TW202212574A - Production of oligosaccharide mixtures by 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 provides a method for the production of a mixture of at least two different oligosaccharides by a cell as well as the purification of at least one of said oligosaccharides from the cultivation. In addition, the present invention provides a method for the production of a mixture of at least two different oligosaccharides by a metabolically engineered cell as well as the purification of at least one of said oligosaccharides from the cultivation.

Description

Making Oligosaccharide Mixtures by Cells

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 provides a method for producing by cells a mixture of at least two different oligosaccharides and purifying at least one of the oligosaccharides from culture. Additionally, the present invention provides a method for producing a mixture of at least two different oligosaccharides by metabolically engineered cells and purifying at least one of the oligosaccharides from the culture.

Oligosaccharides, often present in sugar-bound forms to proteins and lipids, are involved in many biological phenomena such as differentiation, development and biorecognition processes associated with the initiation and progression of fertilization, embryogenesis, inflammation, cancer metastasis and host pathogen adhesion. Oligosaccharides can also be present in body fluids and human milk as unconjugated glycans, where oligosaccharides 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)). Due to the broad functional spectrum of oligosaccharides, there is great scientific and commercial interest in oligosaccharide mixtures. However, the availability of oligosaccharide mixtures is limited because production relies on chemical or chemoenzymatic synthesis or on purification from natural sources such as animal milk. Chemical synthesis methods are laborious and time-consuming, and these methods are difficult to scale up due to the large number of steps involved. Enzymatic routes using glycosyltransferases offer many advantages over chemical synthesis. Glycosyltransferases catalyze the transfer of sugar moieties from activated nucleotide-sugar donors to sugar or non-sugar acceptors (Coutinho et al., J. Mol. Biol. 328 (2003) 307-317). These glycosyltransferases are the source of the synthesis of oligosaccharides by biotechnologists and are used in (chemical) enzymatic pathways as well as in cell-based production systems. However, the stereospecificity and regioselectivity of glycosyltransferases remain a formidable challenge. Additionally, chemoenzymatic pathways require in situ regeneration of nucleotide-sugar donors. Cellular production of oligosaccharides requires tight control of the spatiotemporal availability of sufficient levels of nucleotide-sugar donors in the vicinity of complementary glycosyltransferases. Due to these difficulties, current methods often result in the synthesis of single oligosaccharides rather than mixtures of oligosaccharides.

An object of the present invention is to provide tools and methods by means of which oligosaccharide mixtures comprising at least two different oligosaccharides can be passed through cells in an efficient, time-efficient and cost-effective manner and if necessary in a continuous process, Preferably single cell manufacture.

According to the present invention, this and other objects are achieved by providing cells and methods for producing oligosaccharide mixtures comprising at least two different oligosaccharides, wherein the cells are metabolically engineered for producing the oligosaccharides.

Surprisingly, it has now been found that it is possible to produce oligosaccharide mixtures comprising at least two different oligosaccharides from a single cell. The present invention provides a method for producing an oligosaccharide mixture comprising at least two different oligosaccharides. The method comprises the steps of providing a cell expressing at least one glycosyltransferase and capable of synthesizing a nucleotide-sugar as a donor for the glycosyltransferase, wherein the cell and at least two are used in the manufacture of the oligosaccharide receptors, wherein either of the receptors is a disaccharide or an oligosaccharide. The present invention also provides methods of isolating at least one of the produced oligosaccharides from the oligosaccharide mixture. Furthermore, the present invention provides a cell metabolically engineered for the manufacture of an oligosaccharide mixture comprising at least two different oligosaccharides.

definition

The word group used in this specification to describe the present invention and its various embodiments should be understood not only in the sense of its commonly defined meaning, but also to include structures specifically defined in this specification outside the scope of the commonly defined meaning , material or effect. Thus, if an element can be understood in the context of this specification to include more than one meaning, its use within the scope of the claim must be understood to mean from the ordinary to all possible meanings supported by this specification and the word group itself.

Various implementations and aspects of implementations of the invention disclosed herein are to be construed not only in the order and circumstances specifically described in this specification, but also in any order and any combination thereof. Whenever the situation requires, all words used in the singular will be considered 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 to which this invention belongs. In general, the nomenclature used herein and the experimental procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art, experiments in cell culture Procedures, Molecular Genetics, Organic Chemistry and Nucleic Acid Chemistry and Hybridization. Nucleic acid and peptide synthesis is performed using standard techniques. Generally, purification steps are carried out according to the manufacturer's instructions.

In this 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 not for the purpose of limiting the scope of the present invention, which is set forth in the following patent applications in the range. It must be understood that the illustrated embodiments have been presented for purposes of example only, and should not be construed as limiting the invention. It will be apparent to those skilled in the art that changes, other embodiments, improvements, details and uses can be made in accordance with the letter and spirit of the invention herein and within the scope of the invention, which is limited only by the scope of the patent application, according to Patent law including the doctrine of equivalents is explained. In the following claims, reference characters used to designate claims steps are provided for ease of description only, and such reference characters are not intended to imply any particular order for performing the steps.

In this document and the scope of its patent application, the verb "to comprise" and its inflections are used in their non-limiting sense, meaning to include items following the word group, but not to exclude items not specifically mentioned project. Additionally, the verb "consisting of" may be replaced by "consisting essentially of", meaning that a composition as defined herein may comprise additional component(s) in addition to the specifically identified component(s), the(s) The additional components do not alter the unique characteristics of the present invention. In addition, reference to an element by the indefinite article "a (a/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" usually means "at least one". Throughout this application, unless expressly stated otherwise, the articles "a and an" are preferably replaced by "at least two", more preferably "at least three" , even better by "at least four", even better by "at least five", even better by "at least six", and most Preferably replaced by "at least two".

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

According to the present invention, the term "polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA; DNA that is a mixture of single- and double-stranded regions or a mixture of single-, double- and triple-stranded regions; single- and double-stranded RNA; RNA that is a mixture of stranded and double-stranded regions; hybrid molecules comprising DNA and RNA that may be single-stranded or more commonly double-stranded or triple-stranded regions, or a mixture of single-stranded and double-stranded regions. Additionally, as used herein, the term "polynucleotide" refers to a three-stranded region comprising RNA or DNA or both RNA and DNA. Strands in such regions can be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more generally refer to regions having only some of the molecules. One of the molecules of the triple helix region is often an oligonucleotide. As used herein, the term "polynucleotide" also includes DNA or RNA as described above containing one or more modified bases. Thus, DNA or RNA having a backbone modified for stability or for other reasons is a "polynucleotide" according to the present invention. Furthermore, DNA or RNA comprising uncommon bases (such as inosine) or modified bases (such as tritylated bases) should be understood to be covered by the term "polynucleotide". It will be appreciated that various modifications have been made to DNA and RNA for many suitable purposes known to those of ordinary skill in the art. The term "polynucleotide" as used herein encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as DNA and RNA specific to viruses and cells, including, for example, simple and complex cells the chemical form. The term "polynucleotide" also encompasses short polynucleotides commonly referred to as oligonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to both short chains (often referred to as peptides, oligopeptides and oligomers) and long chains (often referred to as proteins). Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptide" includes polypeptides modified by natural processes, such as processing and other post-translational modifications, as well as by chemical modification techniques. Such modifications are well described in the basic text and in more detailed monographs, as well as in the extensive research literature, and are well known to those of ordinary skill in the art. Modifications of the same type may be present to the same or varying degrees at several sites in a given polypeptide. Furthermore, a given polypeptide may contain various types of modifications. Modifications can occur anywhere in a 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 blood matrix moieties, covalent attachment of nucleotides or nucleotide derivatives, lipids or Covalent attachment of lipid derivatives, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of pyroglutamic acid, methylation , γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid linkage , sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenylation, transfer RNA-mediated addition of amino acids to proteins (such as spermidine) and ubiquitination. Polypeptides may be branched or cyclic with or without branching. Cyclic, branched, and branched cyclic polypeptides can arise from natural post-translational processes, and can also be prepared by fully synthetic methods.

As used herein, the term "polynucleotide encoding a polypeptide" encompasses polynucleotides comprising sequences encoding the polypeptides of the present invention. The term also encompasses polynucleotides that include single contiguous or non-contiguous regions encoding polypeptides (eg interspersed with integrated phage or insert sequences or edits) and other regions that may also contain coding and/or non-coding sequences.

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

As used herein in reference to a cell or host cell, the terms "recombinant" or "transgenic" or "metabolically engineered" or "genetically modified" are interchangeable used, and instructs the cell to replicate a heterologous nucleic acid, or to exhibit a sequence from a heterologous nucleic acid (i.e., a sequence "foreign to said cell" or "foreign to said location or environment in the cell" location or environment in said cell)" sequence) encoded peptide or protein. Such cells are described as being transformed by, or by introducing at least one heterologous or exogenous gene. Metabolically engineered or recombinant or transgenic cells may contain genes not found in the native (non-recombinant) form of the cell. Recombinant cells may also contain the gene found in the cell in its native form, wherein the gene has been modified by artificial means and reintroduced into the cell. These terms also encompass cells containing nucleic acid endogenous to the cell that has been modified or whose expression or activity has been modified without removing the nucleic acid from the cell; such modifications include by gene replacement, promoter replacement ; site-specific mutations; and related art-obtained modifications. Accordingly, a "recombinant polypeptide" is a polypeptide produced by recombinant cells. As used herein, a "heterologous sequence" or "heterologous nucleic acid" is derived from a source foreign to a particular cell (eg, from a different species), or from the same source if from the same A sequence or nucleic acid that has been modified from its original form or position in the genome. Thus, a heterologous nucleic acid operably linked to a promoter is from a source different from the source from which the promoter was derived, or if from the same source, modified from its original form or location in the gene body. Heterologous sequences can be stably introduced into the genome of a host microbial cell, eg, by transfection, transformation, conjugation or transduction, wherein techniques are applied depending on the cell and the sequence to be introduced. Various techniques are known to those of ordinary skill in the art and are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The term "mutant" cell or microorganism as used in the context of the present invention refers to a genetically modified cell or microorganism.

In the context of the present invention, the term "endogenous" refers to anything that is a natural part of the cell and is present at its natural location in the cell's chromosomes and that behaves in contrast to the natural control mechanisms acting on its expression. Control unaltered polynucleotide, polypeptide or protein sequences. The term "exogenous" refers to any polynucleotide, polypeptide that is derived from the outside of the cell under study and not from a natural part of the cell or in its natural location in the cell's chromosome or plastid or protein sequence.

When used in reference to a polynucleotide, gene, nucleic acid, polypeptide or enzyme, the term "heterologous" refers to a polynucleotide derived from a source other than the host organism species or derived from a source other than the host organism species , gene, nucleic acid, polypeptide or enzyme. In contrast, the use of "homologous" polynucleotides, genes, nucleic acids, polypeptides or enzymes herein refers to polynucleotides, genes, nucleic acids, polypeptides or enzymes derived from a species of host organism. When referring to gene regulatory sequences or helper nucleic acid sequences (eg, promoters, 5' untranslated regions, 3' untranslated regions, poly A addition sequences, intron sequences, splice sites, ribosome binding sites, internal ribosome entry sequences, gene body homology regions, recombination sites, etc.), "heterologous" means that a regulatory or auxiliary sequence is not associated with the regulatory or auxiliary nucleic acid sequence in a construct, gene body , chromosomes or episomal bodies with which the genes are naturally associated. Accordingly, a promoter operably linked to a gene that is not operably linked to a promoter in its natural state (ie, in the form of the gene body of a non-genetically engineered organism) is referred to herein as a "heterologous promoter" )" even though the promoter may be derived from the same species (or in some cases, the same organism) as the gene to which it is linked.

The term "modified activity" of a protein or enzyme relates to a change in the activity of a protein or enzyme compared to the wild-type (ie, native) activity of the protein or enzyme. The modified activity can be the activity of the protein or enzyme that is eliminated, attenuated, reduced or delayed compared to the wild-type activity of the protein or enzyme, but can also be the activity of the protein or enzyme compared to the wild-type activity of the protein or enzyme. The accelerated or enhanced activity of an enzyme. The modified activity of a protein or enzyme is obtained by modified expression of the protein or enzyme or by the expression of a modified (ie, mutated) form of the protein or enzyme. The modified activity of the enzyme further relates to the modification of the apparent Michaelis constant Km and/or the apparent maximum velocity (Vmax) of the enzyme.

The term "modified expression" of a gene refers to the change in expression compared to the wild-type expression of the gene at any stage of the production process of the encoded protein. The modified expression is lower or higher than the wild-type expression, wherein in the case of an endogenous gene, the term "higher expression" is also defined as "overexpression" of that gene, Or in the case of a heterologous gene not present in the wild-type strain, it is defined as "expression". Lower expression or reduced expression is obtained by means of common well-known techniques of those of ordinary skill in the art (such as the use of siRNA, CrispR, CrispRi, riboswitches, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutated gene, knockout gene, transposon mutagenesis, ...), these techniques are used to render the gene less capable (i.e. statistically significantly "less capable" compared to a functional wild-type gene) or completely A gene cannot be altered in such a way that a functional end product is produced, such as by knocking out the gene. The term "riboswitch" as used herein is defined as a portion of a messenger RNA that folds into an intricate structure that blocks expression by interfering with translation. Binding of effector molecules induces conformational changes allowing post-transcriptional regulatory expression. The gene of interest is then altered in such a way that lower performance is obtained as described above, also by altering transcription units, promoters, untranslated regions, ribosome binding sites, Shine Dalgarno sequences or transcription terminators for lower performance. Lower expression or reduced expression can be achieved, for example, by mutating one or more base pairs in the promoter sequence or completely changing the promoter sequence to a persistent promoter with a lower expression strength compared to the wild type or An inducible promoter that causes regulated expression or a repressible promoter that causes regulated expression can be obtained. Overexpression or expression is obtained by means of common well-known techniques to those of ordinary skill in the art (such as the use of artificial transcription factors, redesign of promoter sequences, ribosome engineering, introduction or reintroduction of expression modules at eukaryotic , using high-copy-count plastids), in which the gene is part of an "expression cassette" related to the presence of promoter sequences, non-translated region sequences (containing ribosome-binding sequences, summer Indalgano or Kozak sequences), coding sequences, and optionally translation terminators, and any sequence that results in the expression of a functionally active protein. The manifestations are persistent or accommodative.

The term "constitutive expression" is defined as being free under certain growth conditions from transcription factors other than subunits of RNA polymerase (eg, bacterial delta factors such as ^σ70 , ^σ54 , or related σ factors; and Expression of the yeast mitochondrial RNA polymerase-specific factor (MTF1) regulation by co-association with the RNA polymerase core enzyme. Non-limiting examples of such transcription factors are CRP, LacI, ArcA, Cra, IclR in E. coli, or Aft2p, Crzlp, Skn7 in Saccharomyces cerevisiae , or B. subtilis DeoR, GntR, Fur. These transcription factors bind to specific sequences and can block or enhance performance under certain growth conditions. RNA polymerase is the catalytic mechanism for the synthesis of RNA from DNA templates. RNA polymerase binds specific sequences to initiate transcription, eg, via delta factor in prokaryotic hosts or via MTF1 in yeast. Persistent expression provides a constant amount of expression without the need for induction or inhibition.

The term "regulated expression" is defined as an expression regulated by transcription factors other than subunits of RNA polymerase (eg bacterial delta factor) under certain growth conditions. Examples of such transcription factors are described above. Usually performance modulation is obtained by means of an inducer or inhibitor such as (but not limited to) IPTG, arabinose, rhamnose, fucose, allo-lactose, or pH changes, or temperature changes, or carbon depletion or receptors or resulting products or chemical inhibition.

