sold on the market under the name HiMO.

The term "purified" refers to material that is substantially or substantially free of interfering biomolecular active ingredients. With respect to cells, carbohydrates, nucleic acids and polypeptides, the term "purified" refers to a material that is substantially or substantially free of components that normally accompany the material as it exists in its native state. Typically, purified carbohydrates, oligosaccharides, proteins or nucleic acids of the invention are at least about 50%, 55%, 60%, 65% measured by band intensity on silver stained gels or other methods of determining purity , 70%, 75%, 80% or 85% pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure. Purity or homogeneity can be indicated by a number of methods known in the art, such as polyacrylamide gel electrophoresis of protein or nucleic acid samples, followed by visualization upon staining. For some purposes high resolution is required and HPLC or similar purification methods are used. 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 spectrometry.

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 identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence using sequence comparison algorithms or visual measurements. 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 entered into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence, based on the specified program parameters. Percent identity can be calculated collectively from the full-length sequence of the reference sequence, resulting in an overall percent identity score. Alternatively, percent identity can be calculated over a partial sequence of a reference sequence, resulting in a partial percent identity score. The full length of the reference sequence is used in the local sequence alignment, resulting in an overall percent identity score between the test sequence and the reference sequence. Percent identity is determined using different algorithms, such as 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), Clustal Omega method (Sievers et al., 2011, Mol. Syst. Biol. 7:539), MatGAT method (Campanella et al., 2003, BMC Bioinformatics, 4:29) or EMBOSS Needle.

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

Clustal Omega (Clustal W) is a multiple sequence alignment program that uses seed-guided trees and HMM profile-profile techniques to generate alignments between three or more sequences. It produces multiple sequence alignments of biologically meaningful divergent sequences. The web interface of Clustal W is available at https://www.ebi.ac.uk/Tools/msa/clustalo/. The preset parameters for performing multiple sequence alignments and calculating percent identity of protein sequences using the Clustal W method are: enable de-alignment of input sequences: FALSE; enable mbed-like clustering guide-tree: TRUE; enable mbed-like clustering iteration: TRUE; (combined bootstrap tree/HMM) number of iterations: preset (0); max bootstrap tree iteration: preset[-1] ; Maximum HMM Iteration: Preset[-1]; Order: Alignment.

The Matrix Global Alignment Tool (MatGAT) is a computer application that generates a similarity/identity matrix of DNA or protein sequences without pre-aligning the data. The program uses the Myers and Miller global alignment algorithm to perform a series of pairwise alignments, calculate similarity and identity, and then place the results in a distance matrix. The user can specify which type of alignment matrix (eg, BLOSUM50, BLOSUM62, and PAM250) to use for those protein sequence checks.

EMBOSS Needle (https://galaxy-iuc.github.io/emboss-5.0-docs/needle.html) uses the Needleman-Wunsch global permutation algorithm to find the difference between two sequences when considering the overall length of those sequences Optimal arrangement (including gaps). Through dynamic programming methods, all possible permutations are explored and the best one is selected to ensure the best permutation. The Needleman-Wunsch algorithm is a member of a class of algorithms that compute optimal scores and permutations in a sequence of mn steps, (where 'n' and 'm' are the lengths of the two sequences). The gap open penalty (default 10.0) is the point deducted when a gap is created. The default is to putatively use 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. It's a penalty for long vacancies.

As used herein, a polypeptide having an amino acid sequence that has at least 80% sequence identity to the full-length sequence of a reference polypeptide sequence is understood to mean 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% identical to the full-length amino acid sequence of the reference polypeptide sequence. In the present application, unless expressly specified, a polypeptide (or DNA) comprising/having at least 80% sequence identity to the full-length amino acid sequence (or nucleotide sequence) of a reference polypeptide (or nucleotide sequence) sequence), usually represented by SEQ ID NO or UniProt ID or Genbank NO., preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or 99%, more preferably at least 85%, still more preferably at least 90%, most preferably at least 95% identical to the full-length reference sequence.

For purposes of the present invention, percent identity was determined using MatGAT2.01 (Campanella et al. , 2003, BMC Bioinformatics 4:29). The following preset parameters for proteins were used: (1) Gap cost Existence: 12 and extension: 2; (2) The matrix used was BLOSUM65. In preferred embodiments, sequence identity is calculated from the full-length sequence of a given SEQ ID NO, ie, the reference sequence, or a portion thereof. A portion of which preferably refers to at least 50%, 60%, 70%, 80%, 90% or 95% of the complete reference sequence.

The term "culture" refers to a culture medium in which the cells are cultured or fermented, the cells themselves, and disaccharides and/or oligosaccharides with GlcNAc at their reducing ends are produced by the cells of the invention in culture broth, i.e. , the inside (intracellular) as well as the outside (extracellular) of the cell.

As used herein, the term "membrane transporter" refers to a protein that is part of or interacts with a cell membrane and controls the flow of molecules and information across a cell. Therefore, membrane proteins are involved in transport, either as input or output to the cell.

Such membrane transporters may be Porters, P-P-bond-hydrolysis-driven transporters, β-barrel as defined in the Transporter Classification Database. Porins (β-Barrel Porins), auxiliary transport proteins (auxiliary transport proteins), putative transport proteins (putative transport proteins) and phosphotransfer-driven group translocators (phosphotransfer-driven group translocators), the database by the Saier laboratory Operated and curated by the Saier Lab Bioinformatics Group, and available via www.tcdb.org, and provides a functional and systematic classification of membrane transporters. This transport taxonomy repository details the IUBMB-approved comprehensive classification system for membrane transporters, known as the Transport Taxonomy (TC) system. The TCDB taxonomic search described in this article is based on the definition of TCDB.Org published on June 17, 2019.

Transporter is a general term for uniporters, symporters and antiporters that utilize carrier-mediated processes (Saier et al. , Nucleic Acids Res. 44 (2016) D372-D379) . It is an electrochemical potential-driven transporter, also known as a secondary carrier-type facilitator. Carrier-mediated processes catalyze unidirectional transport when membrane transporters are transported in a single species by facilitating diffusion or in a membrane potential-dependent process (if the solute is charged); when two or more species are in a tightly coupled In the process of reverse transport, the use of carrier-mediated processes to catalyze reverse transport without coupling to direct forms of energy other than chemiosmotic energy; and/or when two or more species are in a tight Membrane transporters are included in this classification of secondary carriers when they are transported in the same direction in a coupled process that utilizes carrier-mediated processes to catalyze co-transport without coupling to direct forms of energy other than chemoosmotic energy (Forrest et al. , Biochim. Biophys. Acta 1807 (2011) 167-188). These systems are usually stereospecific. Solutes: Solute back transport is a characteristic property of secondary carriers. The dynamic association of transport proteins and enzymes creates functional membranes that transport the metabolic population of the channel matrix normally obtained from the extracellular compartment directly into its cellular metabolism (Moraes and Reithmeier, Biochim. Biophys. Acta 1818 (2012), 2687-2706). Solutes transported via this transport protein system include, but are not limited to, cations, organic anions, inorganic anions, nucleosides, amino acids, polyols, phosphorylated glycolytic intermediates, osmolytes, siderophores.

A membrane transporter is included in a PP-bond hydrolysis-drive if it hydrolyzes the diphosphate bond of an inorganic pyrophosphate, ATP or another nucleoside triphosphate to drive the active uptake and/or excretion of one or more solutes in the classification of transporters (Saier et al. , Nucleic Acids Res. 44 (2016) D372-D379). Membrane transporters may or may not be transiently phosphorylated, but substrates are not. Substrates transported via PP-bond hydrolysis-driven transporters include, but are not limited to, cations, heavy metals, beta-glucans, UDP-glucose, lipopolysaccharides, teichoic acid.

The β-barrel porin membrane transporter forms transmembrane pores, which normally allow solutes to pass through the membrane independently of energy. The transmembrane portion of these proteins consists entirely of β-chains that form a β-barrel (Saier et al. , Nucleic Acids Res. 44 (2016) D372-D379). These porin-type proteins are present in Gram-negative bacteria, mitochondria, plastids and possibly the outer membrane of acid-fast Gram-positive bacteria. Solutes transported via these β-barrel porins include, but are not limited to, nucleosides, raffinose, glucose, β-glucosides, oligosaccharides.

Cotransporters are defined as proteins that facilitate transport across one or more biological membranes, but are not themselves directly involved in transport. These membrane transporters always function in conjunction with one or more established transport systems such as but not limited to outer membrane factors (OMFs), polysaccharide (PST) transporters, ATP-binding cassette (ABC) type transport protein. It may provide functions related to the energy coupling of transport, play a structural role in the formation of complexes, exert biological or stability functions or regulatory functions (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379) . Examples of cotransporters 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 transporters include families that will be classified elsewhere when the transport function of a member is determined, or deleted from the autotransport classification system if the proposed transport function is overruled. These families include one or more members that are thought to have a transport function, but evidence for this function has not been convincing (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). Examples of putative transporters classified in this group under the TCDB system published on June 17, 2019 include, but are not limited to, copper transporters.

Phosphotransferase-driver group translocator also known as bacterial phosphoenolpyruvate (phosphoenolpyruvate): a PEP-dependent phosphotrans-driver group translocator of the sugar phosphotransferase system (PTS). The product of this reaction, derived from extracellular sugars, is cytoplasmic sugar-phosphate. The enzymatic components that catalyze the phosphorylation of sugars are superimposed on the transport process in a tightly coupled process. The PTS system is concerned with many different aspects, including regulation and chemotaxis, biofilm formation and pathogenic mechanisms (Lengeler, J. Mol. Microbiol. Biotechnol. 25 (2015) 79-93; Saier, J. Mol. Microbiol . Biotechnol. 25 (2015) 73-78). The membrane transporter family of phosphate transfer-driven group translocators classified in the TCDB system released on June 17, 2019 includes transporters related to glucose-glucoside, fructose-mannitol, lactose-N,N'-diacetyl Chitobiose-β-glucoside, sorbitol, galactitol, mannose-fructose-sorbose and ascorbic acid-related PTS systems.

The major facilitator superfamily (MFS) is a superfamily of membrane transporters that catalyze uniport proteins, solute:cation (H+, but rarely Na+) co-transport and/or solute:H+ or solute:solute reverse shipping. Most transporters are 400-600 amino acid residues in length, with 12, 14, or occasionally 24, as defined by the Transporter Taxonomy Database run by the Saier Laboratory Bioinformatics Group (www.tcdb.org). Transmembrane α-helical spanners (TMS).

"SET" or "Sugar Efflux Transporter" as used herein refers to membrane proteins of the SET family, proteins with InterPRO domain IPR004750 and/or proteins belonging to the eggNOGv4.5 family ENOG410XTE9. InterPro domains can be identified by using the online tool at https://www.ebi.ac.uk/interpro/ or by using the default InterProScan (https://www.ebi.ac.uk/interpro/download.html ) for the standalone version. Identification of the heterologous y family in eggNOGv4.5 can be performed using the online version or the standalone version of eggNOG-mapperv1 (http://eggnogdb.embl.de/#/app/home).

The term "Siderophore" as used herein refers to the secondary metabolites of various microorganisms, which are primarily iron ion-specific chelators. These molecules are classified as catecholate, hydroxamate, carboxylate and mixed types. Chelaterin is generally synthesized by the nonribosomal peptide synthetase (NRPS)-dependent pathway or the NRPS-independent pathway (NIS). In the NRPS-dependent chelatin biosynthesis pathway, the most important precursor is chorismate. 2,3-DHBA can be reacted in three steps by isochorismate synthase, isochorismatase and 2,3-dihydroxybenzoic acid-2,3-dehydrogenase (2,3-dihydroxybenzoic acid-2,3-dehydrogenase). 3-dihydroxybenzoate-2, 3-dehydrogenase) forms self-branched hydrochloric acid. Chelaterin can also be formed from salicylic acid, which is formed from isobranched salts by isochorismate pyruvate lyase. When ornithine is used as a precursor of chelatin, biosynthesis depends on the hydroxylation of ornithine catalyzed by L-ornithine N5-monooxygenase. In the NIS pathway, an important step in chelatin biosynthesis is N(6)-hydroxylysine synthase.

A transporter is required to export chelatin out of the cell. During this process, four superfamilies of membrane proteins have been identified: the major facilitator superfamily (MFS); the multidrug/oligo-lipo/polysaccharide flipper superfamily (Multidrug /Oligosaccharidyl-lipid/Polysaccharide Flippase Superfamily, MOP); resistance, nodulation and cell division superfamily (RND); and ABC superfamily. In general, genes involved in chelatin export are clustered with chelatin biosynthesis genes. As used herein, the term "siderophore exporter" refers to such a transporter that is required to export chelatin to the outside of the cell.

The ATP-binding cassette (ABC) superfamily contains intake and excretion transport systems, and these two groups of members are usually loosely clustered. ATP hydrolysis without the need for protein phosphorylation provides energy for transport. There are dozens of families within the ABC superfamily, and families are generally associated with substrate specificity. Members are classified according to class 3.A.1 as defined by the Transport Taxonomy Database run by the Saier Laboratory Bioinformatics Group, available at www.tcdb.org, and which provides a functional and systematic classification of membrane transporters .

The term "enabled efflux" refers to the introduction of transport activity of solutes on the cytoplasmic membrane and/or cell wall. Said transport can be permitted by introducing and/or increasing the expression of the transporters of the invention. The term "enhanced efflux" refers to improving the transport activity of solutes on the cytoplasmic membrane and/or cell wall. The transport of solutes across the cytoplasmic membrane and/or cell wall can be facilitated by introducing and/or increasing the expression of the membrane transporters of the present invention. "Expression" of a membrane transporter is defined as "overexpression" of the gene encoding the membrane transporter if it is an endogenous gene, or if the gene encoding the membrane transporter is a heterologous gene (in wild-type). type strains or cells).

As used herein, the term "precursor" refers to the cellular uptake used to specifically produce disaccharides and/or oligosaccharides (eg, disaccharides or oligosaccharides having an N-acetylglucosamine unit at the reducing end). Incorporated and/or synthesized substances. In this sense, a precursor may be a recipient as defined herein, but may also be another substance, a metabolite, which was first modified in the cell as a disaccharide and/or oligosaccharide (eg, with N at the reducing end - part of the biochemical synthesis pathway of acetylglucosamine units (disaccharides or oligosaccharides). Examples of such precursors include recipients as defined herein, and/or glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, dihydroxyacetone, glucosamine, mannose Amines, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, phosphorylated sugars such as but not limited to glucose-1-phosphate, galactose-1-phosphate Salt (galactose-1-phosphate), glucose-6-phosphate, fructose-1,6-diphosphate, mannose-6-phosphate, mannose-1-phosphate, glycerol-3-phosphate, glycerol Aldehyde-3-phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-acetyl-glucosamine-6-phosphate, N-acetylmannosamine-6-phosphate salts, N-acetylglucosamine-1-phosphate, N-acetylneuraminic acid-9-phosphate and/or nucleotide-active sugars as defined herein, such as UDP-glucose, UDP-galactose, UDP- N-acetylglucosamine, CMP-sialic acid, GDP-mannose, GDP-4-dehydro-6-deoxy-α-D-mannose, GDP-fucose.

The term "acceptor" as used herein refers to a monosaccharide, disaccharide or oligosaccharide that can be modified by a glycosyltransferase. Examples of such recipients include glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, lacto-N-bisaccharide (LNB), lacto-N-trisaccharide, lacto- N-tetrasaccharide (LNT), lacto-N-neotetraose (LNnT), N-acetyl-lactosamine (LacNAc), lacto-N-pentasaccharide (LNP), lacto-N-neopentose, paracetamol -N-pentose, p-lacto-N-neopentose, lacto-N-neopentose I, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNNH), p-lacto-N- Neohexaose (pLNNH), p-lacto-N-heptaose (pLNH), lacto-N-heptaose, lacto-N-neoheptaose, p-lacto-N-heptaose N-neoheptaose (para lacto-N-neoheptaose), para lacto-N-heptaose (para lacto-N-heptaose), lacto-N-octaose (LNO), lacto-N- Neooctose, isolacto-N-octose, p-lacto-N-octose, isolacto-N-neooctaose (iso lacto-N-neooctaose), novo lacto-N -neooctaose), para lacto-N-neooctaose, iso lacto-N-nonaose, novo lacto-N- nonaose), lacto-N-nonaose, lacto-N-decaose, iso lacto-N-decaose, neolacto- N-decaose (novo lacto-N-decaose), lacto-N-neodecaose (lacto-N-neodecaose), galactosyllactose (galactosyllactose), in 1, 2, 3, 4, 5 or more N-acetyllactosamine units and/or lactose extended with 1, 2, 3, 4, 5 or more lacto-N-disaccharide units, and comprising 1 or more N-acetyllactosamine units and /or oligosaccharides of one or more lactose-N-disaccharide units or intermediates thereof in oligosaccharide, fucosylated and sialylated forms.

Detailed Description of the Invention

According to a first aspect, the present invention provides a method for producing a disaccharide or oligosaccharide having an N-acetylglucosamine (GlcNAc) unit at the reducing end by a cell, preferably a single cell. The method includes the following steps: - Provide cells capable of synthesizing nucleotide-sugar and monosaccharide GlcNAc and saccharifying the GlcNAc monosaccharide, - culturing the cell under conditions that allow the production of the disaccharide or oligosaccharide, - Preferably, the disaccharide or oligosaccharide is isolated from the culture.

In the context of the present invention, the words "conditions permissive for producing said di- or oligosaccharide" are to be understood as conditions related to physical or chemical parameters, including but not limited to temperature , pH, pressure, osmotic pressure and product/precursor/acceptor concentrations.

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

In this application, the characteristic "disaccharide or oligosaccharide" is preferably replaced by "oligosaccharide", and the characteristic "disaccharide and/or oligosaccharide" is preferably replaced by "oligosaccharide". According to the present invention, cells are capable of synthesizing GlcNAc and this GlcNAc monosaccharide is further modified by saccharification, which is carried out in the same cell, to synthesize disaccharides or oligosaccharides with GlcNAc at the reducing end. Thereby, the cell expresses a glycosyltransferase to glycosylate the synthetic N-acetylglucosamine to form the disaccharide or oligosaccharide of the present invention with GlcNAc at the reducing end.

Accordingly, the present invention provides a method for the production of disaccharides or oligosaccharides having N-acetylglucosamine (GlcNAc) units at the reducing end by cells, preferably single cells. The method includes the following steps: - provide cells capable of synthesizing nucleotide-sugar and monosaccharide GlcNAc and capable of expressing glycosyltransferases to glycosylate the GlcNAc monosaccharide to form the disaccharide or oligosaccharide, - culturing the cell under conditions that allow the production of the disaccharide or oligosaccharide, - culturing the cell under conditions that allow the production of the disaccharide or oligosaccharide, - Preferably, the disaccharide or oligosaccharide is isolated from the culture.

Glycosyltransferases are enzymes that catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules to form glycosidic bonds. The glycosyltransferases as used herein may be selected from a list comprising but not limited to the following: alpha-1,2-fucosyltransferases, alpha-1,3/1 ,4-fucosyltransferase, alpha-1,6-fucosyltransferase, alpha-2,3-sialyltransferases (alpha-2,3-sialyltransferases), alpha-2,6-sialyltransferase Acid transferase, α-2,8-sialyltransferase, β-1,3-galactosyltransferase, β-1,4-galactosyltransferase, α-1,3-galactosyltransferase , α-1,4-galactosyltransferase, N-acetylglucosamine transferase, N-acetylgalactosamine transferase, glucosyltransferases (glucosyltransferases), mannosyltransferase, N -Acetylmannosyltransferase, xylosyltransferase, glucuronyltransferases, galacturonyltransferases, glucosamine transferases, N-glycolylneuraminyltransferases .

Cells need to produce GlcNAc intracellularly in order to be able to further glycation the synthetic GlcNAc. Intracellular production of GlcNAc needs to be understood as synthesis of GlcNAc within the cell or within the cytoplasm and not within the organelle or organelle membrane or the periplasm of the cell or the cell membrane or cell wall.

In preferred embodiments, the cells described herein express at least one N-acetylglucosamine-6-phosphotransferase and phosphatase to synthesize N-acetylglucosamine. In this preferred embodiment, the N-acetylglucosamine-6-phosphotransferase is an enzyme capable of converting glucosamine-6-phosphate to N-acetylglucosamine-6-phosphate in cells, And this phosphatase is capable of dephosphorylating N-acetylglucosamine-6-phosphate in cells to produce N-acetylglucosamine. In a more preferred embodiment, the phosphatase is an HAD-like phosphatase. Phosphatases from the HAD superfamily and HAD-like family are described in the art. Examples of these families can be found in the genes yqaB , inhX , yniC , ybiV , yidA , ybjI , yigL or cof from E. coli , or include, for example, aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, E. coli genes of any one or more of YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU; or from Pseudomonas putida PsMupP from Pseudomonas putida , ScDOG1 from S. cerevisiae , or enzymes expressed by the BsAraL gene from Bacillus subtilis (as described in WO18122225). A phosphatase catalyzing this reaction was identified in Blastocladiella emersonii . Phosphatases are generally not specific and activities are usually family or structure related. Thus, other examples can be found in all phosphatase families. Specific phosphatases are readily identified and screened by known methods as described by Fahs et al. (ACS Chem. Biol. 11(11), 2944-2961 (2016)).

In the context of the present invention, it is understood that the disaccharide or oligosaccharide according to the present invention having a GlcNAc unit at the reducing end is synthesized intracellularly. Those of ordinary skill in the art will further appreciate that a fraction or substantially all of the synthesized disaccharides or oligosaccharides having a GlcNAc unit at the reducing end remain intracellularly and/or are excreted via passive or active transport.

In preferred embodiments, the cell is capable of expressing at least one glycosyltransferase to saccharify the GlcNAc monosaccharide to form the disaccharide or oligosaccharide. In another preferred embodiment, the cell is capable of expressing at least two, more preferably at least three, still more preferably at least four, still more preferably at least five, and most preferably at least six glycosyltransferases, The disaccharide or oligosaccharide according to the invention is formed by saccharifying the GlcNAc monosaccharide.

In the context of the present invention, the nucleotide-sugar is preferably the supplier for the glycosyltransferase. Preferably, the cell is capable of synthesizing at least two, more preferably at least three, still more preferably at least four, most preferably at least five nucleotide-sugars.

In a preferred embodiment, the disaccharide or oligosaccharide is lacto-N-bisaccharide (LNB) or N-acetyllactosamine (LacNAc), preferably an oligosaccharide containing LNB or LacNAc at the reducing end, more preferably sialylated and/or fucosylated and/or galactosylated and/or GlcNAc-modified forms of LNB or LacNAc, or, more preferably, sialylated and/or oligosaccharides containing LNB or LacNAc at the reducing end or fucosylated and/or galactosylated and/or GlcNAc-modified forms. In another preferred embodiment, the disaccharide or oligosaccharide is a neutral disaccharide or oligosaccharide with N-acetylglucosamine unit at the reducing end, preferably a neutral oligosaccharide containing LNB or LacNAc at the reducing end sugar. Preferably, the neutral oligosaccharide is fucosylated. Alternatively, it is preferred that the neutral oligosaccharide is not fucosylated.

In another preferred embodiment, the present invention provides a method for producing a mixture of disaccharides and/or oligosaccharides having N-acetylglucosamine (GlcNAc) units at the reducing end by a cell, preferably a single cell. The method includes the following steps: - providing cells capable of synthesizing nucleotide-sugar and monosaccharide GlcNAc and capable of expressing a glycosyltransferase to glycosylate the GlcNAc monosaccharide to form the disaccharide and/or oligosaccharide mixture with GlcNAc at its reducing end, - culturing the cell under conditions allowing the production of a mixture of disaccharides and/or oligosaccharides having GlcNAc at the reducing end, - Preferably, the mixture is isolated from the culture.

According to the present invention, the mixture comprises or consists of at least two different disaccharides and/or oligosaccharides having GlcNAc at their reducing ends, preferably at least three different disaccharides and/or oligosaccharides having GlcNAc at their reducing ends or oligosaccharides, more preferably including or consisting of at least four different disaccharides and/or oligosaccharides having GlcNAc at their reducing ends. Preferably, the mixture comprises or consists of neutral disaccharides and/or oligosaccharides. More preferably, the mixture comprises or consists of charged and/or neutral disaccharides and/or oligosaccharides. In a preferred embodiment of the method and/or cell, the charged disaccharides and/or oligosaccharides are sialylated disaccharides and/or oligosaccharides. In preferred embodiments of the method and/or cell, the neutral disaccharides and/or oligosaccharides are fucosylated. In another preferred embodiment of the method and/or cell, the neutral disaccharides and/or oligosaccharides are not fucosylated.

Preferred within the scope of the present invention is a method by a cell, preferably a single cell, to produce a mixture comprising (i) a N-acetylglucosamine (GlcNAc) unit as described herein at the reducing end Disaccharides and/or oligosaccharides, and (ii) one or more lactose-based mammalian milk oligosaccharides (MMOs), preferably one or more lactose-based human milk oligosaccharides (HMOs). The method includes the following steps: - providing cells that are (i) capable of synthesizing nucleotide-sugar and monosaccharide GlcNAc and capable of expressing a glycosyltransferase to saccharify the GlcNAc monosaccharide to produce the disaccharide and/or oligosaccharide, and (ii) wherein the The cell is further capable of expressing one or more glycosyltransferases to saccharify lactose to produce the one or more lactose-based mammalian milk oligosaccharides and wherein the cell is capable of synthesizing one or more nucleotide-sugars for the glycosyl a supplier of transferase, wherein the lactose is produced by cells (preferably intracellularly) or added before and/or after culturing, - culturing the cell under conditions allowing the production of a mixture comprising (i) disaccharides and/or oligosaccharides and (ii) one or more lactose-based mammalian milk oligosaccharides, - Preferably, the mixture is isolated from the culture.

It will be understood by those of ordinary skill in the art that one or more glycosyltransferases involved in the production of disaccharides and/or oligosaccharides with GlcNAc at the reducing end can saccharify lactose to form one or more lactose-based mammalian The glycosyltransferases of lacto-oligosaccharides are the same. Alternatively, the glycosyltransferases involved in the production of the disaccharides and/or oligosaccharides with GlcNAc at the reducing end are different from the glycosyltransferases involved in the production of one or more lactose-based mammalian milk oligosaccharides.

It will also be understood by those of ordinary skill in the art that the one or more nucleotide-sugars involved in the production of disaccharides and/or oligosaccharides with GlcNAc at the reducing end may be associated with the production of one or more lactose-based mammals. The nucleotide-sugar of lacto-oligosaccharide is the same. Alternatively, the one or more nucleotide-sugars involved in the production of the disaccharide and/or oligosaccharide with GlcNAc at the reducing end may be different from the nucleotide-sugars involved in the production of one or more lactose-based mammalian milk oligosaccharides.

Each embodiment disclosed in the content of methods and/or cells for producing disaccharides or oligosaccharides having GlcNAc units at the reducing end and producing mixtures of disaccharides and/or oligosaccharides having GlcNAc units at the reducing end is also disclosed in the production of In the context of the method of a mixture comprising (i) a disaccharide and/or oligosaccharide having a GlcNAc unit at the reducing end, and (ii) one or more lactose-based mammalian milk oligosaccharides, preferably one or more Lactose-based human milk oligosaccharides.

Furthermore, each of the embodiments disclosed herein in terms of methods and/or cells for producing disaccharides or oligosaccharides having GlcNAc units at the reducing end or mixtures comprising disaccharides and/or oligosaccharides having GlcNAc units at the reducing end Also disclosed are mammalian milk oligosaccharides for the production of one or more lactose-based. For example, the amounts and identities of glycosyltransferases and nucleotide-sugars disclosed to produce disaccharides or oligosaccharides with GlcNAc units at the reducing end can also be applied to produce one or more lactose-based mammalian milk oligosaccharides Content. The same applies to other aspects of the invention, such as the use of the cell to produce a disaccharide or oligosaccharide having a GlcNAc unit at the reducing end.

Another embodiment provides a method for producing a disaccharide or oligosaccharide having a GlcNAc unit at the reducing end by genetically modifying a cell, preferably a single genetically modified cell, the method comprising the following steps: - Provide genetically modified cells capable of synthesizing nucleotide-sugar and monosaccharide GlcNAc and saccharifying the GlcNAc monosaccharide, - culturing the cell under conditions that allow the production of the disaccharide or oligosaccharide, - Preferably, the disaccharide or oligosaccharide is isolated from the culture.

In this application, unless expressly stated otherwise, "genetically modified cell" or "metabolically engineered cell" preferably refers to a genetically modified or metabolically engineered cell, respectively, which is used to produce A disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end according to the present invention.

According to a second aspect, there is provided a metabolically engineered cell capable of (i) synthesizing nucleotide-sugars, (ii) synthesizing N-acetylglucosamine, and (iii) saccharifying the N-acetylglucosamine monosaccharide, wherein the cell produces disaccharides or oligosaccharides (or mixtures as disclosed herein) having N-acetylglucosamine units at the reducing end, i.e., the cell is metabolically engineered for production of N-acetylglucosamine at the reducing end Disaccharides or oligosaccharides of amine units (or mixtures as disclosed herein). Disaccharides or oligosaccharides (or mixtures as disclosed herein) with N-acetylglucosamine units at the reducing end preferably do not occur in the wild type of the metabolically engineered cells.

Accordingly, the present invention provides a cell metabolically engineered for the production of disaccharides or oligosaccharides having N-acetylglucosamine units at the reducing end, wherein the cell is capable of (i) synthesizing nucleotide-sugars, (ii) synthesizing N-acetylglucosamine, and (iii) express a glycosyltransferase to saccharify the GlcNAc monosaccharide to form the disaccharide or oligosaccharide. Further specifying any characteristic of the second aspect (eg, the amount and identity of the nucleotide-sugar and glycosyltransferase; the amount and identity of the disaccharide or oligosaccharide comprising mixtures thereof; etc.) Embodiments of the first aspect of the invention are considered to be also disclosed in the context of the second aspect.

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

In the methods and cells of the present invention, the cells are preferably genetically modified for the expression or activity of an enzyme selected from the group consisting of N-acetylglucosamine-6-phosphotransferase, phosphatase, glycosyltransferase , L-glutamine-D-fructose-6-phosphate transaminase or UDP-glucose 4-epimerase. According to the present invention, including N-acetylglucosamine-6-phosphotransferase, phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphotransferase or UDP-glucose 4-epiiso The above-listed enzymes of the constitutive enzymes are modified expression or activity of endogenous proteins, preferably the endogenous proteins are overexpressed; or the enzymes of the above group are heterologous proteins, which can be heterologously expressed by the cell . The heterologously expressed protein is then introduced and expressed, preferably overexpressed. In another embodiment, the endogenous protein can have a modified expression in a cell that also expresses the heterologous protein. Heterologous expression can be derived from the genome of the host or from the vector introduced into the cell as described herein.

The above including N-acetylglucosamine-6-phosphotransferase, phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphotransferase or UDP-glucose 4-epimerase The enumerated enzymes can be produced via expression of polynucleotides produced by recombinant DNA technology using techniques known in the art. Methods known to those of ordinary skill in the art can be used to construct expression vectors containing the coding sequences for the polypeptides of the invention and appropriate transcriptional and/or translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. For example, see 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) techniques described in .

According to another aspect of the present invention, a vector is provided to a cell, the vector comprising an N-acetylglucosamine-6-phosphotransferase, phosphatase, glycosyltransferase, L-glutamine- A polynucleotide of the enzymes listed above for D-fructose-6-phosphate transaminase or UDP-glucose 4-epimerase, wherein the polynucleotide is operable with control sequences recognized by cells transformed with the vector ground connection. In a particularly preferred embodiment, the vector is an expression vector, and according to another aspect of the present invention, the vector can exist in the form of plastids, cosmids, bacteriophages, liposomes or viruses. Thus, a polynucleotide encoding a polypeptide of the present invention may, for example, be contained in a vector that is stably transformed/transfected into a cell. In this vector, the coding sequence for the polypeptides described herein is under the control of a promoter. The promoter can be, for example, an inducible promoter, so that the expression of the gene/polynucleotide can be specifically targeted, in such a way that the gene can be overexpressed if desired. The promoter can also be a persistent promoter. A number of expression systems can be used to produce the polypeptides of the invention. Such vectors include, in particular, chromosomal, episomal and virus-derived vectors, eg, derived from bacterial plastids, phages, transposons, yeast exosomes, insertion elements, yeast chromosomal elements, viruses and vectors derived from combinations thereof, such as those derived from plastids and phage genetic elements, such as cohesoplasts and phagemids. These vectors may contain selectable markers such as, but not limited to, antibiotic markers, helper markers, toxin-antitoxin markers, RNA sense/antisense markers. Expression system constructs may contain control regions that regulate as well as induce expression. In general, any system or vector suitable for maintaining, propagating or expressing polynucleotides and/or expressing polypeptides in a host can be used for performance in this regard. Appropriate DNA sequences can be inserted into the expression system by any of a variety of known and conventional techniques, such as those proposed by Sambrook et al., supra, and the like. For recombinant production, cells can be genetically engineered to incorporate the expression system of the invention, or portions or polynucleotides thereof. Introduction of polynucleotides into cells can be carried out by a number of methods described in standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986) and Sambrook et al., 1989, supra.

According to a further aspect of the invention encoding N-acetylglucosamine-6-phosphotransferase, phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphotransferase or UDP-glucose 4-Epimerase polynucleotides are suitable for codon usage in each cell or expression system.

In another embodiment, the cells used herein comprise N-acetylglucosamine b-1,3-galactosyltransferase or N-acetylglucosamine b-1,4-galactosyltransferase. N-acetylglucosamine b-1,3-galactosyltransferase and N-acetylglucosamine b-1,4-galactosyltransferase are capable of converting galactose units from UDP-galactose donors to Transfer to glycosyltransferases at the GlcNAc acceptor in a β-1,3 and β-1,4-dependent glycosidic linkage, respectively.

In another preferred embodiment, the cells used herein, whether genetically engineered or not, are capable of producing a nucleotide-sugar selected from the group consisting of: UDP-galactose (UDP-Gal), UDP-N-acetylene Glucosamine (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-ethyl Acetamino-2,6-dideoxy--L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-ethyl Acetamino-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-acetamido-L-pneumolysamine (UDP-L-PneNAC or UDP-2-acetamido-2, 6-Dideoxy-L-talose), UDP-N-Acetylmuramic acid, UDP-N-Acetyl-L-Quinosamine (UDP-L-QuiNAc or UDP-2-Acetyl Amino-2,6-dideoxy-L-glucose), GDP-L-isorhamnose, CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N ₃ , CMP-Neu4,5Ac ₂ , CMP-Neu5,7Ac ₂ , CMP-Neu5,9Ac ₂ , CMP-Neu5,7(8,9)Ac ₂ , UDP - Glucuronate, UDP-galacturonate, GDP-rhamnose or UDP-xylose. In further embodiments, cells are genetically engineered for the production of the nucleotide-sugar. In another preferred embodiment, cells are genetically engineered for optimized production of the nucleotide-sugar.

In another embodiment, the cell is capable of producing UDP-galactose. In an optimized embodiment, the cells are optimized for UDP-galactose production. In an optimized embodiment, the cells are modified in the expression or activity of the UDP-glucose 4-epimerase GalE, which is capable of converting UDP-glucose to UDP-galactose.

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

In a preferred embodiment, the disaccharide produced in the cell is lacto-N-disaccharide (Gal-b1,3-GlcNAc) or N-acetyllactosamine (Gal-b1,4-GlcNAc), which in non-reducing The ends contain galactose units linked by β-1,3- or β-1,4-dependent glycosidic linkages, respectively, to the GlcNAc moiety present at the reducing end of the disaccharide. In another preferred embodiment, the oligosaccharide produced in the cell contains lacto-N-disaccharide (Gal-b1,3-GlcNAc) or N-acetyllactosamine (Gal-b1,4-GlcNAc) at the reducing end .

In another preferred embodiment, the cell-synthesized disaccharides or oligosaccharides (or mixtures described herein) according to the present invention do not include chitobiose at the reducing end (ie, GlcNAc-b1,4-GlcNAc) , more preferably excluding N-glycans. In other words, cells are genetically engineered for the production of disaccharides or oligosaccharides (or mixtures described herein) having N-acetylglucosamine (GlcNAc) units at the reducing end, wherein the disaccharides or oligosaccharides are The reducing end does not include chitobiose, more preferably does not include N-glycans.

In another preferred embodiment, the N-acetylglucosamine b-1,3-galactosyltransferase 1) expressed in the cell has the PFAM domain PF00535 and (i) comprises the sequence of SEQ ID NO 01 [AGPS ]XXLN( _Xn )RXDXD, wherein X is any amino acid, wherein n is 12 to 17, or (ii) the sequence comprising SEQ ID NO 02 PXXLN( _Xn )RXDXD( _Xm )[FWY]XX[HKR ]XX[NQST], wherein X is any amino acid, wherein n is 12 to 17 and m is 100 to 115, or (iii) includes according to SEQ ID NO: 03, 04, 05, 06, 07 or 08 Any, preferably any of SEQ ID NOs 03, 04, 05, 06 or 07, more preferably any of SEQ ID NOs 03, 06 or 07, most preferably any of SEQ ID NOs 03 or 06 Any of the polypeptide sequences, or (iv) is any of SEQ ID NOs: 03, 04, 05, 06, 07 or 08, preferably any of SEQ ID NOs 03, 04, 05, 06 or 07 , more preferably any one of SEQ ID NO 03, 06 or 07, most preferably a functional homologue, variant or derivative of any one of SEQ ID NO 03 or 06, which has the same characteristics as SEQ ID NO: Any of 03, 04, 05, 06, 07 or 08, preferably any of SEQ ID NOs 03, 04, 05, 06 or 07, more preferably any of SEQ ID NOs 03, 06 or 07 One, preferably at least 80% overall sequence identity of the full length of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide of any of SEQ ID NO 03 or 06 and having N-acetyl Glucosamine b-1,3-galactosyltransferase activity, or (v) comprises any one from SEQ ID NOs: 03, 04, 05, 06, 07 or 08, preferably SEQ ID NOs 03, 04 , any one of SEQ ID NO 03, 06 or 07, more preferably any one of SEQ ID NO 03, 06 or 07, most preferably at least 8, 9, 10, 11, Oligopeptide sequences of 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b-1,3-galactosyltransferase activity, Or (vi) is any of SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably any of SEQ ID NO 03, 04, 05, 06 or 07, more preferably SEQ ID Any of NO 03, 06 or 07, preferably a functional fragment of any of SEQ ID NO 03 or 06, and having N-acetylglucosamine b-1,3-galactose Glycosyltransferase activity, or (vii) comprises a polypeptide comprising or consisting of any of SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably SEQ ID NO 03, 04, Any of 05, 06 or 07, more preferably any of SEQ ID NOs 03, 06 or 07, and most preferably at least 80% of the full-length amino acid sequence of any of SEQ ID NOs 03 or 06 consisting of an amino acid sequence of identity and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or 2) having a PFAM domain IPR002659, and (i) comprising the sequence of SEQ ID NO 09 KT(X _n )[FY]XXKXDXD(X _m )[FHY]XXG(X, no A, G, S)(X _p )X (no F, H, W, Y)[DE]D[ILV]XX [AG], wherein X is any amino acid, wherein n is 13 to 16, m is 35 to 70, and p is 20 to 45, or (ii) includes according to SEQ ID NO: 10, 11, 12 or 13 A polypeptide sequence of any of, or (iii) a functional homologue, variant or derivative of any of SEQ ID NOs: 10, 11, 12, or 13 having the same properties as SEQ ID NOs: 10, 11 , 12 or 13 of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptides of at least 80% overall sequence identity of any full length and having N-acetylglucosamine b-1,3 - Galactosyltransferase activity, or (iv) comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 from any of SEQ ID NOs: 10, 11, 12, or 13 , an oligopeptide sequence of 18, 19, 20 consecutive amino acid residues and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or (v) is SEQ ID NO: 10, A functional fragment of any one of 11, 12 or 13, and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or (vi) comprising a polypeptide comprising or consisting of a NO: Consists of amino acid sequences of at least 80% sequence identity to the full-length amino acid sequence of any of 10, 11, 12, or 13, and having N-acetylglucosamine b-1,3-galactose Syltransferase activity.

[ ILV][FWY]( _Xn )EDD( _Xm )[ACGST]XXYX[ILMV], wherein X is any amino acid, wherein n is 13 to 15 and m is 50 to 76, or (ii) including according to SEQ Any of ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably any of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably is the polypeptide sequence of any of SEQ ID NO: 17, 18, 20 or 21, or (iii) is any of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23 , preferably any of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably a functional homologue, variant of any of SEQ ID NO: 17, 18, 20 or 21 A body or derivative with any of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NO: 15, 16, 17, 18, 20 or any of 21, more preferably at least 80% of the full length of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide of any of SEQ ID NO: 17, 18, 20 or 21 Overall sequence identity and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (vi) including from SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or any of 23, preferably any of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably at least any of SEQ ID NO: 17, 18, 20 or 21 Oligopeptide sequences of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues, and N-acetylglucosamine b-1,4- Galactosyltransferase activity, or (v) is any of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NOs: 15, 16, 17 , any of 18, 20 or 21, more preferably a functional fragment of any of SEQ ID NO: 17, 18, 20 or 21, and having N-acetylglucosamine b-1,4-galactose Syltransferase activity, or (vi) comprising a polypeptide comprising or consisting of any of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NO: 15, 16, 17, 18, 20 or any of 21, more preferably an amino acid sequence of at least 80% sequence identity to the full-length amino acid sequence of any of SEQ ID NO: 17, 18, 20 or 21, and has N- Acetylglucosamine b-1,4-galactosyltransferase activity, or 2) has a PFAM domain PF00535, and (i) comprises the sequence of SEQ ID NO 24 R[KN]XXXXXXXXGXXXX[FL]XDXD( _Xn )[ FHW]XXX[FHNY](X _m )E[DE], wherein X is any amino acid, wherein n is 50 to 75, m is 10 to 30, or (ii) the sequence R[KN comprising SEQ ID NO 25 ]XXXXXXGXXXXFXDXD(X _n )[FHW]XXX[FHNY](X _m )E[DE](X _p )[FWY]XX[HKR]XX[NQST], where X is any amino acid, where 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 of SEQ ID NOs: 26, 27 or 28, or (iv) is SEQ ID NOs: 58, 59 or a functional homologue, variant or derivative of any of 60 having the N-acetylglucosamine b-1,4-galactosyltransferase of SEQ ID NO: 26, 27 or 28 Any one of the full-length polypeptides has at least 80% overall sequence identity and has N-acetylglucosamine b-1 ,4-galactosyltransferase activity, or (v) comprises a sequence from SEQ ID NO: 26, 27 or 28 An oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues of any of the Amine b-1,4-galactosyltransferase activity, or (vi) is a functional fragment of any of SEQ ID NOs: 26, 27, or 28, and has N-acetylglucosamine b-1,4- Galactosyltransferase activity, or (vii) comprising a polypeptide comprising or consisting of an amino group having at least 80% sequence identity to the full-length amino acid sequence of any of SEQ ID NOs: 26, 27 or 28 consisting of an acid sequence and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or 3) having a PFAM domain PF02709 and not having a PFAM domain PF00535, and (i) comprising SEQ ID NO 29 The sequence [FWY]XX[FY][FWY]( _X23 )[FWY][GQ]X[DE]D, where X is any amino acid, or (ii) the sequence [PV]W comprising SEQ ID NO 30 [GHNP]( _Xn )[FWY][GQ]X[DE]D, wherein X is any amino acid, wherein n is 21 to 24, or (iii) including according to SEQ ID NO: A polypeptide sequence of any of 31, 32, 33, 34, 35 or 36, or (iv) a functional homologue of any of SEQ ID NO: 31, 32, 33, 34, 35 or 36, A variant or derivative having a full-length sequence with any of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides of SEQ ID NO: 31, 32, 33, 34, 35 or 36 At least 80% overall sequence identity and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or (v) including from SEQ ID NO: 31, 32, 33, 34, 35 or 36 Any oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b-1,4-galactosyltransferase activity, or (vi) is a functional fragment of any one of SEQ ID NOs: 31, 32, 33, 34, 35, or 36, and has N-acetylglucosamine b -1,4-galactosyltransferase activity, or (vii) comprising a polypeptide comprising or consisting of a full-length amino acid with any of SEQ ID NO: 31, 32, 33, 34, 35 or 36 The sequence consists of amino acid sequences of at least 80% sequence identity and has N-acetylglucosamine b-1,4-galactosyltransferase activity, or 4) has the PFAM domain PF03808, and (i) includes Sequence of SEQ ID NO 37 [ST][FHY]XN( _Xn )DG(X16)[HKR]X[ST] _FDXX [ST]XA, wherein X is any amino acid, and wherein n is 20 to 25 , or (ii) the sequence comprising SEQ ID NO 38 [ST][FHY]XN( _Xn )DG(X16)[HKR]X[ST] _FDXX [ST]XA( _Xm )[HR]XG[FWY ](X _p )GXGXXXQ[DE], wherein X is any amino acid, wherein n is 20 to 25, m is 40 to 50, and p is 22 to 30, or (iii) includes according to SEQ ID NO:39, The polypeptide sequence of any one of 40 or 41, or (iv) a functional homologue, variant or derivative of any one of SEQ ID NO: 39, 40 or 41 having the same properties as SEQ ID NO: 39 , 40 or 41 of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides of either full-length at least 80% overall sequence identity and having N-acetylglucosamine b-1,4 - Galactosyltransferase activity, or (v) comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 from any of SEQ ID NOs: 39, 40 or 41 , 19, 20 oligopeptide sequences of consecutive amino acid residues with N-acetylglucosamine b-1,4- Galactosyltransferase activity, or (vi) is a functional fragment of any one of SEQ ID NOs: 39, 40 or 41 and has N-acetylglucosamine b-1,4-galactosyltransferase activity, or (vii) comprising a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any of SEQ ID NOs: 39, 40 or 41, and having N-acetylglucosamine b-1,4-galactosyltransferase activity.

The PFAM motifs used in this paper, PF00535, IPR002659, PF01755, PF02709, and PF03808 are protein domains annotated in the PFAM database Pfam version 33.1 released on June 11, 2020. PF00535 belongs to the glycosyltransferase 2 (GT2) family, which includes the conversion of enzymes from UDP-glucose, UDP-N-acetyl-galactosamine, GDP-mannose or CDP-abequose Enzymes that transfer sugars to a range of recipients, including cellulose, dolichol phosphate, and teichoic acid.

IPR002659 belongs to glycosyltransferase family 31 (GH31), which includes enzymes with a number of known activities, including N-acetyllactosamine beta-1,3-N-acetyllactosamine transferase beta-1,3-N-acetylglucosaminyltransferase) (2.4.1.149), beta-1,3-galactosyltransferase (2.4.1), fucose-specific beta-1,3-N-acetylglucosylglucose Glycosaminotransferase (2.4.1), globotriosylceramide β-1,3-GalNAc transferase (2.4.1.79). PF01755 belongs to the glycosyltransferase 25 (GT25) family. This is a family of glycosyltransferases involved in the biosynthesis of lipopolysaccharide (LPS). These enzymes catalyze the transfer of various sugars to growing LPS chains during biosynthesis. PF02709 refers to the Glyco_transf_7C family. This is the N-terminal domain of a family of galactosyltransferases from the broad metazoa (Metazoa), which have three related galactosyltransferase activities, in some cases all possessed by a single sequence : EC: 2.4.1.90, N-acetyllactosamine synthase, EC: 2.4.1.38, β-N-acetylglucosamine-glycopeptide β-1,4-galactosyltransferase, and EC: 2.4.1.22 Lactose synthase. PF03808 refers to the glycosyltransferase 26 (GT26) family, which includes enzymes such as β-N-acetyl mannosaminuronyltransferase (EC 2.4.1.-) , β-N-acetyl-mannosaminyltransferase (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.-) and other active enzymes.

Has the same PFAM domain and motif as assigned to each class of the N-acetylglucosamine b-1,3-galactosyltransferase or the N-acetylglucosamine b-1,4-galactosyltransferase The proteins can be retrieved via RegEx analysis.

RegEx, or Regular Expression, is a special sequence of characters that uses a special syntax in a pattern to help pair or search other strings or sets of strings. Many programs are available for RegEx searches. One of them is the Python module "re", which provides full support for regular expressions in Python. Detailed information, as well as information known to those of ordinary skill in the art, can be obtained from https://towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 published on April 6, 2019 . RegEx analysis for the proteins of the invention has been included in the Examples section herein.

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 to form glycosidic bonds. Glycosyltransferases using nucleotide diphospho-sugars, nucleotide monophospho-sugars, and sugar phosphates and related proteins have been described and classified into different sequence-based family (Campbell et al., Biochem. J. 326, 929-939 (1997)) and available from the CAZy (CArbohydrat-Active EnZymes) website (www.cazy.org).