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

The term "inducible expression upon chemical treatment" is defined as a facultative or regulated expression of a gene that is expressed only upon treatment with a chemical inducer or inhibitor, wherein the inducer and inhibitor comprise ( but not limited to) alcohols (eg ethanol, methanol), carbohydrates (eg glucose, galactose, glycerol, lactose, arabinose, rhamnose, fucose, allolactose), metal ions (eg aluminium, copper, zinc) ), nitrogen, phosphate, IPTG, acetate, formate, xylene. The term "control sequence" refers to a sequence recognized by the cellular transcription and translation system that allows transcription and translation of a polynucleotide sequence into a polypeptide. Such DNA sequences are thus necessary for the expression of the operably linked coding sequence in a particular cell or organism. Such control sequences can be, but are not limited to, promoter sequences, ribosome binding sequences, Schyndalgarno sequences, Kozak sequences, transcription terminator sequences. Control sequences suitable for use in prokaryotes include, for example, promoters, optional operator sequences and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers. The DNA of a presequence or a secretory leader sequence is operably linked to the DNA of a polypeptide if it appears to be involved in the secretion of a pre-protein; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence or operably linked to a coding sequence if the ribosome binding site affects the transcription of the sequence; or operably linked to the coding sequence if the ribosome binding site is positioned so as to facilitate translation . These control sequences may additionally be via inducible promoters or via induction or repression by external chemicals such as, but not limited to, IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose The gene circuit controls the transcription or translation of the polynucleotide into a polypeptide.

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

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

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

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

Throughout this application, unless expressly stated otherwise, the expressions "capable of...<verb>" and "capable to...<verb>" are preferred is replaced by the active voice of the verb, and vice versa. For example, the expression "capable of expressing" is preferably replaced with "express", and vice versa, ie "capable of expressing" is preferably replaced with "capable of expressing".

As used herein, the term "variant" 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. As discussed below, nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Usually, the differences are limited such that the sequence of the reference polypeptide and the sequence of the variant are generally quite similar and identical in many regions. A variant and a 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 a residue encoded by the genetic code. Variants of polynucleotides or polypeptides can be naturally-occurring, such as dual gene variants, or they can be variants not known to be naturally-occurring. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those of ordinary skill in the art.

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 will typically produce the same characteristics to a similar, but not necessarily the same, degree. Functionally homologous proteins produce the same characteristics, wherein the quantitative measure produced by one homologue is at least 10% of the quantitative measure produced by the other; more typically, at least 20% of the quantitative measure produced by the original molecule , between about 30% and about 40%; for example, between about 50% and about 60%; between about 70% and about 80%; or between about 90% and about 95%; % and about 100%, or greater than 100%. Thus, where the molecule has enzymatic activity, the functional homologue will have the enzymatic activity percentages listed above compared to the original enzyme. In the case of a molecule that is a DNA binding molecule (eg, a polypeptide), the homologue will have binding affinity above the recited percentages compared to the original molecule, as measured by the weight of the binding molecule.

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

Heterologous genes are homologous genes in different species that arise by vertical transmission of a single gene from the last common ancestor, where the genes and their major functions are conserved. Homologous genes are genes inherited in two species by a common ancestor.

When used with reference to an amino acid or nucleotide/nucleic acid sequence from a given species, the term "heterolog" refers to the same amino acid or nucleotide/nucleic acid sequence from a different species. It will be understood that two sequences are xenologs of each other when they are derived from a common ancestral sequence through linear transmission, and/or are otherwise closely related with respect to both their sequence and their biological function. Heterologs will typically have a high degree of sequence identity but may not (and usually will not) share 100% sequence identity.

A homologous gene is a homologous gene produced by a gene duplication event. Homologous genes usually belong to the same species, but this is not required. Homologs can be split into in-paralogs (homologous pairs that appear after the speciation event) and out-paralogs (the same species that appear before the speciation event) homologous pair). Between species, an exolog is a pair of homologs that exists between two organisms due to replication prior to speciation. Within a species, an exolog is a pair of homologs that exist in the same organism but whose replication event occurs after speciation. Homologs typically have the same or similar function.

Functional homologues can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologues of the polypeptide of interest, such as biomass-modulating polypeptides, glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis, or membrane trafficking protein. Sequence analysis may involve BLAST, Reciprocal BLAST, or Reciprocal BLAST against non-redundant databases using the amino acid sequences of biomass-modulating polypeptides, glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis, or membrane transport proteins, respectively, as reference sequences. PSI-BLAST analysis. In some cases, the amino acid sequence is deduced from the nucleotide sequence. Typically, those polypeptides in the database with more than 40% sequence identity are candidates for further evaluation as biomass-modulating polypeptides, glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis, or membrane transport proteins, respectively. thing. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue by another hydrophobic residue or substitution of one polar residue by another polar residue or substitution of one acidic amino acid by another acidic amine base acid substitution or substitution of one basic amino acid with another basic amino acid, etc. Preferably, by conservative substitution, is meant a combination of, such as glycine by alanine, and vice versa; valine, isoleucine, and leucine by methionine, and Vice versa; aspartic acid is replaced by glutamic acid and vice versa; aspartic acid is replaced by glutamic acid and vice versa; serine is replaced by threonine and vice versa However; lysine is substituted by arginine, and vice versa; cysteine is substituted by methionine, and vice versa; and phenylalanine and tyrosine are substituted by tryptophan, and vice versa The same is true. If desired, manual inspection of such candidates can be performed in order to limit the number of candidates to be further evaluated. Manual inspection can be performed by selecting candidates that appear to have domains (eg, conserved functional domains) present in productivity-modulating polypeptides. The term "glycosyltransferase" as used herein refers to an enzyme capable of catalyzing the transfer of a sugar moiety from an activating donor molecule to a specific acceptor molecule, thereby forming a glycosidic bond. The oligosaccharides thus synthesized may be of linear type or branched type and may contain multiple monosaccharide building blocks. Glycosyltransferases have been described using nucleotide diphosphate-sugars, nucleotide monophosphate-sugars, and phosphate sugars and related proteins to classify glycosyltransferases into different sequence-based families (Campbell et al., Biochem. J. 326, 929- 939 (1997)) and available on the CAZy (Carbohydrate Active Enzymes) website (www.cazy.org).

As used herein, a glycosyltransferase may be selected from a list including, but not limited to, fucosyltransferases (eg, alpha-1,2-fucosyltransferase, alpha-1,3/1 ,4-fucosyltransferase, alpha-1,6-fucosyltransferase), sialyltransferase (e.g. alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase) acid transferase, alpha-2,8-sialyltransferase), galactosyltransferase (e.g. beta-1,3-galactosyltransferase, beta-1,4-galactosyltransferase, alpha -1,3-galactosyltransferase, α-1,4-galactosyltransferase), N-acetylglucosaminyltransferase, N-acetylgalactosyltransferase, transglucosyltransferase Enzymes, Mannosyltransferase, N-Acetylmannosyltransferase, Xylosyltransferase, Glucuronidase, Galacturonyltransferase, Glucosyltransferase, N-Hydroxytransferase Acetylneuraminotransferase, Rhamnosyltransferase, N-Acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β -L-altrosamine transaminase, UDP-N-acetylglucosamine enolpyruvyl transferase and fucosaminotransferase.

Fucosyltransferases are glycosyltransferases that transfer fucose residues (Fuc) from a GDP-fucose (GDP-Fuc) donor to a glycan acceptor. Fucosyltransferase includes α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,4-fucosyltransferase and α-1, 6-fucosyltransferase, which catalyzes the transfer of Fuc residues from GDP-Fuc to glycan acceptors via an alpha-glycosidic bond. Fucosyltransferases can be found but are not limited to the GT10, GT11, GT23, GT65 and GT68 CAZy families. Sialyltransferases are glycosyltransferases that transfer a sialic acid group (eg, Neu5Ac or Neu5Gc) from a donor (eg, CMP-Neu5Ac or CMP-Neu5Gc) to a glycan acceptor. Sialyltransferases include α-2,3-sialyltransferase, α-2,6-sialyltransferase and α-2,8-sialyltransferase, which catalyze the conversion of The sialic acid group is transferred to the glycan acceptor. Sialyltransferases can be found but are not limited to the GT29, GT42, GT80 and GT97 CAZy families. Galactosyltransferases are glycosyltransferases that transfer a galactosyl group (Gal) from a UDP-galactose (UDP-Gal) donor to a glycan acceptor. Galactosyltransferases include β-1,3-galactosyltransferase, β-1,4-galactosyltransferase, α-1,3-galactosyltransferase and α-1,4-galactosyltransferase Syltransferases that transfer Gal residues from UDP-Gal to glycan acceptors via alpha- or beta-glycosidic linkages. Galactosyltransferases can be found but are not limited to the GT2, GT6, GT8, GT25 and GT92 CAZy families. Glucosyltransferases are glycosyltransferases that transfer glucose groups (Glc) from UDP-glucose (UDP-Glc) donors to glycan acceptors. Glucosyltransferases include α-glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase, and β-1,4-glucosyltransferase, which are produced by α- or β-glucosyltransferase The β-glycosidic bond transfers the Glc residue from UDP-Glc to the glycan acceptor. Glucosyltransferases can be found but are not limited to the GT1, GT4 and GT25 CAZy families. Mannosyltransferases are glycosyltransferases that transfer mannosyl (Man) from a GDP-mannose (GDP-Man) donor to a glycan acceptor. Mannosyltransferases include α-1,2-mannosyltransferase, α-1,3-mannosyltransferase and α-1,6-mannosyltransferase, which convert Mannosyltransferase through α-glycosidic bonds. Residues are transferred from GDP-Man to the glycan acceptor. Mannosyltransferases can be found but are not limited to the GT22, GT39, GT62 and GT69 CAZy families. N-acetylglucosaminyltransferase is a glycosyl group that transfers N-acetylglucosamine (GlcNAc) from a UDP-N-acetylglucosamine (UDP-GlcNAc) donor to a glycan acceptor transferase. N-acetylglucosaminyltransferases can be found but not limited to the GT2 and GT4 CAZy families.

N-Acetylgalactosamine transferase is an enzyme that transfers N-acetylgalactosamine (GalNAc) from a UDP-N-acetylgalactosamine (UDP-GalNAc) donor to a glycan acceptor glycosyltransferase. N-Acetylgalactosaminyltransferases can be found but are not limited to the GT7, GT12 and GT27 CAZy families. N-Acetylmannosylaminotransferase is an enzyme that transfers N-acetylmannosamine (ManNAc) from a UDP-N-acetylmannosamine (UDP-ManNAc) donor to a glycan acceptor glycosyltransferase. Xylosyltransferases are glycosyltransferases that transfer a xylose residue (Xyl) from a UDP-xylose (UDP-Xyl) donor to a glycan acceptor. Xylosyltransferases can be found but are not limited to the GT61 and GT77 CAZy families. Glucuronidase is a glycosyltransferase that transfers glucuronide from a UDP-glucuronide donor to a glycan acceptor via an α- or β-glycosidic bond. Glucuronidyltransferases can be found but are not limited to the GT4, GT43 and GT93 CAZy families.

Galacturonyltransferases are glycosyltransferases that transfer galacturonates from UDP-galacturonate donors to glycan acceptors. N-hydroxyacetylneuraminosyltransferase is a glycosyltransferase that transfers an N-hydroxyacetylneuraminic acid group (Neu5Gc) from a CMP-Neu5Gc donor to a glycan acceptor. Rhamnosyltransferases are glycosyltransferases that transfer rhamnose residues from a GDP-rhamnose donor to a glycan acceptor. Rhamnosyltransferases can be found but are not limited to the GT1, GT2 and GT102 CAZy families. N-Acetylrhamnosyltransferases are sugars that transfer N-acetylrhamnosamine residues from UDP-N-acetyl-L-rhamnosamine donors to glycan acceptors base transferase. UDP-4-Amino-4,6-dideoxy-N-acetyl-β-L-altrosaminotransferase was used for UDP-2-acetamido-2,6-dideoxy --L-arabino-4-hexulose glycosyltransferase for the biosynthesis of pseudoamines, which are sialic sugars used to modify flagellin. UDP-N-Acetylglucosamine enolacetonyltransferase (murA) is used to transfer enolacetonyl from phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UDPAG) A glycosyltransferase to form UDP-N-acetylglucosamine enolpyruvate. Fucosaminotransferases are used to transfer N-acetylfucosamine residues from dTDP-N-acetylfucosamine or UDP-N-acetylfucosamine donors to glycans Glycosyltransferases on receptors.

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-Acetamido-2,6-dideoxy--L-arabinose-4-hexulose, UDP-2-acetamido-2,6-dideoxy--L-lyxose -4-Hexylose, 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-Neomosamine (UDP-L-PneNAC or UDP-2-Acetylamino-2,6-dideoxy-L-talose), UDP -N-Acetylmuramic acid, UDP-N-Acetyl-L-isorhamnosamine (UDP-L-QuiNAc or UDP-2-Acetylamino-2,6-dideoxy-L -glucose), GDP-L-isorhamnose, CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-hydroxyacetylneuraminic 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, GDP-rhamnet sugar or UDP-xylose. Nucleotide-sugars act as sugar donors in glycosylation reactions. These reactions are catalyzed by glycosyltransferases.

As the term is used herein, and as commonly understood in the state of the art, "Oligosaccharide" refers to a sugar polymer containing small amounts, typically three to twenty simple sugars (ie, monosaccharides). A monosaccharide as used herein is a reducing sugar. 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 carbon of the sugar is oxidized to a carboxyl group. Oligosaccharides as used in the present invention may be linear in structure or may include branches. A bond between two sugar units (eg, glycosidic, galactosidic, glucosidic, etc.) may be represented, for example, as 1,4, 1->4, or (1-4), used interchangeably herein. For example, the terms "Gal-b1,4-Glc", "β-Gal-(1->4)-Glc", "Galβ1-4-Glc" and "Gal-b(1-4)-Glc" It has the same meaning, that is, the carbon-1 of galactose (Gal) is linked with the β-glycosidic bond of carbon-4 of glucose (Glc). Each monosaccharide can be in a cyclic form (eg, piperanose in the 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 β 2->6. Oligosaccharides may contain alpha-glycosidic linkages as well as beta-glycosidic linkages or may contain beta-glycosidic linkages only.

"Disaccharide" as the term is used herein, and as commonly understood in the current state of the art, refers to a sugar polymer containing two monosaccharides. Examples of disaccharides include lactose (Gal-b1,4-Glc), lacto-N-disaccharide (Gal-b1,3-GlcNAc), N-acetyllactosamine (Gal-b1,4-GlcNAc), LacDiNAc (GalNAc-b1,4-GlcNAc), N-Acetyl galactosamine glucosamine (GalNAc-b1,4-Glc), Neu5Ac-a2,3-Gal, Neu5Ac-a2,6-Gal and Fucoidan Glycosyl-(1-4)-N-hydroxyacetylneuraminic acid (Fuc-(1-4)-Neu5Gc).

The term "monosaccharide" as used herein refers to sugars that are not broken down by hydrolysis into simpler sugars, classified as aldoses or ketoses, and contain one or more hydroxyl groups per molecule. Monosaccharides are sugars that contain only one simple sugar. Examples of monosaccharides include hexose, D-glucopyranose, D-galactofuranose, D-galactofuranose, L-galactopyranose, D-mannanose, D-alopyranose Sugar (Allopyranose), L-Aldropyranose, D-Gulopyranose (Gulopyranose), L-Idopyranose (Idopyranose), D-Talopyranose (Talopyranose), D-Ribofuranose , D-Ribophenanose, D-Arabinofuranose (Arabinofuranose), D-Arabinofuranose, L-Arabinofuranose, L-Arabinopyranose, D-Xylopyranose, D-Lyxopyranose , D-erythrofuranose (Erythrofuranose), D-threofuranose (Threofuranose), heptose, L-glycerol-D-mannose-heptopyranose (LDmanHep), D-glycerol-D-mannose-heptose Piperanose, (DDmanHep), 6-Deoxy-L-Aldropyranose, 6-Deoxy-D-Gulopranose, 6-Deoxy-D-Talopranose, 6-Deoxy-D-Talopranose Oxy-D-galactopyranose, 6-deoxy-L-galactopyranose, 6-deoxy-D-mannopyranose, 6-deoxy-mannopyranose, 6-deoxy- D-glucopyranose, 2-deoxy-D-arabinose-hexose, 2-deoxy-D-erythro-pentose, 2,6-dideoxy-D-arabinose-hexanose, 3,6-dideoxy-D-arabinose-hexylpyranose, 3,6-dideoxy-L-arabinose-hexylpyranose, 3,6-dideoxy-D-xylose-hexyl Pyranose, 3,6-dideoxy-D-ribose-hexylpyranose, 2,6-dideoxy-D-ribose-hexylpyranose, 3,6-dideoxy-L-xylose -Hexoperanose, 2-amino-2-deoxy-D-glucopyranose, 2-amino-2-deoxy-D-galactopyranose, 2-amino-2-deoxy -D-Mannopyranose, 2-amino-2-deoxy-D-allopyranose, 2-amino-2-deoxy-L-aldropyranose, 2-amino-2- Deoxy-D-guloperanose, 2-amino-2-deoxy-L-iduranose, 2-amino-2-deoxy-D-talopiperanose, 2-ethyl Acetamino-2-deoxy-D-glucopyranose, 2-acetamido-2-deoxy-D-galactopyranose, 2-acetamido-2-deoxy-D- Mannopyranose, 2-Acetamido-2-deoxy-D-allopyranose, 2-acetamido-2-deoxy-L-aldropyranose, 2-acetamido -2-Deoxy-D-gulopranose, 2-acetamido-2-deoxy-L-iduranose, 2-acetamido-2-deoxy-D-tarot pyranose, 2-acetamido-2,6-dideoxy-D-galactopyranose, 2-acetamido-2,6-dideoxy-L-galactopyranose, 2-Acetamido-2,6-dideoxy-L-mannopyranose, 2-acetamido-2,6-dideoxy-D-glucopyranose, 2-acetamido -2,6-dideoxy-L -Aldropyranose, 2-Acetamido-2,6-dideoxy-D-talopyranose, D-glucopyranuronic acid, D-galactopyranuronic acid, D- -Mannopyranuronic acid, D-allopiperanuronic acid, L-aldropyranuronic acid, D-gulopranuronic acid, L-gulopranuronic acid, L-alpine Dupiranuronic acid, D-talopyranuronic acid, sialic acid, 5-amino-3,5-dideoxy-D-glycerol-D-galactose-nonan-2-ketonic acid, 5-Acetylamino-3,5-dideoxy-D-glycerol-D-galactose-nonan-2-ketonic acid, 5-hydroxyacetylamino-3,5-dideoxy- D-glycerol-D-galactose-nonan-2-ketonic acid, erythritol, arabitol, xylitol, ribitol, glucitol, galactitol, mannitol, D-ribose-hex- 2-ketopyranose, D-arabinose-hex-2-ketofuranose (D-fructofuranose), D-arabinose-hex-2-ketopyranose, L-xylose-hex-2-one Pyranose, D-lyxose-hex-2-ketopyranose, D-threose-pentan-2-ketopyranose, D-alzo-hepta-2-ketopyranose, 3-C -(Hydroxymethyl)-D-erythrofuranose, 2,4,6-trideoxy-2,4-diamino-D-glucopyranose, 6-deoxy-3-O-methyl- D-glucose, 3-O-methyl-D-rhamnose, 2,6-dideoxy-3-methyl-D-ribose-hexose, 2-amino-3-O-[(R) -1-Carboxyethyl]-2-deoxy-D-glucopyranose, 2-acetamido-3-O-[(R)-carboxyethyl]-2-deoxy-D-glucopyranose Ranose, 2-hydroxyacetamido-3-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose, 3-deoxy-D-lysole Sugar-hept-2-ketopyranoic acid, 3-deoxy-D-manno-oct-2-ketopyranoic acid, 3-deoxy-D-glycerol-D-galactose-nonan-2-one Phranosic 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-glycerol-L-alzo-non-2-ketopyranoic acid, 5,7-diamino-3,5,7,9-tetrade Oxy-D-glycerol-D-galactose-nonan-2-ketopyranoic acid, 5,7-diamino-3,5,7,9-tetradeoxy-D-glycerol-D-tarot- Non-2-ketopyranoic acid, glucose, galactose, N-acetylglucosamine, glucosamine, mannose, xylose, N-acetylmannosamine, N-acetylneuraminic acid, N-hydroxyacetylneuraminic acid, N-acetylgalactosamine, galactosamine, fucose, rhamnose, glucuronic acid, gluconic acid, fructose and polyols.

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

Oligosaccharides are glycan structures composed of three or more monosaccharide subunits connected to each other in a linear or branched structure via glycosidic bonds. Preferably, the oligosaccharide as described herein contains a monosaccharide selected from the list as used herein above. Examples of oligosaccharides include, but are not limited to, Lewis-type antigenic oligosaccharides, mammalian milk oligosaccharides, and human milk oligosaccharides.

As used herein, "mammalian milk oligosaccharide" (MMO) refers to oligosaccharides such as (but not limited to) the following: 3-fucosyllactose, 2'-fucosyllactose, 6- Fucoyllactose, 2',3-difucoyllactose, 2',2-difucoyllactose, 3,4-difucoyllactose, 6'-sialyllactose, 3'-sialic acid Lactose, 3,6-disialolactose, 6,6'-disialolactose, 8,3-disialolactose, 3,6-disialolacto-N-tetrasaccharide, lactose Difucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III , lacto-N-fucopentose V, lacto-N-fucopentaose VI, sialyl lacto-N-neotetrasaccharide c, sialyl lacto-N-tetrasaccharide b, sialyl lacto-N -Tetrasaccharide a, lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, p-lacto-N-hexaose , monofucosyl monosialyl lacto-N-neotetrasaccharide c, monofucosyl p-lacto-N-hexasaccharide, monofucosyl lacto-N-hexasaccharide III, isofucosyl Glycosylated lacto-N-hexasaccharide III, isofucosylated lacto-N-hexasaccharide I, sialyl lacto-N-hexaose, sialyl lacto-N-neohexaose II, diyan Fucosyl-p-lacto-N-hexasaccharide, difucosyllacto-N-hexasaccharide, difucosyllacto-N-hexasaccharide a, difucosyllacto-N-hexasaccharide c , galactosylated polyglucosamine, fucosylated oligosaccharides, neutral oligosaccharides and/or sialylated oligosaccharides. Mammalian milk oligosaccharides (MMOs) comprise oligosaccharides present in milk found at any stage during lactation, including colostrum from humans (i.e. human milk oligosaccharides or HMOs) and mammals, which Mammals include (but are not limited to) cattle ( Bos Taurus ), sheep ( Ovis aries ), goats ( Capra aegagrus hircus ), Bactrian camels ( Camelus bactrianus ) , horse ( Equus ferus caballus ), pig ( Sus scropha ), dog ( Canis lupus familiaris ), ezo brown bear ( Ursus arctos yesoensis ) , polar bear ( Ursus maritimus ), Japanese black bear (Asian black bear ( Ursus thibetanus japonicus )), striped skunk ( Mephitis mephitis ), hooded seal ( Cystophora cristata ), Asian elephant (Asian Elephant ( Elephas maximus )), African Elephant ( Loxodonta africana ), Giant Anteater ( Myrmecophaga tridactyla ), True Bottlenose Dolphin ( Tursiops truncates ), Arctic Minke Whale ( Balaenoptera acutorostrata ), Eugene Kangaroo ( Macropus eugenii ), Red Kangaroo ( Macropus rufus ), Common Kangaroo ( Trichosurus Vulpecula ), Koala (Koala ( Phascolarctos cinereus )), Eastern quoll ( Dasyurus viverrinus ), Platypus ( Ornithorhynchus anatinus ). Human milk oligosaccharides (HMOs), also known as human homogenous milk oligosaccharides, are chemically identical to human milk oligosaccharides found in human breast milk, but are biotechnologically produced (eg using cell-free systems or cells and containing Bacterial, fungal, yeast, plant, animal or protozoal cell organisms, preferably genetically engineered cells and organisms). Human consensus lacto-oligosaccharide is sold under the name HiMO.

[ Fuca1-4]GlcNAc, or simply 4-FLNB; Lewisb, which is the tetrasaccharide Fuca1-2Galβ1-3[Fucα1-4]GlcNAc, or simply DiF-LNB; Sialyl Lewisa, which is 5-ethyl Acylneuramido-(2-3)-galactoside-(1-3)-(fucopyranosyl-(1-4))-N-acetylglucosamine, or Neu5Acα2- 3Galβ1-3[Fucα1-4]GlcNAc; H2 antigen, which is Fuca1-2Galβ1-4GlcNAc, or otherwise known as 2'fucosyl-N-acetyl-lactosamine, simply 2'FLacNAc; Lewisx, It is the trisaccharide Galβ1-4[Fucα1-3]GlcNAc, or otherwise known as 3-fucosyl-N-acetyl-lactosamine, simply 3-FLacNAc; Lewisy, which is the tetrasaccharide Fuca1-2Galβ1 -4[Fucα1-3]GlcNAc; and Sialyl Lewisx, which is 5-acetylneuramido-(2-3)-galactoside-(1-4)-(fucopyranosyl- (1-3))-N-acetylglucosamine, or Neu5Acα2-3Galβ1-4[Fucα1-3]GlcNAc for short.

As used herein and as generally understood in the current state of the art, a "fucosylated oligosaccharide" is an oligosaccharide bearing a fucose residue. Such fucosylated oligosaccharides are sugar structures comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of the monosaccharide subunits is fucose. Fucosylated oligosaccharides may contain more than one fucose residue, eg, two, three or more fucose residues. Fucosylated oligosaccharides can be neutral oligosaccharides or charged oligosaccharides, eg also containing sialic acid structures. Fucose can be linked to other monosaccharide subunits including glucose, galactose, GlcNAc via α-glycosidic bonds including α-1,2, α-1,3, α-1,4, α-1,6 bond.

Examples include 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), di- Fucosyllactose (diFL), lactodifucotetraose (LDFT), lacto-N-fucopentose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N -Fucopentaose III (LNFP III), Lacto-N-fucopentaose V (LNFP V), Lacto-N-fucopentaose VI (LNFP VI), Lacto-N-neofucopentaose I, Lacto-N-difucohexaose I (LDFH I), lacto-N-difucohexaose II (LDFH II), monofucosyl lacto-N-hexaose III (MFLNH III), difucoid Glycosyl lacto-N-hexasaccharide (DFLNHa), difucosyl-lacto-N-neohexaose.

The terms "alpha-1,2-fucosyltransferase", "alpha 1,2 fucosyltransferase" as used in the present invention , "2-fucosyltransferase (2-fucosyltransferase)", "α-1,2-fucosyltransferase (α-1,2-fucosyltransferase)", "α1,2-fucosyltransferase" Transferase (α 1,2 fucosyltransferase)", "2 fucosyltransferase (2 fucosyltransferase)", "2-FT" or "2FT" are used interchangeably and refer to the catalysis of fucose from the donor GDP-L- A glycosyltransferase that transfers fucose to acceptor molecules in α-1,2-bonds. As used in the present invention, the terms "2' fucosyllactose", "2'-fucosyllactose", "α-1,2-fucosyllactose (α-1,2-fucosyllactose)" -1,2-fucosyllactose)", "α 1,2 fucosyllactose (α 1,2 fucosyllactose)", "α-1,2-fucosyllactose (α-1,2-fucosyllactose)", " α 1,2 fucosyllactose (α 1,2 fucosyllactose)", "Galβ-4(Fucα1-2)Glc", "2FL" or "2'FL" are used interchangeably and refer to α-1 by catalysis , 2-fucosyltransferase transfers fucose residues from GDP-L-fucose to lactose in α-1,2-bonds. The terms "difucosyllactose", "di-fucosyllactose", "lactodifucotetraose", "2',3- Difucosyllactose (2',3-difucosyllactose)", "2',3 difucosyllactose (2',3 difucosyllactose)", "α-2',3-fucosyllactose (α-2 ',3-fucosyllactose)", "α 2',3 fucosyllactose", "Fucα1-2Galβ 1-4(Fucα1-3)Glc", "DFLac", "2',3 diFL", "DFL", "DiFL" or "diFL" are used interchangeably.

The terms "alpha-1,3-fucosyltransferase", "alpha 1,3 fucosyltransferase" as used in the present invention , "3-fucosyltransferase (3-fucosyltransferase)", "α-1,3-fucosyltransferase (α-1,3-fucosyltransferase)", "α1,3fucosyltransferase" Transferase (α 1,3 fucosyltransferase)", "3 fucosyltransferase (3 fucosyltransferase)", "3-FT" or "3FT" are used interchangeably and refer to the catalysis of fucose from the donor GDP-L- Glycosyltransferase that transfers fucose to acceptor molecules in α-1,3-bonds. Terms as used in the present invention "3-fucosyllactose (3-fucosyllactose)", "alpha-1,3-fucosyllactose (alpha-1,3-fucosyllactose)", "alpha 1,3 fucoidose" Alpha 1,3 fucosyllactose”, “α-1,3-fucosyllactose”, “α 1,3 fucosyllactose” , "Galβ-4(Fucα1-3)Glc", "3FL" or "3-FL" are used interchangeably and refer to the conversion of a fucose residue from a fucose residue by a catalytic α-1,3-fucosyltransferase. Product obtained by the transfer of GDP-L-fucose to lactose in α-1,3-bonds.