In another preferred embodiment, the N-acetylglucosamine-6-phosphotransferase is encoded by a heterologous nucleic acid. In other words, the N-acetylglucosamine-6-phosphotransferase is expressed heterologously in the cell. In another preferred embodiment, the N-acetylglucosamine-6-phosphotransferase (i) comprises, preferably a polypeptide sequence from Saccharomyces cerevisiae with UniProt ID P43577, or (ii) is a UniProt A functional homologue, variant or derivative of the polypeptide of ID P43577 having at least 80% overall sequence identity to the full length of the polypeptide of UniProt ID P43577 and having N-acetylglucosamine-6-phosphotransferase activity, or (iii) is a functional fragment of a polypeptide having UniProt ID P43577 and has N-acetylglucosamine-6-phosphotransferase activity, or (iv) a polypeptide comprising or consisting of an amino acid sequence, the amino The acid sequence has at least 80% sequence identity to the full-length amino acid sequence of the polypeptide of UniProt ID P43577 and has N-acetylglucosamine-6-phosphotransferase activity. In another preferred embodiment, the L-glutamine-D-fructose-6-phosphate transaminase (i) comprises, preferably the polypeptide sequence of UniProt ID P17169 from Escherichia coli, or (ii) is Functional homologues, variants or derivatives of the polypeptide with UniProt ID P17169 having at least 80% overall sequence identity with the full length of the polypeptide of UniProt ID P17169 and having L-glutamine-D-fructose-6 - Phosphotransaminase activity, or (iii) is a functional fragment of a polypeptide having UniProt ID P17169 and has L-glutamine-D-fructose-6-phosphate transaminase activity, or (iv) includes or consists of amines Polypeptide composed of amino acid sequence, the amino acid sequence has at least 80% sequence identity with the full-length amino acid sequence of the polypeptide of UniProt ID P17169, and has L-glutamine-D-fructose-6-phosphate transamination enzymatic activity. In an alternative preferred embodiment, the L-glutamine-D-fructose-6-phosphate transaminase (i) comprises, preferably differs from the wild-type E. coli protein of UniProt ID P17169 by having A39T , R250C and G472S mutations, the polypeptide sequence as described by Deng et al. (Biochimie 88, 419-29 (2006)), or (ii) the mutant polypeptide (different from the wild-type E. coli protein of UniProt ID P17169 in that A functional homologue, variant or derivative having the A39T, R250C and G472S mutations) that has the same mutant polypeptide (which differs from the wild-type E. coli protein of UniProt ID P17169 by having the A39T, R250C and G472S mutations) At least 80% overall sequence identity to the full length and having L-glutamine-D-fructose-6-phosphate transaminase activity, or (iii) being the mutant polypeptide (different from the wild-type E. coli protein of UniProt ID P17169) in that it has a functional fragment of A39T, R250C and G472S mutations) and has L-glutamine-D-fructose-6-phosphate transaminase activity, or (iv) a polypeptide comprising or consisting of an amino acid sequence, The amino acid sequence has at least 80% sequence identity to the full-length amino acid sequence of the mutant polypeptide (which differs from the wild-type E. coli protein of UniProt ID P17169 by having the A39T, R250C and G472S mutations) and has L - Glutamine-D-fructose-6-phosphate transaminase activity.

Global sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably using preset parameters, and preferably using mature protein sequences (ie, without regard to secretion signals or transit peptides). Sequence identity is usually higher when only conserved domains or motifs are considered compared to overall sequence identity.

with SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31 , 32, 33, 34, 35, 36, 39, 40, or 41, or UniProt ID P43577 or P17169, or at least any full-length of any of the mutated polypeptides of A39T, R250C, and G472S different from UniProt ID P17169 80% overall sequence identity is to be understood as with SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, respectively , 23, 26, 27, 28, 31, 32, 33, 34, 35, 36, 39, 40, or 41, or UniProt ID P43577 or P17169, or A39T, R250C, and G472S mutations different from UniProt ID P17169 At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% of any of the mutant polypeptides , 94%, 95%, 96%, 97%, 98% or 99% overall sequence identity, as described herein. From SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31 , 32, 33, 34, 35, 36, 39, 40 or 41, or UniProt ID P43577 or P17169, or at least 8 of any of the mutated polypeptides of any of the A39T, R250C and G472S mutations different from UniProt ID P17169 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 oligopeptide sequences of consecutive amino acid residues should be understood as at least 8, 9 of consecutive amino acid residues, respectively , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 any of the oligopeptide sequences within the total number of amino acid residues from SEQ ID NO: 03 , 04, 05, 06, 07, 08, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34 , 35, 36, 39, 40 or 41, or UniProt ID P43577 or P17169, or any of the mutant polypeptides of the A39T, R250C and G472S mutations different from UniProt ID P17169, preferably wherein the oligopeptide does not interact with The PFAM domains completely overlap (if present), preferably wherein the oligopeptide does not overlap the PFAM domains (if present).

In preferred embodiments of the method and/or cell of the present invention, the cell is capable of catabolizing carbon sources selected from the group consisting of glucose, fructose, galactose, lactose, sucrose, maltose, malto-oligosaccharide (malto-oligosaccharide) -oligosaccharides), maltotriose (maltotriose), sorbitol, xylose, rhamnose, mannose, methanol, ethanol, arabinose, trehalose (trehalose), starch, cellulose, hemicellulose, molasses ), corn-steep liquor, high fructose syrup, glycerin, acetate, citrate, lactic acid, pyruvic acid.

In preferred embodiments of the methods and/or cells of the present invention, the cells use lactose in a saccharification reaction to produce oligosaccharides, preferably the lactose-based MMOs described herein. Lactose can be produced by cells (eg, by metabolism of cells and/or after metabolic engineering of cells for this purpose as known to those of ordinary skill in the art), preferably intracellularly, or can be added into cells, which can introduce the lactose by passive or active transport. The production of lactose by cells can be obtained by the expression of N-acetylglucosamine β-1,4-galactosyltransferase and UDP-glucose 4-epimerase. More preferably, the cells are modified to promote lactose production. The modification may be one or more selected from the group consisting of overexpressing N-acetylglucosamine beta-1,4-galactosyltransferase, overexpressing UDP-glucose 4-epimerase.

In preferred embodiments of the methods and/or cells of the present invention, cells using lactose as a recipient in the saccharification reaction preferably have transporters for uptake of lactose from culture. More preferably, the cells are optimized for uptake of lactose. The optimization may be to overexpress a lactose transporter, such as the lactose permease of E. coli or Kluyveromyces lactis . Preferably, the cells continue to express lactose permease. Lactose can be added at the beginning of the culture or immediately after sufficient biomass is formed during the growth phase of the culture, i.e., the lactose-based MMO production phase initiated by adding lactose to the culture is separate from the growth phase. (decoupled). In a preferred embodiment, lactose is added at the beginning and/or during the culturing process, ie, the growth phase and the production phase are not separated.

In preferred embodiments of the methods and/or cells according to the present invention, the cells are resistant to the phenomenon of lactose killing when the cells are grown in an environment in which lactose is combined with one or more other carbon sources. The term "lactose killing" refers to the arrest of cell growth in a medium in which lactose is present along with other carbon sources. In a preferred embodiment, the cells are genetically engineered to retain at least 50% of the lactose influx without lactose killing, even at high lactose concentrations, as described in WO 2016/075243. The genetic modification includes expression and/or overexpression of exogenous and/or endogenous lactose transporter genes by a heterologous promoter that does not result in a lactose-killing phenotype and/or modification of the codon usage of the lactose transporter to produce a Altered manifestations of the lactose transporter of the lactose-killing phenotype. The contents of WO 2016/075243 are incorporated by reference in this regard.

In a further embodiment of the invention, fructose-6-phosphate is the substrate for L-glutamine-D-fructose-6-phosphate transaminase, the L-glutamine-D-fructose-6-phosphate transaminase Aminases are expressed in cells and are able to convert fructose-6-phosphate to glucosamine-6-phosphate as a precursor for the synthesis of GlcNAc. Glucosamine-6-phosphate is a substrate for N-acetylglucosamine-6-phosphotransferase, which is expressed intracellularly and is capable of converting glucosamine-6-phosphate Converted to N-acetylglucosamine-6-phosphate. N-acetylglucosamine-6-phosphate is a substrate for the phosphatase, which is expressed in cells, capable of dephosphorylating N-acetylglucosamine-6-phosphate to synthesize the monosaccharide GlcNAc.

In another preferred embodiment, the cell is incapable of converting N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or incapable of converting glucosamine-6-phosphate to fructose- 6-Phosphate. In cells, N-acetylglucosamine-6-phosphate can be converted to glucosamine-6-phosphate by the activity of N-acetylglucosamine-6-phosphate deacetylases such as nagA, and Glucosamine-6-phosphate can be converted to fructose-6-phosphate by the activity of glucosamine-6-phosphate deaminase (eg nagB). Such N-acetylglucosamine-6-phosphate deacetylase and/or glucosamine-6-phosphate deaminase can be encoded by methods known to those of ordinary skill in the art. Mutagenesis or partial or complete deletion of the corresponding polynucleotide, or mutagenesis of a promoter sequence that controls the expression of the corresponding polynucleotide, results in reduced expression or reduced activity or is deactivated.

In one embodiment, the cell is capable of synthesizing the nucleotide-sugar GDP-fucose. GDP-fucose can be provided by enzymes expressed in cells or by metabolism of cells. This GDP-fucose-synthesizing cell may express an enzyme that converts, for example, fucose added to the cell to GDP-fucose. This enzyme can be, for example, a bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase, such as Fkp from Bacteroides fragilis , or an independent fucose kinase with A combination of independent fucose-1-phosphate guanylate transferase enzymes known from several species including Homo sapiens , Sus scrofa and Rattus norvegicus .

Preferably, the cells are modified to produce GDP-fucose.

More preferably, the cells are modified to promote GDP-fucose production. The modification can be selected from the group consisting of UDP-glucose: undecaprenyl-undecaprenyl-phosphoglucose-1-phosphotransferase-encoding gene knock-out, GDP-L-fucose synthase encoding gene overexpression of genes encoding GDP-mannose 4,6-dehydratase, overexpression of genes encoding mannose-1-phosphate guanylate transferase, overexpression of genes encoding phosphomannose mutase (phosphomannomutase) One or more of the group of overexpression, and overexpression of a gene encoding mannose-6-phosphate isomerase.

In one embodiment, the cell is capable of synthesizing the nucleotide-sugar UDP-galactose. UDP-galactose can be provided by enzymes expressed in cells or by metabolism of cells. Preferably, the cells are modified to synthesize UDP-galactose. More preferably, the cells are modified to promote UDP-galactose production. The modification can be selected from shaving, galactose-1-phosphate uridine transferase (galactose -1-phosphate uridylyltransferase) encoding gene shaving and one or more of the group of overexpression of UDP-galactose 4-epimerase (eg, galE).

In one example, the cell is capable of synthesizing the nucleotide-sugar CMP-N-acetylneuraminic acid (CMP-sialic acid). CMP-N-acetylneuraminic acid can be provided by enzymes expressed in cells or by metabolism of cells. Preferably, the cells are modified to synthesize CMP-N-acetylneuraminic acid. More preferably, the cells are modified to promote CMP-N-acetylneuraminic acid production. The modification can be selected from overexpression of a gene encoding CMP-sialate synthase, overexpression of a gene encoding sialate synthase, and overexpression of a gene encoding N-acetyl-D-glucosamine 2-epimerase One or more of the over-performing groups.

The synthesis of CMP-N-acetylneuraminic acid uses GlcNAc, but GlcNAc in cells as described herein is used for the synthesis of disaccharides or oligosaccharides with a GlcNAc unit at the reducing end. Thus, the production of CMP-N-acetylneuraminic acid in cells may reduce the GlcNAc available for the production of bioproducts of interest, ie, disaccharides or oligosaccharides with GlcNAc units at the reducing end. The production of CMP-N-acetylneuraminic acid and GlcNAc needs to be carefully optimized with each other to ensure high levels of both CMP-N-acetylneuraminic acid and GlcNAc. Optimization may include effectively balancing and fine-tuning the performance levels of polypeptides involved in the synthesis of both CMP-N-acetylneuraminic acid and GlcNAc.

In another preferred embodiment, the cells express at least one other glycosyltransferase to saccharify synthetic N-acetylglucosamine monosaccharides to form oligosaccharides with GlcNAc at the reducing end as described herein. Preferably, at least one glycosyltransferase is expressed to saccharify the GlcNAc monosaccharide to form a disaccharide or oligosaccharide. More preferably, the cell expresses at least two, more preferably at least three, and more preferably at least four glycosyltransferases, and most preferably at least five glycosyltransferases, to saccharify the GlcNAc monosaccharide to form a base. The disaccharide or oligosaccharide of the present invention. The glycosyltransferase may be selected from the list including but not limited to the following: α-1,2-fucosyltransferase, α-1,3/1,4-fucosyltransferase, α-1,2-fucosyltransferase 6-fucosyltransferase, α-2,3-sialyltransferase, α-2,6-sialyltransferase, α-2,8-sialyltransferase, β-1,3-galactose Syltransferase, β-1,4-galactosyltransferase, α-1,3-galactosyltransferase, α-1,4-galactosyltransferase, N-acetylglucosaminyltransferase , N-acetylgalactosamine transferase, glucosyl transferase, mannosyl transferase, N-acetyl mannosyl transferase, xylosyl transferase, glucuronyl transferase, galactose Aldolase, glucosamine transferase, N-glycolyl neuraminidase. In preferred embodiments, the glycosyltransferase is a cellular endogenous protein with a modified expression or activity, preferably the endogenous glycosyltransferase is overexpressed; or the glycosyltransferase is A heterologous protein, which is introduced into and expressed in the cell heterologously, is preferably overexpressed. The endogenous glycosyltransferase can have a modified expression in the cell that also expresses the heterologous glycosyltransferase. The oligosaccharides so synthesized may be of the linear or branched type and may comprise a plurality of monosaccharide building blocks, as explained in the definitions described herein.

如本文所述的寡醣可以線性或分支結構形成，其包含α-和β-醣苷鍵兩者或可僅包含β-醣苷鍵，其中B為N-乙醯葡萄糖胺，且其中A、V、W、X、Y及/或Z不存在，或為半乳糖、葡萄糖、岩藻糖、甘露糖、木糖、葡萄糖醛酸、半乳糖醛酸、艾杜糖醛酸、N-乙醯基神經胺酸、N-乙醇醯神經胺酸、葡萄糖胺、N-乙醯半乳糖胺、N-乙醯甘露糖胺、N-乙醯葡萄糖胺及/或由通式2定義的寡醣結構：

其中B為N-乙醯葡萄糖胺，其中A、V、W、X、Y及/或Z不存在，或為半乳糖、葡萄糖、岩藻糖、甘露糖、木糖、葡萄糖醛酸、半乳糖醛酸、艾杜糖醛酸、N-乙醯基神經胺酸、N-乙醇醯神經胺酸、葡萄糖胺、N-乙醯半乳糖胺、N-乙醯甘露糖胺或N-乙醯葡萄糖胺，且在通式1及通式2中，m是3且可選地v是4，或者m是4且可選地v是3，並且其中p是3及/或4，且其中w是6，並且其中如果n＞1且p是3，x是3且X不是單醣，並且其中如果n＞1且p為4，y是4且Y不是單醣，並且其中z是6，且其中n從1至10。 By combining different active glycosyltransferases in the same cells as described herein, GlcNAc and nucleotide-active sugars comprising UDP-galactose (UDP-Gal), including UDP-galactose (UDP-Gal), are produced. Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-Rock Fucose (GDP-Fuc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy--L-arabino-4- Hexose, UDP-2-acetamido-2,6-dideoxy--L-lytho-4-hexulose, UDP-N-acetamido-L-rhamnosamine (UDP- L-RhaNAc or UDP-2-acetylamino-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP -L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetamido-L-pneumolysamine (UDP-L-PneNAC or UDP- 2-Acetylamino-2,6-dideoxy-L-talose), UDP-N-Acetylmuramic acid, UDP-N-Acetyl-L-Quinosamine (UDP-L -QuiNAc or UDP-2-acetylamino-2,6-dideoxy-L-glucose), GDP-L-isorhamnose, CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolic neuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N ₃ , CMP-Neu4,5Ac ₂ , CMP-Neu5,7Ac ₂ , CMP-Neu5,9Ac ₂ , CMP-Neu5,7 (8,9)Ac ₂ , UDP-glucuronate, UDP-galacturonate, GDP-rhamnose or UDP-xylose, the cells are capable of producing bismuths having GlcNAc units at the reducing end according to formula 1 Sugar or Oligosaccharide:

Oligosaccharides as described herein can be formed in linear or branched structures that contain both alpha- and beta-glycosidic linkages or can contain only beta-glycosidic linkages, wherein B is N-acetylglucosamine, and wherein A, V, W, X, Y and/or Z are absent, or galactose, glucose, fucose, mannose, xylose, glucuronic acid, galacturonic acid, iduronic acid, N-acetylneuron Amino acid, N-glycolylneuraminic acid, glucosamine, N-acetylgalactosamine, N-acetylmannosamine, N-acetylglucosamine and/or oligosaccharide structure as defined by general formula 2:

wherein B is N-acetylglucosamine, wherein A, V, W, X, Y and/or Z are absent, or galactose, glucose, fucose, mannose, xylose, glucuronic acid, galactose uronic acid, iduronic acid, N-acetylneuraminic acid, N-glycolylneuraminic acid, glucosamine, N-acetylgalactosamine, N-acetylmannosamine, or N-acetylglucosamine amines, and in formula 1 and formula 2, m is 3 and optionally v is 4, or m is 4 and optionally v is 3, and wherein p is 3 and/or 4, and wherein w is 6, and where if n > 1 and p is 3, x is 3 and X is not a monosaccharide, and where if n > 1 and p is 4, y is 4 and Y is not a monosaccharide, and where z is 6, and where n is from 1 to 10.

In preferred embodiments, the cells as described herein are modified in the expression or activity of the other glycosyltransferases.

In another preferred embodiment, the oligosaccharides having GlcNAc units at the reducing end as produced by the methods and cells described herein are selected from the list comprising: 2-fucosylacto-N- Disaccharide, 4-fucosyllacto-N-disaccharide, 2-4-difucosyllacto-N-disaccharide, 3'-sialylacto-N-disaccharide, 6'-sialyolide -N-disaccharide, 3',6'-disialo-N-disaccharide, 6,6'-disialo-N-disaccharide, 2'-fucosyl-3'-sialic acid Lacto-N-disaccharide, 2'-fucosyl-6'-sialo-N-disaccharide, 4-fucosyl-3'-sialo-N-disaccharide, 4-fucosyl Glycosyl-6'-Sialylol-N-Disaccharide, 2-Fucosyl-N-Acetyllactosamine, 3'-Fucosyl-N-Acetyllactosamine, 2,3'-Difucosamine Glycosyl N-acetyllactosamine, 3'-sialo-N-acetyllactosamine, 6'-sialo-N-acetyllactosamine, 3',6'-disialo-N-acetyllactosamine, 6 ,6'-Disialo-N-acetyllactosamine, 2'-fucosyl-3'-sialo-N-acetyllactosamine, 2'-fucosyl-6'-sialic acid N-ethyl Glucosamine, 3-fucosyl-3'-sialic acid N-acetyl lactosamine, 3'-fucosyl-6'-sialic acid N-acetyl lactosamine, P1 trisaccharide (P1 trisaccharide) (Gal-a1,4-Gal-b1,4-GlcNAc), xenotransplantation epitope (Gal-a1,3-Gal-b1,4-GlcNAc), Gal-b14-(Galb13)- GlcNAc, poly-N-acetyllactosamine, GalNAc-b1,3-Gal-b1,4-GlcNAc.

In a preferred embodiment of the method and/or cell according to the present invention, the cell expresses a membrane transporter protein or a polypeptide having transport activity to transport compounds across the outer membrane of the cell wall. In a more preferred embodiment of the method and/or cell of the present invention, the cell is modified in the expression or activity of the membrane transporter or polypeptide having transport activity. The membrane transporter or polypeptide with transport activity is a cellular endogenous protein with modified expression or activity, preferably the endogenous membrane transporter or polypeptide with transport activity is overexpressed; or, the membrane transporter A protein or polypeptide having transport activity is a heterologous protein that is introduced into the cell heterologously and is expressed, preferably overexpressed, in the cell. The endogenous membrane transporter or polypeptide with transport activity can have a modified expression in a cell that also expresses the heterologous membrane transporter or polypeptide with transport activity.

In a more preferred embodiment of the method and/or cell of the present invention, the membrane transporter or polypeptide having transport activity is selected from the list including: transporter, P-P-bond-hydrolysis-driven transporter, β-barrel Porins, cotransporters, putative transporters, and phosphotransfer-driver group translocators. In yet more preferred embodiments of the methods and/or cells of the present invention, the transporter proteins include MFS transporters, sugar efflux transporters and chelatin exporters. In another more preferred embodiment of the methods and/or cells of the present invention, the P-P-bond-hydrolysis-driven transporters include ABC transporters and chelatin exporters.

In another preferred embodiment of the method and/or cell of the present invention, a membrane transporter or a polypeptide having transport activity controls the flow of the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end on the outer membrane of the cell wall. In an alternative and/or additional preferred embodiment of the method and/or cell of the present invention, a membrane transporter or a polypeptide having transport activity controls the flow of the disaccharide and oligosaccharide having a GlcNAc unit at the reducing end on the outer membrane of the cell wall . In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, membrane transporters or polypeptides having transport activity control the flow of the one or more lactose-based MMOs on the outer membrane of the cell wall.

In alternative and/or additional preferred embodiments of the methods and/or cells of the present invention, membrane transporters or polypeptides having transport activity control the flow of one or more precursors on the outer membrane of the cell wall for the production of The reducing end has a disaccharide or oligosaccharide of a GlcNAc unit. In alternative and/or additional preferred embodiments of the methods and/or cells of the present invention, membrane transporters or polypeptides having transport activity control the flow of one or more precursors on the outer membrane of the cell wall for the production of The reducing end has disaccharides and oligosaccharides of GlcNAc units. In alternative and/or additional preferred embodiments of the methods and/or cells of the present invention, membrane transporters or polypeptides having transport activity control the flow of one or more precursors on the outer membrane of the cell wall for the production of One or more lactose-based MMOs.

In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, membrane transporters or polypeptides having transport activity control the flow of one or more recipients on the outer membrane of the cell wall for generating The reducing end has a disaccharide or oligosaccharide of a GlcNAc unit. In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, membrane transporters or polypeptides having transport activity control the flow of one or more recipients on the outer membrane of the cell wall for generating The reducing end has disaccharides and oligosaccharides of GlcNAc units. In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, membrane transporters or polypeptides having transport activity control the flow of one or more recipients on the outer membrane of the cell wall for generating the one or more lactose-based MMOs.

In another preferred embodiment of the method and/or cell of the present invention, the membrane transporter or polypeptide having transport activity provides improved production of the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end. In another preferred embodiment of the method and/or cell of the present invention, the membrane transporter or polypeptide having transport activity provides improved production of disaccharides and oligosaccharides having a GlcNAc unit at the reducing end. In another preferred embodiment of the methods and/or cells of the invention, a membrane transporter or polypeptide having transport activity provides improved production of the one or more lactose-based MMOs.

In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, the membrane transporter or polypeptide having transport activity provides for the efflux of disaccharides or oligosaccharides having a GlcNAc residue at the reducing end. In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, membrane transporters or polypeptides having transport activity provide for efflux of disaccharides and oligosaccharides having GlcNAc residues at the reducing end. In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, a membrane transporter or polypeptide having transport activity provides for efflux of the one or more lactose-based MMOs.

In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, the membrane transporter or polypeptide having transport activity provides for facilitating the efflux of the disaccharide or oligosaccharide having a GlcNAc residue at the reducing end. In alternative and/or additional preferred embodiments of the methods and/or cells of the present invention, membrane transporters or polypeptides with transport activity are provided to facilitate the efflux of disaccharides and oligosaccharides having GlcNAc residues at the reducing end. In alternative and/or additional preferred embodiments of the methods and/or cells of the present invention, a membrane transporter or polypeptide having transport activity is provided to facilitate efflux of the one or more lactose-based MMOs.

In another preferred embodiment of the method and/or cell of the present invention, the cell expresses a polypeptide selected from the group comprising lactose transporters, such as LacY or lac12 permease, glucose transporter, galactose transporter, rock Fucose transporter, transporter of nucleotide-active sugars, eg, transporter of UDP-Gal, UDP-GlcNAc, GDP-Fuc or CMP-sialic acid. As such, the transporter will internalize the precursor and/or acceptor added medium for the synthesis of disaccharides or oligosaccharides and/or lactose-based MMOs with GlcNAc units at the reducing end.

In a more preferred embodiment of the method and/or cell of the present invention, the cell expresses a membrane transporter belonging to the MFS transporter family, for example from Escherichia coli (UniProt ID P0AEY8), Cronobacter muytjensii (UniProt ID) MdfA polypeptides of the MdfA family of multidrug transporters of A0A2T7ANQ9), Citrobacter youngae (UniProt ID D4BC23), and Yokenella regensburgei (UniProt ID G9Z5F4) species. In another more preferred embodiment of the method and/or cell of the present invention, the cell expresses a membrane transporter belonging to the family of sugar excretion transporters, such as those derived from Escherichia coli (UniProt ID P31675), Citrobacter koseri ) (UniProt ID A0A078LM16) and SetA polypeptides of the SetA family of species of Klebsiella pneumoniae (UniProt ID A0A0C4MGS7). In another preferred embodiment of the method and/or cell of the present invention, the cell expresses a membrane transporter belonging to the chelatin exporter family, such as E. coli entS (UniProt ID P24077) and E. coli iceT (UniProt ID A0A024L207) . In another preferred embodiment of the method and/or cell of the present invention, the cell expresses a membrane transporter belonging to the ABC transporter family, such as oppF from Escherichia coli (UniProt ID P77737), from Lactococcus lactis subsp. lmrA from Lactococcus lactis subsp. lactis bv. Diacetylactis (UniProt ID A0A1V0NEL4) and Blon_2475 from Bifidobacterium longum subsp. Infantis ( UniProt ID B7GPD4 ) .

According to another embodiment of the method and/or cell of the present invention, the cell is capable of producing phosphoenolpyruvate (PEP). According to another embodiment of the method and/or cell of the present invention, the cell comprises a disaccharide or oligosaccharide production pathway having an N-acetylglucosamine unit at the reducing end, which includes a PEP production pathway. In preferred embodiments of the methods and/or cells of the invention, the cells are modified to facilitate the production and/or supply of PEP compared to unmodified progenitor cells.

In another preferred embodiment, the cell comprises a disaccharide or oligosaccharide production pathway with an N-acetylglucosamine unit at the reducing end, which includes promoting the production of PEP compared to unmodified precursor cells and/or or any one or more modifications of the supply.

In preferred embodiments and as a means of promoting PEP production and/or supply, one or more PEP-dependent sugar transport phosphotransferase systems are disrupted, such as, but not limited to: 1) N-Acetyl-D- Glucosamine Npi-phosphotransferase (EC 2.7.1.193), e.g. encoded by the nagE gene (or cluster nagABCD) in E. coli or Bacillus, 2) encoding the enzyme llMan complex (mannose PTS permease, protein - ManXYZ of Npi-phosphohistidine-D-mannose phosphotransferase) introduces exogenous hexoses (mannose, glucose, glucosamine, fructose, 2-deoxyglucose, mannosamine, N-acetylglucosamine, etc. ) and release phosphate into the cytoplasm, 3) a glucose-specific PTS transporter (e.g. encoded by PtsG/Crr), which takes up glucose and forms glucose-6-phosphate in the cytoplasm, 4) a sucrose-specific PTS transporter , which takes up sucrose and forms sucrose-6-phosphate in the cytoplasm, 5) a fructose-specific PTS transporter (e.g., encoded by the genes fruA and fruB and the kinase fruK, which takes up fructose and forms fructose-1- phosphate and in a second step fructose 1,6-bisphosphate, 6) Lactose PTS transporter (eg, encoded by lacE in Lactococcus casei ), which takes up lactose and forms lactose-6- Phosphate, 7) Galactitol-specific PTS enzymes that absorb galactitol and/or sorbitol and form galactitol-1-phosphate or sorbitol-6-phosphate, respectively, 8) Mannitol-specific PTS Enzymes that take up mannitol and/or sorbitol and form mannitol-1-phosphate or sorbitol-6-phosphate, respectively, and 9) Trehalose-specific PTS enzymes that take up trehalose and form trehalose-6 - Phosphate.

In another and/or further preferred embodiment and as a means of promoting PEP production and/or supply, the PTS system as a whole is disrupted by disrupting the PtsIH/Crr gene cluster. PtsI (enzyme I) is a cytoplasmic protein that acts as a gateway to the phosphoenolpyruvate:sugar phosphotransferase system (PTSsugar) of Escherichia coli K-12. PtsI, one of two (PtsI and PtsH) sugar-nonspecific protein constructs of PTSsugar, works with sugar-specific inner membrane permeases to affect the phosphotransfer cascade leading to coupled phosphorylation of multiple carbohydrate substrates and transfer. HPr (histidine-containing protein) is one of two sugar-nonspecific protein constructs of PTSsugar. It accepts a phosphonium group from phosphorylase I (PtsI-P), which is then transferred to the EIIA domain of any of PTSsugar's many carbohydrate-specific enzymes (collectively referred to as Enzyme II). Crr or EIIAGlc are phosphorylated by PEP in reactions requiring PtsH and PtsI.

In another and/or further preferred embodiment, the cells are further modified to compensate for the absence of the PTS system of the carbon source by introducing and/or overexpressing the corresponding permease. These are, for example, permease or ABC transporters, which include, but are not limited to, specific import of lactose (eg, the transporter encoded by the LacY gene from E. coli, etc.), sucrose (eg, the transporter encoded by the cscB gene from E. coli, etc.) ), glucose (such as the transporter encoded by the galP gene from E. coli, etc.), fructose (such as the transporter encoded by the fruI gene of Streptococcus mutans, etc.), or the sorbitol/mannitol ABC transporter ( For example, transporters encoded by the SmoEFGK cluster of Rhodobacter sphaeroides , etc. ) , trehalose/sucrose/maltose transporters (eg, transporters encoded by the gene cluster ThuEFGK of Sinorhizobium meliloti , etc.) , and the N-acetylglucosamine/galactose/glucose transporter (eg, the transporter encoded by NagP of Shewanella oneidensis ) . Examples of combinations of PTS deletion and alternative transporter overexpression are: 1) deletion of the glucose PTS system (eg, the ptsG gene) combined with introduction and/or overexpression of a glucose permease (eg, galP of glcP), 2) fructose Deletion of a PTS system (eg, one or more fruB, fruA, fruK genes) in combination with the introduction and/or overexpression of a fructose permease (eg, fruI), 3) deletion of a lactose PTS system in combination with a lactose permease (eg, fruI) , LacY) introduction and/or overexpression, and/or 4) deletion of the sucrose PTS system combined with introduction and/or overexpression of a sucrose permease (eg, cscB).

In a further preferred embodiment, by introducing 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)) Modification of cells to compensate for the absence of the PTS system of the carbon source. Examples of combinations of PTS deletion with overexpression of alternative transporters and kinases are: 1) deletion of the glucose PTS system (eg, the ptsG gene) combined with introduction and/or overexpression of a glucose permease (eg, galP of glcP), In conjunction with introduction and/or overexpression of glucokinase (eg, glk), and/or 2) deletion of the fructose PTS system (eg, one or more fruB, fruA, fruK genes) in conjunction with a fructose permease (eg, fruI) ), introduction and/or overexpression of a bound fructokinase (eg, frk or mak).

In another and/or further preferred embodiment and as a means of promoting PEP production and/or supply, the introduction or modification is carried out by including any one or more of the following enumeration: phosphoenolpyruvate synthase activity (e.g. in EC encoded by ppsA in E. coli: 2.7.9.2), phosphoenolpyruvate carboxykinase activity (e.g. EC encoded by PCK in Corynebacterium glutamicum or by pckA in E. coli, respectively) 4.1.1.32 or EC 4.1.1.49), phosphoenolpyruvate carboxylase activity (e.g. in E. coli encoded by ppc EC 4.1.1.31), oxaloacetate decarboxylase (e.g. in E. coli EC 4.1.1.112 encoded by eda in E. coli), pyruvate kinase activity (e.g. EC 2.7.1.40 encoded by pykA and pykF in E. coli), pyruvate carboxylase activity (e.g. EC encoded by pyc in Bacillus subtilis) 6.4.1.1) and malate dehydrogenase activity (eg EC 1.1.1.38 or EC 1.1.1.40 encoded by maeA or maeB in E. coli, respectively).

In a more preferred embodiment, the cells are modified to overexpress any one or more polypeptides including ppsA from E. coli (UniProt ID P23538), PCK from Corynebacterium glutamicum (UniProt ID Q6F5A5), from E. coli 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 further preferred embodiment, the cells are modified to exhibit phosphoenolpyruvate synthase activity, phosphoenolpyruvate carboxykinase activity, oxaloacetate decarboxylase activity or malate dehydrogenase activity Active any one or more polypeptides.

In another and/or further preferred embodiment and as a means of promoting the production and/or supply of PEP, by reducing activity of phosphoenolpyruvate carboxylase activity and/or pyruvate kinase activity, preferably encoding a phosphoene Cell modification by gene deletion of alcohol pyruvate carboxylase, pyruvate carboxylase activity, and/or pyruvate kinase.

In exemplary embodiments, cells are genetically engineered with different adaptations, such as overexpression of phosphoenolpyruvate synthase in combination with deletion of the pyruvate kinase gene, overexpression of phosphoenolpyruvate synthase in combination with phosphoenol Deletion of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with deletion of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase combined with deletion of pyruvate kinase gene, phosphoenolpyruvate Overexpression of carboxykinase in combination with deletion of phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase in combination with deletion of pyruvate carboxylase gene, overexpression of oxaloacetate decarboxylase in combination with pyruvate kinase Gene deletion, overexpression of oxalate decarboxylase combined with deletion of phosphoenolpyruvate carboxylase gene, overexpression of oxalate decarboxylase combined with deletion of pyruvate carboxylase gene, malate dehydrogenase Overexpression of pyruvate kinase gene combined with deletion of pyruvate kinase gene, overexpression of malate dehydrogenase combined with deletion of phosphoenolpyruvate carboxylase gene, and/or overexpression of malate dehydrogenase combined with deletion of pyruvate carboxylase gene .

In another exemplary embodiment, cells are genetically engineered with different adaptations, such as overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase, phosphoenolpyruvate synthase Overexpression combined with overexpression of oxaloacetate decarboxylase, overexpression of phosphoenolpyruvate synthase combined with overexpression of malate dehydrogenase, overexpression of phosphoenolpyruvate carboxykinase combined with oxaloacetate decarboxylase Overexpression of carboxylase, overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of malate dehydrogenase, overexpression of oxalate decarboxylase combined with overexpression of malate dehydrogenase, phosphoenolacetone Overexpression of acid synthase combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase, overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of malate dehydrogenase, overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase Overexpression, overexpression of phosphoenolpyruvate carboxykinase in combination with overexpression of oxaloacetate decarboxylase and overexpression of malate dehydrogenase, and/or overexpression of phosphoenolpyruvate synthase in combination with oxaloacetate Overexpression of salt decarboxylase and overexpression of malate dehydrogenase.

In another exemplary embodiment, cells are genetically modified by different adaptations, such as overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and deletion of the pyruvate kinase gene, phosphoenolpyruvate synthase Overexpression of enolpyruvate synthase combined with overexpression of oxalate decarboxylase and deletion of the pyruvate kinase gene, overexpression of phosphoenolpyruvate synthase combined with overexpression of malate dehydrogenase and pyruvate Deletion of the kinase gene, overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of oxalate decarboxylase and deletion of the pyruvate kinase gene, overexpression of phosphoenolpyruvate carboxykinase combined with malate dehydrogenase Overexpression of pyruvate kinase gene and deletion of pyruvate kinase gene, overexpression of oxalate decarboxylase combined with overexpression of malate dehydrogenase and deletion of pyruvate kinase gene, overexpression of phosphoenolpyruvate synthase combined with phosphate Overexpression of enolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and deletion of the pyruvate kinase gene, overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and Overexpression of malate dehydrogenase and deletion of pyruvate kinase gene, overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxalate decarboxylase and apple Overexpression of acid dehydrogenase and deletion of pyruvate kinase gene, overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and pyruvate kinase gene Deletion of phosphoenolpyruvate synthase, overexpression of phosphoenolpyruvate synthase combined with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and deletion of the pyruvate kinase gene.

In another exemplary embodiment, cells are genetically engineered with different adaptations, such as overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase genes Deletion of phosphoenolpyruvate synthase combined with overexpression of oxalate decarboxylase and deletion of phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with malate decarboxylase Overexpression of hydrogenase and deletion of phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of oxaloacetate decarboxylase and deletion of phosphoenolpyruvate carboxylase gene, Overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of malate dehydrogenase and deletion of the phosphoenolpyruvate carboxylase gene, overexpression of oxalate decarboxylase combined with overexpression of malate dehydrogenase and deletion of phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and phosphoenolpyruvate Deletion of carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of malate dehydrogenase and deletion of phosphoenolpyruvate carboxylase gene, phosphoene Overexpression of alcohol pyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and deletion of the phosphoenolpyruvate carboxylase gene , Overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and deletion of phosphoenolpyruvate carboxylase gene, phosphoenolpyruvate synthase Overexpression of oxalate combined with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and deletion of the phosphoenolpyruvate carboxylase gene.

In another exemplary embodiment, cells are genetically modified with different adaptations, such as overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and deletion of the pyruvate carboxylase gene, Overexpression of phosphoenolpyruvate synthase combined with overexpression of oxalate decarboxylase and deletion of the pyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with overexpression of malate dehydrogenase and Deletion of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of oxaloacetate decarboxylase and deletion of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase combined with apple Overexpression of acid dehydrogenase and deletion of pyruvate carboxylase gene, overexpression of oxalate decarboxylase combined with overexpression of malate dehydrogenase and deletion of pyruvate carboxylase gene, synthesis of phosphoenolpyruvate Enzyme overexpression combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and deletion of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with phosphoenolacetone Overexpression of acid carboxykinase and overexpression of malate dehydrogenase and deletion of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and oxaloacetate Overexpression of decarboxylase and overexpression of malate dehydrogenase and deletion of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of oxalate decarboxylase and malate dehydrogenase Enzyme overexpression and deletion of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and overexpression of pyruvate carboxylase gene missing.

In another exemplary embodiment, cells are genetically engineered with different adaptations, such as overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and pyruvate carboxylase genes and phosphoene Deletion of alcohol pyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with overexpression of oxaloacetate decarboxylase and deletion of pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, phosphoene Overexpression of alcohol pyruvate synthase combined with overexpression of malate dehydrogenase and deletion of pyruvate carboxylase and phosphoenolpyruvate carboxylase genes, and overexpression of phosphoenolpyruvate carboxykinase combined with oxaloacetate Overexpression of decarboxylase and deletion of pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of malate dehydrogenase and pyruvate carboxylase gene and Deletion of phosphoenolpyruvate carboxylase gene, overexpression of oxalate decarboxylase combined with overexpression of malate dehydrogenase and deletion of pyruvate carboxylase and phosphoenolpyruvate carboxylase genes, phosphoene Overexpression of alcohol pyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and deletion of pyruvate carboxylase and phosphoenolpyruvate carboxylase genes, phosphoene Overexpression of alcohol pyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of malate dehydrogenase and deletion of pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, phosphoenolacetone Overexpression of acid synthase combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and pyruvate carboxylase genes and phosphoenolpyruvate carboxylase Deletion of enzyme genes, overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and pyruvate carboxylase and phosphoenolpyruvate carboxylase genes Deletion of phosphoenolpyruvate synthase, overexpression of phosphoenolpyruvate synthase combined with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and deletion of pyruvate carboxylase and phosphoenolpyruvate carboxylase genes .

In another exemplary embodiment, cells are genetically engineered with different adaptations, such as overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and the pyruvate kinase gene and pyruvate carboxykinase Deletion of enzyme gene and phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with overexpression of oxaloacetate decarboxylase and pyruvate kinase gene and pyruvate carboxylase gene and phosphoenol Deletion of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with overexpression of malate dehydrogenase and deletion of pyruvate kinase gene and pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, Overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of oxalate decarboxylase and deletion of pyruvate kinase gene and pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, phosphoenolpyruvate carboxylase Kinase overexpression combined with malate dehydrogenase overexpression and deletion of pyruvate kinase gene and pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, overexpression of oxalate decarboxylase combined with malate decarboxylase Overexpression of hydrogenase and deletion of pyruvate kinase gene and pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and Overexpression of oxalate decarboxylase and deletion of pyruvate kinase gene and pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with phosphoenolpyruvate carboxylase Overexpression of kinases and overexpression of malate dehydrogenase and deletion of pyruvate kinase gene and pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with phosphoenol Overexpression of pyruvate carboxykinase and overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and deletion of pyruvate kinase gene and deletion of pyruvate carboxylase and phosphoenolpyruvate carboxylase genes Deletion, overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and pyruvate kinase genes and pyruvate carboxylase genes and phosphoenolpyruvate carboxylase Deletion of enzyme genes, overexpression of phosphoenolpyruvate synthase combined with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and pyruvate kinase gene and pyruvate carboxylase gene and phosphoenol Deletion of the pyruvate carboxylase gene.

According to another preferred embodiment of the method and/or cell of the present invention, the cell comprises a modification for reducing acetate production compared to an unmodified precursor cell. The modification may be selected from any one or more of the group comprising: an overexpression of acetyl-CoA synthase, a pyruvate dehydrogenase, a fully or partially shave or reduced function pyruvate dehydrogenase, and a fully or partially Shaving or reducing the function of lactate dehydrogenase.

In further embodiments of the methods and/or cells of the invention, the cells are performed with respect to the expression or activity of at least one acetyl-CoA synthase (eg, acs from E. coli, Saccharomyces cerevisiae, Homo sapiens, mice) retouch. In a preferred embodiment, the acetyl-CoA synthase is an endogenous cell protein with a modified expression or activity, preferably the acetyl-CoA synthase is overexpressed; alternatively, the acetyl-CoA A synthetase is a heterologous protein that is introduced into the cell heterologously and is expressed, preferably overexpressed, in the cell. The endogenous acetyl-CoA synthetase can have a modified expression in a cell that also expresses a heterologous acetyl-CoA synthase. In a more preferred embodiment, the cells are modified in the expression or activity of an acetyl-CoA synthase, such as acs from E. coli (UniProt ID P27550). In another and/or further preferred embodiment, the cells are modified in the expression or activity of a functional homologue, variant or derivative of acs from E. coli (UniProt ID P27550), the functional homologue , variant or derivative has at least 80% overall sequence identity to the full length of the polypeptide from Escherichia coli (UniProt ID P27550) and has acetyl-CoA synthase activity.

In alternative and/or additional further embodiments of the methods and/or cells of the invention, the cells express or have activity in at least one pyruvate dehydrogenase (eg, from Escherichia coli, Saccharomyces cerevisiae, Homo sapiens, and brown mouse) modified aspects. In a preferred embodiment, the gene encoding pyruvate dehydrogenase is modified to have at least one partial or complete deletion or mutation by means known to those of ordinary skill in the art, thereby producing at least one less functional or incapacitated gene pyruvate dehydrogenase activity of the protein. In a more preferred embodiment, the cell's poxB-encoding gene is completely shaved, resulting in a lack of pyruvate dehydrogenase activity in the cell.

In alternative and/or additional further embodiments of the methods and/or cells of the present invention, the cells are in the expression or activity of at least one lactate dehydrogenase (eg, from Escherichia coli, Saccharomyces cerevisiae, Homo sapiens, and brown mouse) Make modifications. In a preferred embodiment, the lactate dehydrogenase encoding gene is modified to have at least one partial or complete deletion or mutation by means known to those of ordinary skill in the art, thereby producing at least one less functional or incapacitated gene. A protein with lactate dehydrogenase activity. In a more preferred embodiment, the cell's ldhA-encoding gene is completely shaved, resulting in a lack of lactate dehydrogenase activity in the cell.

According to another preferred embodiment of the method and/or cell of the present invention, the cell comprises lower or reduced expression and/or elimination, impairment, reduction of any one or more proteins compared to unmodified precursor cells or delayed activity, the protein contains: β-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine -6-phosphate deaminase, N-acetylglucosamine arrestin, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undec isoprene-phosphoglucose-1-phosphotransferase ( UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase), L-fuculokinase (L-fuculokinase), L-fucose isomerase, N-acetylneuraminic acid dissociation enzyme (N-acetylneuraminate) lyase), N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1- Phosphate uridine transferase, glucose-1-phosphate adenosyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isoenzyme 1, ATP-dependent 6-phosphofructokinase Isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor protein IclR, lon protease, glucose-specific translocation phosphotransferase IIBC component ptsG, glucose-specific Sex translocation phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIAGlc, β-glucoside-specific PTS enzyme II, fructose-specific PTS multiphosphoryl transfer protein (fructose-specific PTS multiphosphoryl transfer protein) FruA and FruB, ethanol Dehydrogenase Aldehyde dehydrogenase, pyruvate-formate dissociation enzyme, acetate kinase, phosphoacyltransferase (phosphoacyltransferase), phosphate acetyltransferase (phosphate acetyltransferase), pyruvate decarboxylase.

According to another preferred embodiment of the method and/or cell of the present invention, the cell comprises an at least partially inactivated catabolic pathway of a selected monosaccharide, disaccharide or oligosaccharide involved in reduction Required for the production of disaccharides or oligosaccharides with N-acetylglucosamine units at the end and/or for the production of disaccharides or oligosaccharides with N-acetylglucosamine units at the reducing end.

According to another preferred embodiment of the method and/or cell of the present invention, the cell uses a precursor to produce a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end, preferably, the precursor is obtained from the culture medium supply cells. According to more preferred aspects of the method and/or the cell, the cell uses at least two precursors to produce a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end, preferably the precursor is supplied to the cell from the culture medium . According to another preferred aspect of the method and/or cell of the present invention, the cell produces at least one precursor, preferably at least two precursors, to produce a disaccharide having an N-acetylglucosamine unit at the reducing end or Oligosaccharides. In a preferred embodiment of the present method and/or cell, the precursor used by the cell for the production of a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end is completely converted to having N-acetylglucosamine at the reducing end Disaccharides or oligosaccharides of amine units.

According to another preferred embodiment of the method and/or cell of the present invention, the cell has N-acetylglucosamine at the reducing end producing 90 g/L or more in the whole culture medium and/or supernatant units of disaccharides or oligosaccharides. In a more preferred embodiment, the disaccharide or oligosaccharide with N-acetylglucosamine unit at the reducing end produced in the whole culture medium and/or supernatant has a purity of at least 80%, which is sufficient in the whole culture medium and/or Or the total amount of disaccharides or oligosaccharides with N-acetylglucosamine units at the reducing end and their precursors, respectively, produced by the cells in the supernatant.

Another embodiment of the present invention provides a method and cell wherein a disaccharide or oligosaccharide having a GlcNAc unit at the reducing end is produced in and/or by a fungal, yeast, bacterial, insect, animal, plant or protozoan cell as described herein produced. The expression system or cell is selected from a list including bacteria, yeast or fungi, or refers to plant or animal cells. The cells are selected from a list including bacteria, yeast, protozoa or fungi, or plant or animal cells as referred to. The latter bacteria preferably belong to the phylum Proteobacteria or Firmicutes or Cyanobactria or Deinococcus-Thermus. The latter bacteria belonging to the phylum Proteobacteria preferably belong to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacteria preferably refer 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 is used in relation to a cultured E. coli strain - termed E. coli strain K12 strain - which is very suitable for laboratory environments and which, unlike wild-type strains, has lost the ability to reproduce in the intestine. Known examples of E. coli strains K12 strains are K12 wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Accordingly, the present invention particularly relates to a mutated and/or transformed E. coli host cell or strain as described above, wherein the E. coli strain is a K12 strain. More specifically, the E. coli strain K12 strain is E. coli MG1655. The latter bacteria belonging to the phylum Firmicutes preferably belong to the class Bacilli, preferably the order Lactobacilliales, members of which are, for example, Lactobacillus lactis, Leuconostoc mesenteroides, or Order of the Bacillales, members of which are, for example, from the genus Bacillus, eg, Bacillus subtilis or B. amyloliquefaciens . The latter bacteria belonging to the phylum Actinobacteria preferably belong to the family Corynebacteriaceae, whose members are Corynebacterium glutamicum or C. afermentans, or belong to the family Streptomycetaceae, whose members are Members are Streptomyces griseus or S. fradiae . The latter yeast preferably belongs to the phylum Ascomycota or Basidiomycota or Deuteromycota or Zygomycetes. The latter yeast preferably belongs to the genus Saccharomyces (whose members are, for example, Saccharomyces cerevisiae, S. bayanus , S. boulardii ), Zygosaccharomyces , Pichia Pichia (whose members are, for example, Pichia pastoris , P. anomala , P. kluyveri ), Kermagtler Yeast ( Komagataella ), Hansenula ( Hansenula ), Kluyveromyces ( Kluyveromyces ) (whose members are, for example, Kluyveromyces lactis, K. marxianus, Kluyveromyces thermotolerant ( K. thermotolerans )), Debaromyces , Yarrowia (members of which are, for example, Yarrowia lipolytica ), or Todococcus ( Starmerella ) (whose members are, for example, Starmerella bombicola . The latter yeast is preferably selected from Pichia pastoris, Yarrowia lipolytica , Saccharomyces cerevisiae and Kluyveromyces lactis Yeast. The latter fungi preferably belong to the genera Rhizopus , Dictyostelium , Penicillium , Mucor or Aspergillus . Plant cells include flowering and non- Flowering plant cells, as well as algal cells such as Chlamydomonas, Chlorella, etc. Preferably, the plants are tobacco, alfalfa, rice, tomato, cotton, rapeseed, soybean, corn or cereal Plants. The latter animal cells are preferably derived from non-human mammals (eg, cattle, buffalo, pigs, sheep, mice, rats), birds (eg, chickens, ducks, ostriches, turkeys, pheasants), fish ( For example, swordfish, salmon, tuna, sea bass, trout, catfish), invertebrates (for example, lobster, crab, shrimp, clams, oysters, mussels, sea urchins), reptiles (for example, snakes, alligators, turtles) ), amphibians (e.g. frogs) or insects (e.g. flies, nematodes) or are genetically modified cell lines derived from human cells excluding embryonic stem cells. Both human and non-human mammalian cells are preferably selected from epithelial cells containing An enumeration of cells, eg, mammary epithelial cells, embryonic kidney cells (eg, HEK293 or HEK 293T cells), fibroblasts, COS cells, Chinese Hamster Ovary (CHO) cells, murine myeloma cells such as N20, SP2/0 or YB2/0 cells, NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, as described in WO21067641. The latter insect cells are preferably derived from Spodoptera frugiperda, for example, Sf9 or Sf21 cells, Bombyx mori, Mamestra brassicae, Trichoplusia ni, etc., such as BTI- TN-5B1-4 cells or Drosophila melanogaster, eg Drosophila S2 cells. The latter protozoan cells are preferably Leishmania tarentolae cells.