As used herein, "sialylated oligosaccharide" should be understood as a charged sialic acid containing an oligosaccharide (ie an oligosaccharide having a sialic acid residue). It has acidic properties. Sialyl oligosaccharides contain at least one sialic acid monosaccharide subunit, such as, for example, but not limited to, Neu5Ac and Neu5Gc. The sialylated oligosaccharide is a sugar structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of the monosaccharide subunits is sialic acid. The sialylated oligosaccharide may contain more than one sialic acid residue, eg, two, three or more sialic acid residues. The sialic acid can be linked to other monosaccharide subunits comprising galactose, GlcNAc, sialic acid via α-glycosidic bonds comprising α-2,3 bonds, α-2,6 bonds. Some examples are 3-SL (3'-sialyllactose or 3'-SL or Neu5Ac-a2,3-Gal-b1,4-Glc); 3'-sialyllactosamine; 6-SL (6' - Sialylactose or 6'-SL or Neu5Ac-a2,6-Gal-b1,4-Glc); 6'-Sialyllactosamine; Oligosaccharides, including 6'-sialyllactose, 3,6 - Disialolactose (Neu5Ac-a2,3-(Neu5Ac-a2,6)-Gal-b1,4-Glc), 6,6'-disialolactose (Neu5Ac-a2,6-Gal-b1 ,4-(Neu5Ac-a2,6)-Glc), 8,3-disialolactose (Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,4-Glc), SGG hexasaccharide (Neu5Acα - 2,3Galβ- 1,3GalNacβ-1,3Galα-1,4Galβ-1,4Gal), sialylated tetrasaccharide (Neu5Acα- 2,3Galβ- 1,4GlcNacβ-14GlcNAc), pentasaccharide LSTD (Neu5Acα-2, 3Galβ- 1,4GlcNacβ- 1,3Galβ- 1,4Glc), Sialyllacto-N-Triose, Sialyllacto-N-tetraose, Sialyllacto-N-neotetraose, Monosialyl Acid-based lacto-N-hexasaccharide, disialyl lacto-N-hexaose I, monosialo-N-hexose I, monosialo-N-neohexaose II, disialo lactyl lacto-N-neohexasaccharide, disialo lacto-N-tetrasaccharide, disialo lacto-N-hexasaccharide II, disialo lacto-N-tetrasaccharide a, disialo lacto-N - hexasaccharide I, sialyllacto-N-tetrasaccharide b, sialyllacto-N-neotetrasaccharide c, sialyllacto-N-neotetrasaccharide d, 3'-sialyl-3-rock Alginyl lactose, disialo-mono-fuco-N-neohexaose, mono-fuco-mono-sialo-N-octasaccharide (Sialyl Lea), sialyl-milk-N-fucohexa Saccharides II, disialyllacto-N-fucopentose II, monofucosyldisialolacto-N-tetrasaccharides; and oligosaccharides with one or more sialic acid residues, including (but not Limited to): the oligosaccharide moiety of gangliosides selected from GM3 (3' sialyllactose, Neu5Acα-2, 3Galβ-4Glc); and oligosaccharides, including GM3 motif, GD3 Neu5Acα-2, 8Neu5Acα-2, 3Galβ-1,4Glc GT3 (Neu5Acα- 2,8Neu5Acα- 2,8Neu5Acα-2,3Galβ-1,4Glc); GM2 GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc, GM1 Galβ-1, 3GalNAcβ- 1,4 (Neu5Acα- 2,3 )Galβ-1,4Glc, GD1a Neu5Acα-2,3Galβ- 1,3GalNAcβ- 1,4(Neu5Acα- 2,3)Galβ- 1,4Glc, GT1a Neu5Acα-2,8Neu5Acα- 2,3Galβ- 1,3GalNAcβ- 1 ,4(Neu5Acα- 2,3)Galβ-1,4Glc, GD2 GalNAcβ-1,4(Neu5Acα- 2,8Neu5Acα2,3)Galβ- 1,4Glc, GT2 GalNAcβ-1,4(Neu5Acα- 2,8Neu5Acα- 2 ,8Neu5Acα2,3)Galβ-1,4Glc, GD1b, Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc, GT1b Neu5Acα- 2,3Galβ- 1,3GalNAcβ-1, 4(Neu5Aα- 2,8Neu5Aca2,3)Galβ-1,4Glc, GQ1b Neu5Aα- 2,8Neu5Aα- 2,3Galβ-1,3GalNAcβ- 1,4(Neu5Aα-2,8Neu5Aα2,3)Galβ-1,4Glc, GT1c Galβ-1,3GalNAcβ- 1,4(Neu5Acα- 2,8Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc, GQ1c Neu5Acα-2,3Galβ-1,3GalNAc β-1,4(Neu5Acα-2,8Neu5Aα- 2,8Neu5Acα2,3)Galβ-1,4Glc, GP1c Neu5Acα-2,8Neu5Acα-2,3Galβ-1,3GalNAc β-1,4(Neu5Acα- 2,8Neu5Acα- 2,8Neu5Acα2,3)Galβ-1,4Glc, GD1a Neu5Acα-2,3Galβ-1,3(Neu5Acα-2,6)GalNAcβ-1,4Galβ-1,4Glc, Fucoyl-GM1 Fuca-1,2Galβ-1,3GalNAcβ-1,4(Neu5Acα-2, 3) Galβ-1,4Glc; both can be extended to the production of the corresponding gangliosides by reacting the above oligosaccharide moieties with ceramide or synthesizing the above oligosaccharides on ceramide.

As used in the present invention, the terms "alpha-2,3-sialyltransferase (alpha-2,3-sialyltransferase)", "alpha 2,3 sialyltransferase (alpha 2,3 sialyltransferase)", " 3-sialyltransferase (3-sialyltransferase), "α-2,3-sialyltransferase (α-2,3-sialyltransferase)", "α2,3 sialyltransferase (α2 ,3 sialyltransferase)", "3 sialyltransferase (3 sialyltransferase)", "3-ST" or "3ST" are used interchangeably and refer to the catalyzed transfer of sialic acid from the donor CMP-Neu5Ac to α-2,3- The glycosyltransferase of the acceptor molecule in the bond. As used in the present invention, the terms "3' sialyllactose (3' sialyllactose)", "3'-sialyllactose (3'-sialyllactose)", "α-2,3-sialyllactose (alpha) -2,3-sialyllactose)", "α 2,3 sialyllactose (alpha 2,3 sialyllactose)", "α-2,3-sialyllactose (α-2,3-sialyllactose)", " "α2,3 sialyllactose", "3SL" or "3'SL" are used interchangeably and refer to the conversion of sialic acid by catalyzing α-2,3-fucosyltransferase. The product obtained by the transfer of the group from CMP-Neu5Ac to lactose in the α-2,3-bond. As used in the present invention, the terms "alpha-2,6-sialyltransferase (alpha-2,6-sialyltransferase)", "alpha 2,6 sialyltransferase (alpha 2,6 sialyltransferase)", " 6-sialyltransferase (6-sialyltransferase), "α-2,6-sialyltransferase (α-2,6-sialyltransferase)", "α2,6 sialyltransferase (α2 ,6 sialyltransferase)", "6 sialyltransferase (6 sialyltransferase)", "6-ST" or "6ST" are used interchangeably and refer to the catalyzed transfer of sialic acid from the donor CMP-Neu5Ac to α-2,6- Glycosyltransferase of the acceptor in the bond. As used in the present invention, the terms "6' sialyllactose (6' sialyllactose)", "6'-sialyllactose (6'-sialyllactose)", "α-2,6-sialyllactose (alpha) -2,6-sialyllactose)", "α 2,6 sialyllactose (alpha 2,6 sialyllactose)", "α-2,6-sialyllactose (α-2,6-sialyllactose)", " "α2,6 sialyllactose", "6SL" or "6'SL" are used interchangeably and refer to the conversion of sialic acid by catalyzing α-2,6-fucosyltransferase. The product obtained by the transfer of the group from CMP-Neu5Ac to lactose in the α-2,6-bond. As used in the present invention, the terms "alpha-2,8-sialyltransferase (alpha-2,8-sialyltransferase)", "alpha 2,8 sialyltransferase (alpha 2,8 sialyltransferase)", " 8-sialyltransferase (8-sialyltransferase), "α-2,8-sialyltransferase (α-2,8-sialyltransferase)", "α2,8 sialyltransferase (α2 ,8 sialyltransferase)", "8 sialyltransferase (8 sialyltransferase)", "8-ST" or "8ST" are used interchangeably and refer to the catalyzed transfer of sialic acid from the donor CMP-Neu5Ac to α-2,8- Glycosyltransferase of the acceptor in the bond.

As used herein and as generally understood in the current state of the art, a "neutral oligosaccharide" is an oligosaccharide that does not have a negative charge derived from a carboxylic acid group. Examples of such neutral oligosaccharides include 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 2',3-difucosyllactose (diFL), lacto-N- Triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-neofucopentose I, lacto-N-fucopentaose II, Lacto-N-fucopentaose III, Lacto-N-fucopentaose V, Lacto-N-fucopentaose VI, Lacto-N-neofucopentose V, Lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6'-galactosylose, 3'-galactosylose, lacto-N-hexaose, lacto-N-neohexaose, p-lacto-N- Hexose, p-lacto-N-neohexaose, difucosyl-lacto-N-hexasaccharide and difucosyl-lacto-N-neohexaose.

As used herein, the antigens of the human ABO blood group system are oligosaccharides. Such antigens of the human ABO blood group system are not limited to human structures. These structures involve the A determinant GalNAc- present on the disaccharide core structure comprising Gal-β1,3-GlcNAc, Gal-β1,4-GlcNAc, Gal-β1,3-GalNAc and Gal-β1,4-Glc α1,3(Fuc-α1,2)-Gal-, B determinant Gal-α1,3(Fuc-α1,2)-Gal- and H determinant Fuc-α1,2-Gal-.

Terms "LNT II", "LNT-II", "LN3", "lacto-N-triose II", "lacto- N -triose II" as used in the present invention - N -triose II)", "lacto-N-triose", "lacto- N -triose" or " GlcNAcβ1-3Galβ1-4Glc " are used interchangeably .

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

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

As used in the present invention, the terms "LSTa", "LS-Tetrasaccharide a", "Sialyl-lacto-N-tetraose a", "Sialyl-lacto-N-tetraose a", " Sialyllacto-N-tetraose a" or "Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc" are used interchangeably .

The terms "LSTb", "LS-Tetrasaccharide b", "Sialyl-lacto-N-tetraose b", "Sialyl-lacto-N-tetraose b" as used in the present invention Sialyllacto-N-tetraose b” or “Gal-b1,3-(Neu5Ac-a2,6)-GlcNAc-b1,3-Gal-b1,4-Glc” can be Used interchangeably.

As used in the present invention, the terms "LSTc", "LS-Tetrasaccharide c (LS-Tetrasaccharide c)", "Sialyl-lacto-N-tetraose c (Sialyl-lacto-N-tetraose c)", " Sialyllacto-N-tetraose c, "Sialyllacto-N-neotetraose c" or "Neu5Ac-a2,6-Gal" -b1,4-GlcNAc-b1,3-Gal-b1,4-Glc" can be used interchangeably.

As used in the present invention, the terms "LSTd", "LS-Tetrasaccharide d (LS-Tetrasaccharide d)", "Sialyl-lacto-N-tetraose d (Sialyl-lacto-N-tetraose d)", " Sialyllacto-N-tetraose d (sialyllacto-N-tetraose d), "Sialyllacto-N-neotetraose d" or "Neu5Ac-a2,3-Gal" -b1,4-GlcNAc-b1,3-Gal-b1,4-Glc" can be used interchangeably.

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

The terms "DSLNT" and "Disialyllacto-N-tetraose" are used interchangeably and refer to Neu5Ac-a2,6-[Neu5Ac-a2,3-Gal-b1,3-GlcNAc -b1,3]-Gal-b1,4-Glc. Terms "LNFP-I", "lacto-N-fucopentaose I", "LNFP I", "LNF I OH type I determinant", "LNF I", "LNF1", "LNF 1" and "Blood group H antigen pentaose type 1" are used interchangeably and refer to Fuc-a1,2-Gal-b1,3- GlcNAc-b1,3-Gal-b1,4-Glc.

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

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

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

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

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

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

Terms "LNDFH I", "Lacto-N-difucohexaose I", "LNDFH-I", "LDFH I", "Le ^b -lactose" and "Lewis-b six" Sugar (Lewis-b hexasaccharide)" is used interchangeably and refers to Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-Gal-b1,4-Glc.

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

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

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

A "fucosylation pathway" as used herein is a biochemical pathway consisting of enzymes and their respective genes, mannose-6-phosphate isomerase, phosphomannose mutase, Mannose-1-phosphate guanosyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase and/or recycling pathway L-fucose kinase/GDP-fucoidase Sugar pyrophosphorylase, and fucosyltransferase producing alpha 1,2, alpha 1,3, alpha 1,4 or alpha 1,6 fucosylated oligosaccharides.

"Sialylation pathway" is a biochemical pathway consisting of: enzymes and their respective genes, L-glutamic acid-D-fructose-6-phosphate aminotransferase, glucosamine-6 - Phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acetylglucosamine epimerase, UDP-N-acetylglucosamine 2-Epimerase, N-Acetylglucosamine-6P 2-Epimerase, Glucosamine 6-Phosphate N-Acetyltransferase, N-Acetylglucosamine-6-Phosphate Phosphatase, N-acetylmannosamine-6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate uridine Transferase, glucosamine-1-phosphate acetyltransferase, sialic acid synthase, N-acetylneuraminic acid lysase, N-acetylneuraminic acid-9-phosphate synthase, N-acetylneuraminic acid Amino acid-9-phosphate phosphatase and/or CMP-sialic acid synthase, and sialyltransferase producing alpha 2,3, alpha 2,6 or alpha 2,8 sialylated oligosaccharides.

A "galactosylation pathway" as used herein is a biochemical pathway consisting of enzymes and their respective genes, galactose-1-epimerase, galactokinase, glucokinase, galactose -1-phosphate uridine syltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridine syltransferase and/or glucose phosphomutase, and oligosaccharide-producing 2, 3, 4. Galactosyltransferases that bind galactose to alpha or beta on the 6 hydroxyl group.

"N-acetylglucosamine carbohydrate pathway" as used herein is a biochemical pathway consisting of enzymes and their respective genes, L-glutamic acid-D-fructose- 6-Phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine 6-phosphate N-acetyl Syltransferase, N-acetylglucosamine-1-phosphate uridine transferase, glucosamine-1-phosphate acetyltransferase and/or glucosamine-1-phosphate acetyltransferase, and producing A glycosyltransferase that binds N-acetylglucosamine to alpha or beta on the 3, 4, and 6 hydroxyl groups of oligosaccharides.

"N-acetylgalactosaminylation pathway" as used herein is a biochemical pathway comprising at least one of enzymes and their respective genes selected from List containing the following: L-glutamic acid-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N -acetylglucosamine 1-phosphate uridine transferase, glucose Amine-1-phosphate acetyltransferase, UDP - N -acetylglucosamine 4-epimerase, UDP -glucose 4 -epimerase, N -acetylgalactosamine kinase and/or UDP - N -Acetylgalactosamine pyrophosphorylase, and glycosyltransferases that produce GalNAc-modified compounds comprising an alpha- or beta-binding N-acetyl half-saccharide on a mono-, di- or oligosaccharide The mono-, di- or oligosaccharide of lactosamine.

A "mannosylation pathway" as used herein is a biochemical pathway comprising at least one of an enzyme and its respective gene selected from a list comprising: mannose -6-phosphate isomerase, phosphomannose mutase and/or mannose-1-phosphate guanosyltransferase, and glycosyltransferases producing mannosylated compounds contained in mono-, The mono-, di- or oligosaccharide having alpha or beta bound mannose on the di- or oligosaccharide.

"N-acetylmannosaminylation pathway" as used herein is a biochemical pathway comprising at least one of enzymes and their respective genes selected from A list containing the following: L-glutamic acid-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine- 6-Phosphate Deacetylase, Glucosamine 6-Phosphate N-Acetyl Transferase, N-Acetyl Glucosamine-1-Phosphate Uridine Transferase, Glucosamine-1-Phosphate Acetyl Transferase , glucosamine-1-phosphate acetyltransferase, UDP-GlcNAc 2-epimerase and/or ManNAc kinase, and glycosyltransferases that produce ManNAc-modified compounds contained in mono-, di- or The mono-, di- or oligosaccharide having alpha or beta bound N-acetylmannosamine on the oligosaccharide.

Terms "mannose-6-phosphate isomerase", "phosphomannose isomerase", "mannose phosphate isomerase", "phosphate "phosphohexoisomerase", "phosphomannoisomerase", "phosphomannose-isomerase", "phosphohexomutase", " D-mannose-6-phosphate ketol-isomerase (D-mannose-6-phosphate ketol-isomerase)" and "manA" are used interchangeably and refer to catalyzing the reversible conversion of D-fructose 6-phosphate to D-mannose 6-phosphate enzyme.

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

Terms "mannose-1-phosphate guanylyltransferase", "GTP-mannose-1-phosphate guanylyltransferase" , "phosphomannose isomerase-guanosine 5'-diphosphate-D-mannose pyrophosphorylase (phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase; PIM-GMP)", "GDP-mannose Sugar pyrophosphorylase (GDP-mannose pyrophosphorylase), "guanosine 5'-diphospho-D-mannose pyrophosphorylase (guanosine 5'-diphospho-D-mannose pyrophosphorylase)", "guanosine diphosphate mannose Sugar pyrophosphorylase (guanosine diphosphomannose pyrophosphorylase), "guanosine triphosphate-mannose 1-phosphate guanylyltransferase", "mannose 1-phosphate guanylyltransferase" "mannose 1-phosphate guanylyltransferase (guanosine triphosphate)" and "manC" are used interchangeably and refer to the use of GTP to convert D-mannose-1-phosphate to GDP-mannose and Diphosphate enzyme.

Terms "GDP-mannose 4,6-dehydratase", "guanosine 5'-diphosphate-D-mannose oxidoreductase" mannose oxidoreductase)", "guanosine diphosphomannose oxidoreductase", "guanosine diphosphomannose 4,6-dehydratase", "GDP-D -Mannose dehydratase (GDP-D-mannose dehydratase)", "GDP-D-mannose 4,6-dehydratase (GDP-D-mannose 4,6-dehydratase)", "GDP-mannose 4,6 - Hydrogen-dissociation enzyme (GDP-mannose 4,6-hydro-lyase)", "GDP-mannose 4,6-hydro-dissociation enzyme (to form GDP-4-dehydro-6-deoxy-D-mannose) ) (GDP-mannose 4,6-hydro-lyase (GDP-4-dehydro-6-deoxy-D-mannose-forming))” and “gmd” are used interchangeably and refer to GDP-rhamnose and GDP- The enzyme that forms the first step in the biosynthesis of fucose.

Terms "GDP-L-fucose synthase", "GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reduction" Enzyme (GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase)", "GDP-L-fucose:NADP+4-oxidoreductase (3,5-epimerase-4-reductase)" Isomerizing) (GDP-L-fucose:NADP+ 4-oxidoreductase (3,5-epimerizing))" and "fcl" are used interchangeably and refer to the enzyme that forms the second step in the biosynthesis of GDP-fucose .

Terms "L-fucokinase/GDP-fucose pyrophosphorylase (L-fucokinase/GDP-fucose pyrophosphorylase)", "L-fucose kinase/L-fucose-1-P guanosine Syltransferase (L-fucokinase/L-fucose-1-P guanylyltransferase), "GDP-fucose pyrophosphorylase", "GDP-L-fucose pyrophosphorylase (GDP-L-fucose pyrophosphorylase)" GDP-L-fucose pyrophosphorylase)" and "fkp" are used interchangeably and refer to an enzyme that catalyzes the conversion of L-fucose-1-phosphate to GDP-fucose using GTP.

The terms "L-glutamine-D-fructose-6-phosphate aminotransferase", "glutamine-fructose-6-phosphate aminotransferase" Aminase (isomerization) (glutamine---fructose-6-phosphate transaminase (isomerizing))", "hexosephosphate aminotransferase", "glucosamine-6-phosphate isomerase (forming Glutamine-6-phosphate isomerase (glutamine-forming), glutamine-fructose-6-phosphate transaminase (isomerizing) )", "D-fructose-6-phosphate amidotransferase", "glucosaminephosphate isomerase", "glucosamine 6-phosphate synthase" 6-phosphate synthase)", "GlcN6P synthase (GlcN6P synthase)", "GFA" and "glmS" are used interchangeably and refer to the catalysis of the conversion of D-fructose-6-phosphate to D using L-glutamic acid - The enzyme glucosamine-6-phosphate.

Terms "glucosamine-6-P deaminase", "glucosamine-6-phosphate deaminase", "GlcN6P deaminase" ", "glucosamine-6-phosphate isomerase", "glmD" and "nagB" are used interchangeably and refer to catalyzing the reversible isomerization of glucosamine-6-phosphate (GlcN6P) An enzyme that reacts with deamination to form fructose-6-phosphate and ammonium ions.

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

The terms "N-acetylglucosamine-6-P deacetylase", "N-acetylglucosamine-6-phosphate deacetylase" 6-phosphate deacetylase)" and "nagA" are used interchangeably and refer to catalyzing the hydrolysis of the N-acetyl group of N-acetylglucosamine-6-phosphate (GlcNAc-6-P) to yield glucosamine-6 - Phosphate (GlcN6P) and acetate enzymes.

N-Acylglucosamine 2-epimerase is an enzyme that catalyzes the reaction N-Acyl-D-Glucosamine=N-Acyl-D-Mannosamine. Alternative names for this enzyme include N-acetylglucosamine 2-epimerase, N-acetyl-D-glucosamine 2-epimerase, GlcNAc 2-epimerase, N-acetyl- D-glucosamine 2-epimerase and N-acetylglucosamine epimerase.

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

N-Acetylmannosamine-6-phosphate 2-epimerase catalyzes the reaction N-Acetyl-D-glucosamine 6-phosphate = N-Acetyl-D-mannosamine 6-phosphate salt enzyme.

Bifunctional UDP-GlcNAc 2-epimerase/kinase catalyzes the reaction UDP-N-acetyl-D-glucosamine=N-acetyl-D-mannosamine and the reaction N-acetyl-D- Mannosamine+ATP=ADP+N-acetyl-D-mannosamine 6-phosphate bifunctional enzyme.

Glucosamine 6-phosphate N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl-CoA to D-glucosamine-6-phosphate to generate free CoA and N-acetyl-D-glucosamine 6 - Phosphate enzyme. Alternative names include aminodeoxyglucose phosphate acetyltransferase, D-glucosamine-6-P N-acetyltransferase, glucosamine 6-phosphate acetyltransferase, glucosamine 6-phosphate N-acetyltransferase N-acetyltransferase, glucosamine-phosphate N-acetyltransferase, glucosamine-6-phosphate acetylase, N-acetylglucosamine-6-phosphate synthase, phosphoglucosamine acetylase, phosphoric acid Glucosamine N-acetylase phosphoglucosamine N-acetylase, phosphoglucosamine transacetylase, GNA and GNA1.

The term "N-acetylglucosamine-6-phosphate phosphatase" refers to the dephosphorylation of N-acetylglucosamine-6-phosphate (GlcNAc-6-P) This is an enzyme that synthesizes N-acetylglucosamine (GlcNAc).

The term "N-acetylmannosamine-6-phosphate phosphatase" refers to the dephosphorylation of N-acetylmannosamine-6-phosphate (ManNAc-6P) into N-acetylmannosamine (ManNAc) enzyme.

Terms "N-acetylmannosamine-6-phosphate 2-epimerase", "ManNAc-6-P isomerase" )", "ManNAc-6-P 2-epimerase (ManNAc-6-P 2-epimerase)", "N-acetylglucosamine-6P 2-epimerase (N-acetylglucosamine-6P 2 - epimerase)" and "nanE" are used interchangeably and refer to the enzyme that converts ManNAc-6-P to N-acetylglucosamine-6-phosphate (GlcNAc-6-P).

Terms "phosphoacetylglucosamine mutase", "acetylglucosamine phosphomutase", "acetylaminodeoxyglucose phosphomutase" ", "phospho-N-acetylglucosamine mutase (phospho-N-acetylglucosamine mutase)" and "N-acetyl-D-glucosamine 1,6-phosphate mutase (N-acetylglucosamine mutase)" acetyl-D-glucosamine 1,6-phosphomutase)" is used interchangeably and refers to an enzyme that catalyzes the conversion of N-acetyl-glucosamine 1-phosphate to N-acetyl-glucosamine 6-phosphate.

The terms "N-acetylglucosamine 1-phosphate uridine syltransferase", "N-acetylglucosamine 1-phosphate uridine syltransferase" (N-acetylglucosamine 1-phosphate uridine syltransferase) acetylglucosamine-1-phosphate uridyltransferase)", "UDP-N-acetylglucosamine diphosphorylase", "UDP-N-acetylglucosamine pyrophosphorylase (UDP-N-acetylglucosamine diphosphorylase)" -acetylglucosamine pyrophosphorylase)", "uridine diphosphoacetylglucosamine pyrophosphorylase", "UTP:2-acetylglucosamine-2-deoxy-α-D-glucose-1- Uridine phosphate transferase (UTP: 2-acetamido-2-deoxy-alpha-D-glucose- 1-phosphate uridylyltransferase)", "UDP-GlcNAc pyrophosphorylase", "GlmU urine Glysyltransferase (GlmU uridylyltransferase)", "Acetylglucosamine 1-phosphate uridineyltransferase (Acetylglucosamine 1-phosphate uridylyltransferase)", "UDP-acetylglucosamine pyrophosphorylase (UDP- "acetylglucosamine pyrophosphorylase", "uridine diphosphate-N-acetylglucosamine pyrophosphorylase", "uridine diphosphoacetylglucosamine phosphorylase" )" and "acetylglucosamine 1-phosphate uridineyltransferase" are used interchangeably and refer to catalyzed transfer of uridine 5-monophosphate (from uridine 5-triphosphate Phosphate (uridine 5-triphosphate; UTP) transfer) converts N-acetylglucosamine 1-phosphate (GlcNAc-1-P) into UDP-N-acetylglucosamine (UDP-GlcNAc) enzyme.

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

The term "glmU" refers to a pathway that has both N-acetylglucosamine-1-phosphate uridine transferase and glucosamine-1-phosphate acetyltransferase activities and catalyzes the de novo biosynthetic pathway of UDP-GlcNAc Two sequentially reacting bifunctional enzymes. The C-terminal domain catalyzes the transfer of an acetyl group from acetyl-CoA to GlcN-1-P to generate GlcNAc-1-P, which is converted to UDP-GlcNAc by transfer of uridine 5-monophosphate, which is obtained by Reactions catalyzed by the N-terminal domain.

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

Terms "N-acetylneuraminate lyase", "Neu5Ac lyase", "N-acetylneuraminate pyruvate-lyase" )", "N-acetylneuraminic acid aldolase", "NALase", "sialate lyase", "sialic acid aldolase" ", "sialic acid lyase" and "nanA" are used interchangeably and refer to the degradation of N-acetylneuraminate to N-acetylmannosamine (ManNAc) and pyruvate the enzyme.

As used herein, the terms "N-acylneuraminate-9-phosphate synthase", "N-acylneuraminate-9-phosphate synthase" 9-phosphate synthetase)", "NANA synthase (NANA synthase)", "NANAS", "NANS", "NmeNANAS", "N-acetylneuraminic acid pyruvate lyase (pyruvate phosphorylation) ( N-acetylneuraminate pyruvate-lyase (pyruvate-phosphorylating)" is used interchangeably and refers to the ability to use phosphoenolpyruvate (PEP) in a reaction from N-acetylmannosamine-6-phosphate (ManNAc- 6-Phosphate) is an enzyme that synthesizes N-acylneuraminic acid-9-phosphate.

The term "N-acylneuraminate-9-phosphatase" refers to a enzyme capable of dephosphorylating N-acylneuraminic acid-9-phosphate to synthesize N-acylneuraminic acid Amino acid enzyme.

The terms "CMP-sialic acid synthase", "N-acylneuraminate cytidylyltransferase", "CMP-sialic acid synthase" as used herein (CMP-sialate synthase)", "CMP-NeuAc synthase (CMP-NeuAc synthase)", "NeuA" and "CMP-N-acetylneuraminic acid synthase (CMP-N-acetylneuraminic acid synthase)" can be Used interchangeably and refer to an enzyme capable of synthesizing CMP-N-acetylneuraminate from N-acetylneuraminate using CTP in a reaction.

Terms "galactose-1-epimerase", "aldose 1-epimerase", "mutarotase", "aldose 1-epimerase" Gyrase (aldose mutarotase), "galactose mutarotase (galactose mutarotase)", "galactose 1-epimerase (galactose 1-epimerase)" and "D-galactose 1-epimerase (D -galactose 1-epimerase)" is used interchangeably and refers to an enzyme that catalyzes the conversion of β-D-galactose to α-D-galactose.

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

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

Terms "galactose-1-phosphate uridineyltransferase", "Gal-1-P uridineyltransferase", "UDP-glucose" --- Hexose-1-phosphate uridine transferase (UDP-glucose---hexose-1-phosphate uridylyltransferase)", "uridine transferase (uridyl transferas)", "hexose-1- Hexose-1-phosphate uridyltransferase, "uridyltransferase", "hexose 1-phosphate uridyltransferase", "UDP-glucose:alpha-D-galactose-1-phosphate uridineyltransferase", "galB" and "galT" are used interchangeably and Refers to the enzyme that catalyzes the reaction D-galactose 1-phosphate + UDP-D-glucose = D-glucose 1-phosphate + UDP-D-galactose.

Terms "UDP-glucose 4-epimerase", "UDP-galactose 4-epimerase", "uridine diphosphate glucose epimerase" "uridine diphosphoglucose epimerase", "galactowaldenase", "UDPG-4-epimerase", "uridine diphosphate galactose 4-epimerase" Constructing enzyme (uridine diphosphate galactose 4-epimerase)", "uridine diphospho-galactose-4-epimerase (uridine diphospho-galactose-4-epimerase)", "UDP-glucose epimerase (UDP- "glucose epimerase", "4-epimerase", "uridine diphosphoglucose 4-epimerase", "uridine diphosphoglucose 4-epimerase" "uridine diphosphate glucose 4-epimerase" and "UDP-D-galactose 4-epimerase" are used interchangeably and refer to the conversion of UDP-D-glucose into UDP-galactose enzyme.

Terms "glucose-1-phosphate uridine syltransferase (glucose-1-phosphate uridylyltransferase)", "UTP---glucose-1-phosphate uridylyltransferase (UTP---glucose-1-phosphate uridylyltransferase)" )", "UDP glucose pyrophosphorylase", "UDPG phosphorylase", "UDPG pyrophosphorylase", "uridine 5'-diphosphate glucose pyrophosphate" Phosphorylase (uridine 5'-diphosphoglucose pyrophosphorylase), "uridine diphosphoglucose pyrophosphorylase", "uridine diphosphate-D-glucose pyrophosphorylase" "phosphorylase", "uridine-diphosphate glucose pyrophosphorylase" and "galU" are used interchangeably and refer to the catalysis of the conversion of D-glucose-1-phosphate to UDP-glucose using UTP the enzyme.

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

Terms "UDP-N-acetylglucosamine 4-epimerase", "UDP acetylglucosamine epimerase", "uridine "uridine diphosphoacetylglucosamine epimerase", "uridine diphosphate N-acetylglucosamine-4-epimerase", "uridine 5'-diphospho-N-acetylglucosamine-4-epimerase" and "UDP-N-acetylglucosamine-D" - UDP-N-acetyl-D-glucosamine 4-epimerase" is used interchangeably and refers to catalyzing the epimerization of UDP-N-acetyl-glucosamine (UDP-GlcNAc) It is the enzyme of UDP-N-acetylgalactosamine (UDP-GalNAc).

Terms "N-acetylgalactosamine kinase", "GALK2", "GK2", "GalNAc kinase", "N-acetylgalactosamine (GalNAc)-1" -N-acetylgalactosamine (GalNAc)-1-phosphate kinase" and "ATP:N-acetyl-D-galactosamine 1-phosphotransferase" )" is used interchangeably and refers to an enzyme that catalyzes the synthesis of N-acetylgalactosamine 1-phosphate (GalNAc-1-P) from N-acetylgalactosamine (GalNAc) using ATP.

The terms "UDP-N-acetylgalactosamine pyrophosphorylase" and "UDP-GalNAc pyrophosphorylase" are used interchangeably and refer to the use of UTP catalysis The enzyme that converts N-acetylgalactosamine 1-phosphate (GalNAc-1-P) to UDP-N-acetylgalactosamine (UDP-GalNAc).

Terms "N-acetylneuraminate kinase", "ManNAc kinase", "N-acetyl-D-mannosamine kinase" )" and "nanK" are used interchangeably and refer to the enzyme that phosphorylates ManNAc to synthesize N-acetylmannosamine-phosphate (ManNAc-6-P).

Terms "acetyl-coenzyme A synthetase", "acs", "acetyl-CoA synthetase", "AcCoA synthetase", " Acetate--CoA ligase, "acyl-activating enzyme" and "yfaC" are used interchangeably and refer to catalyzing the conversion of acetate to ethyl acetate in an ATP-dependent reaction Acyl-CoA (AcCoA) enzyme.

Terms "pyruvate dehydrogenase", "pyruvate oxidase", "POX", "poxB" and "pyruvate:ubiquinone-8 oxidoreductase" oxidoreductase)" is used interchangeably and refers to an enzyme that catalyzes the oxidative decarboxylation of pyruvate to produce acetate and CO2.

Terms "lactate dehydrogenase (lactate dehydrogenase), "D-lactate dehydrogenase (D-lactate dehydrogenase)", "ldhA", "hslI", "htpH", "D-LDH", "enzymatic lactate dehydrogenase" "fermentative lactate dehydrogenase" and "D-specific 2-hydroxyacid dehydrogenase" are used interchangeably and refer to the enzyme that catalyzes the conversion of lactate to pyruvate, thereby producing NADH. enzymes.