In a preferred embodiment of the method and/or cell of the present invention, the cell is a live Gram-negative bacterium comprising reduced or eliminated poly-N-acetyl-glucosamine ( PNAG), Enterobacterial Common Antigen (ECA), Cellulose, Colanic Acid, Core Oligosaccharide, Osmoregulated Periplasmic Glucan (OPG), Glucosylglycerol, Polyglycerol Synthesis of sugars and/or trehalose.

In more preferred embodiments of the method and/or cell, the reduction or elimination of poly-N-acetyl-glucosamine (PNAG), enterobacterial common antigen (ECA), cellulose, kolaric acid, core oligosaccharide, osmotic The synthesis of pressure-regulated interstitial glucan (OPG), glucosylglycerol, glycan and/or trehalose is involved in the synthesis of the poly-N-acetyl-glucosamine (PNAG), enterobacterial common antigen (ECA), fiber Mutation of any one or more glycosyltransferases synthesized in any one of oxalate, kolaric acid, core oligosaccharide, osmotic pressure-regulating interstitial glucan (OPG), glucosylglycerol, glycan, and/or trehalose. Provided, wherein the mutation provides deletion or lower performance of any one of the glycosyltransferases. The glycosyltransferase includes a glycosyltransferase gene encoding a poly-N-acetyl-D-glucosamine synthase subunit, UDP-N-acetylglucosamine-undec isoprene-phosphate N-ethyl Acetylglucosamine phosphotransferase, Fuc4NAc (4-acetamido-4,6-dideoxy-D-galactose) transferase, UDP-N-acetyl-D-mannosamine uronyltransferase (UDP-N-acetyl-D-mannosaminuronic acid transferase), glycosyltransferase gene encoding the catalytic subunit of cellulose synthase, cellulose biosynthesis protein, colanic acid biosynthesis glucuronosyltransferase ), colaric acid biosynthetic galactosyltransferase, colaric acid biosynthetic fucosyltransferase, UDP-glucose:undec isoprene-phosphoglucose-1-phosphotransferase, putative colonic biosynthesis Glycosyltransferase (putative colanic biosynthesis glycosyl transferase), UDP-glucuronate: LPS (HepIII) glycosyl transferase, ADP-heptose-LPS heptosyltransferase (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 glucose Syltransferase I, lipopolysaccharide core heptosyltransferase 3, β-1,6-galactofuranosyltransferase (β-1,6-galactofuranosyltransferase), undecyisoprene-phosphate 4-deoxy- 4-Methylsulfonamide-L-arabinosyltransferase, lipid IVA 4-amino-4-deoxy-L-arabinosyltransferase, bactoprenol glucosyl transferase, pyrimidine Family 2 glycosyltransferases, osmotic pressure-regulated interstitial glucan (OPG) biosynthesis G, OPG biosynthesis H, glucosylglycerate phosphorylase, glycogen synthase, 1, 4-α-glucan branching enzyme, 4-α-glucanotransferase (4-α-glucanotransferase) and trehalose-6-phosphate synthase. in an exemplary embodiment. Cells containing 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 in any one or more of the glycosyltransferases, wherein the mutation provides the glycosyltransferase Either the absence or lower performance.

In alternative and/or additional preferred embodiments of the method and/or cell, the reduction or elimination of poly-N-acetyl-glucosamine (PNAG) synthesis is by overexpression of a gene encoding a carbon storage regulator, Na+ Deletion of the gene encoding the /H+ reverse transport regulator and/or deletion of the gene encoding the sensor histidine kinase is provided.

Microorganisms or cells as described herein can be cultured on monosaccharides, disaccharides, oligosaccharides, polysaccharides, polyols, glycerol, complex media including molasses, corn steep liquor, proteus, trypsin, yeast extract, or mixtures thereof (eg, mixed Feedstock, preferably mixed monosaccharide feedstock such as hydrolyzed sucrose) as the main carbon source for growth. The term "complex medium" refers to its undefined precise constituent medium. This term primarily refers to the most important carbon source for the biological product of interest, biomass formation, carbon dioxide and/or by-product formation (eg, acids and/or alcohols such as acetate, lactic acid, and/or ethanol), i.e. 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99% of all required carbons come from the carbon sources referred to above. In an embodiment of the present invention, the carbon source is the sole carbon source of the organism, ie 100% of all required carbon is derived from the carbon source referred to above. Usually major carbon sources include but are not limited to glucose, glycerol, fructose, maltose, lactose, arabinose, malto-oligosaccharide, maltotriose, sorbitol, xylose, rhamnose, sucrose, galactose, mannose, Methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn infusion, high fructose syrup, acetate, citrate, lactic acid and pyruvic acid. The term complex medium refers to its undefined precise constituent medium. Examples are molasses, corn steep liquor, egg whites, trypsin or yeast extract.

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

In another embodiment of the method according to the invention, the conditions allowing the production of the disaccharide or oligosaccharide having N-acetylglucosamine units at the reducing end include the use of a medium comprising at least one precursor and/or acceptor for production A disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end. Preferably, the medium comprises at least one precursor selected from the group comprising lactose, galactose, fucose, sialic acid.

According to alternative and/or additional embodiments of the methods of the present invention, the conditions allowing the production of the disaccharide or oligosaccharide having N-acetylglucosamine units at the reducing end include adding to the culture medium at least one precursor and/or acceptor feedstock to For the production of disaccharides or oligosaccharides with N-acetylglucosamine units at the reducing end.

According to an alternative embodiment of the method of the present invention, the conditions allowing the production of the disaccharide or oligosaccharide having N-acetylglucosamine units at the reducing end include culturing the cells of the invention with a medium to produce N-acetylglucosamine units at the reducing end The disaccharide or oligosaccharide, wherein the medium lacks any precursor and/or acceptor for the production of the disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end, and further in combination with adding to the medium at least one Precursor and/or acceptor feedstock for producing the disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end.

In a preferred embodiment, a method for producing a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end, as described herein, comprises at least one of the following steps: i) using a method comprising at least a precursor and/or acceptor medium; ii) adding at least one precursor and/or acceptor feedstock to the medium in a reactor, wherein the total reactor volume ranges from 250 mL (milliliters) to 10,000 m ³ (cubic meters) ), preferably in a continuous manner, and preferably such that the final volume of the medium does not exceed three times the volume of the medium before adding the precursor and/or recipient feedstock, preferably not more than twice, more preferably less than twice; iii) adding at least one precursor and/or acceptor feed to the medium in the reactor, preferably in a continuous manner, and preferably such that the final volume of the medium does not exceed the medium prior to the addition of the precursor and/or acceptor feed three times the volume, preferably no more than twice, more preferably less than twice, and wherein preferably the pH of the precursor and/or recipient feedstock is set between 3 and 7, and wherein preferably the pH The temperature of the precursor and/or recipient feedstock is maintained between 20°C and 80°C; iv) by means of feeding the solution during 1 day, 2 days, 3 days, 4 days, 5 days with adding at least one precursor and/or acceptor feedstock to the culture medium in a continuous manner; v) adding at least one precursor in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of the feeding solution and/or recipient feedstock to the culture medium, and wherein preferably the pH of the feed solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is maintained between 20°C and 80°C Between C; the method produces disaccharides or oligosaccharides having N-acetylglucosamine units at the reducing end at a concentration in the final culture 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 another and/or further preferred embodiment, a method for producing a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end, as described herein, comprises at least one of the following steps: i) Use medium containing at least 50 grams, more preferably at least 75 grams, more preferably at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor volume is 250 mL to 10,000 m ³ (cubic meters); ii) at least one precursor and/or acceptor is added to the culture medium in one pulse delivery or discontinuously (pulse delivery) with a total reactor volume ranging from 250 mL ( milliliters) to 10,000 ^m3 (cubic meters), preferably such that the final volume of the medium is no more than three times, preferably no more than twice the volume of the medium before the addition of the precursor and/or recipient feedstock pulse, more preferably less than twice; iii) adding at least one precursor and/or acceptor feedstock to the culture medium in the reactor in a single pulse delivery or in a discontinuous (pulsed delivery) manner with a total reactor volume ranging from 250 mL (milliliters) to 10,000 ^m3 (cubic meters), preferably such that the final volume of the medium does not exceed three times, preferably not more than twice, more preferably less than twice the volume of the medium before adding the precursor and/or recipient feedstock pulse, And wherein the pH of the pulse of the precursor and/or the recipient feedstock is set between 3 and 7, and wherein preferably, the temperature of the precursor and/or the recipient feedstock pulse is maintained between 20°C and 80°C. iv) by means of feeding solution at 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days Add at least one precursor and/or acceptor feedstock to the culture medium in a discontinuous (pulsed delivery) manner; v) by means of feeding solution at 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, at least one precursor and/or acceptor feedstock is added to the culture medium in a discontinuous (pulsed) manner over the course of 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, and wherein The pH of the feed solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is maintained between 20°C and 80°C; the process produces N-acetylglucose at the reducing end A disaccharide or oligosaccharide of amine units at a concentration in the final culture 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 a further preferred embodiment, a method for producing a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end, as described herein, comprises at least one of the following steps: i) using Medium containing at least 50 grams, more preferably at least 75 grams, more preferably at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor volume ranges from 250 mL up to 10,000 m ³ (cubic meters); ii) addition to contain at least 50 grams, more preferably at least 75 grams, more preferably at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams per liter of initial reactor volume added Lactose feed to medium for lactose, wherein the total reactor volume ranges from 250 mL (milliliters) to 10,000 ^m3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium does not exceed that prior to addition of the lactose feed three times the volume of medium, preferably not more than twice, more preferably less than twice; iii) adding at least 50 grams per liter of initial reactor volume, more preferably at least 75 grams, more preferably at least 100 grams, more preferably Lactose feed to medium of at least 120 grams, more preferably at least 150 grams of lactose, with reactor volumes ranging from 250 mL to 10,000 ^m3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium does not vary. more than three times the volume of the medium before adding the lactose feedstock, preferably no more than twice, more preferably less than twice, and wherein preferably the pH of the lactose feedstock is set between 3 and 7, and wherein preferably The temperature of the lactose feedstock is maintained between 20°C and 80°C; iv) by means of feeding the solution in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days Lactose feedstock to the culture medium; v) adding lactose feedstock to the culture medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of the feed solution, and wherein the concentration of the lactose feed solution 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, better is 225 g/L, better is 250 g/L, better is 275 g/L, better is 300 g/L, better is 325 g/L, better is 350 g/L L, better 375 g/L, better 400 g/L, better 450 g/L, better 500 g/L, still better 550 g/L, better 600 g/L And wherein preferably, the pH of this feed solution is set between 3 and 7, and wherein preferably, the temperature of this feed solution is maintained between 20 ℃ and 80 ℃; This method produces in reducing A disaccharide or oligosaccharide with an N-acetylglucosamine unit at the end at a concentration of at least 50 g/L, preferably at least 75 g/L in the final culture 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 L.

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

In a preferred embodiment, the carbon source (preferably sucrose) is provided in the medium for 3 days or more, preferably up to 7 days; and/or the medium is provided in a continuous manner per liter of initial culture volume at least 100 grams, preferably at least 105 grams, more preferably at least 110 grams, still more preferably at least 120 grams of sucrose, so that the final volume of the medium does not exceed three times, preferably not more than twice, the volume of the medium before culturing, More preferably, it is less than twice.

Preferably, when carrying out the method as described herein, a first step of exponential cell growth is provided by adding a carbon source (preferably glucose or sucrose) to the culture medium prior to adding the precursor to the culture in the second stage. stage.

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

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

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

A method for producing a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end as described herein, further comprising: isolating the disaccharide having a GlcNAc unit at the reducing end from the cell or the medium in which it is grown or oligosaccharide step.

Use and "separating" refer to harvesting, collecting or recovering disaccharides or oligosaccharides or mixtures thereof having GlcNAc units at the reducing end from cells and/or the medium in which they are grown.

Disaccharides or oligosaccharides having GlcNAc units at the reducing end can be isolated from the aqueous medium in which the cells are grown in a conventional manner. In the event that the disaccharide or oligosaccharide having GlcNAc units at the reducing end is still present in the cells producing the disaccharide or oligosaccharide having GlcNAc units at the reducing end, then conventional methods can be used to release or extract the di- or oligosaccharide having GlcNAc units at the reducing end Saccharide or oligosaccharide to extracellular, such as using high pH, heat shock, sonication, French crush, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergent, hydrolysis, etc. to destroy cells. The culture medium and/or cell extracts can then be further used together and individually to isolate disaccharides or oligosaccharides having GlcNAc units at the reducing end. This preferably involves clarification of mixtures containing disaccharides or oligosaccharides with GlcNAc units at the reducing end to remove suspended particles and contaminants, especially cells, cellular components, insoluble metabolites and debris generated by culturing genetically modified cells. In this step, mixtures containing disaccharides or oligosaccharides with GlcNAc units at the reducing end can be clarified in a conventional manner. Preferably, the mixture containing disaccharides or oligosaccharides with GlcNAc units at the reducing end is clarified by centrifugation, coagulation, decantation and/or filtration. The further step of separating the disaccharide or oligosaccharide having GlcNAc units at the reducing end from the mixture comprising the disaccharide or oligosaccharide having GlcNAc units at the reducing end preferably comprises separating the mixture of disaccharides or oligosaccharides having GlcNAc units at the reducing end, Preferably after clarification, substantially all proteins are removed, as well as peptides, amino acids, RNA and DNA and any endotoxins and glycolipids that may interfere with subsequent separation steps. In this step, proteins and related impurities can be removed from a mixture containing disaccharides or oligosaccharides with GlcNAc units at the reducing end in a conventional manner. Preferably, by ultrafiltration, nanofiltration, two-phase partition, reverse osmosis, microfiltration, activated carbon or carbon treatment, nonionic surfactant treatment, enzymatic digestion, tangential flow high performance filtration, Tangential flow ultrafiltration, electrophoresis (eg, using slab-polyacrylamide or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE)), affinity chromatography (using affinity ligands including, eg, DEAE -Sepharose, poly-L-lysine and polymyxin-B (polymyxin-B, endotoxin-selective adsorbent matrix), ion exchange chromatography (such as, but not limited to, cation exchange, anion exchange, mixed bed ion exchange, inside-out ligand attachment), hydrophobic interaction chromatography and/or gel filtration (ie, size exclusion chromatography), especially by chromatography, more specifically Removal of proteins, salts, by-products, pigments, endotoxins and others from mixtures containing disaccharides or oligosaccharides with GlcNAc units at the reducing end by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography related impurities. In addition to size exclusion chromatography, proteins and related impurities are retained by the chromatography medium or membrane of choice, while disaccharides or oligosaccharides with GlcNAc units at the reducing end are retained in mixtures containing disaccharides or oligosaccharides with GlcNAc units at the reducing end .

In a further preferred embodiment, the methods as described herein also provide for further purification of disaccharides or oligosaccharides having a GlcNAc unit at the reducing end. Further purification of the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end can be accomplished by, for example, the use of (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange to remove any residual DNA, protein , LPS, endotoxin or other impurities. 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 by, for example, spray drying, freeze drying, spray freeze drying, freeze spray drying, band dry, belt dry, vacuum band dry, vacuum band dry Disaccharides or oligosaccharides with GlcNAc at the reducing end produced by drying (vacuum belt dry), drum drying, drum drying, vacuum drum drying or vacuum drum drying.

In an exemplary embodiment, isolation and purification of the resulting disaccharide or oligosaccharide having N-acetylglucosamine units at the reducing end is performed in a process comprising the following steps in any order: a) Contact the culture or its clarified form with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da to ensure retention of the produced disaccharides or oligosaccharides and allow at least some of the proteins, salts species, by-products, pigments and other related impurities pass through, b) using the membrane to perform a Diafiltration process on the retentate from step a) with an aqueous solution of inorganic electrolyte, then optionally with pure water to remove excess electrolyte, c) and collecting a retentate rich in disaccharides or oligosaccharides having N-acetylglucosamine units at the reducing end as salts from the cations of this electrolyte, d) Preferably, the retentate is dried.

In an alternative exemplary embodiment, the isolation and purification of the resulting disaccharides or oligosaccharides with N-acetylglucosamine units at the reducing end is performed in a process comprising the following steps in any order: using different Membrane two steps of membrane filtration of the culture or its clarified form, wherein one membrane has a molecular weight cut-off between about 300 and about 500 Daltons and the other membrane has a molecular weight cut-off between about 600 and about 800 Daltons between.

In an alternative exemplary embodiment, isolation and purification of the resulting disaccharide or oligosaccharide having N-acetylglucosamine units at the reducing end is performed in a process comprising the following steps in any order: A step of treating the culture or its clarified form with a strong cation exchange resin in the form and a weak anion exchange resin in the free base form.

In an alternative exemplary embodiment, the isolation and purification of the resulting disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end is performed in the following manner. Cultures comprising the resulting disaccharides or oligosaccharides with N-acetylglucosamine units at the reducing end, biomass, media components and contaminants are suitable for the following isolation and purification steps: i) isolating biomass from the culture, ii) cation exchanger treatment for removal of positively charged materials, iii) Anion exchanger treatment for removal of negatively charged materials, iv) a nanofiltration step and/or an electrodialysis step, Therein is provided a purified solution comprising the produced disaccharide or oligosaccharide having a N-acetylglucosamine unit at the reducing end with a purity of greater than or equal to 80%. Optionally, by being selected from the group consisting of spray drying, freeze drying, spray freeze drying, freeze spray drying, belt drying, conveyor belt drying, vacuum belt drying, vacuum belt drying, drum drying, drum drying, vacuum drum drying Any one or more of the recited drying steps of drying and vacuum drum drying are used to dry the purified solution.

In an alternative exemplary embodiment, isolation and purification of the resulting disaccharide or oligosaccharide having N-acetylglucosamine units at the reducing end is performed in a process that includes the following steps in any order: Enzymatic treatment; biomass removal from culture; ultrafiltration; nanofiltration; and column chromatography steps. Preferably, such column chromatography is a single column or multiple columns. More preferably, the column chromatography step is simulated moving bed chromatography. Such simulated moving bed chromatography preferably comprises i) at least 4 columns, at least one of which 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 eluate comprising water; and/or iv) an operating temperature of 15 to 60 degrees Celsius. Preferably, the process further comprises a method selected from the group consisting of spray drying, freeze drying, spray freeze drying, freeze spray drying, belt drying, conveyor belt drying, vacuum belt drying, vacuum belt drying, drum drying, drum drying, vacuum drying Listed steps for tumble drying and vacuum tumble drying.

In particular embodiments, the present invention provides drying by a method selected from the group consisting of spray drying, freeze drying, spray freeze drying, freeze spray drying, belt drying, conveyor belt drying, vacuum belt drying, vacuum belt drying, tumble drying, drum drying The resulting disaccharide or oligosaccharide bearing GlcNAc at the reducing end, dried to powder by any one or more of the enumerated drying steps of drying, vacuum drum drying and vacuum drum drying, wherein the dry powder contains <15%-wt Water, preferably <10%-wt water, more preferably <7%-wt water, most preferably <5%-wt water.

In a third aspect, the present invention provides the use of metabolically engineered cells as described herein for producing disaccharides or oligosaccharides having N-acetylglucosamine units at the reducing end, preferably for producing LNB or LacNAc, More preferably for the production of oligosaccharides containing LNB or LacNAc at the reducing end, yet more preferably for the production of sialylated and/or fucosylated and/or galactosylated and/or GlcNAc-modified forms of LNB Or LacNAc, or preferably an oligosaccharide containing LNB or LacNAc at the reducing end for the generation of sialylated and/or fucosylated and/or galactosylated and/or GlcNAc modified forms.

In another preferred embodiment of the third aspect, metabolically engineered cells as described herein are used to produce neutral disaccharides or oligosaccharides having N-acetylglucosamine units at the reducing end, more preferably at reducing Neutral oligosaccharides containing LNB or LacNAc at the ends.

In another preferred embodiment of the third aspect, the metabolically engineered cells as described herein are used to produce disaccharides and/or oligosaccharides having N-acetylglucosamine (GlcNAc) units at the reducing end as described herein sugar mixture.

In another preferred embodiment of the third aspect, metabolically engineered cells as described herein are used to generate a mixture comprising (i) a disaccharide having N-acetylglucosamine (GlcNAc) units at the reducing end or oligosaccharides (or mixtures as described herein) and (ii) one or more lactose-based mammalian milk oligosaccharides, preferably one or more lactose-based human milk oligosaccharides as described herein.

To identify disaccharides or oligosaccharides with GlcNAc units at the reducing end, monomeric building blocks (eg, monosaccharide or glycan unit composition), mutated configurations of side chains ( anomeric configuration), presence and position of substituents, degree of polymerization/molecular weight and degree of linkage mode can be identified by standard methods known in the art, such as methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas phase chromatogram) Analysis-Mass Spectrometry), MALDI-MS (Interstitial-Assisted Lightning Desorption/Ionization-Mass Spectrometry), ESI-MS (Electrospray Ionization-Mass Spectrometry), HPLC (High Performance Liquid Phase with UV or Refractive Index Detection) chromatography), HPAEC-PAD (high performance anion exchange chromatography with pulsed current detection), CE (capillary electrophoresis), IR (infrared light)/Raman spectroscopy and NMR (nuclear magnetic resonance) spectroscopy techniques. The crystal structure can be resolved using, for example, solid state NMR, FT-IR (Fourier transform infrared spectroscopy) and WAXS (wide angle X-ray scattering). The degree of polymerization (DP), DP distribution and polydispersity can be determined, for example, by viscometry and SEC (SEC-HPLC, high performance size exclusion chromatography). To identify the monomeric constituents of the saccharide method, for example, acid-catalyzed hydrolysis, HPLC (High Performance Liquid Chromatography) or GLC (Gas Liquid Chromatography) (after conversion to alditol acetate) can be used. To determine glycosidic linkages, methylate the sugar with iodomethane and a strong base in DMSO, perform hydrolysis, complete reduction to partially methylated alditol, perform acetylation to methylated alditol acetate salts and analyzed by GLC/MS (gas-liquid chromatography coupled with mass spectrometry). To determine the oligosaccharide sequence, partial depolymerization is performed using acids or enzymes to determine the structure. To identify the mutator configuration, the oligosaccharide is subjected to enzymatic analysis, for example, by contacting it with an enzyme specific for a particular type of bond (eg, β-galactosidase or α-glucosidase, etc.), Product analysis can be performed using NMR.

including the reducing end with N- Products of disaccharides or oligosaccharides of acetylglucosamine units

In some embodiments, a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end (or a mixture as described herein) produced as described herein is incorporated into food (eg, human food or feed), diet In supplements, pharmaceutical ingredients, cosmetic ingredients or pharmaceuticals. In some embodiments, the disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end is mixed with one or more ingredients suitable for use in food, feed, dietary supplements, pharmaceutical ingredients, cosmetic ingredients, or pharmaceuticals.

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

A "prebiotic" is a substance that promotes the growth of microorganisms that are beneficial to the host, especially in the gastrointestinal tract. In some embodiments, dietary supplements provide a variety of prebiotics, including disaccharides or oligosaccharides with N-acetylglucosamine units at the reducing end produced and/or purified by the processes disclosed in this specification, to promote one or Growth of a variety of beneficial microorganisms. Examples of prebiotic ingredients used in dietary supplements include other prebiotic molecules (eg, HMOs) and plant polysaccharides (eg, inulin, pectin, b-glucans, and xylo-oligosaccharides). "Probiotic" products typically contain live microorganisms that replace or add to the gastrointestinal microbiota to benefit the recipient. Examples of such microorganisms include Lactobacillus species (eg, L. acidophilus and L. bulgaricus ), Lactobacillus species (eg, B. animalis ) , B. longum and B. infantis (e.g. Bi-26)) and Saccharomyces boulardii. In some embodiments, disaccharides or oligosaccharides having N-acetylglucosamine units at the reducing end produced and/or purified by the processes of the present specification are administered orally in combination with such microorganisms.

Examples of other ingredients of dietary supplements include disaccharides (eg, lactose), monosaccharides (eg, glucose and galactose), thickeners (eg, acacia), acidity regulators (eg, trisodium citrate), Water, skim milk and flavouring.

In some embodiments, a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end (or a mixture as described herein) is incorporated into human infant food (eg, infant formula). Infant formula is usually an artificial food used to feed infants as a complete or partial replacement for human breast milk. In some embodiments, the infant formula is sold as a powder and prepared in an infant feeding bottle or cup by mixing with water. The ingredients of infant formula are often designed to roughly mimic human breast milk. In some embodiments, disaccharides or oligosaccharides (or mixtures as described herein) having N-acetylglucosamine units at the reducing end produced and/or purified by the methods of this specification are included in infant formula to Provides nutritional benefits similar to those provided by oligosaccharides in human breast milk. In some embodiments, a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end is mixed with one or more ingredients of the infant formula. Examples of infant formula ingredients include skim milk, carbohydrate sources (eg, lactose), protein sources (eg, whey protein concentrate and casein), fat sources (eg, vegetable oils - eg, palm, high oleic safflower oil, vegetable oil) seed oil, coconut oil, and/or sunflower oil; and fish oil), vitamins (eg, vitamins A, Bb, Bi2, C, and D), minerals (eg, potassium citrate, calcium citrate, magnesium chloride, sodium chloride , sodium citrate and calcium phosphate) and possibly human milk oligosaccharides (HMO). Such HMOs may include, for example, DiFL, lacto-N-trisaccharide II, LNT, LNnT, lacto-N-fucopentaose I, lacto-N-neofucopentose, lacto-N-fucopentaose II, Lacto-N-fucopentaose III, Lacto-N-fucopentaose V, Lacto-N-neofucopentose V, Lacto-N-difucohexaose I, Lacto-N-difucohexaose Sugar II, 6'-galactosylose, 3'-galactosyllose, lacto-N-hexaose and lacto-N-neohexaose.

In some embodiments, the one or more infant formula ingredients include skim milk, carbohydrate sources, protein sources, fat sources, and/or vitamins and minerals.

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

In some embodiments, the concentration of disaccharides or oligosaccharides (or mixtures as described herein) having N-acetylglucosamine units at the reducing end in the infant formula is about the same as the concentration of oligosaccharides typically present in human breast milk. In some embodiments, the disaccharide or oligosaccharide concentration in the infant formula is about the same as the concentration of oligosaccharides typically present in human breast milk.

In some embodiments, a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end (or a mixture as described herein) is incorporated into a feed formulation, wherein the feed is selected from the group consisting of pet food, animal milk replacer , veterinary product, post-weaning feed, or an enumeration of trough feed.

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

"Immunomodulatory" is a substance that alters the immune response or the function of the immune system. "Immunomodulatory" components alter the immune response by increasing (immunostimulatory) or decreasing (immunosuppressive) serum antibody production. Immunostimulants are used to enhance, for example, immune responses against infectious diseases, tumors, primary or secondary immune deficiencies, and altered antibody transfer. Immunosuppressive components are used to reduce the immune response to transplanted organs and to treat autoimmune diseases such as pemphigus, lupus or allergies. In some embodiments, the immunomodulatory component has anti-inflammatory activity. In some embodiments, foods, feeds, dietary supplements, pharmaceutical ingredients and/or drugs provide various immunomodulatory agents including N-acetylglucosamine at the reducing end produced and/or purified by the processes disclosed in this specification Units of disaccharides or oligosaccharides (or mixtures as described herein) to adapt the immune system to normal function. Immunity varies widely across distinguishable life stages. Different food components influence specific immune responses, depending on biased metabolic processes, as well as consumer and patient characteristics. Foods with added immunomodulatory ingredients are also called functional foods. Functional foods are foods that have specific health benefits for specific consumer groups. Examples of immunomodulatory ingredients present in functional foods as well as in feed and dietary supplements include other immunomodulatory molecules such as disaccharides or oligosaccharides and fatty acids (PUFAs) having an N-acetylglucosamine unit at the reducing end as specified in this specification. ), fish oil, amino acids (eg, arginine and glutamine), lectins (eg, sequestrin), vitamins (eg, vitamins A, B6, C, E, thiamine, folic acid), and minerals Substances (eg, zinc). Such disaccharides or oligosaccharides with N-acetylglucosamine units at the reducing end may include LNB, LacNAc, poly-LacNAc and Lewis X, Lewis Y and sialic acid Lewis X epitopes. In addition, examples of immunomodulatory components present in pharmaceutical ingredients and drugs include other immunomodulatory molecules such as, for example, disaccharides or oligosaccharides having N-acetylglucosamine units at the reducing end as specified in this specification, as well as interleukins, lipids Polysaccharides, dextran, interferon gamma and specific antibodies. Such disaccharides or oligosaccharides with N-acetylglucosamine units at the reducing end present in drug mixtures and/or drugs may include LNB, LacNAc, poly-LacNAc and Lewis X, Lewis Y and sialic acid Lewis X epitopes bit.

Embodiments disclosed in the context of one aspect of the invention are also disclosed in the context of all other aspects of the invention, unless expressly stated otherwise.

Unless otherwise defined, 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 invention belongs. In general, the phraseology used herein and the contextual descriptions of laboratory procedures in cell culture, molecular genetics, organic and nucleic acid chemistry, and hybridization are those well known and commonly used in the art. Nucleic acids and peptides are synthesized using standard techniques. In general, purification steps are performed according to the manufacturer's instructions.