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

The term "purified" refers to a substance that is substantially or substantially free of components that interfere with the activity of a biomolecule. With respect to cells, carbohydrates, nucleic acids and polypeptides, the term "purified" refers to a material that is substantially or substantially free of components that normally accompany the material as found in its natural state. Typically, a purified saccharide, oligosaccharide, protein or nucleic acid of the invention is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure, as measured by band intensity based on silver-stained gels or other methods for determining purity Measurement. Purity or homogeneity can be indicated by a variety of means well known in the art, such as polyacrylamide gel electrophoresis of protein or nucleic acid samples followed by observation after staining. For some purposes, high resolution and the use of HPLC or similar means for purification will be required. 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 term "cultivation" refers to the medium in which the cells are cultured or fermented, the cells themselves, and the oligosaccharides produced by the cells in whole culture broth, ie, both internally (intracellularly) and externally (extracellularly).

The term "membrane transporter proteins" as used herein refers to proteins that are part of or interact with the cell membrane and control the flow of molecules and information across the cell. Thus, membrane proteins are involved in transport, whether they are imported into or exported from cells.

Such membrane transporters can be porters as defined by the Transporter Classification Database, transporters driven by P-P-bond hydrolysis, β-Barrel Porin, Accessory Transporters, Putative Transporters, and Phosphotransfer-Driven Group Translocation Proteins, this database is operated and managed by the Saier Lab Bioinformatics Group via www.tcdb.org and provides a functional and phylogenetic classification of membrane transporters. This transporter classification database details the IUBMB-approved comprehensive classification system for membrane transporters, called the Transporter Classification (TC) system. TCDB taxonomy searches as described herein are defined based on TCDB.org as published on June 17, 2019.

Porter is the collective name for uniporter, coporter, and antiporter that utilize carrier-mediated processes (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). It belongs to the class of electrochemical potential-driven transport proteins and is also known as a secondary carrier-type enhancer. Membrane transport proteins are included in this category when using carrier-mediated processes to catalyze unidirectional transport when a single species is transported by facilitated diffusion or in a membrane potential-dependent process (if the solute is charged); when Catalytic reverse transport occurs when two or more species are transported in opposite directions in a tightly coupled process, not coupled to a direct energy form other than chemi-osmotic energy; and/or when two or more species are transported in opposite directions. When they are transported together in the same direction in the process of tight coupling, they are not coupled to direct energy forms other than chemical osmotic energy, and catalyze the transport in the same direction. These proteins belong to secondary carriers (Forrest et al., Biochim. Biophys. Acta 1807 (2011) 167-188). These systems are usually stereospecific. Solute: Solute reverse transport is a typical feature of secondary carriers. Dynamic association of transporters and enzymes produces a functional membrane transport metabolite that directly imports substrates typically obtained from the extracellular compartment into their cellular metabolism (Moraes and Reithmeier, Biochim. Biophys. Acta 1818 (2012). ), 2687-2706). Solutes transported via this transporter system include, but are not limited to, cations, organic anions, inorganic anions, nucleosides, amino acids, polyols, phosphorylated saccharolytic intermediates, osmotic agents, chelating iron.

A membrane transporter is included in P-P-bond hydrolysis-driven transport if it hydrolyzes the diphosphate bond of an inorganic pyrophosphate, ATP, or another nucleoside triphosphate to drive active uptake and/or extrusion of a solute or solutes in the class of proteins (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 by classes of transporters driven by hydrolysis of P-P-bonds include, but are not limited to, cations, heavy metals, beta-glucans, UDP-glucose, lipopolysaccharides, teichoic acid.

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

Auxiliary transport proteins are defined as proteins that facilitate transport across one or more biological membranes but are not themselves directly involved in transport. These membrane transporters consistently function in conjunction with one or more existing transport systems such as (but not limited to) outer membrane factor (OMF), polysaccharide (PST) transporter, ATP-binding cassette (ATP- binding cassette; ABC) type transport protein. It may provide functions associated with energy coupling for transport, play a structural role in complex formation, provide biogenic or stabilizing functions or regulatory functions (Saier et al, Nucleic Acids Res. 44 (2016) D372-D379). Examples of helper transport proteins include, but are not limited to, the family of polysaccharide copolymerases involved in the transport of polysaccharides, the family of membrane fusion proteins involved in the transport of bacteriocins and chemical toxins.

Putative transporters comprise families of which will be classified elsewhere when the transport function of the members is confirmed, or eliminated from the transporter classification system if the proposed transport function proves ineffective. These families include one or more members for which transport functions have been proposed, but evidence for such functions has not been convincing (Saier et al, Nucleic Acids Res. 44 (2016) D372-D379). Examples of putative transport proteins that fall into this group as under the TCDB system published on June 17, 2019 include, but are not limited to, copper transport proteins.

Phosphate transfer-driven group translocation proteins are also known as bacterial phosphoenolpyruvate:sugar phosphotransferase systems (phosphotransferase systems; PTS) PEP-dependent phosphooxyl transfer-driven group translocation proteins. The reaction products derived from extracellular sugars are cytoplasmic phosphate sugars. Enzymatic components that catalyze the phosphorylation of sugars are superimposed on the transport process in a tightly coupled process. The PTS system is involved in many different aspects, including regulation and chemotaxis, biofilm formation and pathogenesis (Lengeler, J. Mol. Microbiol. Biotechnol. 25 (2015) 79-93; Saier, J. Mol. Microbiol. Biotechnol. 25 (2015) 73-78). As in the TCDB system published on June 17, 2019, the membrane transport protein family within the phosphate transfer-driven group translocation proteins includes proteins related to glucose-glucoside, fructose-mannitol, lactose-N,N'-di The PTS system associated with the transport of acetylchitobiose-beta-glucoside, glucitol, galactitol, mannose-fructose-sorbose and ascorbate.

The major facilitator superfamily (MFS) is a superfamily of membrane transport proteins that catalyze unidirectional transport, solute:cation (H+, but hardly Na+) co-transport and/or solute:H+ or solute : Solute reverse transport. Most are 400-600 amido residues in length and have 12, 14 or occasionally 24 transmembrane alpha-helical spanners (TMS), as transported by Saier Lab Bioinformatics Group (www.tcdb.org) Defined by the Protein Taxonomy Database.

"SET" or "Sugar Efflux Transporter" as used herein refers to a membrane protein of the SET family, which is a protein with the InterPRO domain IPR004750 and/or a protein belonging to the eggNOGv4.5 family ENOG410XTE9. InterPro domains can be authenticated by using the online tool at https://www.ebi.ac.uk/interpro/ or the standalone version of InterProScan (https://www.ebi.ac.uk/interpro/download.html) Use preset values. Identification of orthologous families in eggNOGv4.5 can be performed using the online or 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 have been classified as catecholate, hydroxamate, carboxylates and mixed types. Chelaterin is generally synthesized by a nonribosomal peptide synthetase (NRPS)-dependent pathway or an NRPS-independent pathway (NIS). The most important precursor in the NRPS-dependent chelatin biosynthesis pathway is chorismate. 2,3-DHBA can be formed from chorismate by a three-step reaction catalyzed by isochorismate synthase, isochorismate, and 2,3-dihydroxybenzoate-2,3-dehydrogenase. Chelaterin can also be formed from salicylates, which are formed from isochorismates by isochorismate pyruvate lyase. When ornithine is used as a chelating iron precursor, biosynthesis depends on the hydroxylation of ornithine catalyzed by L-ornithine N5-monooxygenase. In the NIS pathway, an important step in the biosynthesis of chelatin is N(6)-hydroxylysine synthase.

Transport proteins are required to export chelatin out of the cell. So far, four superfamilies of membrane proteins have been identified in the process: major facilitator superfamily (MFS); multidrug/oligosaccharidyl-lipid/polysaccharide flippase superfamily (Multidrug/Oligosaccharidyl-lipid/Polysaccharide Flippase Superfamily; MOP ); the resistance, nodulation and cell division superfamily (RND); and the ABC superfamily. In general, genes involved in chelatin export are clustered with chelatin biosynthesis genes. The term "siderophore exporter" as used herein refers to such transport proteins required for the export of chelatin to the outside of the cell.

The ATP-binding cassette (ABC) superfamily contains uptake and efflux transport systems, and members of these two groups are usually loosely clustered together. ATP hydrolysis without 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 Protein Taxonomy Database operated by the Saier Lab Bioinformatics Group, available through www.tcdb.org and providing the functionality and systems of membrane transport proteins Classification occurs.

The term "enabled efflux" means the activity of introducing transport of solutes on the cytoplasmic membrane and/or cell wall. This transport can be achieved by introducing and/or increasing the expression of transport proteins as described in the present invention. The term "enhanced efflux" means an activity that improves the transport of solutes across the cytoplasmic membrane and/or cell wall. Transport of solutes across the cytoplasmic membrane and/or cell wall can be enhanced by introducing and/or increasing the expression of membrane transport proteins as described herein. "Expression" of a membrane transport protein is defined as "overexpression" of the gene encoding the membrane transport protein in the case of an endogenous gene; or in the case of the gene encoding the membrane transport protein being The absence of a heterologous gene in a wild-type strain or cell is defined as "expression".

The term "precursor" as used herein refers to a substance taken up or synthesized by a cell to specifically produce an oligosaccharide according to the invention. In this sense, a precursor may be a receptor as defined herein, but may also be another substance, a metabolite, which is first modified in the cell as part of the biochemical synthesis pathway of the oligosaccharide.

Examples of such precursors include receptors as defined herein; and/or glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, dihydroxyacetone, glucosamine, N-Acetyl-glucosamine, Mannosamine, N-Acetyl-mannosamine, Galactosamine, N-Acetylgalactosamine; Phosphorylated sugars such as, for example, but not limited to, glucose-1-phosphate , galactose-1-phosphate, glucose-6-phosphate, fructose-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-Acetyl Mannosamine-6-Phosphate, N-Acetyl Glucosamine-1-phosphate, N-acetyl-neuraminic acid-9-phosphate; and/or nucleotide-activated sugars as defined herein, such as, for example, UDP-glucose, UDP-galactose, UDP-N- 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 disaccharide or oligosaccharide that is taken up by a cell and modified by the addition of one or more monosaccharide units for oligosaccharide assembly by one or more glycosyltransferases. These receptors are produced by methods including extraction from natural sources, biotechnological processes, physical processes, chemical processes, and combinations thereof. Examples of such receptors include lactose, 2'FL, 3-FL, lacto-N-disaccharide (LNB), lacto-N-triose, lacto-N-tetrasaccharide (LNT), lacto-N-neotetraose Sugar (LNnT), N-Acetyl-Lactosamine (LacNAc), Lacto-N-Pentasaccharide (LNP), Lacto-N-Neopentasaccharide, P-Lacto-N-Pentasaccharide, P-Lacto-N-Pentaose Sugar, lacto-N-novopentaose I (lacto-N-novopentaose I), lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH) ), para lacto-N-neohexaose (pLNnH), para lacto-N-hexaose (pLNH), lacto-N-heptaose, lacto-N- New heptaose, p-lacto-N-heptaose, p-lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-new octaose, isolacto-N- Octose, para-milk-N-octa sugar, isomilk-N-octa sugar, neonatal milk-N-octa sugar, paramilk-N-octa sugar, isomilk-N-nona sugar, neonatal milk-N - nona sugar, lacto-N-nona sugar, lacto-N-deca sugar, isolacto-N-deca sugar, neonatal milk-N-deca sugar, lacto-N-neodeca sugar; galactosyl lactose, which by extension has 1, 2, 3, 4, 5 or more N-acetyllactosamine units and/or 1, 2, 3, 4, 5 or more lactose-N-disaccharide units and lactose containing 1 or more Oligosaccharides of N-acetyllactosamine units and or 1 or more lacto-N-disaccharide units, or intermediates thereof converted to oligosaccharide, fucosylated and sialylated forms.

Throughout this application, unless expressly stated otherwise, the features "synthesize", "synthesized" and "synthesis" may be associated with the features "produce", "produced", respectively " and "production" are used interchangeably. DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect, the present invention provides a method for producing a mixture comprising at least two oligosaccharides. The method includes the following steps: i. Provide a cell, preferably a single cell, capable of expressing a glycosyltransferase and capable of synthesizing a nucleotide-sugar, wherein the nucleotide-sugar is a donor for the glycosyltransferase, and ii. culturing the cell under conditions that allow expression of the glycosyltransferase and synthesis of the nucleotide-sugar, and iii. adding at least two receptors to the culture such that the cells are capable of producing at least two oligosaccharides, preferably any one of these receptors is a disaccharide or an oligosaccharide, iv. Preferably, at least one of the oligosaccharides is isolated from the culture, more preferably, all of the oligosaccharides are isolated from the culture.

Within the scope of the present invention, permissive conditions are 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 a particular embodiment, allowable conditions may include a temperature range of 30 +/- 20 degrees Celsius, a pH range of 7 +/- 3.

In a preferred embodiment of the method of the present invention, the culture is fed with at least two, preferably at least three, more preferably at least four, even more preferably at least five receptors for the synthesis of the receptors in the mixture any of the oligosaccharides. In another preferred embodiment of the method, the culture is fed with at least two, preferably at least three, more preferably at least four, even more preferably at least five receptors for use in synthesizing all of these in the mixture Oligosaccharides. In a more preferred embodiment, the culture is fed with at least three receptors for the synthesis of any or all of the oligosaccharides in the mixture.

According to the present invention, preferably, any one of these receptors is a disaccharide or oligosaccharide, more preferably, each of these receptors is a disaccharide or oligosaccharide, that is, preferably , each receptor is a disaccharide or an oligosaccharide. Those of ordinary skill in the art will therefore understand that, preferably within the scope of the present invention, the receptors added in the method according to the present invention may all be disaccharides, may all be oligosaccharides or may be both disaccharides and oligosaccharides sugar mixture. The disaccharide receptor is preferably selected from the list comprising: lactose (Gal-b1,4-Glc), lacto-N-disaccharide (Gal-b1,3-GlcNAc), N-acetyllactosamine (Gal -b1,4-GlcNAc), LacDiNAc (GalNAc-b1,4-GlcNAc) and N-acetylgalactosamine (GalNAc-b1,4-Glc). The oligosaccharide receptor is preferably a mammalian milk oligosaccharide (MMO) as defined herein, more preferably a human milk oligosaccharide (HMO). The oligosaccharide acceptor is preferably selected from the list comprising: 2'FL, 3-FL, lacto-N-triose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), Lacto-N-pentasaccharide (LNP), lacto-N-neopentasaccharide, p-lacto-N-pentasaccharide, p-lacto-N-neopentasaccharide, lacto-N-neopentasaccharide I, lacto-N-hexasaccharide (LNH), lacto-N-neohexaose (LNnH), p-lacto-N-neohexaose (pLNnH), p-lacto-N-hexose (pLNH), lacto-N-heptaose, lacto-N-neohexaose Heptaose, p-lacto-N-heptaose, p-lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-new octaose, isolacto-N-octaose, p-lacto-N -Octose, Isomilk-N-Octose, Neonatal Milk-N-Octose, Paramilk-N- Neooctaose, Isomilk-N-Nonaose, Neonatal Milk-N-Nonaose, Milk-N - nona, lacto-N-deca, isolacto-N-deca, neonatal lacto-N-deca, lacto-N-neodeca; galactosyl lactose, extended with 1, 2, 3, 4 , 5 or more N-acetyllactosamine units and/or 1, 2, 3, 4, 5 or more lactose-N-disaccharide units and lactose containing 1 or more N-acetyllactosamine Units and oligosaccharides of 1 or more lacto-N-disaccharide units, or intermediates thereof to oligosaccharide, fucosylated and sialylated forms.