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

The present invention relates to the following preferred embodiments: 1. A method for producing a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end by a cell, the method comprising the steps of: a. providing a cell capable of ( i) synthesizing nucleotide-sugar, (ii) synthesizing N-acetylglucosamine, and (iii) saccharifying the N-acetylglucosamine monosaccharide, b. culturing under conditions that allow the production of the disaccharide or oligosaccharide The cells, c. isolate the desired disaccharide or oligosaccharide from the culture. 2. The method of embodiment 1, wherein the cell expresses at least one N-acetylglucosamine-6-phosphotransferase and phosphatase to synthesize the monosaccharide N-acetylglucosamine. 3. The method according to any one of embodiments 1 and 2, wherein the cell expresses at least one glycosyltransferase to glycosylate N-acetylglucosamine. 4. The method according to any one of embodiments 1 to 3, wherein the cell is genetically engineered to produce the disaccharide or oligosaccharide. 5. The method according to any one of embodiments 1 to 4, wherein the cell is modified in the expression or activity of an enzyme selected from the group comprising: N-acetylglucosamine-6-phosphotransferase , phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphate transaminase and UDP-glucose 4-epimerase. 6. The method according to any one of embodiments 1 to 5, wherein the nucleotide-sugar is selected from the group comprising: 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), CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), UDP-glucuronic acid Salt, UDP-galacturonate, GDP-rhamnose, and UDP-xylose. 7. The method according to any one of embodiments 1 to 6, wherein the nucleotide-sugar is UDP-galactose, and the glycosyltransferase is N-acetylglucosamine b-1,3-galactosyl transferase or N-acetylglucosamine b-1,4-galactosyltransferase. 8. The method according to any one of embodiments 1 to 7, wherein the disaccharide is lacto-N-disaccharide (Gal-b1,3-GlcNAc) or N-acetyllactosamine (Gal-b1,4-GlcNAc) ), or the oligosaccharide has lacto-N-disaccharide (Gal-b1,3-GlcNAc) or N-acetyllactosamine (Gal-b1,4-GlcNAc) at the reducing end. 9. The method according to any one of embodiments 1 to 8, wherein the N-acetylglucosamine b-1,3-galactosyltransferase is a glycosyltransferase having: a. PFAM domain PF00535, and i) comprising the sequence of SEQ ID NO 01 [AGPS]XXLN(X _n )RXDXD, wherein X is any amino acid, wherein n is 12 to 17, or ii) comprising the sequence of SEQ ID NO 02 PXXLN(X _n )RXDXD (X _m )[FWY]XX[HKR]XX[NQST], wherein X is any amino acid, wherein n is 12 to 17 and m is 100 to 115, or iii) including according to SEQ ID NOs: 03, 04, A polypeptide sequence of any of 05, 06, 07 or 08, or iv) a functional homologue, variant or derivative of any of SEQ ID NO: 03, 04, 05, 06, 07 or 08 , which has at least 80% of the overall sequence of the full length of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide of any of SEQ ID NOs: 03, 04, 05, 06, 07 or 08 is identical and has N-acetylglucosamine b-1,3-galactosyltransferase activity, or v) comprises at least 8 from any of SEQ ID NOs: 03, 04, 05, 06, 07 or 08 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 oligopeptide sequences of consecutive amino acid residues with N-acetylglucosamine b-1,3- Galactosyltransferase activity, or b. PFAM domain IPR002659, and i) comprising the sequence of SEQ ID NO 09 KT( _Xn )[FY]XXKXDXD( _Xm )[FHY]XXG(X, no A, G, S )(X _p )X (no F, H, W, Y) [DE]D[ILV]XX[AG], wherein X is any amino acid, 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 of SEQ ID NOs: 10, 11, 12 or 13, or iii) is any of SEQ ID NOs: 10, 11, 12 or 13 A functional homologue, variant or derivative having any of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptides of SEQ ID NO: 10, 11, 12 or 13 At least 80% overall sequence identity of full length and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or iv) comprising from any of SEQ ID NOs: 10, 11, 12 or 13 of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 Oligopeptide sequence of 0 consecutive amino acid residues with N-acetylglucosamine b-1,3-galactosyltransferase activity. 10. The method according to any one of embodiments 1 to 8, wherein the N-acetylglucosamine b-1,4-galactosyltransferase is a glycosyltransferase having: a. PFAM domain PF01755, and i) comprising the sequence EXXCXXSHXX[ILV][FWY]( _Xn )EDD( _Xm )[ACGST]XXYX[ILMV] of SEQ ID NO 14, wherein X is any amino acid, wherein n is 13 to 15 and m is 50 to 76, or ii) comprising a polypeptide sequence according to any of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23, or iii) being SEQ ID NOs: 15, 16, A functional homologue, variant or derivative of any of 17, 18, 19, 20, 21, 22 or 23 having the same The N-acetylglucosamine b-1,4-galactosyltransferase polypeptide full-length of any one of 22 or 23 has at least 80% overall sequence identity and has N-acetylglucosamine b-1,4 - galactosyltransferase activity, or iv) comprising at least 8, 9, 10, 11, 12, Oligopeptide sequences of 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues, and N-acetylglucosamine b-1,4-galactosyltransferase activity, or b. PFAM domain PF00535, and i) comprising the sequence of SEQ ID NO 24 R[KN]XXXXXXXGXXXX[FL]XDXD( _Xn )[FHW]XXX[FHNY]( _Xm )E[DE], where X is any amino acid , wherein n is 50 to 75 and m is 10 to 30, or ii) the sequence comprising SEQ ID NO 25 R[KN]XXXXXXXGXXXXFXDXD( _Xn )[FHW]XXX[FHNY]( _Xm )E[DE](X _p ) [FWY]XX[HKR]XX[NQST], wherein X is any amino acid, wherein n is 50 to 75, m is 10 to 30, and p is 20 to 25, or iii) including according to SEQ ID NO : the polypeptide sequence of any one of 26, 27 or 28, or iv) is a functional homologue, variant or derivative of any one of SEQ ID NO: 26, 27 or 28 having the same : at least 80% overall sequence identity of any of the full-length N-acetylglucosamine b-1,4-galactosyltransferase polypeptides of 26, 27 or 28 and having N-acetylglucosamine b-1 ,4-galactosyltransferase activity, or v) comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acids from any of SEQ ID NOs: 26, 27 or 28 oligopeptide sequence of residues with N-acetylglucosamine b-1,4-galactosyltransferase activity, or c. PFAM domain PF02709 and no PFAM domain PF00535, and i) comprising SEQ ID NO 29 the sequence [FWY]XX[FY][FWY]( _X23 )[FWY][GQ]X[DE]D, where X is any amino acid, or ii) the sequence [PV]W[ GHNP]( _Xn )[FWY][GQ]X[DE]D, wherein X is any amino acid, wherein n is 21 to 24, or iii) including according to SEQ ID NOs: 31, 32, 33, 34, The polypeptide sequence of any of 35 or 36, or iv) is a functional homologue, variant or derivative of any of SEQ ID NO: 31, 32, 33, 34, 35 or 36, which has a Any of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides of SEQ ID NO: 31, 32, 33, 34, 35 or 36 have at least 80% overall sequence identity over the full length and have N-acetylglucosamine b-1,4-galactosyltransferase activity, or v) comprising at least 8, 9, 10 from any of SEQ ID NOs: 31, 32, 33, 34, 35, or 36 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 oligopeptide sequences of consecutive amino acid residues with N-acetylglucosamine b-1,4-galactosylation Enzymatic activity, or d. PFAM domain PF03808, and i) comprising the sequence of SEQ ID NO 37 [ST][FHY]XN( _Xn )DG(X16)[HKR]X[ST] _FDXX [ST]XA, wherein X is any amino acid and wherein n is 20 to 25, or ii) the sequence comprising SEQ ID NO 38 [ST][FHY]XN( _Xn )DG(X16)[HKR]X[ST] _FDXX [ ST]XA(X _m )[HR]XG[FWY](X _p )GXGXXXQ[DE], where X is any amino acid, where n is 20 to 25, m is 40 to 50, and p is 22 to 30 , or iii) comprising a polypeptide sequence according to any of SEQ ID NO: 39, 40 or 41, or iv) being a functional homologue, variant or Derivatives having the N with SEQ ID NO: 39, 40 or 41 - At least 80% overall sequence identity of any of the full-length acetylglucosamine b-1,4-galactosyltransferase polypeptides and having N-acetylglucosamine b-1,4-galactosyltransferase activity , or v) comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amine groups from any of SEQ ID NOs: 39, 40, or 41 Oligopeptide sequence of acid residues with N-acetylglucosamine b-1,4-galactosyltransferase activity. 11. The method according to any one of the preceding embodiments, wherein a. the N-acetylglucosamine-6-phosphotransferase is a polypeptide sequence comprising the polypeptide of UniProt ID P43577, or is the functionality of the polypeptide of UniProt ID P43577 A homologue, variant or derivative having at least 80% overall sequence identity with the full-length polypeptide of UniProt ID P43577 and having N-acetylglucosamine-6-phosphotransferase activity, and b. the L- Glutamine-D-fructose-6-phosphate transaminase is a polypeptide sequence comprising the polypeptide of UniProt ID P17169, or is a functional homologue, variant or derivative of the polypeptide having UniProt ID P17169, which has the same At least 80% overall sequence identity to the full length of the polypeptide of ID P17169 and having L-glutamine-D-fructose-6-phosphate transaminase activity; or being mutated A39T, R250C and G472S different from the UniProt ID P17169 polypeptide Retouched version. 12. The method according to any one of the preceding embodiments, wherein the cell can metabolize a carbon source selected from the group comprising the following enumeration: glucose, fructose, galactose, lactose, sucrose, maltose, malto-oligosaccharide, Maltotriose, sorbitol, xylose, rhamnose, mannose, methanol, ethanol, arabinose, trehalose, starch, cellulose, hemicellulose, molasses, corn infusion, high fructose syrup, glycerin, acetic acid Salt, citrate, lactic acid, pyruvic acid. 13. The method according to any one of the preceding embodiments, wherein the cell is unable to convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or is unable to convert glucosamine-6-phosphate Salt is converted to fructose-6-phosphate. 14. The method according to any one of the preceding embodiments, wherein the cell is modified to produce GDP-fucose. 15. The method according to any one of the preceding embodiments, wherein the cell is modified to promote GDP-fucose production, wherein the modification is selected from the group comprising the following enumeration: UDP-glucose:undec isoprene- Shaving of the gene encoding phosphoglucose-1-phosphotransferase, overexpression of the gene encoding GDP-L-fucose synthase, overexpression of the gene encoding GDP-mannose 4,6-dehydratase, mannose-1- Overexpression of a gene encoding phosphoguanylate, overexpression of a gene encoding phosphomannose mutase, or overexpression of a gene encoding mannose-6-phosphate isomerase. 16. The method according to any one of the preceding embodiments, wherein the cell is modified to produce UDP-galactose. 17. The method according to any one of the preceding embodiments, wherein the cell is modified to promote UDP-galactose production, wherein the modification is selected from the group comprising the following enumeration: 5'-nucleotidase/UDP-sugar hydrolysis Shaving of the gene encoding the enzyme or shaving of the gene encoding the galactose-1-phosphate uridine transferase. 18. The method according to any one of the preceding embodiments, wherein the cell is modified to produce CMP-N-acetylneuraminic acid. 19. The method according to any one of the preceding embodiments, wherein the cell is modified to promote CMP-N-acetylneuraminic acid production, wherein the modification is selected from the group comprising the following enumeration: CMP-sialic acid synthase Overexpression of encoding gene, overexpression of gene encoding sialic acid synthase, overexpression of gene encoding N-acetyl-D-glucosamine 2-epimerase. 20. The method according to any one of the preceding embodiments, wherein the cell is capable of expressing at least one other glycosyltransferase, and wherein the other glycosyltransferase is selected from the group comprising: fucosyltransferase, saliva Acid transferase, Galactosyltransferase, Glucosyltransferase, Mannosyltransferase, N-Acetylglucosaminyltransferase, N-Acetylgalactosyltransferase, N-Acetylmannose Aminyltransferase, xylosyltransferase, glucuronidyltransferase, galacturonyltransferase, glucosamine transferase, N-glycolyl neuraminidase, rhamnosyltransferases. 21. The method of embodiment 20, wherein the cell is modified in the expression or activity of the other glycosyltransferase. 22. The method according to any one of the preceding embodiments, wherein the oligosaccharide is selected from the list comprising: 2-fucosyl lacto-N-disaccharide, 4-fucosyl lacto-N-disaccharide, 2-4-Difucosyllacto-N-disaccharide, 3'-Sialylolide-N-disaccharide, 6'-Sialylolide-N-disaccharide, 3',6'-disialolactoide -N-disaccharide, 6,6'-disialo-N-disaccharide, 2'-fucosyl-3'-sialo-N-disaccharide, 2'-fucosyl-6 '-Sialyllacto-N-disaccharide, 4-fucosyl-3'-sialylacto-N-disaccharide, 4-fucosyl-6'-sialylacto-N-disaccharide, 2 - Fucosyl N-acetyllactosamine, 3'-fucosyl N-acetyllactosamine, 2,3'-difucosyl N-acetyllactosamine, 3'-sialic acid N- Acetylactosamine, 6'-Sialyl-N-Acetyl-Lactosamine, 3',6'-Disialo-N-Acetyl-Lactosamine, 6,6'-Disialo-N-Acetyl-Lactosamine, 2' - Fucosyl-3'-sialo-N-acetyllactosamine, 2'-fucosyl-6'-sialo-N-acetyllactosamine, 3-fucosyl-3'-sialic acid N-acetyllactosamine, 3'-fucosyl-6'-sialic acid N-acetyllactosamine, P1 trisaccharide (Gal-a1,4-Gal-b1,4-GlcNAc), allograft antigen Epitope (Gal-a1,3-Gal-b1,4-GlcNAc), Gal-b14-(Galb13)-GlcNAc, poly-N-acetyllactosamine, GalNAc-b1,3-Gal-b1,4-GlcNAc . 23. A metabolically engineered cell capable of (i) synthesizing a nucleotide-sugar, (ii) synthesizing N-acetylglucosamine, and (iii) glycating the N-acetylglucosamine monosaccharide, wherein the cell produces a reduced A disaccharide or oligosaccharide with an N-acetylglucosamine unit at the end. 24. The cell according to embodiment 23, wherein the cell expresses at least one N-acetylglucosamine-6-phosphotransferase and phosphatase to synthesize N-acetylglucosamine. 25. The cell according to any one of embodiments 23 and 24, wherein the cell expresses at least one glycosyltransferase to glycosylate N-acetylglucosamine. 26. The cell according to any one of embodiments 23 to 25, wherein the cell is modified in the expression or activity of an enzyme selected from the group consisting of: N-acetylglucosamine-6-phosphotransferase, phosphoric acid Enzymes, glycosyltransferases, L-glutamine-D-fructose-6-phosphate transaminase and UDP-glucose 4-epimerase. 27. The cell according to any one of embodiments 23 to 26, wherein the nucleotide-sugar is selected from the group comprising: 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), CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), UDP-glucuronic acid Salt, UDP-galacturonate, GDP-rhamnose, and UDP-xylose. 28. The cell according to any one of embodiments 23 to 27, wherein the nucleotide-sugar is UDP-galactose, and the glycosyltransferase is N-acetylglucosamine b-1,3-galactosyl transferase or N-acetylglucosamine b-1,4-galactosyltransferase. 29. The cell according to any one of embodiments 23 to 28, wherein the disaccharide is lacto-N-disaccharide (Gal-b1,3-GlcNAc) or N-acetyllactosamine (Gal-b1,4-GlcNAc) ), or wherein the oligosaccharide has lacto-N-disaccharide (Gal-b1,3-GlcNAc) or N-acetyllactosamine (Gal-b1,4-GlcNAc) at the reducing end. 30. The cell according to any one of embodiments 23 to 29, wherein the N-acetylglucosamine b-1,3-galactosyltransferase is a glycosyltransferase having: a. PFAM domain PF00535, and i) comprising the sequence of SEQ ID NO 01 [AGPS]XXLN(X _n )RXDXD, wherein X is any amino acid, wherein n is 12 to 17, or ii) comprising the sequence of SEQ ID NO 02 PXXLN(X _n )RXDXD (X _m )[FWY]XX[HKR]XX[NQST], wherein X is any amino acid, wherein n is 12 to 17 and m is 100 to 115, or iii) including according to SEQ ID NOs: 03, 04, A polypeptide sequence of any of 05, 06, 07 or 08, or iv) a functional homologue, variant or derivative of any of SEQ ID NO: 03, 04, 05, 06, 07 or 08 , which has at least 80% of the overall sequence of the full length of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide of any of SEQ ID NOs: 03, 04, 05, 06, 07 or 08 is identical and has N-acetylglucosamine b-1,3-galactosyltransferase activity, or v) comprises at least 8 from any of SEQ ID NOs: 03, 04, 05, 06, 07 or 08 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 oligopeptide sequences of consecutive amino acid residues with N-acetylglucosamine b-1,3- Galactosyltransferase activity, or b. PFAM domain IPR002659, and i) comprising the sequence of SEQ ID NO 09 KT( _Xn )[FY]XXKXDXD( _Xm )[FHY]XXG(X, no A, G, S )(X _p )X (no F, H, W, Y) [DE]D[ILV]XX[AG], wherein X is any amino acid, 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 of SEQ ID NOs: 10, 11, 12 or 13, or iii) is any of SEQ ID NOs: 10, 11, 12 or 13 A functional homologue, variant or derivative having any of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptides of SEQ ID NO: 10, 11, 12 or 13 At least 80% overall sequence identity of full length and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or iv) comprising from any of SEQ ID NOs: 10, 11, 12 or 13 of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 An oligopeptide sequence of consecutive amino acid residues with N-acetylglucosamine b-1,3-galactosyltransferase activity. 31. The cell according to any one of embodiments 23 to 29, wherein the N-acetylglucosamine b-1,4-galactosyltransferase is a glycosyltransferase having: a. PFAM domain PF01755, and i) comprising the sequence EXXCXXSHXX[ILV][FWY]( _Xn )EDD( _Xm )[ACGST]XXYX[ILMV] of SEQ ID NO 14, wherein X is any amino acid, wherein n is 13 to 15 and m is 50 to 76, or ii) comprising a polypeptide sequence according to any of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23, or iii) being SEQ ID NOs: 15, 16, A functional homologue, variant or derivative of any of 17, 18, 19, 20, 21, 22 or 23 having the same The N-acetylglucosamine b-1,4-galactosyltransferase polypeptide full-length of any one of 22 or 23 has at least 80% overall sequence identity and has N-acetylglucosamine b-1,4 - galactosyltransferase activity, or iv) comprising at least 8, 9, 10, 11, 12, Oligopeptide sequences of 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b-1,4-galactosyltransferase activity, or b . PFAM domain PF00535 and i) comprising the sequence of SEQ ID NO 24 R[KN]XXXXXXXGXXXX[FL]XDXD( _Xn )[FHW]XXX[FHNY]( _Xm )E[DE], wherein X is any amine group acid, wherein n is 50 to 75 and m is 10 to 30, or ii) comprising the sequence of SEQ ID NO 25 R[KN]XXXXXXXXGXXXXFXDXD( _Xn )[FHW]XXX[FHNY]( _Xm )E[DE]( X _p ) [FWY]XX[HKR]XX[NQST], wherein X is any amino acid, wherein n is 50 to 75, m is 10 to 30, and p is 20 to 25, or iii) including according to SEQ ID The polypeptide sequence of any of NO: 26, 27 or 28, or iv) is a functional homologue, variant or derivative of any of SEQ ID NO: 26, 27 or 28 having the same At least 80% overall sequence identity of any of the full-length N-acetylglucosamine b-1,4-galactosyltransferase polypeptides of NO: 26, 27 or 28 and having N-acetylglucosamine b -1,4-Galactosyltransferase Transferase activity, or v) comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 from any of SEQ ID NOs: 26, 27, or 28 Oligopeptide sequences of consecutive amino acid residues with N-acetylglucosamine b-1,4-galactosyltransferase activity, or c. PFAM domain PF02709 and no PFAM domain PF00535, and i) include SEQ The sequence of ID NO 29 [FWY]XX[FY][FWY]( _X23 )[FWY][GQ]X[DE]D, where X is any amino acid, or ii) the sequence comprising SEQ ID NO 30 [ PV]W[GHNP]( _Xn )[FWY][GQ]X[DE]D, wherein X is any amino acid, wherein n is 21 to 24, or iii) including according to SEQ ID NO: 31, 32, A polypeptide sequence of any of 33, 34, 35 or 36, or iv) a functional homologue, variant or derivative of any of SEQ ID NO: 31, 32, 33, 34, 35 or 36 , which has at least 80% of the overall sequence of any of the full-length N-acetylglucosamine b-1,4-galactosyltransferase polypeptides of SEQ ID NO: 31, 32, 33, 34, 35 or 36 is identical and has N-acetylglucosamine b-1,4-galactosyltransferase activity, or v) comprises at least 8 from any of SEQ ID NOs: 31, 32, 33, 34, 35 or 36 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 oligopeptide sequences of consecutive amino acid residues with N-acetylglucosamine b-1,4- Galactosyltransferase activity, or d. PFAM domain PF03808, and i) comprising the sequence of SEQ ID NO 37 [ST][FHY]XN( _Xn )DG(X16)[HKR]X[ST] _FDXX [ST ]XA, wherein X is any amino acid, and wherein n is 20 to 25, or ii) comprising the sequence of SEQ ID NO 38 [ST][FHY]XN( _Xn )DG( _X16 )[HKR]X[ ST]FDXX[ST]XA(X _m )[HR]XG[FWY](X _p )GXGXXXQ[DE], where X is any amino acid, where 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 of SEQ ID NO: 39, 40 or 41, or iv) is a functional homologue of any of SEQ ID NO: 39, 40 or 41 , variant or derivative having the same as SEQ ID NO: 39, 40 or Any one of the full-length N-acetylglucosamine b-1,4-galactosyltransferase polypeptides of 41 has at least 80% overall sequence identity and has N-acetylglucosamine b-1,4-galactose basotransferase activity, or v) comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 from any of SEQ ID NOs: 39, 40, or 41 An oligopeptide sequence of consecutive amino acid residues with N-acetylglucosamine b-1,4-galactosyltransferase activity. 32. The cell according to any one of embodiments 23 to 31, wherein a. the N-acetylglucosamine-6-phosphotransferase is a polypeptide sequence comprising the polypeptide of UniProt ID P43577, or is a polypeptide of UniProt ID P43577 A functional homologue, variant or derivative having at least 80% overall sequence identity with the full-length polypeptide of UniProt ID P43577 and having N-acetylglucosamine-6-phosphotransferase activity, and b. the L-glutamine-D-fructose-6-phosphate transaminase is a polypeptide sequence comprising the polypeptide of UniProt ID P17169, or is a functional homologue, variant or derivative of the polypeptide having UniProt ID P17169, which has At least 80% overall sequence identity with the full-length polypeptide of UniProt ID P17169, and has L-glutamine-D-fructose-6-phosphate transaminase activity; or A39T, R250C and G472S different from the UniProt ID P17169 polypeptide Modified version of mutation. 33. The cell according to any one of embodiments 23 to 32, wherein the cell can metabolize a carbon source selected from the group comprising the following enumeration: glucose, fructose, galactose, lactose, sucrose, maltose, malt-oligo- Sugar, maltotriose, sorbitol, xylose, rhamnose, mannose, methanol, ethanol, arabinose, trehalose, starch, cellulose, hemicellulose, molasses, corn infusion, high fructose syrup, glycerin , acetate, citrate, lactic acid, pyruvic acid. 34. The cell according to any one of embodiments 23 to 33, wherein the cell is unable to convert N-acetylglucosamine-6-phosphate into glucosamine-6-phosphate, and/or is unable to convert glucosamine-6 - Phosphate conversion to fructose-6-phosphate. 35. The cell according to any one of embodiments 23 to 34, wherein the cell is modified to produce GDP-fucose. 36. The cell according to any one of embodiments 23 to 35, wherein the cell is modified to promote GDP-fucose production, wherein the modification is selected from the group comprising the following enumeration: UDP-glucose: undecylisoprene Shaving of the gene encoding ene-phosphoglucose-1-phosphotransferase, overexpression of the gene encoding GDP-L-fucose synthase, overexpression of the gene encoding GDP-mannose 4,6-dehydratase, mannose- Overexpression of a gene encoding 1-phosphate guanylate transferase, overexpression of a gene encoding phosphomannose mutase, or overexpression of a gene encoding mannose-6-phosphate isomerase. 37. The cell according to any one of embodiments 23 to 36, wherein the cell is modified to produce UDP-galactose. 38. The cell according to any one of embodiments 23 to 37, wherein the cell is modified to promote UDP-galactose production, wherein the modification is selected from the group comprising the following enumeration: 5'-nucleotidase/UDP- Shaving of the gene encoding glycohydrolase or shaving of the gene encoding galactose-1-phosphate uridine transferase. 39. The cell according to any one of embodiments 23 to 38, wherein the cell is modified to produce CMP-N-acetylneuraminic acid. 40. The cell according to any one of embodiments 23 to 39, wherein the cell is modified to promote CMP-N-acetylneuraminic acid production, wherein the modification is selected from the group comprising the following enumeration: CMP-sialic acid Overexpression of the gene encoding synthetase, overexpression of the gene encoding sialic acid synthase, overexpression of the gene encoding N-acetyl-D-glucosamine 2-epimerase. 41. The cell according to any one of embodiments 23 to 40, wherein the cell is capable of expressing at least one other glycosyltransferase, and wherein the other glycosyltransferase is selected from the group comprising: a fucosyltransferase , Sialyltransferase, Galactosyltransferase, Glucosyltransferase, Mannosyltransferase, N-Acetylglucosaminetransferase, N-Acetylgalactosyltransferase, N-Acetyl Mannosaminotransferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosamine transferase, N-glycolyl neuraminotransferase, rhamnosyltransferase. 42. The cell according to embodiment 41, wherein the cell is modified in the expression or activity of the other glycosyltransferase. 43. The cell according to any one of embodiments 23 to 41, wherein the oligosaccharide is selected from the list comprising: 2-fucosyllacto-N-disaccharide, 4-fucosyllacto-N-bisaccharide Sugar, 2-4-Difucosyllacto-N-disaccharide, 3'-Sialyllacto-N-disaccharide, 6'-Sialyllacto-N-disaccharide, 3',6'-Disialo Yogurt-N-disaccharide, 6,6'-disialo-N-disaccharide, 2'-fucosyl-3'-sialoglyco-N-disaccharide, 2'-fucosyl -6'-Sialylacto-N-disaccharide, 4-fucosyl-3'-sialylo-N-disaccharide, 4-fucosyl-6'-sialylo-N-disaccharide , 2-fucosyl N-acetyllactosamine, 3'-fucosyl N-acetyllactosamine, 2,3'-difucosyl N-acetyllactosamine, 3'-sialic acid N-acetyllactosamine, 6'-sialo-N-acetyllactosamine, 3',6'-disialo-N-acetyllactosamine, 6,6'-disialo-N-acetyllactosamine, 2'-fucosyl-3'-sialo-N-acetyllactosamine, 2'-fucosyl-6'-sialo-N-acetyllactosamine, 3-fucosyl-3'- Sialyl N-acetyllactosamine, 3'-fucosyl-6'-sialic acid N-acetyllactosamine, P1 trisaccharide (Gal-a1,4-Gal-b1,4-GlcNAc), heterologous Graft epitopes (Gal-a1,3-Gal-b1,4-GlcNAc), Gal-b14-(Galb13)-GlcNAc, poly-N-acetyllactosamine, GalNAc-b1,3-Gal-b1,4 -GlcNAc. 44. The cell according to any one of embodiments 23 to 43 or the method according to any one of embodiments 1 to 22, wherein the cell is selected from the group consisting of a microorganism, a plant or an animal cell, preferably the Microorganisms are bacteria, fungi or yeasts, preferably, the plants are cotton, rapeseed, soybean, corn or cereal plants, preferably, the animals are insects, fish, birds or non-human mammals; preferably, The cells are E. coli cells. 45. The cell according to any one of embodiments 23 to 44 or the method according to any one of embodiments 1 to 22 or 44, wherein the cell is a bacterial cell, preferably an Escherichia coli strain, more preferably an Escherichia coli strain K12 strain, more preferably, the E. coli strain K12 strain is E. coli MG1655. 46. The cell according to any one of embodiments 23 to 45 or the method according to any one of embodiments 1 to 22, 44 or 45, wherein the cell is a yeast cell. 47. The method according to any one of embodiments 1 to 22, 44 to 46, wherein the separation comprises at least one of clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated carbon or carbon treatment, Tangential flow high performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography. 48. The method of any one of embodiments 1 to 22, 44 to 47, further comprising purifying the disaccharide or oligosaccharide from the cell. 49. The method according to any one of embodiments 1 to 22, 44 to 48, wherein the purification comprises at least one of the following steps: use of activated carbon or carbon, use of carbon, nanofiltration, ultrafiltration or ion exchange, Use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying or lyophilization. 50. A cell according to any one of embodiments 23 to 46 for producing a disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end, preferably for producing LNB or LacNAc, more preferably for producing Use to produce sialylated or fucosylated forms of LNB or LacNAc. In addition, the present invention relates to the following preferred specific embodiments: 1. A method for producing an oligosaccharide or a disaccharide having an N-acetylglucosamine unit at the reducing end by a cell, preferably a single cell, the method comprising the following steps: a. Provide cells capable of: (i) synthesizing the nucleotide-sugar and monosaccharide N-acetylglucosamine (GlcNAc), and (ii) expressing a glycosyltransferase to saccharify the GlcNAc monosaccharide to produce the GlcNAc monosaccharide A disaccharide or oligosaccharide, b. culturing the cell under conditions permitting production of the disaccharide or oligosaccharide, c. preferably, isolating the disaccharide or oligosaccharide from the culture. 2. A method for producing oligosaccharides with N-acetylglucosamine units at the reducing end by cells, the method comprising the steps of: a. providing cells capable of: (i) synthesizing nucleotide-sugars and monosaccharides N-acetylglucosamine (GlcNAc), and (ii) expressing a glycosyltransferase to saccharify the GlcNAc monosaccharide to produce the oligosaccharide, b. culturing the cell under conditions that allow the production of the oligosaccharide, c. comparing Preferably, the oligosaccharide is isolated from the culture. 3. A method by cells for producing a mixture comprising: (i) a disaccharide and/or oligosaccharide having an N-acetylglucosamine unit at the reducing end, and (ii) one or more lactose-based lactose Animal milk oligosaccharides (MMOs), the method comprising the steps of: a. providing cells capable of: (i) synthesizing nucleotide-sugar and monosaccharide N-acetylglucosamine (GlcNAc), and (ii) expressing Glycosyltransferase to glycosylate the GlcNAc monosaccharide to produce disaccharides and/or oligosaccharides having N-acetylglucosamine units at the reducing end, b. culturing the cells under conditions that allow the mixture to be produced, c. comparing Preferably, the mixture is isolated from the culture. 4. The method of any of the above specific embodiments, wherein the cell expresses at least one N-acetylglucosamine-6-phosphotransferase and phosphatase to synthesize the monosaccharide N-acetylglucosamine. 5. The method according to any one of the above specific embodiments, wherein the cell expresses at least one glycosyltransferase to glycosylate N-acetylglucosamine. 6. The method according to any of the above specific embodiments, wherein the cell is genetically modified to produce the disaccharide or oligosaccharide. 7. The method of specific embodiment 6, wherein the cell is modified with one or more gene expression modules, characterized in that the expression of any of the expression modules is constitutive or produced by a natural inducer. 8. The method according to any one of specific embodiments 6 or 7, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein. 9. The method according to any one of the above specific embodiments, wherein the cell is modified in the expression or activity of an enzyme selected from the group consisting of: N-acetylglucosamine-6-phosphotransferase, phosphatase , Glycosyltransferase, L-glutamine-D-fructose-6-phosphate transaminase and UDP-glucose 4-epimerase. 10. The method according to any one of the above specific embodiments, wherein the nucleotide-sugar is selected from the group comprising: 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-acetylamino-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-pneumolysamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy -L-Talose), UDP-N-Acetylmuramic acid, UDP-N-Acetyl-L-Quinosamine (UDP-L-QuiNAc or UDP-2-Acetylamino-2, 6-dideoxy-L-glucose), GDP-L-isorhamnose, CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) , CMP-Neu4Ac, CMP-Neu5Ac9N ₃ , CMP-Neu4,5Ac ₂ , CMP-Neu5,7Ac ₂ , CMP-Neu5,9Ac ₂ , CMP-Neu5,7(8,9)Ac ₂ , UDP-glucuronide , UDP-galacturonate, GDP-rhamnose and UDP-xylose. 11. The method according to any one of the above specific embodiments, wherein the nucleotide-sugar is UDP-galactose and the glycosyltransferase is N-acetylglucosamine b-1,3-galactosyltransferase enzyme or N-acetylglucosamine b-1,4-galactosyltransferase. 12. The method according to any of the above specific embodiments, wherein the oligosaccharide has lacto-N-disaccharide (Gal-b1,3-GlcNAc) or N-acetyllactosamine (Gal-b1,4) at the reducing end -GlcNAc). 13. The method according to any one of the above specific embodiments, wherein the N-acetylglucosamine b-1,3-galactosyltransferase is a glycosyltransferase having: a. PFAM domain PF00535, and i . comprising the sequence of SEQ ID NO 01 [AGPS]XXLN(X _n )RXDXD, wherein X is any amino acid, wherein n is 12 to 17, or ii. comprising the sequence of SEQ ID NO 02 PXXLN(X _n )RXDXD ( X _m ) [FWY]XX[HKR]XX[NQST], wherein X is any amino acid, wherein n is 12 to 17 and m is 100 to 115, or iii. including according to SEQ ID NOs: 03, 04, 05 , any of 06, 07 or 08, preferably any of SEQ ID NOs 03, 04, 05, 06 or 07, more preferably any of SEQ ID NOs 03, 06 or 07, most preferably The polypeptide sequence of any one of SEQ ID NOs 03 or 06, or iv. any one of SEQ ID NOs: 03, 04, 05, 06, 07 or 08, preferably SEQ ID NOs 03, 04, 05, any of 06 or 07, more preferably any of SEQ ID NO 03, 06 or 07, most preferably a functional homologue, variant or derivative of any of SEQ ID NO 03 or 06, It has any of SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably any of SEQ ID NO 03, 04, 05, 06 or 07, more preferably SEQ ID NO 03 Any of , 06 or 07, preferably at least 80% of the overall sequence identity of the full length of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide of any of SEQ ID NOs 03 or 06 and has N-acetylglucosamine b-1,3-galactosyltransferase activity, or v. includes any one from SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably Any of SEQ ID NOs 03, 04, 05, 06 or 07, more preferably any of SEQ ID NOs 03, 06 or 07, most preferably at least 8, Oligopeptide sequences of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b-1,3-half Lactosyltransferase activity, or vi. is any of SEQ ID NOs: 03, 04, 05, 06, 07 or 08, preferably SEQ ID NOs 03, 04, 05, 06 or Any one of 07, more preferably any one of SEQ ID NO 03, 06 or 07, most preferably a functional fragment of any one of SEQ ID NO 03 or 06, and has N-acetylglucosamine b-1 , 3-galactosyltransferase activity, or vii. comprising a polypeptide comprising or consisting of any of SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably SEQ ID NO 03 , any of 04, 05, 06 or 07, more preferably any of SEQ ID NO 03, 06 or 07, most preferably at least the full-length amino acid sequence of any of SEQ ID NO 03 or 06 consisting of an amino acid sequence of 80% sequence identity and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or b. PFAM domain IPR002659, and i. comprising SEQ ID NO 09 Sequence KT(X _n )[FY]XXKXDXD(X _m )[FHY]XXG(X without A,G,S)(X _p )X(without F,H,W,Y)[DE]D[ILV] XX[AG], wherein X is any amino acid, wherein n is 13 to 16, m is 35 to 70, and p is 20 to 45, or ii. The polypeptide sequence of any of , or iii. is a functional homologue, variant or derivative of any of SEQ ID NO: 10, 11, 12, or 13, which has the same properties as SEQ ID NO: 10, 11, At least 80% overall sequence identity of any of the full-length N-acetylglucosamine b-1,3-galactosyltransferase polypeptides of 12 or 13 and having N-acetylglucosamine b-1,3 - Galactosyltransferase activity, or iv. comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, An oligopeptide sequence of 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b-1,3-galactosyltransferase activity, or v. is SEQ ID NO: 10, 11, A functional fragment of any one of 12 or 13, and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or vi. comprising a polypeptide comprising or consisting of a , 11, 12, or 13, consisting of amino acid sequences of at least 80% sequence identity to the full-length amino acid sequence of any one of 11, 12, or 13, and having N-acetylglucosamine b-1,3-galactosyltransferase active. 14. The method according to any one of specific embodiments 1 to 12 above, wherein the N-acetylglucosamine b-1,4-galactosyltransferase is a glycosyltransferase having: a. PFAM domain PF01755 and i. comprises the sequence EXXCXXSHXX[ILV][FWY]( _Xn )EDD( _Xm )[ACGST]XXYX[ILMV] of SEQ ID NO 14, wherein X is any amino acid, wherein n is 13 to 15 and m is from 50 to 76, or ii. including any of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NO: 15, 16, 17, Any one of 18, 20 or 21, more preferably the polypeptide sequence of any one of SEQ ID NO: 17, 18, 20 or 21, or iii. is SEQ ID NO: 15, 16, 17, 18, 19, Any one of 20, 21, 22 or 23, preferably any one of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably one of SEQ ID NO: 17, 18, 20 or 21 A functional homologue, variant or derivative of any of , which has any of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NO: any one of 15, 16, 17, 18, 20 or 21, more preferably the N-acetylglucosamine b-1,4- of any one of SEQ ID NO: 17, 18, 20 or 21 At least 80% overall sequence identity to the full length of the galactosyltransferase polypeptide and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or iv. including from SEQ ID NOs: 15, 16, 17 , any of 18, 19, 20, 21, 22 or 23, preferably any of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably SEQ ID NO: 17, 18 An oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues of any of , 20, or 21, and N- Acetylglucosamine b-1,4-galactosyltransferase activity, or v. is any of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably Any of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably a functional fragment of any of SEQ ID NO: 17, 18, 20 or 21, and having N-acetylglucose Amine b-1,4-galactosyltransferase activity, or vi. Include polypeptides comprising or consisting of any of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NOs: 15, 16, 17, 18 Any of , 20 or 21, more preferably an amino acid sequence of at least 80% sequence identity to the full-length amino acid sequence of any of SEQ ID NO: 17, 18, 20 or 21, and has N-acetylglucosamine b-1,4-galactosyltransferase activity, or b. PFAM domain PF00535, and i. the sequence comprising SEQ ID NO 24 R[KN]XXXXXXXXGXXXX[FL]XDXD( _Xn )[ FHW]XXX[FHNY](X _m )E[DE], wherein X is any amino acid, wherein n is 50 to 75 and m is 10 to 30, or ii. the sequence R[KN] comprising SEQ ID NO 25 XXXXXXXGXXXXFXDXD(X _n )[FHW]XXX[FHNY](X _m )E[DE](X _p )[FWY]XX[HKR]XX[NQST], where X is any amino acid, where 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, 27 or 28, or iv. is in SEQ ID NO: 26, 27 or 28 A functional homologue, variant or derivative of any of SEQ ID NO: 26, 27 or 28 having a At least 80% overall sequence identity of either full length and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or v. including any one from SEQ ID NO: 26, 27, or 28 An oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b- 1,4-galactosyltransferase activity, or vi. is a functional fragment of any one of SEQ ID NOs: 26, 27, or 28, and has N-acetylglucosamine b-1,4-galactosyltransferase Enzymatic activity, or vii. comprising a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any of SEQ ID NOs: 26, 27 or 28, and has N-acetylglucosamine b-1,4-galactosyltransferase activity, or c. PFAM domain PF02709 and does not have PFAM domain PF00535, and i. comprises the sequence of SEQ ID NO 29 [FWY]XX[FY ][FWY](X ₂₃ )[FWY][G Q]X[DE]D, wherein X is any amino acid, or ii. the sequence comprising SEQ ID NO 30 [PV]W[GHNP]( _Xn )[FWY][GQ]X[DE]D, wherein X is any amino acid wherein n is 21 to 24, or iii. comprises a polypeptide sequence according to any of SEQ ID NO: 31, 32, 33, 34, 35 or 36, or iv. is SEQ ID NO: A functional homologue, variant or derivative of any of 31, 32, 33, 34, 35 or 36 having the N- at least 80% overall sequence identity of any of the full-length acetylglucosamine b-1,4-galactosyltransferase polypeptides and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or v. comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, An oligopeptide sequence of 20 consecutive amino acid residues with N-acetylglucosamine b-1,4-galactosyltransferase activity, or vi. are SEQ ID NOs: 31, 32, 33, 34, The functional fragment of any one of 35 or 36, and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or vii. comprising a polypeptide comprising or consisting of having and SEQ ID NO: 31 , 32, 33, 34, 35 or 36, the full-length amino acid sequence of any one of the amino acid sequences of at least 80% sequence identity, and has N-acetylglucosamine b-1,4-half amino acid sequence Lactosyltransferase activity, or d. PFAM domain PF03808, and i. the sequence comprising SEQ ID NO 37 [ST][FHY]XN( _Xn )DG(X16)[HKR]X[ST] _FDXX [ST] XA, wherein X is any amino acid, and wherein n is 20 to 25, or ii. the sequence comprising SEQ ID NO 38 [ST][FHY]XN( _Xn )DG( _X16 )[HKR]X[ST ]FDXX[ST]XA(X _m )[HR]XG[FWY](X _p )GXGXXXQ[DE], where X is any amino acid, where n is 20 to 25, m is 40 to 50, and p is 22 to 30, or iii. comprising a polypeptide sequence according to any one of SEQ ID NO: 39, 40 or 41, or iv. being a functional homologue of any one of SEQ ID NO: 39, 40 or 41, A variant or derivative having any of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides of SEQ ID NO: 39, 40 or 41 in complete agreement with Long at least 80% overall sequence identity and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or v. comprising from any of SEQ ID NOs: 39, 40 or 41 An oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b-1, 4-galactosyltransferase activity, or vi. is a functional fragment of any one of SEQ ID NOs: 39, 40, or 41, and has N-acetylglucosamine b-1,4-galactosyltransferase activity , or vii. comprising a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any of SEQ ID NOs: 39, 40 or 41, and having N-acetylglucosamine b-1,4-galactosyltransferase activity. 15. The method according to any one of the above specific embodiments, wherein a. the N-acetylglucosamine-6-phosphotransferase is a polypeptide sequence comprising the polypeptide of UniProt ID P43577 or a function of the polypeptide of UniProt ID P43577 A sexual homologue, variant or derivative having at least 80% overall sequence identity with the full-length polypeptide of UniProt ID P43577 and having N-acetylglucosamine-6-phosphotransferase activity, and b. the L - Glutamine-D-fructose-6-phosphate transaminase is a polypeptide sequence comprising the polypeptide of UniProt ID P17169, or is a functional homologue, variant or derivative of the polypeptide having UniProt ID P17169, which has a At least 80% overall sequence identity of the full-length polypeptide of UniProt ID P17169 and having L-glutamine-D-fructose-6-phosphate transaminase activity; or A39T, R250C and G472S mutations different from the UniProt ID P17169 polypeptide modified version. 16. The method according to any one of the above specific embodiments, wherein the cell can metabolize a carbon source selected from the group comprising the following enumeration: glucose, fructose, galactose, lactose, sucrose, maltose, malto-oligosaccharide , maltotriose, sorbitol, xylose, rhamnose, mannose, methanol, ethanol, arabinose, trehalose, starch, cellulose, hemicellulose, corn infusion, high fructose syrup, molasses, glycerin, Acetate, citrate, lactic acid, pyruvic acid. 17. The method according to any one of the above specific embodiments, wherein the cell is unable to convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or is unable to convert glucosamine-6-phosphate Phosphate is converted to fructose-6-phosphate. 18. The method according to any of the above specific embodiments, wherein the cell is modified to produce GDP-fucose. 19. The method according to any one of the above specific embodiments, wherein the cell is modified to promote GDP-fucose production, wherein the modification is selected from the group comprising the following enumeration: UDP-glucose:undec isoprene - Shaving of the gene encoding phosphoglucose-1-phosphotransferase, overexpression of the gene encoding GDP-L-fucose synthase, overexpression of the gene encoding GDP-mannose 4,6-dehydratase, mannose-1 - Overexpression of a gene encoding a phosphoguanylate transferase, overexpression of a gene encoding a phosphomannose mutase, or overexpression of a gene encoding a mannose-6-phosphate isomerase. 20. The method according to any of the above specific embodiments, wherein the cell is modified to produce UDP-galactose. 21. The method according to any one of the above specific embodiments, wherein the cell is modified to promote UDP-galactose production, wherein the modification is selected from the group comprising the following enumeration: 5'-nucleotidase/UDP-sugar Shaving of a gene encoding a hydrolase or shaving of a gene encoding a galactose-1-phosphate uridine transferase. 22. The method according to any one of the above specific embodiments, wherein the cell is modified to produce CMP-N-acetylneuraminic acid. 23. The method according to any one of the above specific embodiments, wherein the cell is modified to promote CMP-N-acetylneuraminic acid production, wherein the modification is selected from the group comprising the following enumeration: CMP-sialic acid synthesis Overexpression of gene encoding enzyme, overexpression of gene encoding sialic acid synthase, overexpression of gene encoding N-acetyl-D-glucosamine 2-epimerase. 24. The method according to any one of the above specific embodiments, wherein the cell is capable of expressing at least one other glycosyltransferase, wherein the other glycosyltransferase is selected from the group comprising: fucosyltransferase, saliva Acid transferase, Galactosyltransferase, Glucosyltransferase, Mannosyltransferase, N-Acetylglucosaminyltransferase, N-Acetylgalactosyltransferase, N-Acetylmannose Aminyltransferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosamine transferase, N-glycolyl neuraminosyltransferase, rhamnosyltransferase, N-acetyltransferase Rhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase (UDP-4-amino-4,6 -dideoxy-N-acetyl-beta-L-altrosamine transaminases), UDP-N-acetylglucosamine enolpyruvyl transferases (UDP- N -acetylglucosamine enolpyruvyl transferases) and fucosaminyltransferases (fucosaminyltransferases), - Preferably, the fucosyltransferase is selected from the group consisting of: α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,4- Fucosyltransferases and α-1,6-fucosyltransferases, - preferably, the sialyltransferases are selected from the group consisting of: α-2,3-sialyltransferases, α-2 ,6-sialyltransferase and α-2,8-sialyltransferase, - preferably, the galactosyltransferase is selected from the group consisting of: β-1,3-galactosyltransferase, N- Acetylglucosamine β-1,3-galactosyltransferase, β-1,4-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, α-1, 3-Galactosyltransferase and α-1,4-Galactosyltransferase, - preferably, the glucosyltransferase is selected from the group consisting of α-glucosyltransferase, β-1,2-glucose Syltransferase, β-1,3-glucosyltransferase and β-1,4-glucosyltransferase, - preferably, the mannosyltransferase (mannosyltransferase) is selected from the list including: α-1, 2-mannosyltransferase, α-1,3-mannosyltransferase and α-1,6-mannosyltransferase, - preferably, the N-acetylglucosaminyltransferase is selected from Include the following enumeration: β-1,3-N-acetylglucosaminyltransferase and β-1,6-N-acetylglucosaminyltransferase, - preferably, the N-acetyl half Lactosamine transferase is α-1,3-N-acetylgalactosamine transferase. 25. The method according to specific embodiment 24, wherein the cell is modified in the expression or activity of the other glycosyltransferase. 26. The method according to any one of the above specific embodiments, wherein the cell uses one or more precursors for producing the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end, the precursor being supplied to the cell from a culture medium. 27. The method according to any of the above specific embodiments, wherein the cell produces one or more precursors for producing the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end. 28. The method according to any one of specific embodiments 26 or 27, wherein the precursor used to produce the disaccharide or oligosaccharide is completely converted to a disaccharide or oligosaccharide having a GlcNAc unit at the reducing end. 29. The method according to any one of the above-mentioned specific embodiments, wherein the cell produces a disaccharide or an oligosaccharide with a GlcNAc unit at the reducing end in the cell, and wherein part or substantially all of the generated reducing end has a GlcNAc unit. Disaccharides or oligosaccharides remain intracellular and/or are excreted via passive or active transport. 30. The method according to any one of the above specific embodiments, wherein the cell expresses a membrane transporter or a polypeptide with transport activity to transport the compound through the outer membrane of the cell wall, preferably, the cell is in the membrane transporter or The expression or activity of the polypeptide having transport activity is modified. 31. The method according to particular embodiment 30, wherein the membrane transporter or polypeptide having transport activity is selected from the group consisting of a transporter, PP-bond-hydrolysis-driven transporter, β-barrel porin, assisted transporter protein, putative transporter and phosphate transfer-driven group translocator, preferably, the transporter includes MFS transporter, sugar excretion transporter and chelatin exporter, preferably, the PP-bond-hydrolysis-driven The transporters include the ABC transporter and the chelatin exporter. 32. The method according to any one of specific embodiments 30 or 31, wherein the membrane transporter or the polypeptide with transport activity controls the reducing end with a disaccharide or oligosaccharide and/or one or more precursors and/or a GlcNAc unit. Or the flow of acceptors on the outer membrane of the cell wall, the one or more precursors and/or acceptors for the production of disaccharides or oligosaccharides with GlcNAc units at the reducing end. 33. The method according to any one of specific embodiments 30 or 32, wherein the membrane transporter or the polypeptide with transport activity provides the reducing end with an improved production of disaccharides or oligosaccharides of GlcNAc units and/or allows discharge and/or or promote excretion. 34. The method according to any one of specific embodiments 6 or 33, wherein the cell comprises a modification for reducing acetate production compared to an unmodified precursor cell. 35. The method according to specific embodiment 34, wherein the cell comprises lower or reduced expression and/or eliminated, impaired, reduced or delayed activity of any one or more proteins compared to unmodified precursor cells, the Protein contains: β-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetyl Glucosamine inhibitor protein, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undec isoprene-phosphoglucose-1-phosphotransferase, L-fucosokinase, L-fucoid Carbohydrate isomerase, N-acetylneuraminic acid dissociating enzyme, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man , EIID-Man, ushA, galactose-1-phosphate uridine transferase, glucose-1-phosphate adenosyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isokinase Enzyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration regulatory protein, transcriptional repressor protein IclR, lon protease, glucose-specific translocation phosphotransferase enzyme IIBC component ptsG, glucose specific translocation phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIA ^Glc , β-glucoside specific PTS enzyme II, fructose specific PTS polyphosphoryl transfer protein FruA and FruB, alcohol dehydrogenation Enzymes aldehyde dehydrogenase, pyruvate-formate dissociation enzyme, acetate kinase, phosphoacetyltransferase, phosphoacetyltransferase, pyruvate decarboxylase. 36. The method according to any of the above specific embodiments, wherein the cell is capable of producing phosphoenolpyruvate (PEP). 37. The method according to any one of specific embodiments 6 or 36, wherein the cell has a modification for promoting production and/or supply of phosphoenolpyruvate (PEP) compared to an unmodified precursor cell. 38. The method according to any one of specific embodiments 6 to 37, wherein the cell comprises an at least partially inactivated catabolic pathway of the selected monosaccharide, disaccharide or oligosaccharide involved in the Required for the production of disaccharides or oligosaccharides with GlcNAc units at the reducing end and/or for the production of disaccharides or oligosaccharides with GlcNAc units at the reducing end. 39. The method of any of the above specific embodiments, wherein the cell is resistant to lactose killing when the cell is grown in an environment where lactose is combined with one or more other carbon sources. 40. The method according to any one of the above-mentioned specific embodiments, wherein the cell produces a disaccharide or oligomer of GlcNAc at the reducing end of 90 g/L or more than 90 g/L in whole culture fluid and/or supernatant. Saccharides, and/or disaccharides or oligosaccharides having GlcNAc at the reducing end in the whole culture fluid and/or supernatant, have a purity of at least 80%, respectively, in the whole culture fluid and/or supernatant. The total amount of disaccharides or oligosaccharides with GlcNAc units at the reducing end and their precursors. 41. The method according to any of the above specific embodiments, wherein the cells are stably cultured in culture medium. 42. according to the method of any one of above-mentioned specific embodiment, wherein this condition comprises: - Use the substratum that comprises at least one precursor and/or acceptor, for producing the disaccharide or oligosaccharide that this reducing end has GlcNAc unit , and/or - adding at least one precursor and/or acceptor feedstock to the medium for the production of the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end. 43. The method according to any one of the above specific embodiments, comprising at least one of the following steps: i) using a culture medium comprising at least one precursor and/or acceptor; ii) adding to the culture medium in the reactor At least one precursor and/or recipient feedstock, wherein the total reactor volume ranges from 250 mL (milliliters) to 10,000 ^m3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium does not exceed the Three times the volume of the medium before the precursor and/or recipient feedstock, preferably no more than twice, more preferably less than twice; iii) adding at least one precursor and/or recipient feedstock to the medium in the reactor, wherein the total reactor volume ranges from 250 mL (milliliters) to 10,000 ^m3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium does not exceed that prior to the addition of the precursor and/or recipient feedstock. three times the volume of the medium, preferably no more than twice, more preferably less than twice, wherein preferably the pH of the precursor and/or recipient feedstock is set between 3 and 7, and preferably the precursor The temperature of the material and/or the recipient feedstock is maintained between 20°C and 80°C; iv) by means of feeding the solution in a continuous adding at least one precursor and/or acceptor feedstock to the culture medium in a manner; v) adding at least one precursor in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of the feed solution and/or recipient feedstock to culture medium, wherein preferably the pH of the feed solution is set between 3 and 7, and preferably the temperature of the feed solution is maintained between 20°C and 80°C The method produces a disaccharide or oligosaccharide having a GlcNAc unit at the reducing end, and its concentration in the final culture is 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. 44. The method according to any one of specific embodiments 1 to 42, comprising at least one of the following steps: i) using at least 50 grams per liter of initial reactor volume, more preferably at least 75 grams, more preferably medium of at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams of lactose, wherein the reactor volume ranges from 250 mL to 10,000 m ³ (cubic meters); ii) the addition per liter of initial reactor volume contains at least 50 grams, more preferably at least 75 grams, more preferably at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams of lactose feedstock to culture medium, preferably in a continuous manner, and preferably, such that The final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium before the addition of the lactose feedstock; iii) the addition contains at least 50 grams, more preferably at least 50 grams per liter of initial reactor volume 75 grams, more preferably at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams of lactose feedstock to culture medium, wherein the reactor volume ranges from 250 mL to 10,000 m ³ (cubic meters), preferably In a continuous manner, and preferably, the final volume of the medium is no more than three times the volume of the medium before the addition of the lactose feedstock, preferably no more than two times, more preferably less than two times, wherein preferably, the lactose feedstock is The pH is set between 3 and 7, and preferably, the temperature of the lactose feedstock is maintained between 20°C and 80°C; iv) by means of feeding solution at 1 day, 2 days, 3 days, Lactose feedstock was added to the medium in a continuous manner during 4 days, 5 days; v) Lactose feedstock was added in a continuous manner during 1 day, 2 days, 3 days, 4 days, 5 days by means of feeding solution to culture medium, wherein the concentration of the lactose feed 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, better 350 g/L, better 375 g/L, better 400 g/L, better 450 g/L, better 500 g/L, still better 550 g/L, preferably 600 g/L; wherein preferably, the pH of the feed solution is set between 3 and 7, and preferably, the temperature of the feed solution is maintained at 20°C and 80°C Between C; the method produces a disaccharide or oligosaccharide having a GlcNAc unit at the reducing end, and its concentration in the final culture is at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, More preferably at least 100g/L, more preferably at least 125g/L, more preferably at least 150g/L, more preferably at least 175g/L, More preferably, it is at least 200 g/L. 45. The method according to particular embodiment 44, wherein the lactose feedstock is prepared by adding lactose from the start of the culture at a concentration of at least 5 mM, preferably 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, More preferably this is done by adding lactose at a concentration of >300 mM. 46. The method according to any one of specific embodiments 44 or 45, wherein the lactose feedstock is accomplished by adding lactose to the culture at a concentration such that at least 5 mM is obtained throughout the production stage of the culture, preferably Lactose concentrations of 10 mM or 30 mM. 47. The method according to any of the above specific embodiments, wherein the cells are cultured for at least about 60, 80, 100 or about 120 hours or in a continuous manner. 48. The method according to any of the above specific embodiments, wherein the medium comprises at least one precursor selected from the group comprising lactose, galactose, fucose and sialic acid. 49. The method according to any one of the above specific embodiments, wherein a first stage of exponential cell growth is provided, followed by a second stage of growth by adding a carbonaceous substrate, preferably glucose or sucrose, to the medium comprising the precursor. , only a carbon substrate, preferably glucose or sucrose, is added to the medium. 50. The method according to any one of specific embodiments 1 to 49, wherein a first stage of exponential cell growth is provided by adding a carbonaceous substrate, preferably glucose or sucrose, to the medium comprising the precursor, followed by In the second stage, a carbon-based substrate, preferably glucose or sucrose, and a precursor are added to the medium. 51. The method according to any one of the above-described specific embodiments, wherein the cell produces a mixture comprising at least one charged, preferably sialylated, disaccharide or oligosaccharide comprising a GlcNAc unit at the reducing end. /or oligosaccharides and/or neutral disaccharides and/or oligosaccharides. 52. The method according to any one of the above-described specific embodiments, wherein the cell produces a mixture that is charged, preferably a sialylated oligosaccharide and/or neutral, comprising at least the oligosaccharide of the GlcNAc unit at the reducing end oligosaccharides. 53. The method according to any one of the above specific embodiments, wherein the oligosaccharide is selected from the list comprising: 2-fucosyl lacto-N-disaccharide, 4-fucosyl lacto-N-disaccharide , 2-4-Difucosyllacto-N-disaccharide, 3'-Sialyllacto-N-disaccharide, 6'-Sialyllacto-N-disaccharide, 3',6'-disialo Lacto-N-disaccharide, 6,6'-disialo-Lacto-N-disaccharide, 2'-fucosyl-3'-sialo-N-disaccharide, 2'-fucosyl- 6'-Sialyl lacto-N-disaccharide, 4-Fucosyl-3'-Sialyl lacto-N-disaccharide, 4-fucosyl-6'-Sialyl lacto-N-disaccharide, 2-Fucosyl N-acetyllactosamine, 3'-fucosyl N-acetyllactosamine, 2,3'-difucosyl N-acetyllactosamine, 3'-sialic acid N -Acetylactosamine, 6'-Sialyl-N-Acetylactosamine, 3',6'-Disialo-N-Acetylactosamine, 6,6'-Disialo-N-Acetylactosamine, 2 '-Fucosyl-3'-sialo-N-acetyllactosamine, 2'-fucosyl-6'-sialo-N-acetyllactosamine, 3-fucosyl-3'-sialo Acid N-acetyllactosamine, 3'-fucosyl-6'-sialic acid N-acetyllactosamine, P1 trisaccharide (Gal-a1,4-Gal-b1,4-GlcNAc), allograft Epitope (Gal-a1,3-Gal-b1,4-GlcNAc), Gal-b14-(Galb13)-GlcNAc, poly-N-acetyllactosamine, GalNAc-b1,3-Gal-b1,4- GlcNAc. 54. The method according to any one of specific embodiments 1 or 3 to 53, wherein the disaccharide having a GlcNAc unit at the reducing end excludes chitobiose (GlcNAc-GlcNAc). 55. The method according to any one of the above specific embodiments, wherein the oligosaccharide having a GlcNAc unit at the reducing end does not include chitobiose, preferably N-glycans, at the reducing end. 56. A metabolically engineered cell for producing an oligosaccharide or disaccharide having N-acetylglucosamine units at the reducing end, wherein the cell is capable of: (i) synthesizing nucleotide-sugar and monosaccharide N-acetylglucosamine (GlcNAc), and (ii) express a glycosyltransferase to saccharify the GlcNAc monosaccharide to produce the disaccharide or oligosaccharide. 57. A metabolically engineered cell for producing an oligosaccharide having an N-acetylglucosamine unit at the reducing end, wherein the cell is capable of: (i) synthesizing nucleotide-sugar and monosaccharide N-acetylglucosamine (GlcNAc) , and (ii) expressing a glycosyltransferase to saccharify the GlcNAc monosaccharide to produce an oligosaccharide. 58. A metabolically engineered cell for producing a mixture comprising (i) disaccharides and/or oligosaccharides having N-acetylglucosamine units at the reducing end and (ii) one or more lactose-based mammalian milks Oligosaccharides (MMOs), wherein the cell is capable of: (i) synthesizing the nucleotide-sugar and monosaccharide N-acetylglucosamine (GlcNAc), and (ii) expressing a glycosyltransferase to glycosylate the GlcNAc monosaccharide, and A disaccharide or oligosaccharide with an N-acetylglucosamine unit at the reducing end is produced. 59. The cell according to any one of specific embodiments 56 or 58, wherein the cell is metabolically engineered to produce a disaccharide or oligosaccharide having N-acetylglucosamine at the reducing end. 60. The cell according to specific embodiment 57, wherein the cell is metabolically engineered to produce an oligosaccharide having N-acetylglucosamine at the reducing end. 61. The cell of any one of specific embodiments 56 to 60, wherein the cell is modified with one or more gene expression modules, characterized in that the expression of any of the expression modules is constitutive or induced by a natural inducer. produce. 62. The cell according to any one of specific embodiments 56 to 61, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein. 63. The cell according to any one of specific embodiments 56 to 62, wherein the cell expresses at least one N-acetylglucosamine-6-phosphotransferase and phosphatase to synthesize N-acetylglucosamine. 64. The cell according to any one of specific embodiments 56 to 63, wherein the cell expresses at least one glycosyltransferase to glycosylate N-acetylglucosamine. 65. The cell according to any one of specific embodiments 56 to 64, wherein the cell is modified in the expression or activity of an enzyme selected from the group consisting of N-acetylglucosamine-6-phosphotransferase, Phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphate transaminase and UDP-glucose 4-epimerase. 66. The cell according to any one of specific embodiments 56 to 65, wherein the nucleotide-sugar is selected from the group comprising: 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-acetamide 2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumolysamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-bis Deoxy-L-talose), UDP-N-Acetylmuramic acid, UDP-N-Acetyl-L-Quinosamine (UDP-L-QuiNAc or UDP-2-Acetylamino- 2,6-dideoxy-L-glucose), GDP-L-isorhamnose, CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid (CMP- Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N ₃ , CMP-Neu4,5Ac ₂ , CMP-Neu5,7Ac ₂ , CMP-Neu5,9Ac ₂ , CMP-Neu5,7(8,9)Ac ₂ , UDP-glucaldehyde acid salt, UDP-galacturonate, GDP-rhamnose and UDP-xylose. 67. The cell according to any one of specific embodiments 56 to 66, wherein the nucleotide-sugar is UDP-galactose, and the glycosyltransferase is N-acetylglucosamine b-1,3-galactose Syltransferase or N-acetylglucosamine b-1,4-galactosyltransferase. 68. The cell according to any one of specific embodiments 56 to 67, wherein the oligosaccharide has lacto-N-disaccharide (Gal-b1,3-GlcNAc) or N-acetyllactosamine (Gal-b1) at the reducing end , 4-GlcNAc). 69. The cell according to any one of specific embodiments 56 to 68, wherein the N-acetylglucosamine b-1,3-galactosyltransferase is a glycosyltransferase having: a. PFAM domain PF00535, and i. include the sequence of SEQ ID NO 01 [AGPS]XXLN(X _n )RXDXD, wherein X is any amino acid, wherein n is 12 to 17, or ii. include the sequence PXXLN(X _n ) of SEQ ID NO 02 RXDXD(X _m )[FWY]XX[HKR]XX[NQST], wherein X is any amino acid, wherein n is 12 to 17 and m is 100 to 115, or iii. including according to SEQ ID NOs: 03, 04 , any of 05, 06, 07 or 08, preferably any of SEQ ID NOs 03, 04, 05, 06 or 07, more preferably any of SEQ ID NOs 03, 06 or 07, most Preferably it is the polypeptide sequence of any one of SEQ ID NO 03 or 06, or iv. is any one of SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably SEQ ID NO 03, 04, Any of 05, 06 or 07, more preferably any of SEQ ID NOs 03, 06 or 07, most preferably a functional homologue, variant or derivative of any of SEQ ID NOs 03 or 06 substance, which has any of SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably any of SEQ ID NO: 03, 04, 05, 06 or 07, more preferably SEQ ID Any of NO 03, 06 or 07, preferably at least 80% of the entire length of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide of any of SEQ ID NO 03 or 06 Sequence identity and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or v. including any one from SEQ ID NO: 03, 04, 05, 06, 07, or 08, vs. Preferably any one of SEQ ID NO 03, 04, 05, 06 or 07, more preferably any one of SEQ ID NO 03, 06 or 07, most preferably at least any one of SEQ ID NO 03 or 06 Oligopeptide sequences of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b-1,3 -Galactosyltransferase activity, or vi. is any of SEQ ID NOs: 03, 04, 05, 06, 07 or 08, preferably any of SEQ ID NOs 03, 04, 05, 06 or 07 One, more preferably any one of SEQ ID NO 03, 06 or 07, most preferably S A functional fragment of any one of EQ ID NOs 03 or 06, and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or vii. comprising a polypeptide comprising or consisting of a NO: any one of 03, 04, 05, 06, 07 or 08, preferably any one of SEQ ID NO 03, 04, 05, 06 or 07, more preferably one of SEQ ID NO 03, 06 or 07 any one, preferably the amino acid sequence of at least 80% sequence identity of the full-length amino acid sequence of any one of SEQ ID NO 03 or 06 is formed, and has N-acetylglucosamine b-1, 3-Galactosyltransferase activity, or b. PFAM domain IPR002659, and i. the sequence comprising SEQ ID NO 09 KT( _Xn )[FY]XXKXDXD( _Xm )[FHY]XXG(X, no A, G , S)(X _p )X (no F, H, W, Y) [DE]D[ILV]XX[AG], where X is any amino acid, where n is 13 to 16 and m is 35 to 70 , and p is 20 to 45, or ii. comprises a polypeptide sequence according to any of SEQ ID NOs: 10, 11, 12, or 13, or iii. is any of SEQ ID NOs: 10, 11, 12, or 13 A functional homologue, variant or derivative having the same number as in the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide of SEQ ID NO: 10, 11, 12 or 13 At least 80% overall sequence identity of either full length and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or iv. Included from SEQ ID NO: 10, 11, 12, or 13 Any oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b-1,3-galactosyltransferase activity, or v. is a functional fragment of any one of SEQ ID NOs: 10, 11, 12, or 13, and has N-acetylglucosamine b-1,3- Galactosyltransferase activity, or vi. comprising a polypeptide comprising or consisting of an amine having at least 80% sequence identity to the full-length amino acid sequence of any of SEQ ID NOs: 10, 11, 12, or 13 It is composed of amino acid sequence and has N-acetylglucosamine b-1,3-galactosyltransferase activity. 70. The cell according to any one of specific embodiments 56 to 69, wherein the N-acetylglucosamine b-1,4-galactosyltransferase is a glycosyltransferase having: a. PFAM domain PF01755, and i. comprises the sequence EXXCXXSHXX[ILV][FWY]( _Xn )EDD( _Xm )[ACGST]XXYX[ILMV] of SEQ ID NO 14, wherein X is any amino acid, wherein n is 13 to 15 and m is 50 to 76, or ii. includes according to any of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NO: 15, 16, 17, 18 Any one of , 20 or 21, more preferably the polypeptide sequence of any one of SEQ ID NO: 17, 18, 20 or 21, or iii. is SEQ ID NO: 15, 16, 17, 18, 19, 20 , 21, 22 or 23, preferably any of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably SEQ ID NO: 17, 18, 20 or 21. A functional homologue, variant or derivative of any having the same as any of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NO : any one of 15, 16, 17, 18, 20 or 21, more preferably the N-acetylglucosamine b-1,4-half of any one of SEQ ID NO: 17, 18, 20 or 21 At least 80% overall sequence identity to the full length of the lactosyltransferase polypeptide and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or iv. including from SEQ ID NOs: 15, 16, 17, Any of 18, 19, 20, 21, 22 or 23, preferably any of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably SEQ ID NO: 17, 18, An oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues of any of 20 or 21, and having N- Acetylglucosamine b-1,4-galactosyltransferase activity, or v. is any of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably Any of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably a functional fragment of any of SEQ ID NO: 17, 18, 20 or 21, and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or vi. comprising a polypeptide comprising or consisting of a Any one of 20, 21, 22 or 23, preferably any one of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably one of SEQ ID NO: 17, 18, 20 or 21 Any of the full-length amino acid sequences of at least 80% sequence identity consisting of amino acid sequences, and have N-acetylglucosamine b-1,4-galactosyltransferase activity, or b. PFAM domain PF00535, and i. comprising the sequence of SEQ ID NO 24 R[KN]XXXXXXXGXXXX[FL]XDXD( _Xn )[FHW]XXX[FHNY]( _Xm )E[DE], wherein X is any amino acid, wherein n is 50 to 75, m is 10 to 30, or ii. the sequence comprising SEQ ID NO 25 R[KN]XXXXXXXGXXXXFXDXD( _Xn )[FHW]XXX[ _FHNY ]( _Xm )E[DE](Xp) [FWY]XX[HKR]XX[NQST], wherein X is any amino acid, wherein n is 50 to 75, m is 10 to 30, and p is 20 to 25, or iii. including according to SEQ ID NO: 26 The polypeptide sequence of any of , 27, or 28, or iv. is a functional homologue, variant, or derivative of any of SEQ ID NO: 26, 27, or 28, having the same properties as SEQ ID NO: 26 , 27 or 28 of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides of at least 80% overall sequence identity at least 80% of the full length and having N-acetylglucosamine b-1, 4-galactosyltransferase activity, or v. comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 from any of SEQ ID NOs: 26, 27, or 28 , 19, 20 oligopeptide sequences of contiguous amino acid residues with N-acetylglucosamine b-1,4-galactosyltransferase activity, or vi. is SEQ ID NO: 26, 27, or 28 A functional fragment of any one, and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or vii. comprising a polypeptide comprising or consisting of having and SEQ ID NO: 26, 27 or Any of 28 full-length amino acid sequences consisting of amino acid sequences of at least 80% sequence identity and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or c. PFAM domain PF02709 without PFAM domain PF00535, and i. comprising the sequence of SEQ ID NO 29 [FWY]XX[FY][FWY]( _X23 )[FWY][GQ]X[DE]D, where X is any amino acid, or ii. including SEQ ID NO 30 The sequence of [PV]W[GHNP]( _Xn )[FWY][GQ]X[DE]D, wherein X is any amino acid, wherein n is 21 to 24, or iii. including according to SEQ ID NO: 31 A polypeptide sequence of any of , 32, 33, 34, 35, or 36, or iv. a functional homologue, variant of any of SEQ ID NO: 31, 32, 33, 34, 35, or 36 or a derivative having at least 80% of the full length of any of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides of SEQ ID NO: 31, 32, 33, 34, 35 or 36 % overall sequence identity with N-acetylglucosamine b-1,4-galactosyltransferase activity, or v. includes any one from SEQ ID NO: 31, 32, 33, 34, 35, or 36 An oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b- 1,4-galactosyltransferase activity, or vi. is a functional fragment of any one of SEQ ID NOs: 31, 32, 33, 34, 35, or 36, and has N-acetylglucosamine b-1, 4-galactosyltransferase activity, or vii. comprising a polypeptide comprising or consisting of at least 80 having the full-length amino acid sequence of any of SEQ ID NOs: 31, 32, 33, 34, 35 or 36 consisting of amino acid sequences of % sequence identity and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or d. PFAM domain PF03808, and i. including the sequence of SEQ ID NO 37 [ST][FHY]XN( _Xn )DG(X16)[HKR]X[ST] _FDXX [ST]XA, wherein X is any amino acid, and wherein n is 20 to 25, or ii. including SEQ Sequence of ID NO 38 [ST][FHY]XN( _Xn )DG(X16)[HKR]X[ST] _FDXX [ST]XA( _Xm )[HR]XG[FWY](Xp) _GXGXXXQ [ DE], wherein X is any amino acid, wherein n is 20 to 25, m is 40 to 50, and p is 22 to 30, or iii. including according to any of SEQ ID NOs: 39, 40 or 41 A polypeptide sequence, or iv. is a functional homologue, variant or derivative of any one of SEQ ID NO: 39, 40 or 41 having the N-B with SEQ ID NO: 39, 40 or 41 at least 80% overall sequence identity of any of the full-length acetylglucosamine b-1,4-galactosyltransferase polypeptides and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or v . comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any of SEQ ID NOs: 39, 40, or 41 oligopeptide sequence with N-acetylglucosamine b-1,4-galactosyltransferase activity, or vi. is a functional fragment of any one of SEQ ID NOs: 39, 40 or 41 with N - acetylglucosamine b-1,4-galactosyltransferase activity, or vii. comprising a polypeptide comprising or consisting of a full-length amino acid sequence with any of SEQ ID NO: 39, 40 or 41 It consists of amino acid sequences with at least 80% sequence identity and has N-acetylglucosamine b-1,4-galactosyltransferase activity. 71. The cell according to any one of specific embodiments 56 to 70, wherein a. the N-acetylglucosamine-6-phosphotransferase is a polypeptide sequence comprising a polypeptide of UniProt ID P43577, or is a polypeptide of UniProt ID P43577 A functional homologue, variant or derivative having at least 80% overall sequence identity with the full-length polypeptide of UniProt ID P43577 and having N-acetylglucosamine-6-phosphotransferase activity, and b. The L-glutamine-D-fructose-6-phosphate transaminase is a polypeptide sequence comprising the polypeptide of UniProt ID P17169, or is a functional homologue, variant or derivative of the polypeptide with UniProt ID P17169, which Has at least 80% overall sequence identity with the full-length polypeptide of UniProt ID P17169, and has L-glutamine-D-fructose-6-phosphate transaminase activity; is A39T, R250C and G472S different from the UniProt ID P17169 polypeptide Modified version of mutation. 72. The cell according to any one of specific embodiments 56 to 71, wherein the cell can metabolize a carbon source selected from the group comprising the following enumeration: glucose, fructose, galactose, lactose, sucrose, maltose, malt- Oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, mannose, methanol, ethanol, arabinose, trehalose, starch, cellulose, hemicellulose, corn infusion, high fructose syrup, molasses, Glycerin, acetate, citrate, lactic acid, pyruvic acid. 73. The cell according to any one of specific embodiments 56 to 72, wherein the cell is unable to convert N-acetylglucosamine-6-phosphate into glucosamine-6-phosphate, and/or is unable to convert glucosamine- 6-phosphate is converted to fructose-6-phosphate. 74. The cell according to any one of specific embodiments 56 to 73, wherein the cell is modified to produce GDP-fucose. 75. The cell according to any one of specific embodiments 56 to 74, wherein the cell is modified to promote GDP-fucose production, wherein the modification is selected from the group comprising the following enumeration: UDP-glucose: undecylisoamyl Shaving of the gene encoding diene-phosphoglucose-1-phosphotransferase, overexpression of the gene encoding GDP-L-fucose synthase, overexpression of the gene encoding GDP-mannose 4,6-dehydratase, mannose - Overexpression of a gene encoding 1-phosphate guanylate transferase, overexpression of a gene encoding phosphomannose mutase, or overexpression of a gene encoding mannose-6-phosphate isomerase. 76. The cell according to any one of specific embodiments 56 to 75, wherein the cell is modified to produce UDP-galactose. 77. The cell according to any one of specific embodiments 56 to 76, wherein the cell is modified to promote UDP-galactose production, wherein the modification is selected from the group comprising the following enumeration: 5'-nucleotidase/UDP - Shaving of the gene encoding a glycohydrolase or of the gene encoding a galactose-1-phosphate uridine transferase. 78. The cell according to any one of specific embodiments 56 to 75, wherein the cell is modified to produce CMP-N-acetylneuraminic acid. 79. The cell according to any one of specific embodiments 56 to 78, wherein the cell is modified to promote CMP-N-acetylneuraminic acid production, wherein the modification is selected from the group comprising the following enumeration: CMP-saliva Overexpression of the gene encoding acid synthase, overexpression of the gene encoding sialic acid synthase, overexpression of the gene encoding N-acetyl-D-glucosamine 2-epimerase. 80. The cell according to any one of specific embodiments 56 to 79, wherein the cell is capable of expressing at least one other glycosyltransferase, wherein the other glycosyltransferase is selected from the group comprising: Fucosyltransferase , Sialyltransferase, Galactosyltransferase, Glucosyltransferase, Mannosyltransferase, N-Acetylglucosaminetransferase, N-Acetylgalactosyltransferase, N-Acetyl Mannosyltransferase, xylosyltransferase, glucuronyltransferase, galacturonosyltransferase, glucosamine transferase, N-glycolylneuramidotransferase, rhamnosyltransferase, N- Acetyl rhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosaminotransferase, UDP-N-acetylglucose Amine enolacetonyltransferase and fucosyltransferase, - preferably, the fucosyltransferase is selected from the group consisting of: α-1,2-fucosyltransferase, α-1 , 3-fucosyltransferase, α-1,4-fucosyltransferase and α-1,6-fucosyltransferase, - preferably, the sialyltransferase is selected from the group consisting of the following To enumerate: α-2,3-sialyltransferase, α-2,6-sialyltransferase and α-2,8-sialyltransferase, - preferably, the galactosyltransferase is selected from the group consisting of the following Listed: β-1,3-galactosyltransferase, N-acetylglucosamine β-1,3-galactosyltransferase, β-1,4-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, α-1,3-galactosyltransferase and α-1,4-galactosyltransferase, - preferably, the glucosyltransferase is selected from the group consisting of the following Enumerated: α-glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase and β-1,4-glucosyltransferase, - preferably, the mannose The syltransferase is selected from the group consisting of the following list: α-1,2-mannosyltransferase, α-1,3-mannosyltransferase and α-1,6-mannosyltransferase, - preferably, The N-acetylglucosaminyltransferase is selected from the group consisting of the following list: β-1,3-N-acetylglucosaminyltransferase and β-1,6-N-acetylglucosaminyltransferase , - Preferably, the N-acetylgalactosamine transferase is α-1,3-N-acetylgalactosamine transferase. 81. The cell according to specific embodiment 80, wherein the cell is modified in the expression or activity of the other glycosyltransferase. 82. The cell according to any one of specific embodiments 56 to 81, wherein the cell uses one or more precursors for producing the disaccharide or oligosaccharide having GlcNAc units at the reducing end, and the precursor supplies the cell. 83. The cell according to any one of specific embodiments 56 to 82, wherein the cell produces one or more precursors for producing the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end. 84. The cell according to any one of specific embodiments 82 or 83, wherein the precursor for producing the disaccharide or oligosaccharide is completely converted to a disaccharide or oligosaccharide having a GlcNAc unit at the reducing end. 85. The cell according to any one of specific embodiments 56 to 84, wherein the cell produces a disaccharide or oligosaccharide with a GlcNAc unit at the reducing end in the cell, and wherein part or substantially all of the reducing end produced has GlcNAc The unitary disaccharides or oligosaccharides remain intracellular and/or are excreted via passive or active transport. 86. The cell according to any one of specific embodiments 56 to 85, wherein the cell expresses a membrane transporter or a polypeptide with transport activity to transport the compound through the outer membrane of the cell wall, preferably, the cell is transported in the membrane The expression or activity of the protein or polypeptide having transport activity is modified. 87. The cell according to specific embodiment 86, wherein the membrane transporter or polypeptide having transport activity is selected from the group consisting of the following enumeration: transporter, PP-bond-hydrolysis-driven transporter, β-barrel porin, assisted transporter protein, putative transporter and phosphate transfer-driven group translocator, preferably, the transporter includes MFS transporter, sugar excretion transporter and chelatin exporter, preferably, the PP-bond-hydrolysis-driven The transporters include the ABC transporter and the chelatin exporter. 88. The cell according to any one of specific embodiments 86 or 87, wherein the membrane transporter or the polypeptide with transport activity controls the reducing end with a disaccharide or oligosaccharide of GlcNAc unit and/or one or more precursors and/ Or the flow of acceptors on the outer membrane of the cell wall, the one or more precursors and/or acceptors for the production of disaccharides or oligosaccharides with GlcNAc units at the reducing end. 89. The cell according to any one of specific embodiments 86 to 88, wherein the membrane transporter or the polypeptide with transport activity provides that the reducing end has an improved production of a disaccharide or oligosaccharide of a GlcNAc unit and/or allows to discharge and/or or promote excretion. 90. The cell according to any one of specific embodiments 56 to 89, wherein the cell comprises a modification for reducing acetate production compared to an unmodified precursor cell. 91. The cell according to specific embodiment 90, wherein the cell comprises a lower or reduced expression and/or ablated, impaired, reduced or delayed activity of any one or more proteins compared to an unmodified precursor cell, the Protein contains: β-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetyl Glucosamine inhibitor protein, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undec isoprene-phosphoglucose-1-phosphotransferase, L-fucosokinase, L-fucoid Carbohydrate isomerase, N-acetylneuraminic acid dissociating enzyme, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man , EIID-Man, ushA, galactose-1-phosphate uridine transferase, glucose-1-phosphate adenosyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isokinase Enzyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration regulatory protein, transcriptional repressor protein IclR, lon protease, glucose-specific translocation phosphotransferase enzyme IIBC component ptsG, glucose specific translocation phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIA ^Glc , β-glucoside specific PTS enzyme II, fructose specific PTS polyphosphoryl transfer protein FruA and FruB, alcohol dehydrogenation Enzymes aldehyde dehydrogenase, pyruvate-formate dissociation enzyme, acetate kinase, phosphoacetyltransferase, phosphoacetyltransferase, pyruvate decarboxylase. 92. The cell according to any one of specific embodiments 56 to 91, wherein the cell is capable of producing phosphoenolpyruvate (PEP). 93. The cell according to any one of specific embodiments 56 to 92, having a modification for the enhanced production and/or supply of phosphoenolpyruvate (PEP) compared to an unmodified precursor cell. 94. The cell according to any one of specific embodiments 56 to 93, wherein the cell comprises an at least partially inactivated catabolic pathway of the selected monosaccharide, disaccharide or oligosaccharide involved in the Required for the production of disaccharides or oligosaccharides with GlcNAc units at the reducing end and/or for the production of disaccharides or oligosaccharides with GlcNAc units at the reducing end. 95. The cell according to any one of specific embodiments 56 to 94, wherein the cell is resistant to the phenomenon of lactose killing when the cell is grown in an environment in which lactose is combined with one or more other carbon sources. 96. The cell according to any one of specific embodiments 56 to 95, wherein the cell produces a disaccharide with GlcNAc at this reducing end of 90 g/L or more than 90 g/L in whole culture fluid and/or supernatant Or oligosaccharides, and/or disaccharides or oligosaccharides with GlcNAc at this reducing end in the whole culture fluid and/or supernatant have a purity of at least 80% in the whole culture fluid and/or supernatant The total amount of disaccharides or oligosaccharides with GlcNAc units at the reducing end and their precursors, respectively, is calculated. 97. The cell according to any one of specific embodiments 56 to 96, wherein the cell produces a mixture comprising at least one charged, preferably sialylated, disaccharide or oligosaccharide comprising at least one reducing end having a GlcNAc unit. Saccharides and/or oligosaccharides and/or neutral disaccharides and/or oligosaccharides. 98. The cell according to any one of specific embodiments 56 to 97, wherein the cell produces a mixture comprising at least the charged, preferably sialylated oligosaccharide of an oligosaccharide having a GlcNAc unit at the reducing end Neutral oligosaccharides. 99. The cell according to any one of specific embodiments 56 to 98, wherein the oligosaccharide is selected from the list comprising: Disaccharide, 2-4-Difucosyllacto-N-disaccharide, 3'-Sialylolacto-N-disaccharide, 6'-Sialyllacto-N-disaccharide, 3',6'-Disaccharide Sialylolacto-N-disaccharide, 6,6'-disialolacto-N-disaccharide, 2'-fucosyl-3'-sialyolacto-N-disaccharide, 2'-fucose yl-6'-sialyol-N-disaccharide, 4-fucosyl-3'-sialyol-N-disaccharide, 4-fucosyl-6'-sialyol-N-bisaccharide Sugar, 2-fucosyl N-acetyllactosamine, 3'-fucosyl N-acetyllactosamine, 2,3'-difucosyl N-acetyllactosamine, 3'-saliva acid N-acetyllactosamine, 6'-sialo-N-acetyllactosamine, 3',6'-disialo-N-acetyllactosamine, 6,6'-disialo-N-acetyllactosamine , 2'-fucosyl-3'-sialic acid N-acetyllactosamine, 2'-fucosyl-6'-sialic acid N-acetyllactosamine, 3-fucosyl-3' -Sialyl N-acetyllactosamine, 3'-fucosyl-6'-sialic acid N-acetyllactosamine, P1 trisaccharide (Gal-a1,4-Gal-b1,4-GlcNAc), iso Source graft epitope (Gal-a1,3-Gal-b1,4-GlcNAc), Gal-b14-(Galb13)-GlcNAc, poly-N-acetyllactosamine, GalNAc-b1,3-Gal-b1, 4-GlcNAc. 100. The cell according to any one of specific embodiments 56 or 58 to 99, wherein the disaccharide having a GlcNAc unit at the reducing end excludes chitobiose (GlcNAc-GlcNAc). 101. The cell according to any one of specific embodiments 56 to 100, wherein the oligosaccharide having a GlcNAc unit at the reducing end excludes chitobiose, preferably N-glycans, at the reducing end. 102. The cell according to any one of specific embodiments 56 to 101 or the method according to any one of specific embodiments 1 to 55, wherein the cell is a bacterium, fungus, yeast, plant cell, animal cell or protozoan cell , - preferably, the bacteria are Escherichia coli strains, more preferably Escherichia coli strains K12 strains, and more preferably, the Escherichia coli strains K12 strains are Escherichia coli MG1655, - preferably, the fungi are selected from the group consisting of black Genus (genus) of the group of Mold, Reticulinum, Penicillium, White mold or Koji, - preferably, the yeast belongs to a genus selected from the group consisting of Saccharomyces, Zygomyces, Bis A genus of the group of Escherichia, Kermagtella, Hansenula, Ascomyces, Bacteroides, Kluyveromyces or Debaryomyces, - compared Preferably, the plant cells are algal cells or derived from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soybean, corn or cereal plants, - preferably, the animal cells are derived from non-human mammals, birds, fish , invertebrates, reptiles, amphibians or insects, or genetically modified cell lines derived from human cells excluding embryonic stem cells, more preferably, the human and non-human mammalian cells are epithelial cells, embryonic kidney cells, Fibroblasts, COS cells, Chinese hamster ovary (CHO) cells, murine myeloma cells, NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, more preferably, the insect cells are derived from autumn armyworm, silkworm, Cabbage Spodoptera, Trichoderma or Drosophila melanogaster, - preferably, the protozoan cell is a Leishmania tarantula cell. 103. The cell according to specific embodiment 102 or the method according to specific embodiment 102, wherein the cell is a live Gram-negative bacteria comprising a reduction or elimination of poly-N-acetylene compared to unmodified precursor cells - Glucosamine (PNAG), Enterobacterial Common Antigen (ECA), Cellulose, Koramic Acid, Core Oligosaccharide, Osmotic Pressure Regulating Interstitial Glucan (OPG), Glucosylglycerol, Polysaccharide and/or Trehalose synthesis. 104. The method according to any one of specific embodiments 1 to 55, 102 or 103, wherein the separation comprises at least one of the following: clarification, ultrafiltration, nanofiltration, two-phase partition, reverse osmosis, microfiltration, Activated carbon or carbon treatment, nonionic surfactant treatment, 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. 105. The method of any one of specific embodiments 1 to 55, 102 to 104, further comprising purifying the disaccharide or oligosaccharide from the cell. 106. The method according to any one of specific embodiments 1 to 55, 102 to 105, wherein the purification comprises at least one of the following steps: use of activated carbon or carbon, use of carbon, nanofiltration, ultrafiltration, electrophoresis, Enzyme treatment or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying, freeze drying, spray freeze drying, freeze spray drying, belt drying, conveyor belt drying, vacuum belt drying, vacuum conveyor drying, tumble drying, tumble drying, vacuum tumble drying or vacuum tumble drying. 107. A cell according to any one of specific embodiments 56 to 103 or a method according to any one of specific embodiments 1 to 55, 102 to 106 for producing a disaccharide having an N-acetylglucosamine unit at the reducing end or oligosaccharides. 108. A cell according to any one of specific embodiments 56, 58 to 103 or the method according to any one of specific embodiments 1, 3 to 55, 102 to 106 for producing a reducing end with N-acetylglucosamine Use of oligosaccharides of units, preferably for the production of charged or neutral oligosaccharides having N-acetylglucosamine units at the reducing end, more preferably for the production of sialylated or fucosylated forms of LNB or LacNAc .