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

In a preferred embodiment of the method according to the invention, one of the receptors added to the culture has a degree of polymerization of two (DP3), preferably the receptor is lactose, and the polymerization of the other receptors added to the culture A degree of at least three (DP3). The degree of polymerization (DP) of the acceptor refers to the number of monosaccharide units present in the acceptor structure. In another embodiment of the method according to the invention, any one of the receptors added to the culture has a degree of polymerization of at least three (DP3), preferably all receptors have a degree of polymerization of at least three (DP3). ). In an alternative embodiment of the method according to the invention, the DP of any of the recipients added to the culture is higher than three. Receptors with a DP higher than three are understood to include tetrasaccharides constructed from 4 monosaccharide subunits (DP4), pentasaccharides constructed from 5 monosaccharide subunits (DP5), pentasaccharides constructed from DP6 or higher up to DP20 Oligosaccharide structure. In a preferred alternative embodiment, all receptors added to the culture have a DP higher than three. For example, all receptors added to the culture are tetrasaccharides and/or longer oligosaccharides built from 5, 6 or more monosaccharide subunits. In an alternative embodiment of the method according to the invention, any of the receptors added to the culture has a degree of polymerization of three (DP3). Receptors with DP3 are understood to be oligosaccharides built up from three monosaccharide subunits, also known as trisaccharides. In a preferred embodiment of the present invention, all of these receptors have a DP3 structure. Preferably, therefore, the culture is fed with a mixture of oligosaccharides comprising at least two receptors, all of which are trisaccharides composed of three monosaccharide subunits. In an alternative embodiment of the method according to the invention, any, preferably all, of the receptors added to the culture have a DP higher than four, preferably higher than five.

In a preferred embodiment of the method according to the invention, all receptors added to the culture have different DPs. For example, two receptors can be fed into the culture, wherein the first receptor is a disaccharide and the second receptor is a trisaccharide. Alternatively, the culture can be fed with two receptors, wherein the first receptor is a disaccharide and the second receptor is a tetrasaccharide or a longer saccharide of 5, 6 or more subunits. Two receptors can also be fed into the culture, wherein the first receptor is a trisaccharide and the second receptor is a tetrasaccharide or a longer saccharide of 5, 6 or more subunits. In a preferred embodiment, the culture is fed with more than two receptors, all of which are constructed with different numbers of monosaccharide subunits.

In another embodiment of the invention, the culture is fed with three or more receptors for oligosaccharide synthesis, wherein the receptors may have the same or different DPs relative to each other.

According to the present invention, the method for producing a mixture comprising at least two oligosaccharides may utilize cells that are not metabolically engineered or may utilize cells that are metabolically engineered, ie metabolically engineered for the manufacture of a mixture comprising at least two oligosaccharides. cells of the mixture of the two oligosaccharides. Throughout the application, unless expressly stated otherwise, "genetically modified cell" or "metabolically engineered cell" preferably means genetically modified or metabolically engineered, respectively Cells engineered for the manufacture of this mixture comprising at least two oligosaccharides according to the invention. In the context of the present invention, at least two oligosaccharides in the mixture as disclosed herein are preferably not present in wild-type precursor cells of the metabolically engineered cell.

According to a preferred embodiment of the method of the invention, metabolically engineered cells for oligosaccharide synthesis are modified with gene expression modules, wherein the expression from any of the expression modules is persistent or borrowed Produced by natural inducers.

These expression modules are also referred to as transcriptional units and comprise polynucleotides for expression of recombinant genes including the coding gene sequence and appropriate transcriptional and/or translational control signals operably linked to the coding gene. Such control signals include promoter sequences, non-translated regions, ribosome binding sites, terminator sequences. These expression modules may contain elements for expression of a single recombinant gene, but may also contain elements for expression of more recombinant genes or may be organized in operon structures for integrated expression of two or more recombinant genes middle. Such polynucleotides can be produced by recombinant DNA technology using techniques well known in the art. Methods of constructing expression modules well known to those of ordinary skill in the art include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. See, eg, techniques described in: Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, CSH, New York or Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989 and updated annually).

According to a preferred embodiment of the present invention, the cells are modified with one or more expression modules. The expression module can be integrated into the genome of the cell or can be presented to the cell on a vector. The vector may be in the form of a plastid, cosmid, phage, liposome or virus, which will be stably transformed/transfected into the metabolically engineered cell. Among them, such vectors include chromosomal, episomal, and virus-derived vectors, for example, vectors derived from bacterial plastids, phages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, and vectors derived therefrom. Combination vectors, such as those derived from plastid and phage genetic elements such as cosmids and phages. Such vectors may contain selectable markers such as, but not limited to, antibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNA sense/antisense markers. A performance system construct may contain control areas that regulate and generate performance. In general, any system or vector suitable for maintaining, amplifying or expressing polynucleotides and/or expressing polypeptides in a host can be used for expression in this regard. Appropriate DNA sequences can be inserted into the expression system by any of a variety of well-known and conventional techniques, such as, for example, those described in Sambrook et al., supra. For recombinant production, cells can be genetically engineered to incorporate an expression system or portion thereof or a polynucleotide of the invention. Introduction of polynucleotides into cells can be accomplished by methods described in a number of standard laboratory manuals, such as Davis et al, Basic Methods in Molecular Biology, (1986) and Sambrook et al, 1989, supra.

As used herein, expression modules comprise polynucleotides for expression of at least one recombinant gene. The recombinant gene is involved in the expression of a polypeptide that functions in the synthesis of the oligosaccharide mixture; or the recombinant gene is associated with other pathways in the host cell that are not involved in the synthesis of the mixture of three or more oligosaccharides . The recombinant genes encode endogenous proteins with modified expression or activity, preferably the endogenous proteins are overexpressed; or the recombinant genes encode heterologous introduction and expression in the modified cells, preferably overexpressed heterologous protein. An endogenous protein can have a modified expression in cells that also express the heterologous protein.

According to a preferred embodiment of the present invention, the expression of each of the expression modules is either persistent or produced by natural inducers. As used herein, persistent expression is understood to mean the expression of genes that are continuously transcribed in an organism. Expression by a natural inducer is to be understood as a facultative or regulatory expression of a gene that is expressed only under certain natural conditions of the host (such as an organism during parturition or during lactation), as a response to environmental changes (such as including ( but not limited to) responses to hormones, heat, cold, light, oxidative or osmotic stress/signaling), or depending on the location of the developmental stage or the cell cycle of the host cell, including but not limited to apoptosis and autonomic body phagocytosis.

According to a preferred embodiment of the present invention, the cells preferably contain multiple copies of the same coding DNA sequence encoding a protein. In the context of the present invention, the protein may be a glycosyltransferase, a membrane transport protein or any other protein as disclosed herein. Throughout this application, the feature "multiple" means at least 2, preferably at least 3, more preferably at least 4, even better at least 5.

According to a preferred embodiment, the present invention provides a method for the manufacture of at least two kinds, preferably at least three kinds, more preferably at least four kinds, even better at least five kinds, even better at least six kinds, even better at least seven kinds Method for mixtures of different oligosaccharides. The method comprises steps as previously disclosed herein. In this context, it is preferred to add various receptors to the culture and/or to provide cells capable of expressing various glycosyltransferases, wherein each desired nucleotide-sugar is preferably synthesized by the cells.

Preferably, the cell manufactures a mixture comprising three different oligosaccharides or more than three different oligosaccharides.

According to another preferred embodiment, the cells produce a mixture of different oligosaccharides comprising at least two oligosaccharides according to the invention which differ in the degree of polymerization.

According to a more preferred embodiment of the method of the present invention, at least one, preferably at least two, more preferably all of the oligosaccharides in the mixture produced according to the present invention are MMOs, preferably HMOs, as defined herein. Throughout this application, unless expressly stated otherwise, the feature "mixture comprising at least two oligosaccharides" is preferably referred to as "mixture comprising at least two MMOs, preferably HMOs" at least two MMOs, preferably HMOs)" is replaced by "mixture comprising at least three MMOs, preferably HMOs" with "mixture comprising at least three MMOs, preferably HMOs" mixture comprising at least three oligosaccharides)”.

In one embodiment of the method according to the invention, the glycosyltransferase is selected from the list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, Mannosyltransferase, N-Acetylglucosaminyltransferase, N-Acetylgalactosaminyltransferase, N-Acetylmannosyltransferase, Xylosyltransferase, Glucuronic acid Glycosyltransferase, galacturonyltransferase, glucosaminyltransferase, N-hydroxyacetylneuraminosyltransferase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP -4-Amino-4,6-dideoxy-N-acetyl-β-L-altrosaminotransferase, UDP-N-acetylglucosamine enolacetonyltransferase and rock Fucosaminyltransferase, as defined herein.

In a preferred embodiment, the fucosyltransferase is selected from the list comprising: α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,4-fucosyltransferase and α-1,6-fucosyltransferase. In another preferred embodiment, the sialyltransferase is selected from the list comprising: α-2,3-sialyltransferase, α-2,6-sialyltransferase and α- 2,8-Sialyltransferase. In another preferred embodiment, the galactosyltransferase is selected from the list comprising: β-1,3-galactosyltransferase, N-acetylglucosamine β-1,3-half Lactosyltransferase, β-1,4-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, α-1,3-galactosyltransferase and α- 1,4-Galactosyltransferase. In another preferred embodiment, the glucosyltransferase is selected from the list comprising: α-glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase enzyme and β-1,4-glucosyltransferase. In another preferred embodiment, the mannosyltransferase is selected from the list comprising α-1,2-mannosyltransferase, α-1,3-mannosyltransferase and α- 1,6-Mannosyltransferase. In another preferred embodiment, the N-acetylglucosaminyltransferase is selected from the list comprising: galactoside beta-1,3-N-acetylglucosaminyltransferase and beta -1,6-N-Acetylglucosaminyltransferase. In another preferred embodiment, the N-acetylgalactosaminyltransferase is α-1,3-N-acetylgalactosaminyltransferase.

In a preferred embodiment, the cells express more than one glycosyltransferase as described herein. Preferably, the cells exhibit at least two, more preferably at least three, even more preferably at least 4 glycosyltransferases, even more preferably at least 5 glycosyltransferases, even more preferably at least 6 glycosyltransferases as described herein transferase.

In another embodiment of the method of the invention, the cell is modified in the expression or activity of the glycosyltransferase. In a preferred embodiment, the glycosyltransferase is an endogenous protein of the cell that has been modified for expression or activity, preferably the endogenous glycosyltransferase is overexpressed; or the glycosyltransferase is heterogeneously introduced and expressed In such cells, overexpressed heterologous proteins are preferred. The endogenous glycosyltransferase can have a modified expression in cells that also express the heterologous glycosyltransferase.

In another embodiment of the method of the invention, the glycosyltransferase is a fucosyltransferase and the cell is capable of synthesizing GDP-Fuc. GDP-fucose can be provided by enzymes expressed in cells or by cellular metabolism. Such cells producing GDP-fucose may express enzymes that convert, for example, fucose added to the cell to GDP-fucose. This enzyme can be, for example, a bifunctional fucosokinase/fucose-1-phosphate guanosyltransferase, such as Fkp from Bacteroides fragilis , or a respective fucose kinase together with a respective A combination of allofucose-1-phosphate guanosyltransferases, as 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 for enhanced GDP-fucose production. The modification may be any one or more selected from the group comprising: UDP-glucose: undecaprenyl-phosphoglucose-1-phosphotransferase-encoding gene deletion, GDP-L-rock Overexpression of the gene encoding alucose synthase, overexpression of the gene encoding GDP-mannose 4,6-dehydratase, overexpression of the gene encoding mannose-1-phosphate guanosyltransferase, phosphomannose mutase Overexpression of encoding genes and overexpression of genes encoding mannose-6-phosphate isomerase.

In another embodiment of the method of the invention, the glycosyltransferase is a sialyltransferase and the cell is capable of synthesizing CMP-Neu5Ac. CMP-Neu5Ac can be provided by enzymes expressed in cells or by cellular metabolism. Such cells producing CMP-Neu5Ac may express enzymes that convert, for example, sialic acid added to the cell to CMP-Neu5Ac. This enzyme can be a CMP-sialic acid synthase, such as N-acylneuraminic acid cytidine acyltransferase from several species including Homo sapiens, Neisseria meningitidis, and Pasteurella multocida . Preferably, the cells are modified to produce CMP-Neu5Ac. More preferably, the cells are modified for enhanced CMP-Neu5Ac production. The modification may be any one or more selected from the group consisting of gene deletion of N-acetylglucosamine-6-phosphate deacetylase, gene deletion of glucosamine-6-phosphate deaminase, Overexpression of a gene encoding sialic acid synthase and overexpression of a gene encoding N-acetyl-D-glucosamine-2-epimerase.

In another embodiment of the method of the invention, the glycosyltransferase is N-acetylglucosaminyltransferase and the cell is capable of synthesizing UDP-GlcNAc. UDP-GlcNAc can be provided by enzymes expressed in cells or by cellular metabolism. Such UDP-GlcNAc-producing cells can express, for example, enzymes that convert GlcNAc to UDP-GlcNAc. Such enzymes may be N-acetyl-D-glucosamine kinase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase from several species including Homo sapiens, Escherichia coli and N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase. Preferably, the cells are modified to produce UDP-GlcNAc. More preferably, the cells are modified for enhanced UDP-GlcNAc production. The modification may be any one or more selected from the group comprising: deletion of N-acetylglucosamine-6-phosphate deacetylase, L-glutamic acid-D-fructose-6-phosphate Overexpression of aminotransferase, overexpression of phosphoglucosamine mutase, and overexpression of N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase .

In another embodiment of the method of the invention, the glycosyltransferase is a galactosyltransferase and the cell is capable of synthesizing UDP-Gal. UDP-Gal can be provided by enzymes expressed in cells or by cellular metabolism. Such cells that produce UDP-Gal may express enzymes that convert, for example, UDP-glucose to UDP-Gal. This enzyme may for example be the UDP-glucose-4-epimerase GalE as known from several species including Homo sapiens, E. coli and brown rat. Preferably, the cells are modified to produce UDP-Gal. More preferably, the cells are modified for enhanced UDP-Gal production. The modification may be any one or more selected from the group comprising: gene deletion of bifunctional 5'-nucleotidase/UDP-glycohydrolase encoding gene, galactose-1-phosphate uridine group Knockout of transferase-encoding gene and overexpression of UDP-glucose-4-epimerase-encoding gene.

In another embodiment of the method of the present invention, the glycosyltransferase is N-acetylgalactosaminyltransferase and the cell is capable of synthesizing UDP-GalNAc. UDP-GalNAc can be provided by enzymes expressed in cells or by cellular metabolism. Such cells producing UDP-GalNAc can express enzymes that convert, for example, UDP-GlcNAc to UDP-GalNAc. This enzyme may be, for example, UDP-N-acetylglucosamine 4-epimerase, such as, for example, wbgU from Plesiomonas shigelloides , gne from Yersinia enterocolitica , or wbpP from Pseudomonas aeruginosa serotype O6. Preferably, the cells are modified to produce UDP-GalNAc. More preferably, the cells are modified for enhanced UDP-GalNAc production. This modification may be an overexpression of UDP-N-acetylglucosamine 4-epimerase.

In another embodiment of the method of the invention, the glycosyltransferase is N-acetylmannosylaminotransferase and the cell is capable of synthesizing UDP-ManNAc. UDP-ManNAc can be provided by enzymes expressed in cells or by cellular metabolism. Such cells producing UDP-ManNAc may express enzymes that convert, for example, UDP-GlcNAc to UDP-ManNAc. This enzyme may be eg a UDP-GlcNAc 2-epimerase such as eg cap5P from Staphylococcus aureus, RffE from Escherichia coli, Cps19fK from S. pneumoniae or from Salmonella enterica RfbC of ( S. enterica ). Preferably, the cells are modified to produce UDP-ManNAc. More preferably, the cells are modified for enhanced UDP-ManNAc production. This modification may be an overexpression of UDP-GlcNAc 2-epimerase.