The following examples are intended to further illustrate and clarify the invention and are not intended to limit the invention.

Example

Example 1 : Materials and Methods Escherichia coli

culture medium

Luria Broth (LB) medium consisted of 1% trypsin (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). Medium for 96-well plate culture experiments or shake flask experiments contains 2.00g/L NH4Cl, 5.00g/L (NH4)2SO4, 2.993g/L KH2PO4, 7.315g/L K2HPO4, 8.372g/L MOPS, 0.5g /L NaCl, 0.5g/L MgSO4.7H2O, 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 described 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 precursors. The pH of the medium was set to 7 with 1M KOH. The vitamin solution consists of 3.6g/L FeCl2.4H2O, 5g/L CaCl2.2H2O, 1.3g/L MnCl2.2H2O, 0.38g/L CuCl2.2H2O, 0.5g/L CoCl2.6H2O, 0.94g/L ZnCl2, 0.0311g /L H3BO4, 0.4g/L Na2EDTA.2H2O and 1.01g/L thiamine hydrochloride. The molybdate solution contained 0.967g/L NaMoO4.2H2O. The selenium solution contains 42g/L SeO2.

The minimal medium used for fermentation contained 6.75g/L NH4Cl, 1.25g/L(NH4)2SO4, 2.93g/L KH2PO4 and 7.31g/L KH2PO4, 0.5g/L NaCl, 0.5g/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 above. As described 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 minimal medium for fermentation as precursors.

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

plastid

pKD46 (Red helper plastid, ampicillin resistance), pKD3 (containing FRT flanked chloramphenicol resistance (cat) gene), pKD4 (containing FRT flanked kanamycin resistance (kan) gene) and The pCP20 (expressing FLP recombinase activity) plastid was obtained from Prof. R. Cunin (Vrije Universiteit Brussel, Belgium in 2007).

Plastids were maintained in host E. coli DH5α (F ^- , phi80d lacZΔ M15, Δ( lacZYA - argF ) U169, deoR , recA1 , endA1 , hsdR17(rk ^- , mk ⁺ ), phoA , supE44 , lambda ^- , purchased from Invitrogen thi -1, gyrA96 , rel A1).

Strains and Mutations

E. coli K12 MG1655 [λ ⁻ , F ⁻ , rph-1] was obtained from Coli Genetic Stock Center (US), CGSC strain #: 7740 in March 2007. Gene disruption, gene introduction and gene replacement were performed using techniques published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). This technology is based on antibiotic selection after homologous recombination by lambda Red recombinase. Subsequent catalysis by flippase recombinase ensures removal of the antibiotic selection cassette in the final production strain.

Transformants carrying the Red helper plastid pKD46 were grown in 10 ml LB medium containing ampicillin (100 mg/L) and L-arabinose (10 mM) at 30 °C to an OD ₆₀₀ nm of 0.6. Cells were made electrocompetent by a first wash with 50 ml ice-cold water and a second wash with 1 ml of ice-cold water. Then, cells were resuspended in 50 μl of ice-cold water. Electroporation was performed with 50 μl of cells and 10-100 ng of linear double-stranded DNA product using a Gene Pulser™ (BioRad) (600Ω, 25 μFD and 250 volts).

After electroporation, cells were added to 1 mL of LB medium for 1 h at 37°C, and finally plated on LB agar containing 25 mg/L chloramphenicol or 50 mg/L kanamycin to select for antibiotic-resistant transfectants. Selected mutants were verified by PCR with primers upstream and downstream of the modified region and grown in LB agar at 42°C to lose helper plastids. Mutants were tested for ampicillin sensitivity.

Linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and their derivatives as templates. The primers used have one part of the sequence complementary to the template and another part complementary to the side of the chromosomal DNA on which recombination must occur. For gene knockout, homology regions were designed to be 50-nt upstream and 50-nt downstream of the start and stop codons of the target gene. For knock-in, transcription start (+1) must be considered. PCR products were PCR purified, Dpnl digested, repurified from agarose gels, and suspended in elution buffer (5 mM Tris, pH 8.0).

Selected mutants were transformed with pCP20 plastids, which are ampicillin and chloramphenicol-resistant plastids, which show temperature-sensitive replication and heat-induced FLP synthesis. Ampicillin resistant transformants were selected at 30°C, then a few colonies were purified in LB at 42°C and then tested for all antibiotic resistance and loss of FLP helper plastids. Gene shaves and knock-ins were checked with control primers.

In one example of GDP-fucose and fucosylated oligosaccharide production, the mutant strain is derived from E. coli K12 MG1655, which includes shaving of the E. coli wcaJ and thyA genes and knockout of the persistent expression construct In, the construct contains a sucrose transporter (eg, CscB (UniProt ID E0IXR1) from E. coli W), fructokinase (eg, frk (UniProt ID from Zymomonas mobilis , ZmFrk) Q03417)), sucrose phosphorylase (eg, BaSP from Bifidobacterium adolescentis (UniProt ID A0ZZH6)), additionally including expressing plastids with α-1,2-fucosyl Transferase (eg, HpFutC from Helicobacter pylori (GenBank No. AAD29863.1)) and/or α-1,3-fucosyltransferase (eg, HpFucT from Helicobacter pylori (UniProt ID O30511)) and has a persistent expression construct for E. coli thyA (UniProt ID P0A884) as a selectable marker. The fucosyltransferase gene can also be present in mutant E. coli strains via gene knock-in. GDP-fucose production in mutant E. coli strains can be further optimized by genetically shaving E. coli genes including glgC , agp , pfkA , pfkB , pgi , arcA , iclR , pgi and lon , as described in WO2016075243 and WO2012007481 . GDP-fucose production can additionally be optimized including mannose-6-phosphate isomerase (eg, manA from E. coli (UniProt ID P00946)), phosphomannose mutase (eg, from E. coli manB (UniProt ID P24175)), mannose-1-phosphate guanylate transferase (eg, manC from E. coli (UniProt ID P24174)), GDP-mannose 4,6-dehydratase (eg, from Knock-in of persistent expression constructs of gmd from E. coli (UniProt ID P0AC88)) and GDP-L-fucose synthase (eg, fcl from E. coli (UniProt ID P32055)). GDP-fucose production can also be achieved by gene shaving of E. coli fucK and fucI with fucose permeases (eg, fucP from E. coli (UniProt ID P11551)) and with fucokinase/fuco Obtained by knock-in of a persistent expression construct of a bifunctional enzyme with sugar-1-phosphate guanylate transferase activity (eg, fkp from Bacteroides fragilis (UniProt ID SUV40286.1)). If a GDP-fucose-producing mutant strain is intended to make fucosylated lactose structures, the strain also needs to genetically shave the E. coli LacZ, LacY, and LacA genes and for lactose permease (e.g., large intestine Bacillus LacY (UniProt ID P02920)) was modified by knock-in of a persistent expression construct. Alternatively, and/or in addition, GDP-fucose and/or fucosylated oligosaccharide production can be further optimized in mutant E. coli strains by gene knock-in of constitutive transcriptional units, which Transcription units comprise membrane transporters such as MdfA from Enterobacter sakazakii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter johnsonii (UniProt ID D4BC23), MdfA from Escherichia coli (UniProt ID P0AEY8), MdfA from Lebsiella (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207) or iceT from Citrobacter japonicus (UniProt ID D4B8A6).

In one example of sialic acid production, the mutant strain is derived from E. coli K12 MG1655, which includes a knock-in of a persistent transcription unit containing one or more replicates of N-acetylglucosamine-6 - Phosphotransferase (eg, GNA1 from Saccharomyces cerevisiae (UniProt ID P43577)), N-acetylglucosamine 2-epimerase (eg, AGE from Bacteroides ovatus (UniProt ID A7LVG6) )), N-acetylneuraminic acid synthase (eg, from Neisseria meningitidis (UniProt ID E0NCD4) or Campylobacter jejuni (UniProt ID Q93MP9)).

Alternatively, and/or additionally, sialic acid production can be obtained by gene knock-in of a persistent transcription unit containing UDP-N-acetylglucosamine 2-epimerase (eg, from Bacillus NeuC (UniProt ID Q93MP8)) and N-acetylneuraminic acid synthase (eg, from Neisseria meningitidis (UniProt ID E0NCD4) or Aspergillus jejuni (UniProt ID Q93MP9)).

Alternatively and/or additionally, sialic acid production can be obtained by gene knock-in of a persistent transcription unit containing a phosphoglucosamine mutase (e.g., glmM (UniProt ID) from E. coli. P31120)), N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase (eg, glmU from E. coli (UniProt ID P0ACC7)), UDP-N - Acetylglucosamine 2-epimerase (eg, NeuC from Aspergillus jejuni (UniProt ID Q93MP8)) and N-acetylneuraminic acid synthase (eg, from Neisseria meningitidis (UniProt ID E0NCD4) or Aspergillus jejuni (UniProt ID Q93MP9)).

Alternatively, and/or additionally, sialic acid production can be obtained by gene knock-in of a persistent transcription unit containing a bifunctional UDP-GlcNAc2-epimerase/N-acetylmannosamine kinase ( For example, from mouse (strain C57BL/6J) (UniProt ID Q91WG8), N-acylneuraminate-9-phosphate synthetase (for example, from Pseudomonas spp. ( Pseudomonas sp.) UW4 (UniProt ID K9NPH9) and N-acylneuraminic acid-9-phosphatase (eg, 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 gene knock-in of a persistent transcription unit containing a phosphoglucosamine mutase (eg, glmM from E. coli (UniProt ID P31120)) , N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase (eg, glmU from E. coli (UniProt ID P0ACC7)), bifunctional UDP-GlcNAc 2 - epimerase/N-acetylmannosamine kinase (e.g., from mouse (strain C57BL/6J) (UniProt ID Q91WG8), N-acylneuraminic acid-9-phosphate synthase (e.g., from sham) Unimonas UW4 (UniProt ID K9NPH9) and N-acylneuraminic acid-9-phosphatase (eg, from Candidatus Magnetomorum sp. HK-1 (UniProt ID KPA15328.1) or Bacteroides (UniProt ID Q8A712)).

By gene shaving of E. coli genes (including one or more nagA , nagB , nagC , nagD , nagE , nanA , nanE , nanK , manX , manY and manZ ) as described in WO18122225, and/or E. coli genes ( Gene shaving, including one or more nanT , poxB , ldhA , adhE , aldB , pflA , pflC , ybiY , ackA and/or pta ), and gene knock-in of persistent transcription units can be further optimized in mutant E. coli strains sialic acid production, this persistent transcription unit includes one or more replicated L-glutamine-D-fructose-6-phosphate transaminase (e.g., mutant glmS*54 from E. coli (with UniProt ID P17169 The wild-type E. coli glmS differs by having the A39T, R250C and G472S mutations, as described by Deng et al. (Biochimie 88, 419-29 (2006))), preferably a phosphatase (e.g., from a phosphatase 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 Any one or more of the E. coli genes of YbiU, or PsMupP from Pseudomonas putida, ScDOG1 from Saccharomyces cerevisiae, or BsAraL from Bacillus subtilis) and acetyl-CoA synthase (eg, from E. coli acs (UniProt ID P27550)) as described in WO18122225.

For sialylated oligosaccharide production, the sialic acid producing strain was further modified to express N-acylneuraminate cytidylyltransferase (eg, NeuA enzyme from Aspergillus jejuni (UniProt ID Q93MP7) , NeuA enzyme (GenBank No. AGV11798.1) from Haemophilus influenzae (GenBank No. AGV11798.1) or NeuA enzyme (GenBank No. AMK07891.1) from Pasteurella multocida and expressing one or more replications β-galactoside α-2,3-sialyltransferase (eg, PmultST3 from Pasteurella haemorrhagic (UniProt ID Q9CLP3) or by a β-galactoside α-2,3-sialyltransferase PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of active UniProt ID Q9CLP3, NmeniST3 from Neisseria meningitidis (GenBank No. ARC07984.1 ) or from Pasteurella hemorrhagic subsp. . multocida subsp. multocida str.) PmultST2 of Pm70 (GenBank No. AAK02592.1)), β-galactoside α-2,6-sialyltransferase (eg, PdST6 from Photobacterium damselae ) (UniProt ID O66375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID O66375 with β-galactoside α-2,6-sialyltransferase activity or from Photobacterium JT- P-JT-ISH-224-ST6 of ISH-224 (UniProt ID A8QYL1) or amino acid residues 18 to 514 of UniProt ID A8QYL1 with β-galactoside α-2,6-sialyltransferase activity composed of P-JT-ISH-224-ST6-like polypeptide) and/or α-2,8-sialyltransferase (eg, from mouse (UniProt ID Q64689). N-Acyl neuraminic acid cytidine The persistent transcription units of acyltransferase and sialyltransferase can be delivered to mutant strains by knock-in or expressing plastids. If mutants producing sialic acid and CMP-sialic acid are designed to sialylate the lactose structure, the strain By genetically shaving the E. coli LacZ , LacY , and LacA genes and by genomically knocking in the continuous transcription unit of lactose permease (e.g., E. coli LacY (UniProt ID). P02920)) for additional modifications. All mutants producing sialic acid, CMP-sialic acid, and/or sialylated oligosaccharides are selectively mediated via a sucrose transporter (e.g., CscB derived from E. coli W (UniProt ID E0IXR1)), fructokinase (e.g., Knock-in of persistent transcription units from Z. mobilis Frk (UniProt ID Q03417)) and sucrose phosphorylase (eg, BaSP from B. adolescentis (UniProt ID A0ZZH6)) adapted to sucrose growth.

Alternatively, and/or additionally, sialic acid and/or sialylated oligosaccharide production can be further optimized for mutant E. coli strains by gene knock-in of a persistent transcription unit including a continuous transcription unit, such as a membrane transporter such as sialic acid Transporters such as 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 from E. coli nanT from E. albertii (UniProt ID B1EFH1), or transport proteins such as 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 Enterobacter sakazakii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter johnsonii ( UniProt ID D4BC23), MdfA from Escherichia coli (UniProt ID P0AEY8), MdfA from Klebsiella pneumoniae (UniProt ID G9Z5F4), iceT from Escherichia coli (UniProt ID A0A024L207), iceT from Citrobacter japonicus (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 such as from E. coli oppF (UniProt ID P77737), lmrA (UniProt ID A0A1V0NEL4) from Lactococcus lactis subsp. lactis diacetate biomutant, or Blon_2475 (UniProt ID B7GPD4) from Lactobacillus bifidum subsp. infantis.

In an example used to promote UDP-galactose production, the E. coli K12 MG1655 strain was transformed by shaving the genes of one or more of the E. coli ushA , galT , ldhA and agp genes and by E. coli UDP-glucose - Gene knock-in modification of a persistent expression construct of 4-epimerase (galE) (UniProt ID P09147).

In an example for promoting UDP-GlcNAc production, the E. coli K12 MG1655 strain was treated with L-glutamine-D-fructose-6-phosphate transaminase (such as mutant glmS*54 from E. coli, which The wild-type E. coli glmS protein, which differs from UniProt ID P17169, has the A39T, R250C and G472S mutations, modified by knock-in of the persistent transcription unit as described by Deng et al. (Biochimie 2006, 88: 419-429).

In the example for the production of lacto-N-trisaccharide (LN3, GlcNAc-b1,3-Gal-b1,4-Glc), the mutant strain was derived from E. coli K12 MG1655 and obtained by combining E. coli lacZ , lacY , The lacA and nagB genes are shaving off and expressed by lactose permease (eg, Escherichia coli LacY (UniProt ID P02920)) and galactoside β-1,3-N-acetylglucosaminyltransferase (eg, from meninges The gene knock-in modification of the persistent transcription unit of IgtA (UniProt ID Q9JXQ6) of Neisseria pyogenes.

In an example for the production of LN3-derived oligosaccharides (eg, lacto-N-tetrasaccharide) (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), mutated LN3 The production strain is further transformed by gene knock-in via N-acetylglucosamine β-1,3-galactosyltransferase (eg, wbgO with SEQ ID NO 03 from E. coli 055:H7) or by the formation of expression plastids. Modifications are made in the manner in which the persistent transcription unit is delivered to the strain.

In an example for the production of LN3-derived oligosaccharides (eg, lacto-N-neotetraose) (LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), the mutated The LN3-producing strain is further characterized by gene knock-in via N-acetylglucosamine β-1,4-galactosyltransferase (eg, lgtB with SEQ ID NO 15 from Neisseria meningitidis) or the formation of expression plastids. Modifications are made in the manner in which the persistent transcription unit is delivered to the strain.

In an example for the production of lacto-N-bisaccharide (LNB, Gal-b1,3-GlcNAc) and LNB-derived oligosaccharides, the strains were further modified by N-acetylglucosamine-6 for one or more replications - Phosphotransferase (eg, GNA1 from Saccharomyces cerevisiae (UniProt ID P43577)) and N-acetylglucosamine beta-1,3-galactosyltransferase (selected from including from SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12, and 13) gene knock-in or modified expression plasmids containing persistent transcription units.

In the examples for the production of N-acetyllactosamine (LacNAc, Gal-b1,4-GlcNAc) and LacNAc-derived oligosaccharides, the strains were further modified by N-acetylglucosamine-6 for one or more replications - Phosphotransferases (eg, GNA1 from Saccharomyces cerevisiae (UniProt ID P43577)) and N-acetylglucosamine beta-1,4-galactosyltransferases (selected from including from SEQ ID NO: 15, 16, 17, 18, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35, 36, 39, 40, or 41) knock-in or containing a persistent transcription unit Expression plastids are modified.

Mutant LNB, LacNAc, LN3, LNT, and LNnT-producing E. coli strains can also selectively via sucrose transporters (e.g., CscB from E. coli W (UniProt ID E0IXR1)), fructokinase (e.g., from locomotor) Zymomonas Frk (UniProt ID Q03417)) and sucrose phosphorylase (eg, BaSP from Bifidobacterium adolescentis (UniProt ID A0ZZH6)) were knocked in for growth on sucrose.

Alternatively, and/or additionally, the production of LN3, LNT, LNnT, LNB and LacNAc and their derived oligosaccharides can be further optimized in mutant E. coli strains by gene knock-in of a persistent transcription unit comprising a membrane Transporters such as MdfA from Enterobacter sakazakii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter johnsonii (UniProt ID D4BC23), MdfA from Escherichia coli (UniProt ID P0AEY8), Klebsiella pneumoniae MdfA (UniProt ID G9Z5F4), iceT from Escherichia coli (UniProt ID A0A024L207) or iceT from Citrobacter japonicus (UniProt ID D4B8A6).

Preferably, but not necessarily, any one or more of glycosyltransferases, nucleotide-active sugar-synthesizing proteins, and/or membrane transporters are fused at the N- and/or C-terminus to a solubility enhancer tag, e.g., SUMO tags, MBP tags, His, FLAG, Strep-II, Halo-tag, NusA, thioredoxin, GST and /Fh8 tags to improve 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).

Alternatively, mutant E. coli strains are modified by gene knock-in of a persistent transcription unit encoding a chaperone protein (e.g., DnaK, DnaJ, GrpE, or the GroEL/ES chaperone protein). The 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).

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

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

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

All genes were ordered synthetically at Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com) and codon usage was adjusted using the vendor's tools.

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

Table 1: Summary of SEQ ID NOs described in the present invention SEQ ID NO organism source Country of origin of the digital sequence information 01 NA synthesis artificial sequence 02 NA synthesis artificial sequence 03 Escherichia coli O55:H7 synthesis Germany 04 Pseudogulbenkiania ferrooxidans synthesis U.S. 05 Serotype Salmonella enterica subsp. salamae serovar synthesis Australia 06 Corynebacterium glutamicum synthesis Japan 07 Photobacterium leiognathi synthesis U.S. 08 Chromobacterium violaceum synthesis U.S. 09 NA synthesis artificial sequence 10 Arabidopsis ( A. thaliana ) synthesis U.S. 11 Trypanosoma brucei synthesis Scotland (UK) 12 mouse synthesis U.S. 13 Homo sapiens synthesis unknown 14 NA synthesis artificial sequence 15 Meningococcus MC58 synthesis U.K. 16 Meningococcus MC58 synthesis U.K. 17 Helicobacter pylori synthesis Australia 18 Helicobacter pylori synthesis Australia 19 Aggregatibacter aphrophilus synthesis U.K. 20 Helicobacter pylori synthesis Australia twenty one Pasteurella haemorrhagic synthesis Australia twenty two Haemophilus influenzae synthesis U.S. twenty three Kingella denitrificans synthesis unknown twenty four NA synthesis artificial sequence 25 NA synthesis artificial sequence 26 Streptococcus pneumoniae synthesis U.S. 27 Streptococcus agalactiae synthesis U.K. 28 Hafnia alvei synthesis unknown 29 NA synthesis artificial sequence 30 NA synthesis artificial sequence 31 Steroidobacter denitrificans synthesis Germany 32 Parachlamydiaceae bacterium HS-T3 synthesis Japan 33 Coxiella sp . DG_40 synthesis U.S. 34 Bacteroides fragilis synthesis U.K. 35 Corallococcus exercitus synthesis U.K. 36 Hypericibacter adhaerens synthesis Germany 37 NA synthesis artificial sequence 38 NA synthesis artificial sequence 39 Bacteroides vulgatus synthesis U.K. 40 Prevotella copri synthesis U.S. 41 Pseudomonas fluorescens 90F12-2 synthesis U.S.

Culture conditions

Pre-incubation of 96-well microtiter plate experiments was initiated from frozen vials in 150 μL of LB overnight at 37°C on an orbital shaker at 800 rpm. This culture was used as an inoculum in a 96-well square microtiter plate, diluted 400-fold with 400 μL of MMsf medium. These final 96-well plates were then incubated at 37°C on an orbital shaker at 800 rpm for 72 h or less or longer. To determine the sugar concentration at the end of the culture experiment, a whole broth sample (=intracellular and extracellular sugar concentration average) was taken from each well by boiling the medium at 60°C for 15 minutes before centrifuging the cells.

Bioreactor precultures were started from whole 1 mL cryovials of specific strains, inoculated in 250 mL or 500 mL MMsf medium in 1 L or 2.5 L shake flasks, and grown at 37°C for 24 h on an orbital shaker at 200 rpm. A 5 L bioreactor was then inoculated (250 mL in 2 L batch medium); the process was controlled by MFCS control software (Sartorius StedimBiotech, Melsungen, Germany). Culture conditions were set at 37°C with maximum agitation; pressure gas flow rate was dependent on strain and bioreactor. The pH was controlled at 6.8 using 0.5M H2SO4 and 20% NH4OH. Cool the exhaust gas. Add 10% organosilicon defoamer solution when foaming during fermentation.

Optical density

Cultures were frequently monitored for cell density by measuring optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or Spark 10M Microplate Reader, Tecan, Switzerland).

Analytical analysis

Standards such as, but not limited to, sucrose, glucose, N-acetylglucosamine, N-acetyllactosamine, lacto-N-disaccharide, fucosylated N-acetyllactosamine (2'FLAcNAc, 3 -FlacNAc), fucosylated lacto-N-disaccharides (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 using in-house developed standards.

N-acetylglucosamine and N-acetyllactosamine were analyzed using pulsed amperometric detection (PAD) on the Dionex HPAEC system. Inject 5 µL of sample into Dionex CarboPac PA1 Column 4x250mm and Dionex CarboPac PA1 Guard Column 4x50mm. The column temperature was 20°C. The separation was performed using 3 eluates: A) deionized water, B) 200 mM sodium hydroxide, and C) 500 mM sodium acetate. The dissolution profiles were applied as follows: 0-10 minutes 50% A and 50% B; 10-18 minutes 50-44% A and 50% B; 18-28 minutes 44% A and 50% B; 28-32 minutes 44- 30.8%A and 50%B; 32-39 minutes 30.8%A and 50%B; 39-40 minutes 30.8-2%A and 50%B; 40-43 minutes 2%A and 50%B; 43-44 minutes 2-50%A and 50%B; 44-50 minutes 50%A and 50%B. The flow rate was 1.0 mL/min.

N-acetylglucosamine, N-acetyllactosamine, lacto-N-disaccharide, fucosylated N-acetyllactosamine, and fucosylated LNB with evaporative light scattering detector (ELSD) ) or on a Waters Acquity H-stage UPLC with a Refractive Index (RI) detector. A volume of 0.7 µL of the sample was injected onto a Waters Acquity UPLC BEH Amide column (2.1 x 100mm; 130Å; 1.7 µm) and an Acquity UPLC BEH Amide VanGuard column, 130Å, 2.1x 5mm. The column temperature was 50°C. The mobile phase consisted of ¼ water and ¾ acetonitrile solution with addition of 0.2% triethylamine. The method is isocratic at a flow rate of 0.130 mL/min. The drift tube temperature of the ELS detector was 50°C, the N gas pressure was 50 psi, the gain was ₂₀₀ , and the data rate was 10pps. The temperature of the RI detector was set to 35°C.

Sialylated N-acetyllactosamine and sialylated lacto-N-disaccharide were analyzed on a Waters Acquity H-stage UPLC equipped with a refractive index (RI) detector. A volume of 0.5 µL of sample was injected into a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm with 1.7 µm particle size) with a mobile phase containing 70 mL of acetonitrile, 150 mM ammonium acetate in 26 mL of ultrapure water, and 4 mL was added with 0.05% pyrrolidine in methanol. The method is isocratic at a flow rate of 0.150 mL/min. The column temperature was set to 50°C.

Low concentrations (below 50 mg/L) of sugars were analyzed using pulsed amperometric detection (PAD) on the Dionex HPAEC system. A volume of 5 µL of sample was injected into a Dionex CarboPac PA200 column 4 x 250 mm and a Dionex CarboPac PA200 guard column 4 x 50 mm. The column temperature was set to 30°C. A gradient was used where eluent A was deionized water, where eluent B was 200 mM sodium hydroxide and where eluent C was 500 mM sodium acetate. Oligosaccharides were separated in 60 min while maintaining a constant ratio of 25% eluent B using the following gradient: an initial isocratic step with 75% eluent A for 10 min and an initial increase in eluent C over 8 min From 0 to 4%, the second isocratic step holds 71% of eluent A and 4% of eluant C for 6 minutes, and a second increase of eluent C from 4% to 12% in 2.6 minutes, The third isocratic step held 63% eluate A and 12% eluate C for 3.4 minutes, with a third increase in eluate C from 12% to 48% in 5 minutes. As a washing step, 48% solution C was used to wash for 3 minutes. For column equilibration, the initial conditions of 75% eluent A and 0% eluent C were recovered within 1 minute and held for 11 minutes. The applied flow rate was 0.5 mL/min.

data normalization

For all types of culture conditions, data obtained from mutant strains were normalized to data obtained under the same culture conditions as the reference strain.

Example 2 : in a modified E. coli host GlcNAc generation

In this example, first, wild-type E. coli K-12 MG1655 can be identified by homologous E. coli N-acetylglucosamine-6-phosphate deacetylase ( nagA ) gene and E. coli glucosamine-6- Modification by knockout of the phosphate deaminase ( nagB ) gene, followed by further transformation with expression plastids including N-acetylglucosamine-6-phosphotransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577 ) of the persistent performance box. The mutant E. coli strains thus obtained produced GlcNAc in whole broth samples when evaluated in growth experiments according to the culture conditions in Example 1, wherein the medium contained glycerol.

Example 3 : in a modified E. coli host GlcNAc generation

Mutant E. coli strains modified to produce GlcNAc as described in Example 2 can be further transformed with a second compatible expression plastid comprising L-glutamine from E. coli— Persistent expression cassette of a mutant variant of the D-fructose-6-phosphate transaminase glmS*54, which differs from the wild-type glmS protein (UniProt ID P17169) by the A39T, R250C and G472S mutations, as described in Deng et al. described (Biochimie 2006: 88, 419-429). The novel strain also produced GlcNAc in whole broth samples when evaluated in growth experiments according to the culture conditions in Example 1 (wherein the medium contained glycerol), and the GlcNAc titers obtained in this strain were higher than those obtained in the GlcNAc titers obtained from mutant strains generated in Example 2 and lacking mutant glmS*54.

Example 4 : in a modified E. coli host GlcNAc generation

Wild-type E. coli K-12 MG1655 was modified by deletion of the E. coli nagA and nagB genes and knock-in of a persistent cassette of GNA1 (UniProt ID P43577) from Saccharomyces cerevisiae. The mutant E. coli strains thus obtained produced GlcNAc in whole broth samples when evaluated in growth experiments according to the culture conditions in Example 1, wherein the medium contained glycerol.

Example 5 : in a modified E. coli host GlcNAc generation

Mutant E. coli strains modified to produce GlcNAc as described in Example 4 can be further transformed with an expression plastid comprising a persistent expression cassette of glmS*54 from E. coli, which is combined with The wild-type glmS protein (UniProt ID P17169) differs by the A39T, R250C and G472S mutations as described by Deng et al. (Biochimie 2006: 88, 419-429). The novel strain also produced GlcNAc in whole broth samples when evaluated in growth experiments according to the culture conditions in Example 1 (wherein the medium contained glycerol), and the GlcNAc titers obtained in this strain were higher than those obtained in the GlcNAc titers obtained from mutant strains generated in Example 4 and lacking mutant glmS*54.

Example 6 : in a modified E. coli host GlcNAc generation

Mutant E. coli strains modified to produce GlcNAc as described in Example 4 can be further transformed with a gene knock-in comprising a persistent expression cassette of glmS*54 from E. coli, which is combined with the wild-type glmS protein (UniProt ID P17169) differed by the A39T, R250C and G472S mutations as described by Deng et al. (Biochimie 2006: 88, 419-429). The novel strain also produced GlcNAc in whole broth samples when evaluated in growth experiments according to the culture conditions in Example 1 (wherein the medium contained glycerol), and the GlcNAc titers obtained in this strain were higher than those obtained in the GlcNAc titers obtained from mutant strains generated in Example 4 and lacking mutant glmS*54.

Example 7 : in a modified E. coli host GlcNAc generation

Mutant E. coli strains modified to produce GlcNAc as described in Example 2 can be further transformed with a gene knock-in comprising a persistent expression cassette of glmS*54 from E. coli, which is combined with the wild-type glmS protein (UniProt ID P17169) differed by the A39T, R250C and G472S mutations as described by Deng et al. (Biochimie 2006: 88, 419-429). The novel strain also produced GlcNAc in whole broth samples when evaluated in growth experiments according to the culture conditions in Example 1 (wherein the medium contained glycerol), and the GlcNAc titers obtained in this strain were higher than those obtained in the GlcNAc titers obtained from mutant strains generated in Example 2 and lacking mutant glmS*54.

Example 8 : in a modified E. coli host LacNAc or LNB generation

As described in Examples 2 and 4 to 7, with nagAB KO and expressing GNA1 (UniProt ID P43577) with or without additional mutant glmS*54 expression (different from wild-type glmS protein (UniProt ID P17169) in A39T, R250C and the G472S mutation, as described by Deng et al. (Biochimie 2006: 88, 419-429), the mutant GlcNAc-producing E. coli K-12 MG1655 strain can be further transformed with plastids containing a persistent transcription unit to express Meningococcus N-acetylglucosamine β1,4-galactosyltransferase LgtB of SEQ ID NO 15 or N-acetylglucosamine β1,3- from Escherichia coli O55:H7 with SEQ ID NO 03 Galactosyltransferase WbgO.

Each of the novel strains expressing LgtB of SEQ ID NO 15 produced GlcNAc and LacNAc in whole broth samples when evaluated in growth experiments according to the culture conditions in Example 1, wherein the medium contained glycerol. GlcNAc and LacNAc titers were higher in the novel strain expressing SEQ ID NO 15 and also expressing the mutant glmS*54 compared to the modified strain expressing SEQ ID NO 15 but lacking the mutant glmS*54.

Each of the novel strains expressing WbgO of SEQ ID NO 03 produced GlcNAc and LNB in whole broth samples when evaluated in growth experiments according to the culture conditions in Example 1, wherein the medium contained glycerol. GlcNAc and LNB titers were higher in the novel strain expressing SEQ ID NO 03 and also expressing the mutant glmS*54 compared to the modified strain expressing the WbgO of SEQ ID NO 03 but lacking the mutant glmS*54.

Example 9 : in a modified E. coli host GlcNAc generation

As described in Example 1, the E. coli mutant K-12 MG1655 strain optimized for GDP-fucose production can be further mutated to additionally produce GlcNAc. The mutations included shaving of the E. coli nagA and nagB genes and knock-in of a persistent expression construct containing the mutation glmS*54 (which differs from the wild-type glmS protein (UniProt ID P17169) by A39T, R250C and G472S mutation) and GNA1 from Saccharomyces cerevisiae (UniProt ID P43577). The novel strains were evaluated in growth experiments and compared to their parental strains according to the culture conditions provided in Example 1 (wherein the medium contained 30 g/L sucrose). Each strain was grown in multiple wells of a 96-well plate for 72 hours. Experiments showed that 0.90 ± 0.10 g/L GlcNAc could be measured in whole broth samples of the novel mutant strain, whereas no GlcNAc production was detected in whole broth samples of the original parental strain. The experiments further demonstrated that the mutations added for GlcNAc production did not affect GDP-fucose production by the novel strain compared to the original parental strain.

Example 10 : in a modified E. coli host GlcNAc and LacNAc generation

As described in Example 1, the E. coli mutant K-12 MG1655 strain optimized for GDP-fucose production can shave the E. coli nagA and nagB genes with N-acetylglucose from N. meningitidis having SEQ ID NO 15 Knock-in of a persistent expression construct of amine β1,4-galactosyltransferase (LgtB) was further mutated. Next, cells of this mutant strain were transformed with different persistent expression vectors constructed from different transcription units (TUs) containing the mutant glmS*54 from E. coli (which is identical to the wild-type glmS protein (UniProt ID P17169) The differences are A39T, R250C and G472S mutations) and GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), as listed in Tables 2 and 3. All novel strains (AH) were evaluated in growth experiments according to the culture conditions provided in Example 1. Table 2 shows the yields of GlcNAc (g/L) and LacNAc (g/L) in whole broth samples taken from each novel mutant E. coli strain after 72 hours of culture in minimal medium containing 30 g/L sucrose . The data demonstrate that all new strains are capable of producing both GlcNAc and LacNAc without supplemental addition of GlcNAc to the medium. In contrast, a reference strain of the same genetic background but lacking glmS*54 and GNA1 expressing plastids could only produce LacNAc in medium supplemented with GlcNAc (results not shown). Table 2 also shows that the production titers of both GlcNAc and LacNAc in all novel strains can vary depending on the transcription unit selected in expression vector AH to express SEQ ID NOs 15 and 19.

Table 2: Production of GlcNAc (g/L) and LacNAc (g/L) in whole broth samples taken from mutant E. coli strains after 72 hours in minimal medium containing 30 g/L sucrose strain TU of glmS*54 (*) TU of GNA1 ( UniProt ID P43577 ) GlcNAc ( g/L ) ( ±sd ) LacNAc ( g/L ) ( ±sd ) A TU45 TU44 0.98 (± 0.07) 1.22 (± 0.12) B TU53 TU44 1.90 (± 0.14) 1.53 (± 0.13) C TU53 TU52 2.08 (± 0.06) 1.45 (± 0.08) D TU47 TU55 2.14 (± 0.25) 1.86 (± 0.25) E TU46 TU44 2.40 (± 0.22) 1.74 (± 0.23) F TU45 TU57 2.81 (± 0.02) 1.60 (± 0.08) G TU47 TU54 3.02 (± 0.17) 2.19 (± 0.17) H TU45 TU54 3.21 (± 0.54) 2.26 (± 0.46) *Differs from wild-type glmS protein (UniProt ID P17169) by A39T, R250C and G472S mutations

Table 3: Promoter and UTR sequences used for transcription units (TU) to express glmS*54 as shown in Table 2 (which differs from wild-type glmS protein (UniProt ID P17169) by A39T, R250C and G472S mutations) and GNA1 (UniProt ID P43577) TU promoter * UTR* terminator TU44 Mutalik_P11 GalE_BCD18 rnpB_T1** TU45 Mutalik_P5 Gene10_LeuL T7early*** TU46 Mutalik_P5 GalE_LeuAB T7early*** TU47 Mutalik_P9 Gene10_LeuL T7early*** TU52 Mutalik_P11 ThrA_BCD2 rnpB_T1** TU53 Mutalik_P9 GalE_LeuAB T7early*** TU54 Mutalik_P10 GalE_BCD18 rnpB_T1** TU55 Mutalik_P10 GalE_BCD18 rnpB_T1** TU57 Mutalik_P10 ThrA_BCD2 rnpB_T1** *sequence taken from Mutalik et al. (Nat. Methods 2013, 10, 354-360) **sequence taken from Kim and Lee (FEBS letters 1997, 407(3), 353-356) *** taken from Dunn et al. Human sequence (Nucleic Acids Res. 1980, 8(10), 2119-2132)

Example 11 : Evaluation of modified E. coli hosts in LacNAc produce

In the next experiment, the E. coli K-12 MG1655 strain that was optimized for GDP-fucose production and that produced GlcNAc as described in Example 9 was further treated with N-acetylglucosamine of N. meningitidis having SEQ ID NO 15 Modified by knock-in of β1,4-galactosyltransferase (LgtB). This novel strain, which shares nagAB shaving and LgtB knock-in with the reference strain, was evaluated in growth experiments as described in Example 1 but expresses glmS*54 from the same transcriptional unit (which is identical to the wild-type glmS protein (UniProt ID P17169) The difference is that A39T, R250C and G472S mutations) and GNA1 (UniProt ID P43577) are presented to the plastid-derived strain. Table 4 shows the production of GlcNAc (g/L) and LacNAc (g/L) in whole broth samples from each mutant E. coli strain collected after 72 hours of culture in minimal medium with 30 g/L sucrose. The data show that both mutant strains produce GlcNAc and LacNAc independent of how both GNA1 and glmS*54 are presented to the strain.

Table 4: Production of GlcNAc (g/L) and LacNAc (g/L) in whole broth samples collected from mutant E. coli strains after 72 hours of culture in minimal medium including 30 g/L sucrose Expression of transcription units of GNA1(*) and glmS *54 (**) GlcNAc ( g/L ) ( ±sd ) LacNAc ( g/L ) ( ±sd ) from knock-in 1.62 (± 0.12) 1.60 (± 0.06) from expressing plastids 6.71 (± 0.57) 2.73 (± 0.23) *UniProt ID P43577 **Differs from wild-type glmS protein (UniProt ID P17169) by A39T, R250C and G472S mutations

Example 12 : in a modified E. coli host GlcNAc and LacNAc generation

In the next experiment, the E. coli K-12 MG1655 strain was further modified by knock-in of N-acetylglucosamine β1,4-galactosyltransferase (LgtB) of N. meningitidis with SEQ ID NO 15, The E. coli K-12 MG1655 strain produces sialic acid as described in Example 1 and contains shaving of the E. coli nagA and nagB genes and glmS*54 (which differs from the wild-type glmS protein (UniProt ID P17169) in A39T, R250C and G472S mutations), GNA1 (UniProt ID P43577), N-acetylglucosamine 2-epimerase (AGE) from Bacteroides ovale (UniProt ID A7LVG6) and N-acetyl from Neisseria meningitidis Knock-in of a persistent expression construct of neuraminidine synthase (NeuB) (UniProt ID E0NCD4).

Also, a persistent expression construct of UDP-glucose-4-epimerase (galE) (UniProt ID P09147) with genetic shaving of E. coli ushA and galT genes and knock-in of E. coli as described in Example 1 Body optimization promoted UDP-galactose production and the E. coli K-12 MG1655 strain was additionally mutated by shaving the E. coli nagB gene and knocking in a persistent expression construct for the gene encoding SEQ ID NO 15. Novel strains were evaluated in growth experiments and compared to their respective parental strains according to the culture conditions provided in Example 1 (wherein the medium contained sucrose). Each strain was grown in multiple wells of a 96-well plate for 72 hours. The experiments confirmed that, in the whole broth samples, each novel strain produced both GlcNAc and LacNAc, while neither parent strain produced LacNAc.

Example 13 : Production of galactosylated oligosaccharides in modified E. coli hosts

The E. coli K-12 MG1655 strain optimized to promote UDP-galactose production and produce GlcNAc and LacNAc as described in Example 12 can be further modified with N-acetylglucosamine from E. coli O55:H7 containing SEQ ID NO 3 The expression vector for the persistent expression construct of β1,3-galactosyltransferase WbgO was additionally transformed. When evaluated in growth experiments according to the culture conditions provided in Example 1 (wherein the medium contained sucrose), in addition to GlcNAc, LacNAc and LNB, the novel strain also produced Gal-b14-(Galb13)-GlcNAc, which contained two β-1,3 and β-1,4 were linked to the galactose moiety of GlcNAc.

Example 14 : Fucosylation in modified E. coli hosts LacNAc generation

The E. coli K-12 MG1655 mutant strain H that was optimized for GDP-fucose production and produced GlcNAc and LacNAc as described in Example 10 may contain the al,2-fucosyltransferase HpFutC (GenBank No. AAD29863.1) or the expression plastids of the persistent expression construct of a1,3-fucosyltransferase HpFucT (UniProt ID O30511) underwent additional transformation. Novel strains were evaluated in growth experiments according to the culture conditions provided in Example 1. Table 5 shows the production of 2'FLacNAc (g/L) or 3-FLacNAc (g/L) in whole broth samples from two mutant strains collected after 72 hours of culture in minimal medium with 30 g/L sucrose. The data confirmed that each novel strain produced GlcNAc, LacNAc, and fucosylated forms of LacNAc, with 2'FLacNAc produced in a strain expressing HpFutC (GenBank NO. AAD29863.1) and 3-FLacNAc in a strain expressing HpFucT (UniProt ID O30511 ) strains. Difucosylated LacNAc was not detected in this experiment. Fucosylated LacNAc variants could not be detected in whole broth samples of the parental strain of the same genetic background lacking the fucosyltransferase expression vector.

Table 5: Production of 2'FLacNAc (g/L) and 3-FLacNAc (g/L) in whole broth samples collected from mutant E. coli strains after 72 hours of culture in minimal medium including 30 g/L sucrose Expression of mutant Escherichia coli LacNAc- producing strains 2'FLacNAc ( g/L ) ( ±sd ) 3-FLacNAc ( g/L ) ( ±sd ) reference ( = afucosyltransferase ) 0 0 a1,2 -fucosyltransferase HpFutC ( GenBank NO. AAD29863.1 ) 0.46 (± 0.02) 0 a1,3/4- fucosyltransferase HpFucT ( UniProt ID O30511 ) 0 0.53 (± 0.02)

Example 15 : Difucosylation in a modified E. coli host LacNAc generation

E. coli K-12 MG1655 mutant strain H that is optimized for GDP-fucose production and produces GlcNAc and LacNAc as described in Example 10 may contain the fucosyltransferases HpFutC (GenBank NO. AAD29863.1) and HpFucT (UniProt ID O30511) with additional transformation of the presentation plastids of the persistent presentation construct. The novel strain produced 2'-fucosylated, 3 - Fucosylated and difucosylated LacNAc.

Example 16 : Sialylation in modified E. coli hosts LacNAc generation

The sialic acid-producing E. coli K-12 MG1655 strain as described in Example 1 was further mutated with a knock-in of a persistent expression construct of LgtB having SEQ ID NO 15 and transformed with an expression plastid that The body contains N-acylneuraminic acid cytidine transferase (NeuA) (GenBank NO. AMK07891.1) from Pasteurella haemorrhage and a A persistent expression construct of a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of active UniProt ID Q9CLP3. The novel strains were evaluated in growth experiments and compared to their parental strains according to the culture conditions provided in Example 1 (wherein the medium contained 30 g/L sucrose). Each strain was grown in multiple wells of a 96-well plate for 72 hours. The data showed that, in the whole broth samples, the novel strain produced 0.32 ± 0.02 g/L a2,3-sialylated LacNAc in addition to GlcNAc and LacNAc. Disialyl LacNAc could not be detected. The sialylated LacNAc variant could not be detected in whole broth samples of the parental strain of the same genetic background lacking the sialyltransferase expression vector.

Similarly, the sialic acid-producing E. coli K-12 MG1655 strain as described in Example 1 can be further mutated with a knock-in of a persistent expression construct of LgtB having SEQ ID NO 15 and transformed with an expression plasmid , the expressing plastid contains NeuA from Pasteurella haemorrhagic (GenBank NO. AMK07891.1) and amino acid residues from UniProt ID O66375 with β-galactoside α-2,6-sialyltransferase activity Persistent expression constructs of PdST6-like polypeptides composed of bases 108 to 497. The novel strain produced GlcNAc and LacNAc, as well as a2,6-sialylated LacNAc, in the whole broth sample when evaluated in growth experiments according to the culture conditions provided in Example 1 (wherein the medium contained sucrose).

Example 17 : Disialylation in a modified E. coli host LacNAc generation

The CMP-sialic acid-producing E. coli K-12 MG1655 strain as described in Example 1 can be further mutated with a knock-in of a persistent expression construct of LgtB having SEQ ID NO 15 and transformed with an expression plasmid, The expressing plastid contains a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 with β-galactoside α-2,3-sialyltransferase activity and a PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity. A persistent expression construct of a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 with alpha-2,6-sialyltransferase activity. The novel strain produced GlcNAc and LacNAc, as well as 3'-sialylation, 6'-sialylation, and di-sialylation in whole broth samples, when evaluated in growth experiments according to the culture conditions provided in Example 1 (wherein the medium contained sucrose) Sialyl LacNAc.

Example 18 : modified in E. coli hosts LacNAc generation

The E. coli K-12 MG1655 strain optimized for GDP-fucose production and producing GlcNAc and LacNAc as described in Example 11 may contain bl,3-N-acetyl-hexosaminotransferases both from N. meningitidis The expressing plastids of the persistent expressing construct of the enzyme lgtA (GenBank NO. AAM33849.1) underwent additional transformation. By subsequent action of mutant glmS*54, which differs from wild-type glmS protein (UniProt ID P17169) by A39T, R250C and G472S mutations, homologous EcGlmM and EcGlmU, and heterologous NmLgtA (GenBank NO. AAM33849.1), The resulting strain converts fructose-6-phosphate to UDP-GlcNAc intracellularly, and uses this Intracellular modification of LacNAc was performed to result in the production of GlcNAc-bl,3-Gal-bl,4-GlcNAc in whole broth samples. This strain also produces a poly-LacNAc structure, (Gal-b1,4-GlcNAc)n, which is constructed from repeating N-acetyllactosamine by having SEQ ID The alternative activity of LgtB at NO 15 and LgtA expressed in the strain (GenBank NO. AAM33849.1) linked beta1,3 to each other.

The E. coli K-12 MG1655 strain optimized for UDP-galactose production and producing GlcNAc and LacNAc as described in Example 12 was modified to persistently express the UDP-GlcNAc epimerase wbpP from Pseudomonas aeruginosa ( UniProt ID Q8KN66) and the glycosyltransferase lgtD from Haemophilus influenzae (UniProt ID A0A2X4DBP3). Subsequent action by mutant E. coli glmS*54 (which differs from wild-type glmS protein (UniProt ID P17169) by A39T, R250C and G472S mutations), homologous E. coli GlmM and GlmU, and Pseudomonas aeruginosa wbpP (UniProt ID Q8KN66) , fructose-6-phosphate is intracellularly converted to UDP-GalNAc via the intermediate compounds glucosamine-6-phosphate, glucosamine-1-phosphate and UDP-GlcNAc. By subsequent action of the newly expressed LgtD enzyme (UniProt ID A0A2X4DBP3), the novel strain was able to modify cells with GalNAc when evaluated in growth experiments according to the culture conditions provided in Example 1 (wherein the medium contained 30 g/L sucrose). LacNAc was produced in the whole broth sample, yielding 0.12 ± 0.02 g/L GalNAc-b1,3-Gal-b1,4-GlcNAc in the whole broth sample.

Example 19 : sialylated polysaccharides in modified E. coli LacNAc generation

E. coli strains producing LacNAc and sialylated forms of LacNAc in whole broth samples as described in Examples 16 and 17 can be further transformed with expression plastids containing LgtA (GenBank NO. AAM33849.1) The persistent expressive construct. By having the replacement activities of LgtB and LgtA transferases with SEQ ID NO 15 and GenBank NO. AAM33849.1, respectively, the novel strains were evaluated in growth experiments according to the culture conditions provided in Example 1 (wherein the medium contained sucrose) A poly-LacNAc structure was additionally generated, namely (Gal-b1,4-GlcNAc)n, which consisted of repeating N-acetyllactosamine, which was sialylated together with the Gal in which Gal was sialylated PolylacNAc structure, linking β1,3- to each other.

Example 20 : in a modified E. coli host LNB generation

In the next experiment, the E. coli K-12 MG1655 strain optimized for GDP-fucose production and producing GlcNAc as described in Example 9 can be further characterized with SEQ ID NO 03 or WbgO from plastids or from knock-in Persistent performance is modified. According to the culture conditions provided in Example 1, the novel strain was evaluated in growth experiments and compared to the parental strain lacking the expression construct of SEQ ID NO 03. Table 6 shows LNB (g/L) production in whole broth samples of each mutant strain obtained after 72 hours of culture in minimal medium containing 30 g/L sucrose. The data confirmed that both novel strains produced LNB in whole broth samples regardless of how WbgO was presented to the strains.

Table 6: Production of LNB (g/L) in whole broth samples collected from mutant E. coli strains after 72 hours of culture in minimal medium including 30 g/L sucrose Expression of the transcription unit of EcWbgO with SEQ ID NO 03 LNB ( g/L ) ( ±sd ) from knock-in 0.63 (± 0.12) from expressing plastids 2.81 (± 0.11)

Example twenty one : Fucosylation in modified E. coli hosts LNB generation

The E. coli K-12 MG1655 strain that is optimized for GDP-fucose production and that produces GlcNAc and LNB as described in Example 20 (wherein WbgO with SEQ ID NO 03 is expression from knock-in) may contain both derived from Helicobacter pylori The expression plastids of the persistent expression constructs of the bacillus a1,2-fucosyltransferase HpFutC (GenBank NO. AAD29863.1) or a1,3-fucosyltransferase HpFucT (UniProt ID O30511) were additionally Transform. Novel strains were evaluated in growth experiments according to the culture conditions provided in Example 1. Table 7 shows the production of 2' FLNB (g/L) or 4-FLNB (g/L) in whole broth samples from two mutant strains collected after 72 hours of culture in minimal medium with 30 g/L sucrose. The data confirmed that each novel strain produced fucosylated forms of LNB in whole broth samples, with 2'FLNB measured in a strain expressing HpFutC (GenBank NO. AAD29863.1) and 4-FLNB in a strain expressing HpFucT ( UniProt ID O30511) strains. Difucosylated LNB was not detected in this experiment. Fucosylated LNB variants could not be detected in whole broth samples of the parental strain of the same genetic background lacking the fucosyltransferase expression vector.

Table 7: Production of 2'FLNB (g/L) and 4-FLNB (g/L) in whole broth samples collected from mutant E. coli strains after 72 hours of culture in minimal medium including 30 g/L sucrose Expression of mutant E. coli LNB- producing strains 2'FLNB ( g/L ) ( ±sd ) 4-FLNB ( g/L ) ( ±sd ) reference ( = afucosyltransferase ) 0 0 a1,2 -fucosyltransferase HpFutC ( GenBank NO. AAD29863.1 ) 0.19 (± 0.03) 0 a1,3 -fucosyltransferase HpFucT ( UniProt ID O30511 ) 0 0.04 (± 0.01)

Example twenty two : Difucosylation in a modified E. coli host LNB generation

E. coli K-12 MG1655 strains that are optimized for GDP-fucose production and that produce GlcNAc and LNB as described in Example 20 (wherein WbgO with SEQ ID NO 03 is expression from knock-in) may contain a1,2- The expressing plastids of the persistent expressing constructs of both fucosyltransferase HpFutC (GenBank NO. AAD29863.1) and a1,3-fucosyltransferase HpFucT (UniProt ID O30511) underwent additional transformation. The novel strain produced GlcNAc and LNB as well as 2'-fucosylated, 4-fucosylated in whole broth samples when evaluated in growth experiments according to the culture conditions provided in Example 1 (wherein the medium contained sucrose) and difucosylated LNB.

Example twenty three : galactosylation in modified E. coli hosts LNB generation

The E. coli K-12 MG1655 strain optimized for UDP-galactose production as described in Example 1 can be further mutated by shaving the E. coli nagB gene and further mutated to have SEQ ID NO 03 either from plastid or from The persistent expression of the knock-in WbgO was further modified. The novel strains all produced LNB in the whole broth samples when evaluated in growth experiments according to the culture conditions provided in Example 1 (wherein the medium contained sucrose). When using α-1,2-galactosyltransferase and/or α-1,3-galactosyltransferase and/or β-1,3-galactosyltransferase and/or β-1,4- When additionally transforming novel LNB-producing strains with a galactosyltransferase expressing construct, each novel strain was in a whole broth sample in a growth experiment evaluated according to the culture conditions provided in Example 1 (wherein the medium contained sucrose). GlcNAc and LNB as well as galactosylated forms of LNB are produced.

Example twenty four : Sialylation in modified E. coli hosts LNB generation

The CMP-sialic acid-producing E. coli K-12 MG1655 strain as described in Example 1 can be further mutated with a knock-in of a persistent expression construct of WbgO having SEQ ID NO 03 and transformed with an expression plastid, The expression plasmid contains a persistent expression construct of a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having β-galactoside α-2,3-sialyltransferase activity. The novel strain produced the sialylated LacNAc LNB form in the whole broth sample, in addition to GlcNAc and LNB, when evaluated in growth experiments according to the culture conditions provided in Example 1 (wherein the medium contained sucrose). Similarly, WbgO with SEQ ID NO 03 and a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 with β-galactoside α-2,6-sialyltransferase activity are additionally represented The E. coli K-12 MG1655 CMP-sialic acid-producing strain produced GlcNAc and LNB as well as sialylation in whole broth samples when grown according to the growth experiments assessed according to the culture conditions provided in Example 1 (wherein the medium contained sucrose) LNB form.

Example 25 : Disialylation in a modified E. coli host LNB generation

The CMP-sialic acid-producing E. coli K-12 MG1655 strain as described in Example 1 can be further mutated with a knock-in of a persistent expression construct of WbgO having SEQ ID NO 03 and transformed with an expression plastid, The expressing plastid contains a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 with β-galactoside α-2,3-sialyltransferase activity and/or a PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity A persistent expression construct of a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 with lactoside alpha-2,6-sialyltransferase activity. The novel strain produced GlcNAc and LNB, as well as mono- and/or disialylated LNB in whole broth samples when evaluated in growth experiments according to the culture conditions provided in Example 1 (wherein the medium contained sucrose).

Example 26 : Fermentative production with mutant E. coli hosts LacNAc

GDP-fucose production was optimized and as described in Example 11 by glmS*54 presented to the host from an expression plastid (which differs from the wild-type glmS protein (UniProt ID P17169) by the A39T, R250C and G472S mutations) and GNA1 (UniProt ID P43577) mutant E. coli K-12 MG1655 strain producing GlcNAc and LacNAc was evaluated in a 5 L bioreactor scale fed batch fermentation according to the conditions provided in Example 1. In this example, sucrose was used as the carbon source. As described in Example 1, samples were periodically sampled and measured for LacNAc production.

Example 27 : in modified E. coli hosts when grown on carbon sources other than sucrose LacNAc or LNB generation

Mutant E. coli strains modified to produce GlcNAc and LacNAc as described in Examples 10 to 12 or as described in Example 20 when evaluated in growth experiments according to the culture conditions provided in Example 1 (wherein the medium contained glycerol) The mutant E. coli strains modified to produce GlcNAc and LNB produce GlcNAc and LacNAc or GlcNAc and LNB, respectively. When used as described in Example 1, one or more of the following carbon sources are used, but not limited to: glycerol, glucose, fructose, lactose, arabinose, maltotriose, sorbitol, xylose, rhamnose and This mutant strain also produced GlcNAc and LacNAc or GlcNAc and LNB, respectively, when evaluated in a bioreactor-scale fed-batch fermentation.

Example 28 : Materials and Methods Saccharomyces cerevisiae

culture medium

The strains were grown in agar containing 6.7 g/L yeast nitrogen-free amino acids (YNB w/o AA, Difco), 20 g/L agar (Difco) (solid culture), 22 g/L glucose monohydrate or 20 g/L Synthetic Defined Yeast Medium (MP Biomedical) with Complete Supplementation Mix (SD CSM) or CSM drop-out (SD CSM-Ura) of L lactose with 0.79 g/L CSM or 0.77 g/L CSM-Ura grow on.

strain

Saccharomyces cerevisiae BY4742 created by Bachmann et al. (Yeast (1998) 14:115-32) was obtained from the Euroscarf Culture Center. All mutant strains were generated by homologous recombination or plastid transformation using the method of Gietz (Yeast 11:355-360, 1995).

plastid

The yeast expression plastid p2a_2µ (Chan 2013, Plasmid 70, 2-17) was used to express foreign genes in Saccharomyces cerevisiae. This plastid contains an ampicillin resistance gene and a bacterial origin of replication to allow selection and maintenance in E. coli. The plastids further contained 2μ yeast ori and Ura3 selectable markers for selection and maintenance in yeast.

In one example, the yeast expressed plastid p2a_2µ can be modified to obtain the mutant fructose-6-phosphate transaminase glmS*54 from E. coli as described in WO18122225 (which differs from the wild-type glmS protein (UniProt ID P17169) in A39T, R250C and G472S mutations), N-acetylglucosamine-6-phosphotransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), including for example 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 of any one or more of the E. coli genes, Or the phosphatase of PsMupP from Pseudomonas putida, ScDOG1 from Saccharomyces cerevisiae or BsAraL from Bacillus subtilis, as described in WO18122225. The modified plastids can be further modified to obtain N-acetylglucosamine b-1,3-galactosyltransferases selected from the group consisting of SEQ ID NOs: 03, 04, 05, 06, 07, 08, 10, 11, 12 and 13 and/or selected from the group consisting of N-acetylglucosamine b-1,4-galactosyltransferase SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21 , 22, 23, 26, 27, 28, 31, 32, 33, 34, 35, 36, 39, 40 and 41.

In one example for the production of GDP-fucose, yeast expressed plastids such as p2a_2µ_Fuc (Chan 2013, Plasmid 70, 2-17) can be modified with a persistent transcription unit for, e.g., from lactate gram Lactose permease from LAC12 (UniProt ID P07921) of K. lactis , GDP-mannose 4,6-dehydratase from gmd (UniProt ID P0AC88) from E. coli, and e.g. from E. coli GDP-L-fucose synthase of fcl (UniProt ID P32055). The yeast expression plastid p2a_2μ_Fuc2 can be used as an alternative to the p2a_2μ_Fuc plastid containing the ampicillin resistance gene, bacterial ori, 2μ yeast ori, and Ura3 selectable marker persistent transcription units, which are used, for example, from Lactose permease from LAC12 of Kluyveromyces lactis (UniProt ID P07921), fucose permease from eg fucP from Escherichia coli (UniProt ID P11551), and eg fkp from Bacteroides fragilis (UniProt ID SUV40286. 1) A bifunctional enzyme having fucose kinase/fucose-1-phosphate guanylate transferase activity. For further production of fucosylated oligosaccharides, p2a_2µ_Fuc and its variant p2a_2µ_Fuc2 also contain a continuous transcription unit for, for example, α-1,2- Fucosyltransferase and/or alpha-1,3-fucosyltransferase such as HpFucT (UniProt ID 030511) from Helicobacter pylori.

In one example for the production of UDP-galactose, yeast expressed plastids can be derived from the pRS420-plastid series (Christianson et al. , 1992, Gene 110: 119-122), which contains the HIS3 selectable marker and a continuous transcription unit for UDP-glucose-4-epimerase such as galE from E. coli (UniProt ID P09147). This plastid can be further treated with lactose permease such as LAC12 from Kluyveromyces lactis (UniProt ID P07921) and galactoside β-1,3-N such as lgtA from Neisseria meningitidis (UniProt ID Q9JXQ6). - The persistent transcription unit of acetylglucosaminyltransferase activity is modified to generate LN3. For further production of LN3-derived oligosaccharides, such as LNT, mutant LN3 producing strains are further selected from the list of N-B Modification of the continuous transcription unit of glucosamine beta-1,3-galactosyltransferase. For further production of LN3-derived oligosaccharides, such as LNnT, mutant LN3 producing strains are further selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31 , 32, 33, 34, 35, 36, 39, 40, and 41, the continuous transcription unit of the enumerated N-acetylglucosamine beta-1,4-galactosyltransferase was modified.

In one example for the production of sialic acid and CMP-sialic acid, yeast expressed plastids can be derived from the pRS420-plastid series (Christianson et al. , 1992, Gene 110: 119-122), which contains TRP1 Selectable markers and persistent transcription units for one or more replicated L-glutamine-D-fructose-6-phosphate transaminase enzymes, e.g., mutant glmS*54 from Escherichia coli (different from wild-type E. coli glmS of UniProt ID P17169 with A39T, R250C, and G472S mutations, as described by Deng et al. (Biochimie 88, 419-29 (2006)), a phosphatase, e.g., 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 Any one or more of the E. coli genes of YbiU, or PsMupP from Pseudomonas putida, ScDOG1 from Saccharomyces cerevisiae, or BsAraL from Bacillus subtilis, as described in WO18122225, such as AGE from Bacteroides ovale ( N-acetylglucosamine 2-epimerase of UniProt ID A7LVG6, eg N-acetylneuraminic acid synthase from Neisseria meningitidis (UniProt ID EONCD4) and eg NeuA enzyme from Aspergillus jejuni (UniProt ID Q93MP7), NeuA from Haemophilus influenzae (GenBank No. AGV11798.1) or NeuA from Pasteurella haemorrhagic (GenBank No. AMK07891.1) ). Optionally, a continuous transcription unit comprising one or more replicated N-acetylglucosamine-6-phosphotransferase enzymes is further added, for example, GNA1 from Saccharomyces cerevisiae (UniProt ID P43577). To generate sialylation An oligosaccharide, the plastid further comprising a persistent transcription unit for, for example, the lactose permease from LAC12 of Kluyveromyces lactis (UniProt ID P07921 ), and one or more replicated β-galactoside α- 2,3-Sialyltransferases such as PmultST3 from Pasteurella haemorrhagic (UniProt ID Q9CLP3) or amino acids from UniProt ID Q9CLP3 with β-galactoside α-2,3-sialyltransferase activity The PmultST3-like polypeptide consisting of residues 1 to 268 and, from meningococcal Coccus NmeniST3 (GenBank No. ARC07984.1) or PmultST2 (GenBank No. AAK02592.1) from Pasteurella haemorrhagic subsp. haemorrhagic strain Pm70, β-galactoside α-2,6-sialyltransferase , such as PdST6 (UniProt ID O66375) from Photobacillus marina or PdST6 consisting of amino acid residues 108 to 497 of UniProt ID O66375 with β-galactoside α-2,6-sialyltransferase activity Like polypeptide or P-JT-ISH-224-ST6 (UniProt ID A8QYL1) from Photobacterium sp JT-ISH-224 or by having β-galactoside α-2,6-sialyltransferase activity A P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 and/or e.g. α-2,8-sialyltransferase from mouse (UniProt ID Q64689) .

Preferably, but not necessarily, glycosyltransferase proteins and/or proteins involved in nucleotide-activated sugar synthesis are N-terminally fused to a SUMOstar tag (eg, obtained from pYSUMOstar, Life Sensors, Malvern, PA) to improve solubility.

Optionally, to encode e.g. Hsp31, Hsp32, Hsp33, Sno4, Kar2, Ssb1, Sse1, Sse2, Ssa1, Ssa2, Ssa3, Ssa4, Ssb2, Ecm10, Sscl, 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) Knock-in modification of the persistent transcription unit of the chaperone protein mutant yeast strains .

Plastids were maintained in host E. coli DH5α (F ^- , phi80d lacZ deltaM15, delta( lacZYA - argF )U169, deoR , recA1 , endA1 , hsdR17(rk ^- , mk ⁺ ), phoA , supE44 , lambda ^- , purchased from Invitrogen thi -1, gyrA96 , rel A1).

Heterologous and Homologous Expression

The genes to be expressed, whether from the plastid or the genome, are synthesized synthetically by one of the following companies: DNA2.0, Gen9, IDT or Twist Bioscience.

Expression can be further facilitated by optimizing codon usage for the codon usage of the expression host. Genes were optimized using vendor tools.

Culture conditions

Generally, yeast strains are initially grown on SD-CSM plates to obtain single colonies. The plates were grown at 30°C for 2-3 days.

Starting from a single colony, the preculture was grown overnight in 5 mL at 30°C with shaking at 200 rpm. Subsequent 125 ml shake flask experiments were inoculated with 2% of these precultures in 25 mL of medium. The flasks were incubated at 30°C using orbital shaking at 200 rpm.

gene expression promoter

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

Example 29 : In Saccharomyces cerevisiae GlcNAc and LacNAc ;or GlcNAc and LNB generation

Another embodiment provides the use of eukaryotic organisms in the form of Saccharomyces cerevisiae for practicing the present invention. Using the strains, plastids and methods as described in Example 28, mutant S. cerevisiae strains producing GlcNAc and LacNAc were generated. Such modifications included an additional persistent expression unit for the mutant fructose-6-phosphate transaminase glmS*54 of E. coli (different from wild-type E. coli glmS with UniProt ID P17169 with A39T, R250C and G472S mutation), Saccharomyces cerevisiae N-acetylglucosamine-6-phosphotransferase GNA1 (UniProt ID P43577), a monophosphatase selected from the list 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 E. coli genes One or more, or PsMupP from Pseudomonas putida, ScDOG1 from Saccharomyces cerevisiae and BsAraL from Bacillus subtilis as described in WO18122225 and N-acetylglucose from Neisseria meningitidis of SEQ ID NO 15 Amine b-1,4-galactosyltransferase LgtB. Mutant S. cerevisiae strains are able to grow on glucose or glycerol as carbon source, convert the carbon source to fructose-6-phosphate, and then use the newly expressed fructose-6-phosphate transaminase, N-acetylglucosamine-6 - The activities of phosphotransferase, phosphatase and N-acetylglucosamine b-1,4-galactosyltransferase convert it to GlcNAc and subsequently LacNAc.

A pre-culture of this strain was performed in 5 mL of defined synthetic medium SD-CSM containing 22 g/L glucose and grown at 30°C as described in Example 28. This preculture was then inoculated into 25 mL of medium in shake flasks containing 10 g/L glucose as the sole carbon source and grown at 30°C. Samples were taken periodically and the production of GlcNAc and LacNAc was measured as described in Example 1 . In a similar experiment, the N-acetylglucosamine b-1,3-galactosyltransferase WbgO of E. coli O55:H7 contained SEQ ID NO 03, but not the N-acetylglucosamine b of SEQ ID NO 15 - Similar yeast strains of the 1,4-galactosyltransferase LgtB are capable of producing GlcNAc and LNB.

Example 30 : for having PFAM area PF00535 of N- Acetyl Glucosamine b-1,3- galactosyltransferase gene RegEx retrieve

RegEx analysis was performed on the N-acetylglucosamine b-1,3-galactosyltransferase gene with PFAM domain PF00535 to find members comprising [AGPS]XXLN( _Xn )RXDXD with SEQ ID NO 01, wherein X is any amino acid where n is 12 to 17, or a member is found comprising the sequence PXXLN( _Xn )RXDXD( _Xm )[FWY]XX[HKR]XX[NQST] having SEQ ID NO 02, wherein X is any amino acid where n is 12 to 17 and m is 100 to 115. To this end, all N-acetylglucosamine b-1,3-galactosyltransferase genes with PFAM domain PF00535 as annotated in Pfam database version Pfam 33.1 (released June 11, 2020) were obtained from the UniProt database (2020 Published 03 Jul 2019) downloaded and analyzed the model according to the method as obtained from https://towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (Published 06 Apr 2019) the existence of the body.來自該RegEx檢索的相應成員包括：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, A0A3D3MDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28TX and A0A538.

Example 31 : for other N- Acetyl Glucosamine b-1,3- Galactosyltransferase or N- Acetyl Glucosamine b-1,4- galactosyltransferase gene RegEx retrieve

RegEx analysis can be performed on the N-acetylglucosamine b-1,3-galactosyltransferase gene with PFAM domain IPR002659 to find a sequence KT( _Xn )[FY]XXKXDXD(X with SEQ ID NO 09 _m ) [FHY]XXG(X, no A, G, S) (X _p ) a member of X (no F, H, W, Y) [DE]D[ILV]XX[AG], where X is any amine base acid, 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 with PFAM domain IPR002659 as annotated in Pfam database version Pfam 33.1 (released June 11, 2020) were obtained from the UniProt database (2020 Published 03 Jul 2019) downloaded and analyzed the model according to the method as obtained from https://towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (Published 06 Apr 2019) the existence of the body.

A similar RegEx analysis can be performed on the N-acetylglucosamine b-1,4-galactosyltransferase gene with PFAM domain PF01755 to find the sequence EXXCXXSHXX[ILV][FWY]( _Xn with SEQ ID NO 14 A member of )EDD( _Xm )[ACGST]XXYX[ILMV], wherein X is any amino acid, wherein n is 13 to 15 and m is 50 to 76. To this end, all N-acetylglucosamine b-1,4-galactosyltransferase genes with PFAM domain PF01755 as annotated in Pfam database version Pfam 33.1 (released June 11, 2020) were obtained from the UniProt database (2020 Published 03 Jul 2019) downloaded and analyzed the model according to the method as obtained from https://towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (Published 06 Apr 2019) the existence of the body.

Similarly, RegEx analysis can be performed on the N-acetylglucosamine b-1,4-galactosyltransferase gene with PFAM domain PF00535 to find the sequence R[KN]XXXXXXXGXXXX[FL]XDXD with SEQ ID NO 24 A member of (X _n )[FHW]XXX[FHNY](X _m )E[DE], wherein X is any amino acid, wherein n is 50 to 75, m is 10 to 30, or having SEQ ID NO 25 A member of the sequence R[KN]XXXXXXXGXXXXFXDXD( _Xn )[FHW]XXX[FHNY]( _Xm )E[DE](Xp)[ _FWY ]XX[HKR]XX[NQST], where X is any amino acid , where 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 with PFAM domain PF00535 as annotated in Pfam database version Pfam 33.1 (released June 11, 2020) were obtained from the UniProt database (2020 Published 03 Jul 2019) downloaded and analyzed the model according to the method as obtained from https://towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (Published 06 Apr 2019) the existence of the body.

A similar RegEx analysis can be performed on the N-acetylglucosamine b-1,4-galactosyltransferase gene with PFAM domain PF02709 but not PFAM domain PF00535 to find the sequence with SEQ ID NO 29 [FWY]XX A member of [FY][FWY]( _X23 )[FWY][GQ]X[DE]D, where X is any amino acid, or has the sequence of SEQ ID NO 30 [PV]W[GHNP]( _Xn )[FWY][GQ]X[DE]D where X is any amino acid and n is 21 to 24. For this purpose, all N-acetylglucosamine b-1,4-galactosyltransferase genes with PFAM domain PF02709 but not PFAM domain PF00535 as annotated in Pfam database version Pfam 33.1 (released June 11, 2020) Both downloaded from the UniProt database (published 03 Jul 2020) and obtained according to e.g. https://towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (published 06 Apr 2019) ) method to analyze the existence of the motif.

Finally, RegEx analysis can be performed on the N-acetylglucosamine b-1,4-galactosyltransferase gene with the PFAM domain PF03808 to find the sequence [ST][FHY]XN( _Xn with SEQ ID NO 37 ) DG(X ₁₆ )[HKR]X[ST]FDXX[ST]XA, wherein X is any amino acid, and wherein n is 20 to 25, or has the sequence of SEQ ID NO 38 [ST][FHY A member of ]XN( _Xn )DG(X16)[HKR]X[ST] _FDXX [ST]XA( _Xm )[HR]XG[FWY](Xp) _GXGXXXQ [DE], where X is any amine base acid, 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 with PFAM domain PF03808 as annotated in Pfam database version Pfam 33.1 (released June 11, 2020) were obtained from the UniProt database (2020 Published 03 Jul 2019) downloaded and analyzed the model according to the method as obtained from https://towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (Published 06 Apr 2019) the existence of the body.

Example 32 : Produced in a modified E. coli host containing 6'-SL , LacNAc , sialylation LacNAc , LN3 , sialylation LN3 , LNnT and LSTc oligosaccharide mixture

E. coli K-12 strain MG1655 modified to produce sialic acid as described in Example 1, which includes shaving and continuous transcription of the E. coli nagA, nagB, nanA, nanT, nanE, nanK, LacZ, LacY and LacA genes Knock-in of units encoding lactose permease (LacY) from E. coli (UniProt ID P02920), sialic acid transporter (nanT) from E. coli (UniProt ID P41036), The mutant L-glutamine-D-fructose-6-phosphate transaminase glmS*54 (different from wild-type E. coli glmS with UniProt ID P17169 with A39T, R250C and G472S mutations), from Saccharomyces cerevisiae N-acetylglucosamine-6-phosphotransferase (GNA1) (UniProt ID P43577), N-acetylglucosamine 2-epimerase (AGE) from Bacteroides ovale (UniProt ID A7LVG6), from Aspergillus jejuni N-acetylneuraminic acid synthase (NeuB) (UniProt ID Q93MP9), sucrose transporter (CscB) from Escherichia coli W (UniProt ID E0IXR1), fructokinase (Frk) from Zymomonas mobilis ) (UniProt ID Q03417) and the gene for sucrose phosphorylase (BaSP) (UniProt ID A0ZZH6) from Bifidobacterium adolescentis. Therefore, the obtained sialic acid-producing mutant E. coli strain was further modified with a gene knock-in of a persistent transcription unit to express the N-acylneuraminic acid cytidine transferase NeuA (UniProt ID Q93MP7) from A. jejuni. ) and the α-2,6-sialyltransferase PdbST (UniProt ID O66375) from P. damselae to produce 6′-sialyllactose. In the next step, the mutant strain was further modified with a knock-in comprising a persistent transcription unit for galactoside β-1,3-N-acetylglucose from Neisseria meningitidis Aminyltransferase IgtA (UniProt ID Q9JXQ6) and selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35 , 36, 39, 40 and 41 to enumerate N-acetylglucosamine beta-1,4-galactosyltransferases to produce 6'-SL, LacNAc, sialylated LacNAc, LN3, sialylated LN3, LNnT and LSTc (Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc) oligosaccharide mixture. Novel strains were evaluated in growth experiments according to the culture conditions provided in Example 1 (wherein the medium contained sucrose and lactose). This strain was grown in four biological replicates in 96-well plates. After 72 hours of incubation, the culture broth was collected and analyzed for carbohydrates by UPLC.

Example 33 : Produced in a modified E. coli host LN3 , sialylation LN3 , LNT , LNB , sialylation LNB , 3’-SL and LSTa oligosaccharide mixture

E. coli modified as described in Example 32 to produce sialic acid (Neu5Ac) was further modified with a gene knock-in of a persistent transcription unit to express an N-acyl neuraminic acid cytidine transferase from A. jejuni NeuA (UniProt ID Q93MP7) and α-2,3-sialyltransferase PmultST3 (UniProt ID Q9CLP3) from Pasteurella haemorrhagic to produce 3'-siallactose. In the next step, the mutant strain was further modified with a knock-in comprising a persistent transcription unit for galactoside β-1,3-N-acetylglucose from Neisseria meningitidis Aminyltransferase IgtA (UniProt ID Q9JXQ6) and N-acetylglucosamine beta-1,3 selected from the list comprising SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12 or 13 -Galactosyltransferase to generate LN3, 3'-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc), LNT, LNB, sialylated LNB, 3' - Oligosaccharide mixture of SL and LSTa (Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc). The novel strains were evaluated in growth experiments according to the culture conditions provided in Example 1, wherein the medium contained sucrose and lactose. This strain was grown in four biological replicates in 96-well plates. After 72 hours of incubation, the culture broth was collected and analyzed for carbohydrates by UPLC.

Example 34 : Materials and Methods Bacillus subtilis

culture medium

Two different media, named rich Luria broth (LB) and minimal media for shake flasks (MMsf), were used. Minimal medium uses a mixture of trace elements.

The trace element mixture consisted of 0.735 g/L CaCl2.2H2O, 0.1 g/L MnCl2.2H2O, 0.033 g/L CuCl2.2H2O, 0.06 g/L CoCl2.6H2O, 0.17 g/L ZnCl2, 0.0311 g/L H3BO4, 0.4 g /L Na2EDTA.2H2O and 0.06 g/L Na2MoO4. The ferric citrate solution contained 0.135 g/L FeCl3.6H2O, 1 g/L sodium citrate (Hoch 1973 PMC1212887).

Luria broth (LB) medium consisted of 1% trypsin (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). Luria Broth Agar (LBA) plates consisted of LB medium with 2 g/L agar (Difco, Erembodegem, Belgium).

The minimal medium (MMsf) used for shake flask experiments consisted of 2.00 g/L (NH4)2SO4, 7.5 g/L KH2PO4, 17.5 g/L K2HPO4, 1.25 g/L sodium citrate, 0.25 g/L MgSO4.7H2O, 0.05 g/L tryptophan, 10 to 30 g/L glucose or other carbon sources including but not limited to fructose, maltose, sucrose, glycerol and maltotriose (when specified in the examples), 10 ml/L trace Elemental mixture and 10 ml/L ferric citrate solution. The pH of the medium was set to 7 using 1M KOH. Depending on the experiment, sialic acid or lactose can be added as precursors.

Complex media such as LB are sterilized by autoclaving (121°C, 21') and minimal media by filtration (0.22 µm Sartorius). When necessary, the medium was made selective by adding antibiotics such as zeocin (20 mg/L).

Strains, plastids and mutations

Bacillus subtilis 168 was obtained from the Bacillus Genetic Stock Center (Ohio, USA).

Plastids for gene deletion via Cre/lox were constructed as described by Yan et al. (Appl. & Environm. Microbial., Sept 2008, p5556-5562). Gene disruption is accomplished via homologous recombination with linear DNA and transformation via electroporation as described by Xue et al. (J. Microb. Meth. 34 (1999) 183-191). Liu et al. (Metab. Engine. 24 (2014) 61-69) describe a method for genetic shaving. This method uses 1000 bp of homology upstream and downstream of the gene of interest.

The integration vector described by Popp et al. (Sci. Rep., 2017, 7, 15158) was used as an expression vector and further used for gene integration if necessary. Suitable promoters for expression can be obtained from the component repository (iGem): sequence id: Bba_K143012, Bba_K823000, Bba_K823002 or Bba_K823003. Colonization can be performed using Gibson assemblies, Golden Gate assemblies, Cliva assemblies, LCR or restriction ligation.

In the LNB-producing example, the B. subtilis mutant was modified with a genomic knock-in including a persistent transcription unit against glmS*54 from E. coli (compared to wild-type E. coli with UniProt ID P17169 glmS differs by having the A39T, R250C and G472S mutations as described by Deng et al. (Biochimie 88, 419-29 (2006)), the N-acetylglucosamine-6-phosphotransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), a monophosphatase selected from the following list including: comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, Any one or more of the E. coli genes of AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU, or PsMupP from Pseudomonas putida, DOG1 from Saccharomyces cerevisiae or AraL from Bacillus subtilis, as described in WO18122225, and selected from N-acetylglucosamine beta from the enumeration comprising SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12 or 13 -1,3-Galactosyltransferase.

In the examples for the production of LacNAc, the B. subtilis mutant was modified with a genomic knock-in including a persistent transcription unit against glmS*54 from E. coli (compared to wild-type E. coli with UniProt ID P17169 glmS differs by having the A39T, R250C and G472S mutations as described by Deng et al. (Biochimie 88, 419-29 (2006)), the N-acetylglucosamine-6-phosphotransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), a monophosphatase selected from the following list including: comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, Any one or more of the E. coli genes of AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU, or PsMupP from Pseudomonas putida, DOG1 from Saccharomyces cerevisiae or AraL from Bacillus subtilis, as described in WO18122225, and selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33 , 34, 35, 36, 39, 40 and 41 to enumerate the N-acetylglucosamine beta-1,4-galactosyltransferase. In order to further fucosylate the LNB or LacNAc, the LNB or LacNAc producing strain is further modified with a persistent transcription unit directed against, for example, alpha of HpFutC (GenBank No. AAD29863.1) from Helicobacter pylori -1,2-fucosyltransferase and/or α-1,3-fucosyltransferase from HpFucT (UniProt ID O30511) from Helicobacter pylori.

In an example of the production of lactose-like oligosaccharides, mutant strains of B. subtilis were created to contain the gene encoding the lactose import protein (eg, E. coli LacY of UniProt ID P02920). For 2'FL, 3FL and diFL production, a-1,2-fucosyltransferase and/or a-1,3-fucosyltransferase expression constructs were additionally added to the strains.

In the examples producing lacto-N-trisaccharides (LNT-II, LN3, GlcNAc-b1,3-Gal-b1,4-Glc), the Bacillus subtilis strain was modified with a gene knock-in of a persistent transcription unit, The persistent transcription unit comprises a lactose importer (eg, E. coli LacY of UniProt ID P02920) and a galactoside β-1,3-N-acetylglucosaminyltransferase, such as from meningococcal LgtA of cocci (GenBank: AAM33849.1). For LNT production, the LN3 producing strain was further modified with a persistent transcription unit for a persistent transcription unit selected from the list comprising SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12 or 13 N-acetylglucosamine beta-1,3-galactosyltransferase, which can be presented to strains via gene knock-in or self-expressing plastids. For LNnT production, the LN3-producing strain was further modified with a persistent transcription unit directed against the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27 , 28, 31, 32, 33, 34, 35, 36, 39, 40 and 41 of the enumerated N-acetylglucosamine beta-1,4-galactosyltransferases. To further fucosylate LN3, LNT or LNnT, the mutant strain was further modified with a persistent transcription unit directed to, for example, α-1 of HpFutC (GenBank No. AAD29863.1) from H. pylori ,2-fucosyltransferase and/or α-1,3-fucosyltransferase from HpFucT (UniProt ID O30511) from Helicobacter pylori.

In the example of sialic acid production, mutant B. subtilis strains were created by overexpressing the native fructose-6-P-transaminase (UniProt ID POCI73) to promote intracellular glucosamine-6-phosphate pools. In addition, the enzymatic activities of the nagA, nagB, and gamA genes were disrupted by gene shaving and glucosamine-6-P-transaminase GNA1 (UniProt ID P43577) from Saccharomyces cerevisiae, N-acetyltransferase from Bacteroides ovale Glucosamine-2-epimerase (UniProt ID A7LVG6) and N-acetylneuraminic acid synthase (UniProt ID Q93MP9) from Aspergillus jejuni were genomically overexpressed. To allow the production of sialylated oligosaccharides, the sialic acid producing strains are further modified with expression constructs including an N-acylneuraminic acid cytidine transferase, such as the NeuA enzyme from Aspergillus jejuni (UniProt ID Q93MP7), the NeuA enzyme from H. influenzae (GenBank No. AGV11798.1) or the NeuA enzyme from Pasteurella haemorrhagic (GenBank No. AMK07891.1), and one or more Replicated β-galactoside α-2,3-sialyltransferase, such as PmultST3 (UniProt ID Q9CLP3) from Pasteurella haemorrhage or by a β-galactoside α-2,3-sialyltransferase PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of active UniProt ID Q9CLP3 and NmeniST3 from Neisseria meningitidis (GenBank No. ARC07984.1) or from Pasteurella hemorrhagic subsp. hemorrhagic strain Pm70 PmultST2 (GenBank No. AAK02592.1), β-galactoside α-2,6-sialyltransferase, such as PdST6 (UniProt ID O66375) from Photobacterium A PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID O66375 with 2,6-sialyltransferase activity or P-JT-ISH-224-ST6 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 with β-galactoside α-2,6-sialyltransferase activity and /or eg alpha-2,8-sialyltransferase from mouse (UniProt ID Q64689). In an example of the production of lactose-sialylated oligosaccharides, the B. subtilis mutant strain is further modified with a persistent transcription unit directed against a lactose import protein (eg, E. coli LacY of UniProt ID P02920).

For growth on sucrose, mutant strains can be additionally modified with gene knock-in of persistent transcription units including the sucrose transporter (CscB) (UniProt ID E0IXR1) from E. Fructokinase (Frk) from Bifidobacterium spp. (UniProt ID Q03417) and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (UniProt ID A0ZZH6).

Heterologous and Homologous Expression

The genes to be expressed, whether from the plastid or the genome, are synthesized synthetically by one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.

Expression can be further facilitated by optimizing codon usage for the codon usage of the expression host. Genes were optimized using vendor tools.

Culture conditions

Pre-incubation of 96-well microtiter plate experiments was initiated from a single colony in a frozen vial or LB plate, in 150 μL of LB and incubated overnight at 37°C on an orbital shaker at 800 rpm. This culture was used as an inoculum in a 96-well square microtiter plate, diluted 400-fold with 400 μL of MMsf medium. Each strain was grown in biological replicates in multiple wells of a 96-well plate. These final 96-well plates were then incubated at 37°C on an orbital shaker at 800 rpm for 72 h or less or longer. At the end of the culture experiment, samples were taken from each well to measure the supernatant concentration (extracellular sugar concentration after 5 min of spinning down cells), or by placing the culture broth at 90° before centrifuging the cells Boil for 15 minutes at C or 60 minutes at 60°C (= whole broth concentration, intracellular and extracellular sugar concentration as defined herein).

Again, the cultures were diluted to measure the optical density at 600 nm. The Cell Performance Index or CPI is determined by dividing the oligosaccharide concentration by the biomass, as a relative percentage compared to a reference strain. Biomass was empirically determined to be about 1/3 the optical density measured at 600 nm.

Example 35 : Produced in a modified Bacillus subtilis strain 2’FLNB

Bacillus subtilis strains were first modified for LNB production and growth on sucrose by gene shaving of nagB, glmS, and gamA genes and knock-in of a persistent transcription unit that included encoding a native fructose- 6-P-Transaminase (UniProt ID P0CI73), glmS*54 from E. coli (different from wild-type E. coli glmS with UniProt ID P17169 with A39T, R250C and G472S mutations as described in Deng et al. (Biochimie 88, 419-29 (2006))), N-acetylglucosamine-6-phosphotransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), phosphatase AraL from Bacillus subtilis (UniProt ID P94526) , N-acetylglucosamine beta-1,3-galactosyltransferase selected from the list comprising SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12 or 13, from the large intestine Sucrose transporter (CscB) from Bacillus W (UniProt ID E0IXR1), fructokinase (Frk) from Zymomonas mobilis (UniProt ID Q03417) and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (UniProt ID A0ZZH6) gene. In the next step, the LNB-producing strain is transfected with an expressing plastid comprising a persistent transcription unit for, for example, α-1,2-fucosyltransferase from HpFutC (GenBank No. AAD29863.1) from H. pylori shape.

The novel strains were evaluated for 2&apos; FLNB production in growth experiments in MMsf medium lacking the precursor according to the culture conditions provided in Example 34. After 72 hours of incubation, the culture broth was collected and analyzed for carbohydrates by UPLC.

Example 36 : Produced with modified Bacillus subtilis strains including 6'-SL , LacNAc , sialylation LacNAc , LN3 , sialylation LN3 , LNnT and LSTc oligosaccharide mixture

In a first step, the Bacillus subtilis strain is modified with gene shaving of the nagA, nagB and gamA genes and knock-in of a persistent transcription unit including a gene encoding a native fructose- 6-P-Transaminase (UniProt ID P0CI73), Glucosamine-6-P-Transaminase from Saccharomyces cerevisiae (UniProt ID P43577), N-Acetyl Glucosamine-2-Table from Bacteroides ovale Isomerase (UniProt ID A7LVG6), and genes for N-acetylneuraminic acid synthase (UniProt ID Q93MP9) from Aspergillus jejuni. In the next step, the mutant strain was further modified with gene knock-in of a persistent transcription unit including NeuA (UniProt ID Q93MP7) encoding the N-acylneuraminic acid cytidine transferase from Aspergillus jejuni and the gene for the α-2,6-sialyltransferase PdbST (UniProt ID O66375) from P. damselae to produce 6′-sialyllactose. In the next step, the mutant strain was further modified to include knock-in of a persistent transcription unit for galactoside β-1,3-N-acetylglucosamine transfer from Neisseria meningitidis Enzyme IgtA (UniProt ID Q9JXQ6) and selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35, 36 , 39, 40 and 41 enumerated N-acetylglucosamine beta-1,4-galactosyltransferase. According to the culture conditions provided in Example 34, novel strains including 6'-SL, LacNAc, sialylated LacNAc, LN3, sialylated LN3, LNnT and LSTc (Neu5Ac- a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc) oligosaccharide mixture. After 72 hours of incubation, the culture broth was collected and analyzed for carbohydrates by UPLC.

Example 37 : Materials and Methods Corynebacterium glutamicum

culture medium

Two different media were used, named trypsin enriched-yeast extract (TY) medium and minimal medium for shake flasks (MMsf). Minimal medium uses 1000x stock trace element mix.

The trace element mixture consists 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.

Minimal medium (MMsf) for shake flask experiments contains 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 L MOPS, 10 to 30 g/L glucose or other carbon sources including but not limited to fructose, maltose, sucrose, glycerol and maltotriose (when specified in the examples), and 1 ml/L trace element mixture. According to experiments, lactose and/or sialic acid can be added as precursors.

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

Complex media such as TY are sterilized by autoclaving (121°C, 21') and minimal media by filtration (0.22 µm Sartorius). When necessary, media were made selective by the addition of antibiotics (eg, kanamycin, ampicillin).

Strains and Mutations

Corynebacterium glutamicum ATCC 13032 was obtained from the American Type Culture Collection.

Construction of integrating plasmid vectors based on the Cre/loxP technology described by Suzuki et al. (Appl. Microbiol. Biotechnol., 2005 Apr, 67(2):225-33) and by Okibe et al. (Journal of Microbiological Methods 85, 2011 , 155-163) described temperature-sensitive shuttle vectors for gene deletion, mutation and insertion. Suitable promoters for (heterologous) gene expression can be obtained from Yim et al. (Biotechnol. Bioeng., 2013 Nov, 110(11):2959-69). Colonization can be performed using Gibson assemblies, Golden Gate assemblies, Cliva assemblies, LCR or restriction ligation.

In the LNB-producing example, C. glutamicum was modified with genomic knock-in of persistent expression units including glmS*54 from E. coli (with wild-type E. coli with UniProt ID P17169 glmS differs by having the A39T, R250C and G472S mutations as described by Deng et al. (Biochimie 88, 419-29 (2006)), the N-acetylglucosamine-6-phosphotransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), a monophosphatase selected from the following list including: comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, Any one or more of the E. coli genes of AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU, or PsMupP from Pseudomonas putida, ScDOG1 from Saccharomyces cerevisiae or BsAraL from Bacillus subtilis, as described in WO18122225, and selected from N-acetylglucosamine beta from the enumeration comprising SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12 or 13 -1,3-Galactosyltransferase.

In the LacNAc-producing example, C. glutamicum strains were modified with genomic knock-in of persistent expression units including glmS*54 from E. coli (with wild-type large intestine with UniProt ID P17169) Bacillus glmS differs by having the A39T, R250C and G472S mutations as described by Deng et al. (Biochimie 88, 419-29 (2006)), N-acetylglucosamine-6-phosphotransferase from Saccharomyces cerevisiae GNA1 (UniProt ID P43577), a monophosphatase selected from the list comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL , any or more of the E. coli genes of AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU, or PsMupP from Pseudomonas putida, Saccharomyces cerevisiae ScDOG1 or BsAraL from Bacillus subtilis as described in WO18122225 and selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, The N-acetylglucosamine beta-1,4-galactosyltransferases enumerated in 33, 34, 35, 36, 39, 40 and 41. In order to further fucosylate the LNB or LacNAc, the LNB or LacNAc producing strain is further modified with a persistent transcription unit directed against, for example, alpha of HpFutC (GenBank No. AAD29863.1) from Helicobacter pylori -1,2-fucosyltransferase and/or α-1,3-fucosyltransferase from HpFucT (UniProt ID O30511) from Helicobacter pylori.

In an example of the production of lactose-like oligosaccharides, a mutant Corynebacterium glutamicum strain was created to contain a gene encoding a lactose import protein (eg, E. coli LacY of UniProt ID P02920). For 2'FL, 3FL and diFL production, e.g. α-1,2-fucosyltransferase from HpFutC (GenBank No. AAD29863.1) from Helicobacter pylori and/or HpFucT from Helicobacter pylori (UniProt ID O30511) α-1,3-fucosyltransferase was additionally added to the strain.

In the examples of the production of lacto-N-trisaccharides (LNT-II, LN3, GlcNAc-b1,3-Gal-b1,4-Glc), the Corynebacterium glutamicum strain was knocked-in with a persistent transcription unit Modified to include a lactose importin (eg, E. coli LacY from UniProt ID P02920) and a galactoside β-1,3-N-acetylglucosaminyltransferase such as from Neisseria meningitidis LgtA (GenBank: AAM33849.1). For LNT production, the LN3 producing strain was further modified with a persistent transcription unit for a persistent transcription unit selected from the list comprising SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12 or 13 N-acetylglucosamine beta-1,3-galactosyltransferase, which can be presented to strains via gene knock-in or self-expressing plastids. For LNnT production, the LN3-producing strain was further modified with a persistent transcription unit directed against the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27 , 28, 31, 32, 33, 34, 35, 36, 39, 40 and 41 of the enumerated N-acetylglucosamine beta-1,4-galactosyltransferases. To further fucosylate LN3, LNT or LNnT, the mutant strain was further modified with a persistent transcription unit directed to, for example, α-1 of HpFutC (GenBank No. AAD29863.1) from H. pylori ,2-fucosyltransferase and/or α-1,3-fucosyltransferase from HpFucT (UniProt ID O30511) from Helicobacter pylori.

In an example of sialic acid production, a mutant Corynebacterium glutamicum strain was created by overexpressing the native fructose-6-P-transaminase (UniProt ID Q8NND3) to promote intracellular glucosamine-6-phosphate library. In addition, the enzymatic activities of the Corynebacterium glutamicum genes ldh, cgl2645, nagB, gamA and nagA were disrupted by gene shaving and the glucosamine-6-P-transaminase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577) , N-acetylglucosamine-2-epimerase from Bacteroides ovale (UniProt ID A7LVG6) and N-acetylneuraminic acid synthase from Aspergillus jejuni (UniProt ID Q93MP9) are genomically overrepresented . To allow the production of sialylated oligosaccharides, the sialic acid producing strains are further modified with expression constructs including an N-acylneuraminic acid cytidine transferase, such as the NeuA enzyme from Aspergillus jejuni (UniProt ID Q93MP7), the NeuA enzyme from H. influenzae (GenBank No. AGV11798.1) or the NeuA enzyme from Pasteurella haemorrhagic (GenBank No. AMK07891.1), and one or more Replicated β-galactoside α-2,3-sialyltransferase, such as PmultST3 (UniProt ID Q9CLP3) from Pasteurella haemorrhage or by a β-galactoside α-2,3-sialyltransferase PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of active UniProt ID Q9CLP3 and NmeniST3 from Neisseria meningitidis (GenBank No. ARC07984.1) or from Pasteurella hemorrhagic subsp. hemorrhagic strain Pm70 PmultST2 (GenBank No. AAK02592.1), β-galactoside α-2,6-sialyltransferase, such as PdST6 (UniProt ID O66375) from Photobacterium A PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID O66375 with 2,6-sialyltransferase activity or P-JT-ISH-224-ST6 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 with β-galactoside α-2,6-sialyltransferase activity and /or eg alpha-2,8-sialyltransferase from mouse (UniProt ID Q64689). In an example of the production of lactose-sialylated oligosaccharides, the C. glutamicum mutant strain is further modified with a persistent transcription unit directed against a lactose importin (eg, E. coli LacY of UniProt ID P02920) .

For growth on sucrose, mutant strains can be additionally modified with gene knock-in of persistent transcription units including the sucrose transporter (CscB) (UniProt ID E0IXR1) from E. Fructokinase (Frk) from Bifidobacterium spp. (UniProt ID Q03417) and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (UniProt ID A0ZZH6).

Heterologous and Homologous Expression

The genes to be expressed, whether from the plastid or the genome, are synthesized synthetically by one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.

Expression can be further facilitated by optimizing codon usage for the codon usage of the expression host. Genes were optimized using vendor tools.

Culture conditions

Pre-cultures for 96-well microtiter plate experiments were initiated from a single colony in a frozen vial or TY plate, in 150 μL of TY and incubated overnight at 37°C on an orbital shaker at 800 rpm. This culture was used as an inoculum in a 96-well square microtiter plate, diluted 400-fold with 400 μL of MMsf medium. Each strain was grown in biological replicates in multiple wells of a 96-well plate. These final 96-well plates were then incubated at 37°C on an orbital shaker at 800 rpm for 72 h or less or longer. At the end of the culture experiment, samples were taken from each well to measure the supernatant concentration (extracellular sugar concentration after 5 min of spinning down cells), or by heating the culture broth at 60°C before centrifuging the cells Boil for 15 minutes at C (= whole broth concentration, intracellular and extracellular sugar concentration as defined herein).

Again, the cultures were diluted to measure the optical density at 600 nm. The Cell Performance Index or CPI is determined by dividing the measured oligosaccharide concentration in the whole broth by the biomass, as a relative percentage compared to a reference strain. Biomass was empirically determined to be about 1/3 the optical density measured at 600 nm.

Example 38 : Produced with a modified strain of Corynebacterium glutamicum 2’FLNB

Corynebacterium glutamicum strains were first modified for LNB production and growth on sucrose by gene shaving of the ldh, cgl2645 and nagB genes, and knock-in of a persistent transcription unit comprising codes from glmS*54 of E. coli (different from wild-type E. coli glmS with UniProt ID P17169 with A39T, R250C and G472S mutations as described by Deng et al. (Biochimie 88, 419-29 (2006))), from Saccharomyces cerevisiae N-acetylglucosamine-6-phosphotransferase GNA1 (UniProt ID P43577), phosphatase AraL (UniProt ID P94526) from Bacillus subtilis, selected from the group consisting of SEQ ID NOs: 03, 04, 05, N-acetylglucosamine beta-1,3-galactosyltransferase enumerated in 06, 07, 08, 10, 11, 12 and 13, sucrose transporter (CscB) from Escherichia coli W (UniProt ID E0IXR1) , fructokinase (Frk) from Zymomonas mobilis (UniProt ID Q03417) and genes for sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (UniProt ID A0ZZH6). In the next step, the LNB-producing strain is transfected with an expressing plastid comprising a persistent transcription unit for, for example, α-1,2-fucosyltransferase from HpFutC (GenBank No. AAD29863.1) from H. pylori shape. According to the culture conditions provided in Example 37, the novel strains were evaluated for 2&apos; FLNB production in growth experiments in MMsf medium lacking the precursor. After 72 hours of incubation, the culture broth was collected and analyzed for carbohydrates by UPLC.

Example 39 : Produced by a modified strain of Corynebacterium glutamicum including sialylation LacNAc mixture

Corynebacterium glutamicum was first modified with gene shaving of the ldh, cgl2645, nagB, nagA, and gamA genes and knock-in of a persistent transcription unit for the production of LacNAc and growth on sucrose, including Encoding native fructose-6-P-transaminase (UniProt ID Q8NND3), mutant glmS*54 from E. coli (different from wild-type E. coli glmS with UniProt ID P17169 with A39T, R250C and G472S mutations, (Biochimie 88, 419-29 (2006)), N-acetylglucosamine-6-phosphotransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), phosphatase from Bacillus subtilis as described by Deng et al. (Biochimie 88, 419-29 (2006)) AraL (UniProt ID P94526), selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35, 36, N-acetylglucosamine beta-1,4-galactosyltransferase enumerated in 39, 40 and 41, sucrose transporter (CscB) from Escherichia coli W (UniProt ID E0IXR1), from Zymomonas mobilis The fructokinase (Frk) (UniProt ID Q03417) and sucrose phosphorylase (BaSP) (UniProt ID A0ZZH6) genes from Bifidobacterium adolescentis. In the next step of sialic acid synthesis, the mutant strain was further modified with gene knock-in of a persistent transcription unit including the encoding for N-acetylglucosamine-2-epiisomer from Bacteroides ovale Enzyme (UniProt ID A7LVG6) and genes for N-acetylneuraminic acid synthase (UniProt ID Q93MP9) from Aspergillus jejuni. In the next step, the novel strain is transformed with an expression plastid comprising a persistent transcription unit comprising the gene encoding the NeuA enzyme (UniProt ID Q93MP7) from A. Bacillus β-galactoside α-2,3-sialyltransferase PmultST3 (UniProt ID Q9CLP3) or β-galactoside α-2,6-sialyltransferase PdST6 (UniProt ID O66375) ) gene combination.

The novel strains were evaluated for LacNAc, sialic acid and sialylated LacNAc production in growth experiments in MMsf medium lacking precursors according to the culture conditions provided in Example 37. It was also assessed whether the mutant strain additionally produced 3'-SL or 6'-SL depending on the expression of α-sialyltransferase when lactose was added as a precursor to the MMsf medium. After 72 hours of incubation, the culture broth was collected and analyzed for carbohydrates by UPLC.

Example 40 : Materials and Methods Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii )

culture medium

Chlamydomonas reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7.0). TAP medium uses a 1000x Hutner trace element mix. The Hutner trace element mixture consists 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 It is composed of L CoCl2.6H2O, 1.6 g/L CuSO4.5H2O and 1.1 g/L (NH4)6MoO3.

1.6 g/L CuSO4.5H2O contains 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 precursors for saccharide synthesis, precursors such as galactose, glucose, fructose and/or fucose can be added. The medium was sterilized by autoclaving (121°C, 21'). For stock cultures on agar slants, use TAP medium containing 1% agar (purified high strength, 1000 g/cm2).

Strains, plastids and mutations

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

Expression plasmids derived from pSI103 or from the Chlamydomonas Resource Center. Colonization can be performed using Gibson assemblies, Golden Gate assemblies, Cliva assemblies, LCR or restriction ligation. Suitable promoters for (heterologous) gene expression can be obtained, for example, from Scranton et al. (Algal Res. 2016, 15: 135-142). Modification of the target gene (eg, gene knockout or gene replacement) can be performed using the Crispr-Cas technology described by Jiang et al. (Eukaryotic Cell 2014, 13(11): 1465-1469).

Transformation can be performed via electroporation as described by Wang et al. (Biosci. Rep. 2019, 39: BSR2018210). Cells were grown in liquid TAP medium under constant aeration and continuous light at a light intensity of 8000 Lx until cell density reached 1.0 to 2.0 x 10 ⁷ cells/mL. Next, cells were seeded into fresh liquid TAP medium at a concentration of 1.0 x 10 ⁶ cells/mL and grown under continuous light for 18-20 hours until the cell density reached 4.0 x 10 ⁶ cells/mL. Cells were then harvested by centrifugation at 1250 g for 5 min at room temperature, washed and resuspended in pre-chilled liquid TAP medium containing 60 mM sorbitol (Sigma, USA), and frozen for 10 min. Next, place 250 µL of the cell suspension (corresponding to 5.0 × 10 ⁷ cells) with 100 ng of plastid DNA (400 ng/mL) into a pre-cooled 0.4 cm electroporation colorimetric tube. Electroporation was performed using a BTX ECM830 electroporation device (1575 Ω, 50 μFD) with 6 pulses of 500 V, each with a pulse length of 4 ms and a pulse interval of 100 ms. Immediately after electroporation, place the cuvette on ice for 10 minutes. Finally, transfer the cell suspension to a 50 mL conical centrifuge tube containing 10 mL of fresh liquid TAP medium and 60 mM sorbitol and recover with slow shaking overnight in dim light. After overnight recovery, cells were re-harvested and seeded by starch embedding into selective 1.5% (w/v) containing ampicillin (100 mg/L) or chloramphenicol (100 mg/L). ) on agar-TAP dishes. Plates were then incubated at 23+-0.5°C under continuous illumination with a light intensity of 8000 Lx. Cells were analyzed after 5-7 days.

In an example for the production of UDP-galactose, the Chlamydomonas reinhardtii cells are modified with a transcription unit comprising a gene encoding a galactokinase (KIN, UniProt ID Q9SEE5) from Arabidopsis thaliana , and Gene encoding UDP-sugar pyrophosphorylase (USP) (UniProt ID Q9C5I1) from Arabidopsis.

In an embodiment of producing LNB, the C. reinhardtii cell modified to produce UDP-galactose is modified with a presentation plastid comprising a plastid selected from the group consisting of SEQ ID NOs: 03, 04, 05, 06, 07, 08 , 10, 11, 12 and 13 of the transcription unit of N-acetylglucosamine beta-1,3-galactosyltransferase. In addition, mutant C. reinhardtii cells can be modified by expressing plastids including, for example, α-1,2-fucosyltransferase and/or HpFutC (GenBank No. AAD29863.1) from Helicobacter pylori Transcription unit of α-1,3-fucosyltransferase from HpFucT (UniProt ID O30511) from Helicobacter pylori.

In the embodiment of producing LacNAc, the Chlamydomonas reinhardtii cell modified to produce UDP-galactose is further modified with a presentation plastid comprising a plastid selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, N-acetylglucosamine beta-1,4-galactosylation enumerated in 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35, 36, 39, 40 and 41 The transcription unit of an enzyme. In addition, mutant C. reinhardtii cells can be modified by expressing plastids including, for example, α-1,2-fucosyltransferase and/or HpFutC (GenBank No. AAD29863.1) from Helicobacter pylori Transcription unit of α-1,3-fucosyltransferase from HpFucT (UniProt ID O30511) from Helicobacter pylori.

In an example of CMP-sialic acid synthesis, Chlamydomonas reinhardtii cells are modified with a persistent transcription unit directed to, for example, UDP-N-acetylglucosamine- 2-Epimerase/N-acetylmannosamine kinase or mutant forms of human GNE polypeptides including the R263L mutation, such as N-acylneuraminic acid-9-phosphate from NANS (UniProt ID Q9NR45) from Homo sapiens Synthetase and eg N-acylneuraminocytidine cytidine transferase from Homo sapiens CMAS (UniProt ID Q8NFW8). In an example of production of sialylated oligosaccharides, C. reinhardtii cells have high levels of CMP sialic acid transporters such as CST from mouse (UniProt ID Q61420), and species selected from, e.g., Homo sapiens, mouse, brown rat Golgi-localised sialyltransferase for modification.

Heterologous and Homologous Expression

The genes to be expressed, whether from the plastid or the genome, are synthesized synthetically by one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.

Expression can be further facilitated by optimizing codon usage for the codon usage of the expression host. Genes were optimized using vendor tools.

Culture conditions

Chlamydomonas reinhardtii cells were cultured in selective TAP agar plates at 23 +/- 0.5°C with a 14/10 hour light/dark cycle at a light intensity of 8000 Lx. Cell analysis was performed after 5 to 7 days in culture.

For high-density culture, cells can be grown in a closed system, for example, vertically as described by Chen et al. (Bioresour. Technol. 2011, 102: 71-81) and Johnson et al. Or horizontal tube photobioreactor, stirred tank photobioreactor or flat plate photobioreactor (Bioresour. Technol. 2011, 102: 71-81).

Example 41 : produced in modified Chlamydomonas reinhardtii cells LNB and 2’FLNB

As described in Example 40, C. reinhardtii cells were engineered to produce UDP-Gal with knock-in of a persistent transcription unit including galactokinase (KIN, UniProt ID Q9SEE5) from Arabidopsis and Arabidopsis UDP-sugar pyrophosphorylase (USP) (UniProt ID Q9C5I1). In the next step, the mutant cells are transformed with an expression plasmid comprising a transcription unit comprising a transcription unit selected from the group consisting of SEQ ID NOs: 03, 04, 05, 06, 07, 08, 10, 11, 12 and 13 The exemplified N-acetylglucosamine beta-1,3-galactosyltransferase and the alpha-1,2-fucosyltransferase HpFutC from Helicobacter pylori (GenBank No. AAD29863.1). The novel strains were evaluated in culture experiments on TAP agar plates including galactose as a precursor according to the culture conditions provided in Example 40. After 5 days in culture, cells were harvested and analyzed by UPLC for LNB and 2&apos;FLNB production.

Example 42 : produced in modified Chlamydomonas reinhardtii cells LacNAc and 3'- Fucosylation LacNAc

As described in Example 40, C. reinhardtii cells were engineered to produce UDP-Gal with knock-in of a persistent transcription unit including galactokinase (KIN, UniProt ID Q9SEE5) from Arabidopsis and Arabidopsis UDP-sugar pyrophosphorylase (USP) (UniProt ID Q9C5I1). In the next step, the mutant cells are transformed with an expression plasmid comprising a transcription unit comprising a transcription unit selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26 , 27, 28, 31, 32, 33, 34, 35, 36, 39, 40 and 41 enumerated N-acetylglucosamine beta-1,4-galactosyltransferases and, for example, HpFucT from Helicobacter pylori (UniProt ID O30511) α-1,3-fucosyltransferase. The novel strains were evaluated in culture experiments on TAP agar plates including galactose as a precursor according to the culture conditions provided in Example 40. After 5 days in culture, cells were harvested and analyzed by UPLC for LacNAc and 3&apos;-fucosylated LacNAc production.

Example 43 : Materials and Methods Animal Cells

Isolation of mesenchymal stem cells from adipose tissue of different mammals

Fresh adipose tissue was obtained from slaughterhouses (eg, cattle, pigs, sheep, chickens, ducks, catfish, snakes, frogs) or liposuctioned (eg, in the case of humans, after informed consent) and stored in antibiotic-supplemented in phosphate buffered saline. Adipose tissue is enzymatically digested and then centrifuged to isolate mesenchymal stem cells. Transfer the isolated mesenchymal stem cells to cell culture flasks and grow under standard growth conditions, such as 37°C, 5% CO2. Initial media included DMEM-F12, RPMI and A-MEM media (supplemented with 15% fetal bovine serum) and 1% antibiotics. After the first passage, the medium was subsequently replaced with medium supplemented with 10% FBS (fetal bovine serum). For example, Ahmad and Shakoori (2013, Stem Cell Regen. Med. 9(2): 29-36), which is incorporated herein by reference in its entirety, describe some variations of the methods described herein in this Example.

Isolation of mesenchymal stem cells from milk

This example illustrates the isolation of mesenchymal stem cells from milk collected under sterile conditions from a human or any other mammal as described herein. An equal volume of phosphate buffered saline was added to the diluted milk, followed by centrifugation for 20 minutes. The cell pellets were washed three times with phosphate buffered saline, and the cells were seeded in cell culture flasks in DMEM-F12, RPMI and A-MEM medium supplemented with 10% fetal bovine serum and 1% antibiotics under standard culture conditions. For example, Hassiotou et al. (2012, Stem Cells. 30(10): 2164-2174), which is incorporated herein by reference in its entirety, describe some variations of the methods described herein in this example.

use 2D and 3D culture system differentiated stem cells

Isolated mesenchymal cells can be differentiated into mammary-like epithelial cells and luminal cells in 2D and 3D culture systems. For example, see 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; all incorporated herein by reference in their entirety for all purposes.

For 2D culture, isolated cells were initially seeded in culture dishes in growth medium supplemented with 10 ng/ml epithelial growth factor and 5 pg/ml insulin. At confluence, cells were fed for 48 h 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. To induce differentiation, 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 was removed from the complete induction medium.

For 3D culture, isolated cells were trypsinized and cultured in Matrigel, hyaluronic acid, or ultra-low attachment surface culture dishes for 6 days and supplemented with 10 ng/ml epithelial growth factor and 5 Growth medium with pg/ml insulin induces differentiation and lactate. At confluence, cells were fed 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, 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 was removed from the complete induction medium.

Method for preparing mammary gland-like cells

Mammalian cells are rendered pluripotency by reprogramming of viral vectors encoding Oct4, Sox2, Klf4 and c-Myc. Next, the resulting reprogrammed cells were grown in Mammocult medium (available from Stem Cell Technologies) or breast cell enrichment medium (DMEM, 3% FBS, estrogen, progesterone, heparin, hydrocortisone, insulin, EGF) Cultured to resemble mammary glands, from which performance of selected milk components can be induced. Alternatively, epigenetic remodelling using a remodeling system such as CRISPR/Cas9 to persistently activate selected genes of interest such as casein, alpha-lactalbumin to allow their respective proteins Selected endogenous genes are expressed, and/or down-regulated and/or knocked out, as described, for example, in WO21067641, which is hereby incorporated by reference in its entirety for all purposes.

nourish

Complete growth medium including high glucose DMEM/F12, 10% FBS, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, and 5 pg/m hydrocortisone . Complete lactation medium including 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 ml prolactin (5ug/ml in Hyunh 1991). Cells were seeded into complete growth medium on collagen-coated flasks at a density of 20,000 cells/cm2 and allowed to adhere and expand in complete growth medium for 48 hours, after which the medium was switched to complete lactation medium. After exposure to lactation medium, cells begin to differentiate and stop growing. Within about a week, cells begin to secrete lactation products such as milk fat, lactose, casein and whey into the medium. The lactation medium can be concentrated or diluted by ultrafiltration to obtain the desired concentration of lactation medium. The desired salt balance of the lactation medium can be accomplished by dialysis, eg, to remove unwanted metabolites from the medium. Hormones and other growth factors used can be selectively extracted by resin purification, such as the use of nickel resins to remove His-tagged growth factors, to further reduce contaminant levels in the lactic acid product.

Example 44 : Evaluation of non-mammary adult stem cells in 2'FL , LNFP-I and 2'FLNB generation

Isolated stromal cells and reprogrammed to mammary-like cells as described in Example 43 were modified with CRISPR-CAS to overexpress the group consisting of 03, 04, 05, 06, 07, 08, 10, 11, 12, and 13 Enumerated codon-optimized N-acetylglucosamine beta-1,3-galactosyltransferase, GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), and codons from Helicobacter pylori Optimized α-1,2-fucosyltransferase (GenBank No. AAD29863.1). Cells were seeded into complete growth medium on collagen-coated flasks at a density of 20,000 cells/cm2 and allowed to adhere and expand in complete growth medium for 48 hours, after which the medium was switched to complete lactation medium for approximately 7 days. After culturing as described in Example 43, cells were subjected to UPLC to analyze 2'FL, LNFP-I (lacto-N-fucopentaose I, Fuc-a1, 2-Gal-b1, 3-GlcNAc-b1, 3-Gal-b1,4-Glc) and 2'FLNB production.

Example 45 : Evaluation of non-mammary adult stem cells in LacNAc , sialylation LacNAc and sialic acid - Lewis x ( sialyl-Lewis x ) is produced

Isolated mesenchymal cells and reprogrammed to mammary-like cells as described in Example 43 were modified with CRISPR-CAS to overexpress selected from the group consisting of 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, N-acetylglucosamine beta-1,4-galactosyltransferase enumerated in 27, 28, 31, 32, 33, 34, 35, 36, 39, 40 and 41, GDP-fucoid from Homo sapiens Carbohydrate synthase GFUS (UniProt ID Q13630), galactoside alpha-1,3-fucosyltransferase FUT3 (UniProt ID P21217) from Homo sapiens, N-acyl neuraminic acid cytidine from mouse Transferase (UniProt ID Q99KK2) and CMP-N-acetylneuraminic acid-β-1,4-galactoside α-2,3-sialyltransferase ST3GAL3 (UniProt ID Q11203) from Homo sapiens. All genes introduced into cells are codon-optimized for the host. Cells were seeded into complete growth medium on collagen-coated flasks at a density of 20,000 cells/cm2 and allowed to adhere and expand in complete growth medium for 48 hours, after which the medium was switched to complete lactation medium for approximately 7 days. After incubation as described in Example 43, cells were subjected to UPLC to analyze the production of LacNAc, sialylated LacNAc and sialic acid-Lewis x.

none.

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Claims (108)

A method for producing oligosaccharides or disaccharides with N-acetylglucosamine units at the reducing end by a cell, preferably a single cell, the method comprising the steps of: a. Provide cells capable of: (i) synthesizing the nucleotide-sugar and monosaccharide N-acetylglucosamine (GlcNAc) and (ii) expressing a glycosyltransferase to glycosylate the GlcNAc monosaccharide to produce the double sugar or oligosaccharide, b. Cultivate the cell under conditions that allow the production of the disaccharide or oligosaccharide, c. Preferably, the disaccharide or oligosaccharide is isolated from the culture. A method for producing oligosaccharides with N-acetylglucosamine units at the reducing end by cells, the method comprising the steps of: a. Provide cells capable of: (i) synthesizing the nucleotide-sugar and monosaccharide N-acetylglucosamine (GlcNAc) and (ii) expressing a glycosyltransferase to glycosylate the GlcNAc monosaccharide to produce the oligo sugar, b. culturing the cell under conditions that allow the production of the oligosaccharide, c. Preferably, the oligosaccharide is isolated from the culture. A method for producing a mixture by a cell, the mixture comprising (i) a disaccharide and/or oligosaccharide having an N-acetylglucosamine unit at the reducing end, and (ii) one or more lactose-based mammalian milk oligosaccharides ( MMOs), the method includes the following steps: a. Provide cells capable of: (i) synthesizing the nucleotide-sugar and monosaccharide N-acetylglucosamine (GlcNAc) and (ii) expressing a glycosyltransferase to glycosylate the GlcNAc monosaccharide to produce the reduction disaccharides and/or oligosaccharides having N-acetylglucosamine units at the ends, b. culturing the cell under conditions that allow production of the mixture, c. Preferably, the mixture is isolated from the culture. The method of any one of the preceding claims, wherein the cell expresses at least one N-acetylglucosamine-6-phosphotransferase and phosphatase to synthesize the monosaccharide N-acetylglucosamine. The method of any one of the preceding claims, wherein the cell expresses at least one glycosyltransferase to glycosylate N-acetylglucosamine. The method of any one of the preceding claims, wherein the cell is genetically modified to produce the disaccharide or oligosaccharide. The method of claim 6, wherein the cell is modified with one or more gene expression modules, wherein the expression of any of the expression modules is constitutive or produced by natural inducers. The method of any one of claims 6 or 7, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein. The method of any one of the preceding claims, wherein the cell is modified in the expression or activity of an enzyme selected from the group consisting of N-acetylglucosamine-6-phosphotransferase, phosphatase, Glycosyltransferase, L-glutamine-D-fructose-6-phosphate transaminase and UDP-glucose 4-epimerase. The method of any one of the preceding claims, wherein the nucleotide-sugar is selected from the group comprising: 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-acetylamino-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-pneumolysamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy- L-talose), UDP-N-Acetylmuramic acid, UDP-N-Acetyl-L-Quinosamine (UDP-L-QuiNAc or UDP-2-Acetylamino-2,6 -Dideoxy-L-glucose), GDP-L-isorhamnose, CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N ₃ , CMP-Neu4,5Ac ₂ , CMP-Neu5,7Ac ₂ , CMP-Neu5,9Ac ₂ , CMP-Neu5,7(8,9)Ac ₂ , UDP-glucuronate, UDP-galacturonate, GDP-rhamnose and UDP-xylose. The method of any one of the preceding claims, wherein the nucleotide-sugar is UDP-galactose, and the glycosyltransferase is N-acetylglucosamine b-1,3-galactosyltransferase or N-acetylglucosamine b-1,4-galactosyltransferase. The method of any one of the preceding claims, wherein the oligosaccharide has lacto-N-disaccharide (Gal-b1,3-GlcNAc) or N-acetyllactosamine (Gal-b1,4) at the reducing end -GlcNAc). The method of any one of the preceding claims, wherein the N-acetylglucosamine b-1,3-galactosyltransferase is a glycosyltransferase having: a. PFAM domain PF00535, and i. comprising the sequence of SEQ ID NO 01 [AGPS]XXLN(X _n )RXDXD, wherein X is any amino acid, wherein n is 12 to 17, or ii. comprising the sequence of SEQ ID NO 02 PXXLN(X _n )RXDXD(X _m ) [FWY]XX[HKR]XX[NQST], wherein X is any amino acid, wherein n is 12 to 17 and m is 100 to 115, or iii. including according to SEQ ID NOs: 03, 04, 05, Any of 06, 07 or 08, preferably any of SEQ ID NOs 03, 04, 05, 06 or 07, more preferably any of SEQ ID NOs 03, 06 or 07, most preferably SEQ ID NOs The polypeptide sequence of any one of ID NO 03 or 06, or iv. any one of SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably SEQ ID NO 03, 04, 05, 06 or any of 07, more preferably any of SEQ ID NO 03, 06 or 07, most preferably a functional homologue, variant or derivative of any of SEQ ID NO 03 or 06, which With any of SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably any of SEQ ID NO 03, 04, 05, 06 or 07, more preferably SEQ ID NO 03, Any of 06 or 07, preferably at least 80% overall sequence identity of the full length of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide of any of SEQ ID NOs 03 or 06 and has N-acetylglucosamine b-1,3-galactosyltransferase activity, or v. includes any one from SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably SEQ ID NO: 03, 04, 05, 06, 07 or 08 Any one of ID NO 03, 04, 05, 06 or 07, more preferably any one of SEQ ID NO 03, 06 or 07, most preferably at least 8, 9 of any one of SEQ ID NO 03 or 06 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 oligopeptide sequences of consecutive amino acid residues with N-acetylglucosamine b-1,3-galactose Syltransferase activity, or vi. is any of SEQ ID NOs: 03, 04, 05, 06, 07 or 08, preferably any of SEQ ID NOs 03, 04, 05, 06 or 07, more Preferably any one of SEQ ID NO 03, 06 or 07, the best is SEQ ID NO 03 or 0 The functional fragment of any one of 6, and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or vii. comprising a polypeptide comprising or consisting of having and SEQ ID NOs: 03, 04 , any of 05, 06, 07 or 08, preferably any of SEQ ID NOs 03, 04, 05, 06 or 07, more preferably any of SEQ ID NOs 03, 06 or 07, most It is preferably composed of amino acid sequences of at least 80% sequence identity to the full-length amino acid sequence of any one of SEQ ID NOs 03 or 06, and has N-acetylglucosamine b-1,3-galactosyl Transferase activity, or b. PFAM domain IPR002659, and i. comprising the sequence of SEQ ID NO 09 KT( _Xn )[FY]XXKXDXD( _Xm )[FHY]XXG(X, no A,G,S)(X _p )X (No F, H, W, Y) [DE]D[ILV]XX[AG], where X is any amino acid, where n is 13 to 16, m is 35 to 70, and p is 20 to 45, or ii. comprising a polypeptide sequence according to any one of SEQ ID NOs: 10, 11, 12 or 13, or iii. being a functionally equivalent of any one of SEQ ID NOs: 10, 11, 12 or 13 A source, variant or derivative having at least a full-length equivalent to any of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptides of SEQ ID NO: 10, 11, 12 or 13 80% overall sequence identity with N-acetylglucosamine b-1,3-galactosyltransferase activity, or iv. Comprising at least one from any of SEQ ID NOs: 10, 11, 12, or 13 Oligopeptide sequences of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b-1,3 - Galactosyltransferase activity, or v. is a functional fragment of any of SEQ ID NOs: 10, 11, 12 or 13 and has N-acetylglucosamine b-1,3-galactosyltransferase activity, or vi. comprising a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any of SEQ ID NOs: 10, 11, 12 or 13 , and has N-acetylglucosamine b-1,3-galactosyltransferase activity. The method of any one of claims 1 to 12, wherein the N-acetylglucosamine b-1,4-galactosyltransferase is a glycosyltransferase having: a. PFAM domain PF01755, and i. The sequence EXXCXXSHXX[ILV][FWY]( _Xn )EDD( _Xm )[ACGST]XXYX[ILMV] comprising SEQ ID NO 14, wherein X is any amino acid, wherein n is 13 to 15 and m is 50 to 76, or ii. including according to any of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NO: 15, 16, 17, 18, Any one of 20 or 21, more preferably the polypeptide sequence of any one of SEQ ID NO: 17, 18, 20 or 21, or iii. is SEQ ID NO: 15, 16, 17, 18, 19, 20, Any of 21, 22 or 23, preferably any of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably any of SEQ ID NO: 17, 18, 20 or 21 A functional homologue, variant or derivative of 1, which has any of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NO: Any one of 15, 16, 17, 18, 20 or 21, more preferably the N-acetylglucosamine b-1,4-galactose of any one of SEQ ID NO: 17, 18, 20 or 21 At least 80% overall sequence identity of full length in a syltransferase polypeptide and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or iv. including from SEQ ID NOs: 15, 16, 17 , any of 18, 19, 20, 21, 22 or 23, preferably any of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably SEQ ID NO: 17, 18 An oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 contiguous amino acid residues of any of , 20, or 21 with N - acetylglucosamine b-1,4-galactosyltransferase activity, or v. is any of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably is any one of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably a functional fragment of any one of SEQ ID NO: 17, 18, 20 or 21, and has N-acetylglucose Amine b-1,4-galactosyltransferase activity, or vi. including polypeptides that contain including or consisting of any of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NO: 15, 16, 17, 18, 20 or 21 any, more preferably an amino acid sequence of at least 80% sequence identity of the full-length amino acid sequence of any of SEQ ID NO: 17, 18, 20 or 21, and has N-acetylglucose Amine b-1,4-galactosyltransferase activity, or b. PFAM domain PF00535, and i. the sequence comprising SEQ ID NO 24 R[KN]XXXXXXXXGXXXX[FL]XDXD( _Xn )[FHW]XXX[FHNY ](X _m )E[DE], wherein X is any amino acid, wherein n is 50 to 75 and m is 10 to 30, or ii. the sequence comprising SEQ ID NO 25 R[KN]XXXXXXXXGXXXXFXDXD(X _n ) [FHW]XXX[FHNY](X _m )E[DE](X _p )[FWY]XX[HKR]XX[NQST], where X is any amino acid, where n is 50 to 75 and m is 10 to 75 30, and p is 20 to 25, or iii. comprises a polypeptide sequence according to any one of SEQ ID NOs: 26, 27 or 28, or iv. is a function of any one of SEQ ID NOs: 26, 27 or 28 A sexual homologue, variant or derivative having at least a full-length equivalent to any of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides of SEQ ID NO: 26, 27 or 28 80% overall sequence identity and have N-acetylglucosamine b-1,4-galactosyltransferase activity, or v. comprising at least 8, Oligopeptide sequences of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b-1,4-half Lactosyltransferase activity, or vi. is a functional fragment of any one of SEQ ID NOs: 26, 27, or 28, and has N-acetylglucosamine b-1,4-galactosyltransferase activity, or vii . Include 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 of SEQ ID NOs: 26, 27 or 28, and having N-ethyl Glucosamine b-1,4-galactosyltransferase activity, or c. PFAM domain PF02709 without PFAM domain PF00535, and viii. the sequence comprising SEQ ID NO 29 [FWY]XX[FY][FWY]( X ₂₃ )[FWY][GQ]X[DE]D, where X is any amino acid, or ix. comprising the sequence of SEQ ID NO 30 [PV]W[GHNP]( _Xn )[FWY][GQ]X[DE]D, wherein X is any amino acid, wherein n is 21 to 24, or x. comprises a polypeptide sequence according to any one of SEQ ID NO: 31, 32, 33, 34, 35 or 36, or xi. is in SEQ ID NO: 31, 32, 33, 34, 35 or 36 A functional homologue, variant or derivative of any one having the N-acetylglucosamine b-1,4-galactose of SEQ ID NO:31, 32, 33, 34, 35 or 36 At least 80% overall sequence identity of any of the full-length syltransferase polypeptides and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or xii. Comprising from SEQ ID NO: 31, Oligopeptides of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues of any of 32, 33, 34, 35, or 36 sequence, and has N-acetylglucosamine b-1,4-galactosyltransferase activity, or xiii. is a functional fragment of any one of SEQ ID NOs: 31, 32, 33, 34, 35, or 36, and has N-acetylglucosamine b-1,4-galactosyltransferase activity, or xiv. comprises a polypeptide comprising or consisting of a Any of the full-length amino acid sequences consisting of amino acid sequences of at least 80% sequence identity and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or d. PFAM domain PF03808, and i. comprise the sequence of SEQ ID NO 37 [ST][FHY]XN( _Xn )DG(X16)[HKR]X[ST] _FDXX [ST]XA, wherein X is any amino acid, and wherein n is 20 to 25, or ii. the sequence comprising SEQ ID NO 38 [ST][FHY]XN( _Xn )DG(X16)[HKR]X[ST] _FDXX [ST]XA( _Xm )[ HR]XG[FWY](Xp) _GXGXXXQ [DE], wherein X is any amino acid, wherein n is 20 to 25, m is 40 to 50, and p is 22 to 30, or iii. including according to SEQ ID The polypeptide sequence of any of NO: 39, 40 or 41, or iv. is a functional homologue, variant or derivative of any of SEQ ID NO: 39, 40 or 41 having the same At least 80% of the overall sequence of any of the full-length N-acetylglucosamine b-1,4-galactosyltransferase polypeptides of NO: 39, 40 or 41 is identical to Monolithic and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or v. comprising at least 8, 9, 10, Oligopeptide sequences of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b-1,4-galactosyltransferase activity, or vi. is a functional fragment of any one of SEQ ID NOs: 39, 40, or 41, and has N-acetylglucosamine b-1,4-galactosyltransferase activity, or vii. comprises a polypeptide, The polypeptide comprises or consists of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any of SEQ ID NOs: 39, 40 or 41, and has N-acetylglucosamine b -1,4-galactosyltransferase activity. A method as claimed in any preceding claim, wherein a. The N-acetylglucosamine-6-phosphotransferase is a polypeptide sequence comprising the polypeptide of UniProt ID P43577, or is a functional homologue, variant or derivative of the polypeptide of UniProt ID P43577, which has the same The full-length polypeptide of ID P43577 has at least 80% overall sequence identity and has N-acetylglucosamine-6-phosphotransferase activity, and b. The L-glutamine-D-fructose-6-phosphate transaminase is a polypeptide sequence comprising the polypeptide of UniProt ID P17169, or a functional homologue, variant or derivative of the polypeptide with UniProt ID P17169 , which has at least 80% overall sequence identity with the full-length polypeptide of UniProt ID P17169, and has L-glutamine-D-fructose-6-phosphate transaminase activity; or is an A39T, Modified versions of the R250C and G472S mutations. The method of any one of the preceding claims, wherein the cell is capable of metabolizing a carbon source selected from the group consisting of glucose, fructose, galactose, lactose, sucrose, maltose, malto-oligo- Sugar, maltotriose, sorbitol, xylose, rhamnose, mannose, methanol, ethanol, arabinose, trehalose, starch, cellulose, hemicellulose, corn infusion, high fructose syrup, molasses, glycerin , acetate, citrate, lactic acid, pyruvic acid. The method of any one of the preceding claims, wherein the cell is unable to convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or is unable to convert glucosamine-6-phosphate Salt is converted to fructose-6-phosphate. The method of any one of the preceding claims, wherein the cell is modified to produce GDP-fucose. The method of any one of the preceding claims, wherein the cell is modified to promote GDP-fucose production, and the modification is selected from the group comprising: UDP-glucose:undecisoprene- Shaving of the gene encoding phosphoglucose-1-phosphotransferase, overexpression of the gene encoding GDP-L-fucose synthase, overexpression of the gene encoding GDP-mannose 4,6-dehydratase, mannose-1- Overexpression of a gene encoding phosphoguanylate, overexpression of a gene encoding phosphomannose mutase, or overexpression of a gene encoding mannose-6-phosphate isomerase. The method of any one of the preceding claims, wherein the cell is modified to produce UDP-galactose. The method of any one of the preceding claims, wherein the cell is modified to promote UDP-galactose production, and the modification is selected from the group comprising: 5'-nucleotidase/UDP-sugar hydrolysis Shaving of the gene encoding the enzyme or shaving of the gene encoding the galactose-1-phosphate uridine transferase. The method of any one of the preceding claims, wherein the cell is modified to produce CMP-N-acetylneuraminic acid. The method of any one of the preceding claims, wherein the cell is modified to promote CMP-N-acetylneuraminic acid production, and the modification is selected from the group comprising: CMP-sialic acid synthase Overexpression of encoding gene, overexpression of gene encoding sialic acid synthase, overexpression of gene encoding N-acetyl-D-glucosamine 2-epimerase. The method of any one of the preceding claims, wherein the cell is capable of expressing at least one other glycosyltransferase, and the other glycosyltransferase is selected from the group consisting of: fucosyltransferase, sialic acid Transferase, Galactosyltransferase, Glucosyltransferase, Mannosyltransferase, N-Acetyl Glucosamine Transferase, N-Acetyl Galactosamine Transferase, N-Acetyl Mannosamine Transferase, xylosyltransferase, glucuronidyltransferase, galacturonosyltransferase, glucosamine transferase, N-glycolyl neuraminidase, rhamnosyltransferase, N-acetyltransferase Rhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP- N- acetylglucosamine enol Acetoyltransferase and fucosyltransferase, - preferably, the fucosyltransferase is selected from the group including the following list: α-1,2-fucosyltransferase, α-1,3- Fucosyltransferases, α-1,4-fucosyltransferases and α-1,6-fucosyltransferases, - preferably, the sialyltransferases are selected from the list including: α -2,3-sialyltransferase, α-2,6-sialyltransferase and α-2,8-sialyltransferase, - Preferably, the galactosyltransferase is selected from the group consisting of: β -1,3-Galactosyltransferase, N-acetylglucosamine β-1,3-galactosyltransferase, β-1,4-galactosyltransferase, N-acetylglucosamine β-1 ,4-galactosyltransferase, α-1,3-galactosyltransferase and α-1,4-galactosyltransferase, - preferably, the glucosyltransferase is selected from the group comprising the following list: α -glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase and β-1,4-glucosyltransferase, - preferably, the mannosyltransferase is selected from the enumeration including α-1,2-mannosyltransferase, α-1,3-mannosyltransferase and α-1,6-mannosyltransferase, - preferably, the N- Acetylglucosaminylase selected from the group consisting of the following enumeration: β-1,3-N-acetylglucosaminyltransferase and β-1,6-N-acetylglucosaminyltransferase, - Preferably, the N-acetylgalactosamine transferase is α-1,3-N-acetylgalactosamine transferase. The method of claim 24, wherein the cell is modified in the expression or activity of the other glycosyltransferase. The method of any one of the preceding claims, wherein the cell uses one or more precursors for producing the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end, the precursor being supplied to the cell from a culture medium. The method of any one of the preceding claims, wherein the cell produces one or more precursors for producing the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end. The method of any one of claims 26 or 27, wherein the precursor used to generate the disaccharide or oligosaccharide is completely converted to a disaccharide or oligosaccharide having a GlcNAc unit at the reducing end. The method of any one of the preceding claims, wherein the cell produces intracellularly a disaccharide or oligosaccharide having a GlcNAc unit at the reducing end, and wherein part or substantially all of the reducing end produced has a GlcNAc unit The disaccharide or oligosaccharide remains within the cell and/or is excreted from the cell via passive or active transport. The method of any one of the preceding claims, wherein the cell expresses a membrane transporter or a polypeptide having transport activity to transport compounds across the outer membrane of the cell wall, Preferably, the cell is modified in the expression or activity of the membrane transporter or polypeptide having transport activity. The method of claim 30, wherein the membrane transporter or polypeptide having transport activity is selected from the group consisting of: transporter, P-P-bond-hydrolysis-driven transporter, β-barrel porin, cotransporter , putative transporters and phosphotransfer-driver translocators, Preferably, the transporter includes MFS transporter, sugar excretion transporter and chelating ferritin exporter, Preferably, the P-P-bond-hydrolysis-driven transporter includes ABC transporter and chelatin exporter. The method of any one of claims 30 or 31, wherein the membrane transporter or polypeptide with transport activity controls the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end and/or one or more precursors and/or The flow of acceptors on the outer membrane of the cell wall, the one or more precursors and/or acceptors are used to generate disaccharides or oligosaccharides with GlcNAc units at the reducing end. The method of any one of claims 30 to 32, wherein the membrane transporter or polypeptide with transport activity provides improved production of disaccharides or oligosaccharides having a GlcNAc unit at the reducing end and/or allows for excretion and/or promote excretion. The method of any one of claims 6 to 33, wherein the cell comprises a modification for reducing acetate production compared to an unmodified precursor cell. The method of claim 34, wherein the cell comprises a lower or reduced expression and/or ablated, impaired, reduced or delayed activity of any one or more proteins compared to unmodified precursor cells, the protein Contains: Beta-Galactosidase, Galactoside O-Acetyl Transferase, N-Acetyl Glucosamine-6-Phosphate Deacetylase, Glucosamine-6-Phosphate Deaminase, N-Acetyl Glucosamine arrestin, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undec isoprene-phosphoglucose-1-phosphotransferase, L-fucose kinase, L-fucose Isomerase, N-acetylneuraminic acid dissociating enzyme, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridine transferase, glucose-1-phosphate adenosyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isoenzyme 1. ATP-dependent 6-phosphofructokinase isoenzyme 2, glucose-6-phosphate isomerase, aerobic respiration regulatory protein, transcriptional repressor protein IclR, lon protease, glucose-specific translocation phosphotransferase IIBC Component ptsG, glucose specific translocation phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIA ^Glc , β-glucoside specific PTS enzyme II, fructose specific PTS polyphosphoryl transfer protein FruA and FruB, alcohol dehydrogenase Aldehyde dehydrogenase, pyruvate-formate dissociating enzyme, acetate kinase, phosphoacetyltransferase, phosphoacetyltransferase, pyruvate decarboxylase. The method of any one of the preceding claims, wherein the cell is capable of producing phosphoenolpyruvate (PEP). The method of any one of claims 6 to 36, wherein the cell has a modification for promoting production and/or supply of phosphoenolpyruvate (PEP) compared to an unmodified precursor cell. The method of any one of claims 6 to 37, wherein the cell comprises an at least partially inactivated catabolic pathway of the selected monosaccharide, disaccharide or oligosaccharide involved in the reduction Required for the production of disaccharides or oligosaccharides with GlcNAc units at the end and/or for the production of disaccharides or oligosaccharides with GlcNAc units at the reducing end. The method of any one of the preceding claims, wherein the cell is resistant to lactose killing when the cell is grown in an environment in which lactose is combined with one or more other carbon sources. The method of any one of the preceding claims, wherein the cell produces 90 g/L or more of a disaccharide or oligosaccharide with GlcNAc at the reducing end in the whole culture broth and/or supernatant , and/or wherein the disaccharide or oligosaccharide having GlcNAc at this reducing end in the whole culture fluid and/or supernatant has a purity of at least 80%, which are respectively in the whole culture fluid and/or supernatant with this The total amount of disaccharides or oligosaccharides with GlcNAc units at the reducing end and their precursors. The method of any one of the preceding claims, wherein the cells are stably cultured in culture medium. The method of any one of the preceding claims, wherein the condition comprises: - the use of a medium comprising at least one precursor and/or acceptor for the production of the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end, and/or - Add at least one precursor and/or acceptor feedstock to the medium for the production of the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end. The method of any one of the preceding claims, comprising at least one of the following steps: vi) using a medium comprising at least one precursor and/or acceptor; vii) adding at least one medium to the medium in the reactor A precursor and/or recipient feedstock wherein the total reactor volume ranges from 250 mL (milliliters) to 10,000 ^m3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium does not exceed the Three times, preferably not more than twice, more preferably less than twice the volume of the medium before the precursor and/or recipient feedstock; viii) adding at least one precursor and/or recipient feedstock to the culture medium in the reactor, wherein The total reactor volume ranges from 250 mL (milliliters) to 10,000 ^m3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium does not exceed the medium prior to addition of the precursor and/or recipient feedstock three times the volume, preferably no more than twice, more preferably less than two times the volume, and preferably, the pH of the precursor and/or recipient feedstock is set between 3 and 7, and preferably, the precursor and/or the temperature of the recipient feedstock is maintained between 20°C and 80°C; ix) in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of feeding the solution Add at least one precursor and/or acceptor feedstock to the culture medium; x) add at least one precursor and/or in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of feeding solutions /or recipient feedstock to culture medium, and preferably, the pH of the feed solution is set between 3 and 7, and preferably, the temperature of the precursor and/or recipient feedstock is maintained between 20°C and 80°C Between ℃; This method produces the disaccharide or oligosaccharide with GlcNAc unit in this reducing end, its concentration in final culture is at least 50g/L, preferably at least 75g/L, more preferably at least 90g/L , more preferably at least 100g/L, more preferably at least 125g/L, more preferably at least 150g/L, more preferably at least 175g/L, more preferably at least 200g/L. The method of any one of claims 1 to 42, comprising at least one of the following steps: vi) using an initial reactor volume comprising at least 50 grams per liter, more preferably at least 75 grams, more preferably Medium of at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams of lactose, wherein the reactor volume ranges from 250 mL to 10,000 ^m3 (cubic meters); vii) containing at least 50 per liter of initial reactor volume grams, more preferably at least 75 grams, more preferably at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams of lactose lactose feedstock is added to the culture medium, wherein the reactor volume ranges from 250 mL to 10,000 m ³ ( cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium is no more than three times, preferably no more than twice, and more preferably less than twice the volume of the medium before adding the lactose feedstock; viii ) Lactose feedstock containing at least 50 grams, more preferably at least 75 grams, more preferably at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams of lactose per liter of initial reactor volume is added to the culture medium, wherein the reaction The volume of the vessel ranges from 250 mL to 10,000 ^m3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium does not exceed three times the volume of the medium prior to the addition of the lactose feedstock, preferably not more than twice, more preferably less than twice, and preferably, the pH of the lactose feedstock is set between 3 and 7, and preferably, the temperature of the lactose feedstock is maintained between 20°C and 80°C; ix) Lactose feedstock was added to the culture medium in a continuous manner during the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of feeding solution; x) At 1 day, 2 days by means of feeding solution In the course of days, 3 days, 4 days and 5 days, the lactose raw material is added to the culture medium in a continuous manner, wherein the concentration of the lactose feed solution is 50 g/L, preferably 75 g/L, more preferably 100 g/L. L, better is 125 g/L, better is 150 g/L, better is 175 g/L, better is 200 g/L, better is 225 g/L, better is 250 g/L, better 275 g/L, better 300 g/L, better 325 g/L, better 350 g/L, better 375 g/L, better 400 g/L, better is 450 g/L, more preferably 500 g/L, still more preferably 550 g/L, and most preferably 600 g/L; and wherein preferably, the pH of the feed solution is set between 3 and 7 , and preferably, the temperature of the precursor and/or the acceptor raw material is maintained between 20°C and 80°C; the method produces a disaccharide or oligosaccharide with a GlcNAc unit at the reducing end, which is in the final culture is 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. The method of claim 44, wherein the lactose feedstock is prepared by adding lactose at a concentration of at least 5 mM, preferably 30, 40, 50, 60, 70, 80, 90, 100, 150 mM from the beginning of the culture, more This is preferably done with the addition of lactose at concentrations >300 mM. The method of any one of claims 44 or 45, wherein the lactose feedstock is accomplished by adding lactose to the culture at a concentration such that at least 5 mM is obtained throughout the production phase of the culture, preferably Lactose concentration of 10 mM or 30 mM. The method of any one of the preceding claims, wherein the cells are cultured for at least about 60, 80, 100, or about 120 hours or in a continuous manner. The method of any one of the preceding claims, wherein the medium comprises at least one precursor selected from the group consisting of lactose, galactose, fucose and sialic acid. The method of any one of the preceding claims, wherein a first stage of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the medium comprising the precursor, followed by a second stage, Only the carbonaceous substrate, preferably glucose or sucrose, is added to the medium. The method of any one of claims 1 to 49, wherein a first stage of exponential cell growth is provided by adding a carbonaceous substrate, preferably glucose or sucrose, to the medium comprising the precursor, followed by a A second stage in which a carbon-based substrate, preferably glucose or sucrose, and a precursor are added to the medium. The method of any one of the preceding claims, wherein the cell produces a mixture comprising at least one charged disaccharide or oligosaccharide having a GlcNAc unit at the reducing end, preferably a sialylated disaccharide and/or Or oligosaccharides and/or neutral disaccharides and/or oligosaccharides. A method as claimed in any one of the preceding claims, wherein the cell produces a mixture comprising charged, preferably sialylated and/or neutral oligosaccharides comprising GlcNAc units at least at the reducing end Oligosaccharides. The method of any one of the preceding claims, wherein the oligosaccharide is selected from the list comprising: 2-fucosyllacto-N-disaccharide, 4-fucosyllacto-N-disaccharide, 2-4-Difucosyllacto-N-disaccharide, 3'-Sialylolide-N-disaccharide, 6'-Sialylolide-N-disaccharide, 3',6'-disialolactoide -N-disaccharide, 6,6'-disialo-N-disaccharide, 2'-fucosyl-3'-sialo-N-disaccharide, 2'-fucosyl-6 '-Sialyllacto-N-disaccharide, 4-fucosyl-3'-sialylacto-N-disaccharide, 4-fucosyl-6'-sialylacto-N-disaccharide, 2 - Fucosyl N-acetyllactosamine, 3'-fucosyl N-acetyllactosamine, 2,3'-difucosyl N-acetyllactosamine, 3'-sialic acid N- Acetylactosamine, 6'-Sialyl-N-Acetyl-Lactosamine, 3',6'-Disialo-N-Acetyl-Lactosamine, 6,6'-Disialo-N-Acetyl-Lactosamine, 2' - Fucosyl-3'-sialo-N-acetyllactosamine, 2'-fucosyl-6'-sialo-N-acetyllactosamine, 3-fucosyl-3'-sialic acid N-acetyllactosamine, 3'-fucosyl-6'-sialic acid N-acetyllactosamine, P1 trisaccharide (Gal-a1,4-Gal-b1,4-GlcNAc), allograft antigen Epitope (Gal-a1,3-Gal-b1,4-GlcNAc), Gal-b14-(Galb13)-GlcNAc, poly-N-acetyllactosamine, GalNAc-b1,3-Gal-b1,4-GlcNAc . The method of any one of claims 1 or 3 to 53, wherein the disaccharide having a GlcNAc unit at the reducing end does not include chitobiose (GlcNAc-GlcNAc). The method of any one of the preceding claims, wherein the oligosaccharide having a GlcNAc unit at the reducing end does not include chitobiose, preferably N-glycans, at the reducing end. A metabolically engineered cell for producing oligosaccharides or disaccharides with N-acetylglucosamine units at the reducing end, wherein the cell is capable of: (i) synthesizing nucleotide-sugar and monosaccharide N-acetylglucosamine (GlcNAc ), and (ii) expressing a glycosyltransferase to saccharify the GlcNAc monosaccharide to produce the disaccharide or oligosaccharide. A metabolically engineered cell for producing oligosaccharides having N-acetylglucosamine units at the reducing end, wherein the cell is capable of: (i) synthesizing nucleotide-sugar and monosaccharide N-acetylglucosamine (GlcNAc), and (ii) expressing a glycosyltransferase to saccharify the GlcNAc monosaccharide to produce the oligosaccharide. A metabolically engineered cell for producing a mixture comprising: (i) a disaccharide and/or oligosaccharide having an N-acetylglucosamine unit at the reducing end, and (ii) one or more lactose-based mammalian milk oligosaccharides sugars (MMOs), wherein the cell is capable of: (i) synthesizing the nucleotide-sugar and monosaccharide N-acetylglucosamine (GlcNAc), and (ii) expressing a glycosyltransferase to glycosylate the GlcNAc monosaccharide, producing A disaccharide or oligosaccharide having an N-acetylglucosamine unit at the reducing end. The cell of any one of claims 56 or 58, wherein the cell is metabolically engineered to produce a disaccharide or oligosaccharide having N-acetylglucosamine at the reducing end. The cell of claim 57, wherein the cell is metabolically engineered to produce an oligosaccharide having N-acetylglucosamine at the reducing end. The cell of any one of claims 56 to 60, wherein the cell is modified with one or more gene expression modules, characterized in that the expression of any of the expression modules is constitutive or produced by natural inducers . The cell of any one of claims 56 to 61, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein. The cell of any one of claims 56 to 62, wherein the cell expresses at least one N-acetylglucosamine-6-phosphotransferase and phosphatase to synthesize N-acetylglucosamine. The cell of any one of claims 56 to 63, wherein the cell expresses at least one glycosyltransferase to glycosylate N-acetylglucosamine. The cell of any one of claims 56 to 64, wherein the cell is modified in the expression or activity of an enzyme selected from the group consisting of N-acetylglucosamine-6-phosphotransferase, phosphate Enzymes, glycosyltransferases, L-glutamine-D-fructose-6-phosphate transaminase and UDP-glucose 4-epimerase. The cell of any one of claims 56 to 65, wherein the nucleotide-sugar is selected from the group comprising: 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-acetylamino- 2,6-Dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylfucosamine) -2,6-Dideoxy-L-galactose), UDP-N-Acetyl-L-Pneumolysamine (UDP-L-PneNAC or UDP-2-Acetylamino-2,6-Diesel Oxy-L-talose), UDP-N-Acetylmuramic acid, UDP-N-Acetyl-L-Quinosamine (UDP-L-QuiNAc or UDP-2-Acetylamino-2 ,6-dideoxy-L-glucose), GDP-L-isorhamnose, CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc ), CMP-Neu4Ac, CMP-Neu5Ac9N ₃ , CMP-Neu4,5Ac ₂ , CMP-Neu5,7Ac ₂ , CMP-Neu5,9Ac ₂ , CMP-Neu5,7(8,9)Ac ₂ , UDP-glucuronic acid Salt, UDP-galacturonate, GDP-rhamnose and UDP-xylose. The cell of any one of claims 56 to 66, wherein the nucleotide-sugar is UDP-galactose, and the glycosyltransferase is N-acetylglucosamine b-1,3-galactosyl transferase or N-acetylglucosamine b-1,4-galactosyltransferase. The cell of any one of claims 56 to 67, wherein the oligosaccharide has lacto-N-disaccharide (Gal-b1,3-GlcNAc) or N-acetyllactosamine (Gal-b1,3-GlcNAc) at the reducing end 4-GlcNAc). The cell of any one of claims 56 to 68, wherein the N-acetylglucosamine b-1,3-galactosyltransferase is a glycosyltransferase having: c. a PFAM domain PF00535, and viii. comprising the sequence of SEQ ID NO 01 [AGPS]XXLN(X _n )RXDXD, wherein X is any amino acid, wherein n is 12 to 17, or ix. comprising the sequence of SEQ ID NO 02 PXXLN(X _n )RXDXD (X _m )[FWY]XX[HKR]XX[NQST], wherein X is any amino acid, wherein n is 12 to 17 and m is 100 to 115, or x. including according to SEQ ID NOs: 03, 04, Any of 05, 06, 07 or 08, preferably any of SEQ ID NOs 03, 04, 05, 06 or 07, more preferably any of SEQ ID NOs 03, 06 or 07, the best is the polypeptide sequence of any one of SEQ ID NOs 03 or 06, or xi. is any one of SEQ ID NOs: 03, 04, 05, 06, 07 or 08, preferably SEQ ID NOs 03, 04, 05 Any of , 06 or 07, more preferably any of SEQ ID NO 03, 06 or 07, most preferably a functional homologue, variant or derivative of any of SEQ ID NO 03 or 06 , which has any of SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably any of SEQ ID NO 03, 04, 05, 06 or 07, more preferably SEQ ID NO Any of 03, 06 or 07, preferably at least 80% of the overall sequence of the full length of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide of any of SEQ ID NOs 03 or 06 is identical and has N-acetylglucosamine b-1,3-galactosyltransferase activity, or xii. includes any one from SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably is any of SEQ ID NOs 03, 04, 05, 06 or 07, more preferably any of SEQ ID NOs 03, 06 or 07, and most preferably at least 8 of any of SEQ ID NOs 03 or 06 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 oligopeptide sequences of consecutive amino acid residues with N-acetylglucosamine b-1,3- Galactosyltransferase activity, or xiii. is any of SEQ ID NOs: 03, 04, 05, 06, 07 or 08, preferably SEQ ID NOs 03, 04, 05, 06 Or any of 07, more preferably any of SEQ ID NO 03, 06 or 07, and most preferably a functional fragment of any of SEQ ID NO 03 or 06, and has N-acetylglucosamine b- 1,3-Galactosyltransferase activity, or xiv. comprising a polypeptide comprising or consisting of any of SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably SEQ ID NO Any of 03, 04, 05, 06 or 07, more preferably any of SEQ ID NO 03, 06 or 07, and most preferably of the full-length amino acid sequence of any of SEQ ID NO 03 or 06 consisting of amino acid sequences of at least 80% sequence identity and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or d. PFAM domain IPR002659, and vii. including SEQ ID NO 09 The sequence of KT(X _n )[FY]XXKXDXD(X _m )[FHY]XXG(X,without A,G,S)(X _p )X(without F,H,W,Y)[DE]D[ILV ]XX[AG], wherein X is any amino acid, wherein n is 13 to 16, m is 35 to 70, and p is 20 to 45, or viii. Included in SEQ ID NO: 10, 11, 12, or 13 The polypeptide sequence of any of , or ix. is a functional homologue, variant, or derivative of any of SEQ ID NO: 10, 11, 12, or 13, which has the same properties as SEQ ID NO: 10, 11, At least 80% overall sequence identity of any of the full-length N-acetylglucosamine b-1,3-galactosyltransferase polypeptides of 12 or 13 and having N-acetylglucosamine b-1,3 - Galactosyltransferase activity, or x. Comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, An oligopeptide sequence of 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b-1,3-galactosyltransferase activity, or xi. are SEQ ID NOs: 10, 11, A functional fragment of any one of 12 or 13, and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or xii. comprising a polypeptide comprising or consisting of a , 11, 12, or 13, consisting of amino acid sequences of at least 80% sequence identity to the full-length amino acid sequence of any one of 11, 12, or 13, and having N-acetylglucosamine b-1,3-galactosyltransferase active. The cell of any one of claims 56 to 69, wherein the N-acetylglucosamine b-1,4-galactosyltransferase is a glycosyltransferase having: a. a PFAM domain PF01755, and vii. The sequence EXXCXXSHXX[ILV][FWY]( _Xn )EDD( _Xm )[ACGST]XXYX[ILMV] comprising SEQ ID NO 14, wherein X is any amino acid, wherein n is 13 to 15 and m is 50 to 76, or viii. including according to any of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NO: 15, 16, 17, 18, Any one of 20 or 21, more preferably the polypeptide sequence of any one of SEQ ID NO: 17, 18, 20 or 21, or ix. is SEQ ID NO: 15, 16, 17, 18, 19, 20, Any of 21, 22 or 23, preferably any of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably any of SEQ ID NO: 17, 18, 20 or 21 A functional homologue, variant or derivative of 1, which has any of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NO: Any one of 15, 16, 17, 18, 20 or 21, more preferably the N-acetylglucosamine b-1,4-galactose of any one of SEQ ID NO: 17, 18, 20 or 21 At least 80% overall sequence identity to the full length of the syltransferase polypeptide and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or x. Including from SEQ ID NOs: 15, 16, 17, 18 , 19, 20, 21, 22 or 23, preferably any of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably SEQ ID NO: 17, 18, 20 Or an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 contiguous amino acid residues of any of 21 with N-ethyl Glucosamine b-1,4-galactosyltransferase activity, or xi. is any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NO: 1 Any of ID NO: 15, 16, 17, 18, 20 or 21, more preferably a functional fragment of any of SEQ ID NO: 17, 18, 20 or 21, and having N-acetylglucosamine b -1,4-galactosyltransferase activity, or xii. including polypeptides, The polypeptide comprises or consists of any of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably SEQ ID NO: 15, 16, 17, 18, 20 or Any of 21, more preferably an amino acid sequence of at least 80% sequence identity to the full-length amino acid sequence of any of SEQ ID NO: 17, 18, 20 or 21, and having N-ethyl Glucosamine b-1,4-galactosyltransferase activity, or b. PFAM domain PF00535, and viii. the sequence comprising SEQ ID NO 24 R[KN]XXXXXXXXGXXXX[FL]XDXD( _Xn )[FHW]XXX [FHNY](X _m )E[DE], wherein X is any amino acid, wherein n is 50 to 75, m is 10 to 30, or ix. The sequence comprising SEQ ID NO 25 R[KN]XXXXXXXGXXXXFXDXD(X _n )[FHW]XXX[FHNY](X _m )E[DE](X _p )[FWY]XX[HKR]XX[NQST], where X is any amino acid, where n is 50 to 75 and m is 10 to 30, and p is 20 to 25, or x. comprises a polypeptide sequence according to any of SEQ ID NOs: 26, 27, or 28, or xi. is any of SEQ ID NOs: 26, 27, or 28 A functional homologue, variant or derivative having the same full length as any of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptides of SEQ ID NO: 26, 27 or 28 at least 80% overall sequence identity with N-acetylglucosamine b-1,4-galactosyltransferase activity, or xii. including at least one from any of SEQ ID NOs: 26, 27, or 28 Oligopeptide sequences of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues with N-acetylglucosamine b-1,4 - galactosyltransferase activity, or xiii. is a functional fragment of any one of SEQ ID NOs: 26, 27 or 28 and has N-acetylglucosamine b-1,4-galactosyltransferase activity, xiv. Include 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 of SEQ ID NOs: 26, 27 or 28, and having N- Acetylglucosamine b-1,4-galactosyltransferase activity, or c. PFAM domain PF02709 without PFAM domain PF00535, and viii. including SEQ The sequence of ID NO 29 [FWY]XX[FY][FWY]( _X23 )[FWY][GQ]X[DE]D, where X is any amino acid, or ix. including the sequence of SEQ ID NO 30 [ PV]W[GHNP]( _Xn )[FWY][GQ]X[DE]D, where X is any amino acid, where n is 21 to 24, or x. Including SEQ ID NOs: 31, 32, 33 The polypeptide sequence of any of , 34, 35, or 36, or xi. is a functional homologue, variant, or derivative of any of SEQ ID NOs: 31, 32, 33, 34, 35, or 36, It has at least 80% overall sequence identity to any of the full length of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide of SEQ ID NO: 31, 32, 33, 34, 35 or 36 and has N-acetylglucosamine b-1,4-galactosyltransferase activity, or xii. Comprising at least 8 from any of SEQ ID NOs: 31, 32, 33, 34, 35, or 36 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 oligopeptide sequences of consecutive amino acid residues with N-acetylglucosamine b-1,4- Galactosyltransferase activity, or xiii. is a functional fragment of any one of SEQ ID NOs: 31, 32, 33, 34, 35, or 36, and has N-acetylglucosamine b-1,4-galactose Syltransferase activity, or xiv. comprising a polypeptide comprising or consisting of at least 80% sequence identity to the full-length amino acid sequence of any of SEQ ID NOs: 31, 32, 33, 34, 35, or 36 consisting of the amino acid sequence of , and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or d. PFAM domain PF03808, and viii. comprising the sequence of SEQ ID NO 37 [ST][ FHY]XN( _Xn )DG(X16)[HKR]X[ST] _FDXX [ST]XA, wherein X is any amino acid, and wherein n is 20 to 25, or ix. including SEQ ID NO 38 The sequence [ST][FHY]XN( _Xn )DG(X16)[HKR]X[ST] _FDXX [ST]XA( _Xm )[HR]XG[FWY](Xp) _GXGXXXQ [DE], where X is any amino acid, wherein n is 20 to 25, m is 40 to 50, and p is 22 to 30, or x. including according to SEQ ID NO: 39, 40 or 41 The polypeptide sequence of any, or xi. is a functional homologue, variant or derivative of any of SEQ ID NO: 39, 40 or 41 having the same as SEQ ID NO: 39, 40 or 41 At least 80% overall sequence identity of any of the full-length N-acetylglucosamine b-1,4-galactosyltransferase polypeptides with N-acetylglucosamine b-1,4-galactosyltransferase Enzymatic activity, or xii. comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive from any of SEQ ID NOs: 39, 40, or 41 An oligopeptide sequence of amino acid residues with N-acetylglucosamine b-1,4-galactosyltransferase activity, or xiii. is the function of any of SEQ ID NOs: 39, 40, or 41 fragment and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or xiv. comprising a polypeptide comprising or consisting of a The full-length amino acid sequence consists of amino acid sequences with at least 80% sequence identity and has N-acetylglucosamine b-1,4-galactosyltransferase activity. The cell of any one of claims 56 to 70, wherein c. The N-acetylglucosamine-6-phosphotransferase is a polypeptide sequence comprising the polypeptide of UniProt ID P43577, or is a functional homologue, variant or derivative of the polypeptide of UniProt ID P43577, which has the same The full-length polypeptide of ID P43577 has at least 80% overall sequence identity and has N-acetylglucosamine-6-phosphotransferase activity, and d. The L-glutamine-D-fructose-6-phosphate transaminase is a polypeptide sequence comprising the polypeptide of UniProt ID P17169, or a functional homologue, variant or derivative of the polypeptide of UniProt ID P17169, It has at least 80% overall sequence identity with the full-length polypeptide of UniProt ID P17169, and has L-glutamine-D-fructose-6-phosphate transaminase activity; or is A39T, R250C different from the UniProt ID P17169 polypeptide and a modified version of the G472S mutation. The cell of any one of claims 56 to 71, wherein the cell can metabolize a carbon source selected from the group consisting of glucose, fructose, galactose, lactose, sucrose, maltose, malt - Oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, mannose, methanol, ethanol, arabinose, trehalose, starch, cellulose, hemicellulose, corn infusion, high fructose syrup, molasses , glycerol, acetate, citrate, lactic acid, pyruvic acid. The cell of any one of claims 56 to 72, wherein the cell is unable to convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or is unable to convert glucosamine-6 - Phosphate conversion to fructose-6-phosphate. The cell of any one of claims 56 to 73, wherein the cell is modified to produce GDP-fucose. The cell of any one of claims 56 to 74, wherein the cell is modified to promote GDP-fucose production, and the modification is selected from the group comprising: UDP-glucose:undecylisoprene Shaving of the gene encoding ene-phosphoglucose-1-phosphotransferase, overexpression of the gene encoding GDP-L-fucose synthase, overexpression of the gene encoding GDP-mannose 4,6-dehydratase, mannose- Overexpression of a gene encoding 1-phosphate guanylate transferase, overexpression of a gene encoding phosphomannose mutase, or overexpression of a gene encoding mannose-6-phosphate isomerase. The cell of any one of claims 56 to 75, wherein the cell is modified to produce UDP-galactose. The cell of any one of claims 56 to 76, wherein the cell is modified to promote UDP-galactose production, and the modification is selected from the group comprising: 5'-nucleotidase/UDP- Shaving of the gene encoding glycohydrolase or shaving of the gene encoding galactose-1-phosphate uridine transferase. The cell of any one of claims 56 to 75, wherein the cell is modified to produce CMP-N-acetylneuraminic acid. The cell of any one of claims 56 to 78, wherein the cell is modified to promote CMP-N-acetylneuraminic acid production, and the modification is selected from the group comprising: CMP-sialic acid Overexpression of the gene encoding synthetase, overexpression of the gene encoding sialic acid synthase, overexpression of the gene encoding N-acetyl-D-glucosamine 2-epimerase. The cell of any one of claims 56 to 79, wherein the cell is capable of expressing at least one other glycosyltransferase, and the other glycosyltransferase is selected from the group comprising: fucosyltransferase, Sialyltransferase, Galactosyltransferase, Glucosyltransferase, Mannosyltransferase, N-Acetylglucosaminyltransferase, N-Acetylgalactosyltransferase, N-Acetylmannan Glycosyltransferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosamine transferase, N-glycolyl neuraminotransferase, rhamnosyltransferase, N-ethyl Acylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP-N-acetylglucosamine Enolacetonyltransferase and Fucosaminotransferase, - Preferably, the fucosyltransferase is selected from the group consisting of: α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,4- Fucosyltransferase and α-1,6-fucosyltransferase, - Preferably, the sialyltransferase is selected from the group consisting of: α-2,3-sialyltransferase, α-2,6-sialyltransferase and α-2,8-sialyltransferase, - Preferably, the galactosyltransferase is selected from the group consisting of: β-1,3-galactosyltransferase, N-acetylglucosamine β-1,3-galactosyltransferase, β-1 ,4-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, α-1,3-galactosyltransferase and α-1,4-galactosyltransferase , - Preferably, the glucosyltransferase is selected from the group consisting of: α-glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase and β-1,4 -glucosyltransferase, - Preferably, the mannosyltransferase is selected from the group consisting of: α-1,2-mannosyltransferase, α-1,3-mannosyltransferase and α-1,6-mannosyltransferase transferase, - Preferably, the N-acetylglucosaminyltransferase is selected from the group consisting of: β-1,3-N-acetylglucosaminyltransferase and β-1,6-N-acetylglucosaminyltransferase glucosamine transferase, - Preferably, the N-acetylgalactosamine transferase is α-1,3-N-acetylgalactosamine transferase. The cell of claim 80, wherein the cell is modified in the expression or activity of the other glycosyltransferase. The cell of any one of claims 56 to 81, wherein the cell uses one or more precursors for the production of the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end, the precursor being supplied to the cell from a culture medium . The cell of any one of claims 56 to 82, wherein the cell produces one or more precursors for producing the disaccharide or oligosaccharide having a GlcNAc unit at the reducing end. The cell of any one of claims 82 or 83, wherein the precursor for producing the disaccharide or oligosaccharide is completely converted to a disaccharide or oligosaccharide having a GlcNAc unit at the reducing end. The cell of any one of claims 56 to 84, wherein the cell produces intracellularly a disaccharide or oligosaccharide having a GlcNAc unit at the reducing end, and wherein part or substantially all of the produced reducing end has a GlcNAc unit The disaccharides or oligosaccharides remain intracellular and/or are excreted via passive or active transport. The cell of any one of claims 56 to 85, wherein the cell expresses a membrane transporter or a polypeptide having transport activity to transport compounds across the outer membrane of the cell wall, Preferably, the cell is modified in the expression or activity of the membrane transporter or polypeptide having transport activity. The cell of claim 86, wherein the membrane transporter or polypeptide having transport activity is selected from the group consisting of transporters, P-P-bond-hydrolysis-driven transporters, β-barrel porins, cotransporters , putative transporters and phosphotransfer-driver translocators, Preferably, the transporter includes MFS transporter, sugar excretion transporter and chelating ferritin exporter, Preferably, the P-P-bond-hydrolysis-driven transporter includes ABC transporter and chelatin exporter. The cell according to any one of claims 86 or 87, wherein the membrane transporter or polypeptide with transport activity controls a disaccharide or oligosaccharide having a GlcNAc unit at the reducing end and/or one or more precursors and/or The flow of acceptors on the outer membrane of the cell wall, the one or more precursors and/or acceptors are used to generate disaccharides or oligosaccharides with GlcNAc units at the reducing end. The cell of any one of claims 86 to 88, wherein the membrane transporter or polypeptide with transport activity provides improved production of disaccharides or oligosaccharides having a GlcNAc unit at the reducing end and/or allows excretion and/or promote excretion. The cell of any one of claims 56 to 89, wherein the cell comprises a modification for reducing acetate production compared to an unmodified precursor cell. The cell of claim 90, wherein the cell comprises a lower or reduced expression and/or eliminated, impaired, reduced or delayed activity of any one or more proteins compared to unmodified precursor cells, the protein Contains: Beta-Galactosidase, Galactoside O-Acetyl Transferase, N-Acetyl Glucosamine-6-Phosphate Deacetylase, Glucosamine-6-Phosphate Deaminase, N-Acetyl Glucosamine arrestin, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undec isoprene-phosphoglucose-1-phosphotransferase, L-fucose kinase, L-fucose Isomerase, N-acetylneuraminic acid dissociating enzyme, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridine transferase, glucose-1-phosphate adenosyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isoenzyme 1. ATP-dependent 6-phosphofructokinase isoenzyme 2, glucose-6-phosphate isomerase, aerobic respiration regulatory protein, transcriptional repressor protein IclR, lon protease, glucose-specific translocation phosphotransferase IIBC Component ptsG, glucose specific translocation phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIA ^Glc , β-glucoside specific PTS enzyme II, fructose specific PTS polyphosphoryl transfer protein FruA and FruB, alcohol dehydrogenase Aldehyde dehydrogenase, pyruvate-formate dissociating enzyme, acetate kinase, phosphoacetyltransferase, phosphoacetyltransferase, pyruvate decarboxylase. The cell of any one of claims 56 to 91, wherein the cell is capable of producing phosphoenolpyruvate (PEP). The cell of any one of claims 56 to 92, wherein the cell has a modification for promoting production and/or supply of phosphoenolpyruvate (PEP) compared to an unmodified precursor cell. The cell of any one of claims 56 to 93, wherein the cell comprises an at least partially inactivated catabolic pathway of the selected monosaccharide, disaccharide or oligosaccharide involved in the reduction Required for the production of disaccharides or oligosaccharides with GlcNAc units at the end and/or for the production of disaccharides or oligosaccharides with GlcNAc units at the reducing end. The cell of any one of claims 56 to 94, wherein the cell is resistant to lactose killing when the cell is grown in an environment where lactose is combined with one or more other carbon sources. The cell of any one of claims 56 to 95, wherein the cell produces 90 g/L or more of the reducing end disaccharide with GlcNAc or Oligosaccharides, and/or disaccharides or oligosaccharides having GlcNAc at the reducing end in the whole culture fluid and/or supernatant, have a purity of at least 80%, respectively, in the whole culture fluid and/or supernatant Based on the total amount of disaccharides or oligosaccharides with GlcNAc units at the reducing end and their precursors. The cell of any one of claims 56 to 96, wherein the cell produces a mixture comprising at least one charged, preferably sialylated disaccharide comprising at least one disaccharide or oligosaccharide having a GlcNAc unit at the reducing end and/or oligosaccharides and/or neutral disaccharides and/or oligosaccharides. A cell as claimed in any one of claims 56 to 97, wherein the cell produces a mixture comprising charged, preferably sialylated oligosaccharides and/or neutral oligosaccharides comprising GlcNAc units at least at the reducing end Sexual oligosaccharides. The cell of any one of claims 56 to 98, wherein the oligosaccharide is selected from the list comprising: 2-fucosyllacto-N-bisaccharide, 4-fucosyllacto-N-bisaccharide Sugar, 2-4-Difucosyllacto-N-disaccharide, 3'-Sialyllacto-N-disaccharide, 6'-Sialyllacto-N-disaccharide, 3',6'-Disialo Yogurt-N-disaccharide, 6,6'-disialo-N-disaccharide, 2'-fucosyl-3'-sialoglyco-N-disaccharide, 2'-fucosyl -6'-Sialylacto-N-disaccharide, 4-fucosyl-3'-sialylo-N-disaccharide, 4-fucosyl-6'-sialylo-N-disaccharide , 2-fucosyl N-acetyllactosamine, 3'-fucosyl N-acetyllactosamine, 2,3'-difucosyl N-acetyllactosamine, 3'-sialic acid N-acetyllactosamine, 6'-sialo-N-acetyllactosamine, 3',6'-disialo-N-acetyllactosamine, 6,6'-disialo-N-acetyllactosamine, 2'-fucosyl-3'-sialo-N-acetyllactosamine, 2'-fucosyl-6'-sialo-N-acetyllactosamine, 3-fucosyl-3'- Sialyl N-acetyllactosamine, 3'-fucosyl-6'-sialic acid N-acetyllactosamine, P1 trisaccharide (Gal-a1,4-Gal-b1,4-GlcNAc), heterologous Graft epitopes (Gal-a1,3-Gal-b1,4-GlcNAc), Gal-b14-(Galb13)-GlcNAc, poly-N-acetyllactosamine, GalNAc-b1,3-Gal-b1,4 -GlcNAc. The cell of any one of claims 56 or 58 to 99, wherein the disaccharide having a GlcNAc unit at the reducing end does not include chitobiose (GlcNAc-GlcNAc). The cell of any one of claims 56 to 100, wherein the oligosaccharide having a GlcNAc unit at the reducing end does not include chitobiose, preferably N-glycans, at the reducing end. The cell of any one of claims 56 to 101 or the method of any one of claims 1 to 55, wherein the cell is a bacterium, fungus, yeast, plant cell, animal cell or protozoan cell , - Preferably, the bacteria is Escherichia coli strain, more preferably Escherichia coli strain K12 strain, and more preferably, the Escherichia coli strain K12 strain is Escherichia coli MG1655, - preferably, the fungus belongs to a genus selected from the group consisting of Black mold, Reticulomyces, Penicillium, White mold or Koji mold, - Preferably, the yeast belongs to the genus including Saccharomyces cerevisiae, Zygomyces spp., Pichia spp., Kermagtella spp., Hansenula spp., Ascomyces spp., Bacteroides spp., a genus of the group Kluyveromyces or Debaryomyces, - preferably, the plant cells are algal cells or derived from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soybean, corn or cereal plants, - Preferably, the animal cells are derived from non-human mammals, birds, fish, invertebrates, reptiles, amphibians or insects, or are genetically modified cell lines derived from human cells excluding embryonic stem cells, more preferably Typically, the human and non-human mammalian cells are epithelial cells, embryonic kidney cells, fibroblasts, COS cells, Chinese hamster ovary (CHO) cells, murine myeloma cells, NIH-3T3 cells, non-mammary adult stem cells, or the like. Derivatives, more preferably, the insect cells are derived from Fall Armyworm, Silkworm, Spodoptera cabbage, Trichoderma or Drosophila melanogaster, - Preferably, the protozoan cells are tarantula Leishmania cells. The cell of claim 102 or the method of claim 102, wherein the cell is a live Gram-negative bacterium comprising a reduction or elimination of poly-N-acetylene compared to unmodified precursor cells - Glucosamine (PNAG), Enterobacterial Common Antigen (ECA), Cellulose, Koramic Acid, Core Oligosaccharide, Osmotic Pressure Regulating Interstitial Glucan (OPG), Glucosylglycerol, Polysaccharide and/or Trehalose synthesis. The method of any one of claims 1 to 55, 102, or 103, wherein the separation comprises at least one of the following: clarification, ultrafiltration, nanofiltration, two-phase partition, reverse osmosis, microfiltration, activated carbon or carbon treatment, nonionic surfactant treatment, enzymatic digestion, tangential flow high performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, Body exchange chromatography. The method of any one of claims 1 to 55, 102 to 104, further comprising purifying the disaccharide or oligosaccharide from the cell. The method of any one of claims 1 to 55, 102 to 105, wherein the purification comprises at least one of the following steps: use of activated carbon or carbon, use of carbon, nanofiltration, ultrafiltration, electrophoresis, enzymes Treatment or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying, freeze drying, spray freeze drying, freeze spray drying, belt drying, conveyor belt drying, vacuum belt drying Drying, vacuum conveyor drying, tumble drying, tumble drying, vacuum tumble drying or vacuum tumble drying. A cell according to any one of claims 56 to 103, or a method according to any one of claims 1 to 55, 102 to 106, for the production of a bicarbonate having an N-acetylglucosamine unit at the reducing end Use of sugars or oligosaccharides. A cell according to any one of claims 56, 58 to 103, or a method according to any one of claims 1, 3 to 55, 102 to 106 for producing a reducing end with N-acetylglucose Oligosaccharides of amine units, preferably charged or neutral oligosaccharides for the production of N-acetylglucosamine units at the reducing end, more preferably for the production of sialylated or fucosylated forms of LNB or LacNAc. use.