In another embodiment of the method of the invention, the cell is capable of synthesizing any of the nucleotide-sugars selected from the list comprising: GDP-Fuc, CMP-Neu5Ac , UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man) , UDP-glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy--L-arabinose-4-hexulose, UDP-2-acetamido-2, 6-dideoxy--L-lyxose-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-acetamide 2,6-dideoxy-L-galactose), UDP-N-acetamido-L-neumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6- Dideoxy-L-talose), UDP-N-Acetylmuramic acid, UDP-N-Acetyl-L-Isorhamnosamine (UDP-L-QuiNAc or UDP-2-Acetyl Amino-2,6-dideoxy-L-glucose), GDP-L-isorhamnose, CMP-N-hydroxyacetylneuraminic 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 ester, UDP-galacturonic acid ester, GDP-rhamnose, UDP-xylose. In a preferred embodiment, the cell is metabolically engineered for the manufacture of nucleotide-sugars. In another preferred embodiment, the cells are modified and/or engineered for optimal production of nucleotide-sugars.

According to the method of the present invention, at least one of the oligosaccharides of the mixture is fucosylated, sialylated, galactosylated, glycosylated, xylosylated, mannosylated, containing N-acetylglucosamine, containing N-acetylneuraminic acid, containing N-hydroxyacetylneuraminic acid, containing N-acetylgalactosamine, containing rhamnose, containing glucuronide, Contains galacturonic acid esters and/or N-acetylmannosamine.

In a preferred embodiment, the oligosaccharide mixture comprises charged oligosaccharides. More preferably, at least one of the charged oligosaccharides is a sialylated oligosaccharide. Alternatively and or additionally, the oligosaccharide mixture comprises neutral oligosaccharides.

In another preferred embodiment, the oligosaccharide mixture comprises fucosylated neutral oligosaccharides. Alternatively and or additionally, the oligosaccharide mixture comprises unfucosylated neutral oligosaccharides.

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

Alternatively or additionally, the oligosaccharide mixture comprises at least one sialylated oligosaccharide as defined herein.

Alternatively or additionally, the mixture of oligosaccharides comprises at least one oligosaccharide having three or more monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of the monosaccharide residues is a GlcNAc residue. The oligosaccharide may contain more than one GlcNAc residue, eg, two, three or more. The oligosaccharide may be neutral or charged, eg also comprising a sialic acid structure. GlcNAc can be present at the reducing end of the oligosaccharide. The GlcNAc can also be present at the non-reducing end of the oligosaccharide. The GlcNAc can also be present within the oligosaccharide structure. GlcNAc can be linked to other monosaccharide subunits including galactose, fucose, Neu5Ac, Neu5Gc.

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

Preferably, all oligosaccharides present in the oligosaccharide mixture are fucosylated oligosaccharides. More preferably, the oligosaccharide mixture comprises three fucosylated oligosaccharides as defined herein.

According to a preferred embodiment, the present invention provides a method for the manufacture of at least two kinds, preferably at least three kinds, more preferably at least four kinds, even better at least five kinds, even better at least six kinds, even better at least seven kinds A method of a mixture of different oligosaccharides: (i) oligosaccharides, wherein the mixture consists of charged, preferably sialylated and neutral oligosaccharides (preferably neutral fucosylated, or preferably neutral unrocked) (ii) neutral fucosylated oligosaccharides; (iii) sialylated oligosaccharides; and/or (iv) neutral unfucosylated oligosaccharides. The method comprises steps as previously disclosed herein. In this context, preferably at least two, more preferably at least three receptors are added to the culture and/or cells capable of expressing a variety of glycosyltransferases are provided, wherein each desired nucleotide-sugar is preferably synthesized by the cells .

In this regard, for the production of the mixture according to (i) above, it is preferred to provide a cell, preferably a single cell, capable of expressing a sialyltransferase and at least one additional glycosyltransferase as described herein, synthesizing a nucleotide- The sugar CMP-N-acetylneuraminic acid (CMP-Neu5Ac) and one or more nucleotide-sugars as described herein for use in the additional glycosyltransferase Donors, and preferably at least three, more preferably at least four, even more preferably at least five acceptors are added to the culture for the synthesis of any of the oligosaccharides in the mixture, preferably All of the oligosaccharides in the mixture were synthesized.

In this regard, for the production of the mixture according to (ii) above, it is preferred to provide a cell, preferably a single cell, capable of expressing a fucosyltransferase and at least one additional glycosyltransferase as described herein, synthesizing nucleotides - the sugar GDP-fucose (GDP-Fuc) and one or more nucleotide-sugars as described herein which are donors for the additional glycosyltransferase, and Preferably at least three, more preferably at least four, even more preferably at least five receptors are added to the culture for the synthesis of any of the oligosaccharides in the mixture, preferably for the synthesis of the mixture all of these oligosaccharides.

In this regard, for producing the mixture according to (iii) above, preferably a cell, preferably a single cell is provided, which is capable of expressing a sialyltransferase and at least one additional glycosyltransferase as described herein, synthesizing a nucleotide- The sugar CMP-N-acetylneuraminic acid (CMP-Neu5Ac) and one or more nucleotide-sugars as described herein for use in the additional glycosyltransferase Donors, and preferably at least three, more preferably at least four, even more preferably at least five acceptors are added to the culture for the synthesis of any of the oligosaccharides in the mixture, preferably All of the oligosaccharides in the mixture were synthesized.

In this regard, for the production of the mixture according to (iv) above, it is preferred to provide a cell, preferably a single cell, capable of expressing at least two glycosyltransferases as described herein, synthesizing one or more nucleosides as described herein Acid-sugars, the one or more nucleotide-sugars are donors for the additional glycosyltransferases, and preferably at least three, more preferably at least four, even more preferably at least five acceptors are added to the Culturing for the synthesis of any of the oligosaccharides in the mixture, preferably for the synthesis of all of the oligosaccharides in the mixture.

In the context of the present invention, it is understood that the mixture comprising at least two oligosaccharides according to the present invention is preferably synthesized intracellularly. Those of ordinary skill in the art will further appreciate that some or substantially all of the synthesized oligosaccharides remain intracellularly and/or are excreted extracellularly via passive or active transport.

In a preferred embodiment of the method of the invention, any of the oligosaccharides, more preferably all of the oligosaccharides, is consumed by passive transport, ie without the aid of an active transport system, from the cell energy is displaced outside the cell.

In another preferred embodiment of the method of the present invention, the cells are further metabolically engineered for use in i) Modified expression of endogenous membrane proteins, and/or ii) the modified activity of endogenous membrane proteins, and/or iii) Representation of homologous membrane proteins, and/or iv) performance of heterologous membrane proteins, wherein the membrane protein is involved in the extracellular secretion of any of the oligosaccharides. The cell may express one of the membrane proteins involved in the secretion of either of the oligosaccharides from the cell to the outside of the cell. The cell may also express more than one of the membrane proteins. Any of the membrane proteins can translocate one or more of the oligosaccharides outside the cell. The cell producing a mixture of at least two, preferably at least three, oligosaccharides can translocate any of the oligosaccharides including passive diffusion, channel membrane proteins, membrane transport proteins , Membrane carrier protein.

In one embodiment, the cells are further metabolically engineered for use in i. Modified expression of endogenous membrane proteins, and/or ii. Modified activity of endogenous membrane proteins, and/or iii. Representation of homologous membrane proteins, and/or iv. Representation of heterologous membrane proteins, wherein the membrane protein is involved in the uptake of precursors for the synthesis of any of the oligosaccharides in the mixture, preferably wherein the membrane protein is involved in the uptake of all desired precursors, more preferably wherein the membrane protein is involved in uptake all such receptors.

In another preferred embodiment of the method of the present invention, the cells are further metabolically engineered for use in i) Modified expression of endogenous membrane proteins, and/or ii) the modified activity of endogenous membrane proteins, and/or iii) Representation of homologous membrane proteins, and/or iv) performance of heterologous membrane proteins, wherein the membrane protein is involved in uptake of receptors for the synthesis of any of the oligosaccharides. As explained in the definitions disclosed herein, the term "acceptor" should be understood. The cell may express one of the membrane proteins involved in uptake of any type of receptor used to synthesize any of the oligosaccharides. The cell may also express more than one of the membrane proteins involved in the uptake of at least one of the receptors. The cells can be modified for uptake of more than one receptor for the synthesis of any of the oligosaccharides.

In a more preferred embodiment of the method of the present invention, the membrane protein is selected from the list comprising: a transporter protein, a P-P-bond hydrolysis driven transporter protein, a β-barrel porin, an accessory transporter protein, a putative transporter protein and phosphotransfer driven group translocation proteins. In an even more preferred embodiment of the method of the invention, the transporter protein comprises MFS transporter, carbohydrate efflux transporter and chelatin exporter. In another more preferred embodiment of the method of the present invention, the P-P-bond hydrolysis-driven transporter comprises ABC transporter and chelatin exporter.

In another preferred embodiment of the method of the invention, the membrane protein provides for the improved manufacture of this mixture of at least two oligosaccharides. In an alternative and/or additional preferred embodiment of the method of the present invention, the membrane protein provides for the efflux of this mixture of at least two oligosaccharides.

In a more preferred embodiment of the method of the present invention, the cells express membrane proteins belonging to the MFS transporter family, such as for example derived from Escherichia coli (UniProt ID P0AEY8), Cronobacter muytjensii MdfA polypeptides of the MdfA family of multidrug transport proteins of species (UniProt ID A0A2T7ANQ9), Citrobacter youngae (UniProt ID D4BC23) and Yokenella regensburgei (UniProt ID G9Z5F4). In another more preferred embodiment of the method of the invention, the cells express membrane proteins belonging to the family of sugar efflux transporters, such as those derived from, for example, Escherichia coli (UniProt ID P31675), Citrobacter koseri ) (UniProt ID A0A078LM16) and SetA polypeptides of the SetA family of species Klebsiella pneumoniae (UniProt ID A0A0C4MGS7). In another more preferred embodiment of the method of the invention, the cells express membrane proteins belonging to the chelatin export protein family, such as eg E. coli entS (UniProt ID P24077) and E. coli iceT (UniProt ID A0A024L207) . In another more preferred embodiment of the method of the invention, the cells express membrane proteins belonging to the ABC transporter family, such as eg oppF from Escherichia coli (UniProt ID P77737), from Lactococcus lactis subsp. lactis II lmrA (UniProt ID A0A1V0NEL4) of Lactococcus lactis subsp. lactis bv. diacetylactis and Blon_2475 (UniProt ID B7GPD4) from Bifidobacterium longum subsp. infantis.

Optionally, the cell is transformed to comprise at least one nucleic acid sequence encoding a protein selected from the group consisting of: lactose transporter; N-acetylneuraminic acid transporter, fucose transporter; for nucleotides Activates a carbohydrate transporter, wherein the transporter is internalized into the medium to which precursors or receptors are added for oligosaccharide synthesis.

In a preferred embodiment of the method according to the present invention, cells are resistant to lactose killing when they are grown in an environment where lactose is combined with one or more other carbon sources. In the context of the term "lactose killing" it is meant that cell growth in a medium in which lactose and another carbon source are present is stunted. In a preferred embodiment, the cells are genetically modified such that they maintain at least 50% influx of lactose without undergoing lactose kill, even at high lactose concentrations, as described in WO 2016/075243. The genetic modification comprises expression and/or overexpression of an exogenous and/or endogenous lactose transporter gene by a heterologous promoter that does not cause a lactose-killing phenotype and/or the codon of the lactose transporter Modifications used by the gene to produce altered performance of the lactose transporter protein that do not cause the lactose-killing phenotype. The contents of WO 2016/075243 are incorporated by reference in this regard.

In another embodiment of the present invention, the receptors are produced by methods comprising extraction from natural sources; biotechnological processes comprising by the cell or another producing the oligosaccharide mixture according to the present invention Cellular synthesis that does not produce the oligosaccharide mixture according to the invention; physical processes; chemical processes and combinations thereof.

In another preferred embodiment of the method, the cell produces a receptor for the synthesis of any of the oligosaccharides. In a more preferred embodiment, the cell produces one or more receptors for the synthesis of the oligosaccharide mixture, more preferably, the cell produces all receptors for the synthesis of the oligosaccharide mixture according to the invention. In an even more preferred embodiment, the cell is modified to optimize the production of any of the receptors for the synthesis of any of the oligosaccharides, more preferably, the cell is modified to optimize All the receptors are generated for the synthesis of the oligosaccharide mixture according to the invention.

In another preferred embodiment of the method, the receptor added to the culture is not produced by the cells provided in the method according to the invention.

In a preferred embodiment of the method of the present invention, the cell confers enhanced phage resistance. This enhancement of phage resistance may result from reduced expression of endogenous membrane proteins and/or mutations in genes encoding endogenous membrane proteins. The terms "phage insensitive" or "phage resistant" or "phage resistance" or "phage resistant profile" should be understood to mean and/or growth inhibition of infection and/or killing of less susceptible and preferably less susceptible bacterial strains. As used herein, the term "anti-phage activity" or "resistant to infection by at least one phage" refers to a system that exhibits a functional phage resistance system Increased resistance of bacterial cells to infection by at least one family of bacteriophages, as can be achieved by, for example, bacterial viability, compared to bacterial cells of the same species at the same developmental stage (eg, in culture) that do not exhibit a functional phage resistance system , phage lysogenicity, phage genome replication, and phage genome degradation. Phages can be lytic phages or mild (lysogenic) phages. Membrane proteins involved in phage resistance of cells include OmpA, OmpC, OmpF, OmpT, BtuB, TolC, LamB, FhuA, TonB, FadL, Tsx, FepA, YncD, PhoE and NfrA and their homologues.

In a preferred embodiment of the method of the present invention, the cells impart reduced viscosity. Reduction in cell viscosity can be obtained by biosynthesis of modified cell walls. Cell wall biosynthesis can be modified to include, for example, poly-N-acetyl-glucosamine, enterobacterial common antigen, cellulose, colanic acid, nuclear oligosaccharides, osmoregulatory periplasmic glucans, and glycerol The synthesis of glucosides, polysaccharides and cocoon honey is reduced or eliminated.

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

In a preferred embodiment and as a means for enhanced production and/or supply of PEP, one or more PEP-dependent, sugar transport phosphotransferase systems are disrupted, such as (but not limited to): 1) N- Acetyl-D-glucosamine Npi-phosphotransferase (EC 2.7.1.193), which is encoded by, for example, the nagE gene (or cluster nagABCD) in E. coli or Bacillus species, 2) ManXYZ, which encodes imported extraneous Source hexose (mannose, glucose, glucosamine, fructose, 2-deoxyglucose, mannosamine, N-acetylglucosamine, etc.) and the enzyme ll Man complex (mannose PTS permease, protein-Npi-phosphohistidine-D-mannose phosphotransferase), 3) glucose-specific PTS transporter (encoded by PtsG/Crr for example), which takes up glucose in the cytoplasm and forms glucose -6-phosphate, 4) sucrose-specific PTS transporter protein, which takes up sucrose in the cytoplasm and forms sucrose-6-phosphate, 5) fructose-specific PTS transporter protein (encoded for example by the genes fruA and fruB and a kinase Coding for fruK, the kinase fruK takes up fructose and forms fructose-1-phosphate in a first step and fructose 1,6 diphosphate in a second step, 6) Lactose PTS transporter (eg by Lactococcus casei) (lacE code in Lactococcus casei), which absorbs lactose and forms lactose-6-phosphate, 7) galactitol-specific PTS enzymes, which absorbs galactitol and/or sorbitol and forms galactitol-1, respectively - Phosphate or sorbitol-6-phosphate, 8) Mannitol-specific PTS enzymes that absorb mannitol and/or sorbitol and form mannitol-1-phosphate or sorbitol-6-phosphate, respectively salt, and 9) molasses-specific PTS enzymes that absorb molasses and form molasses-6-phosphate.

In another and/or additional preferred embodiment and as a means for enhanced production and/or supply of PEP, the complete PTS system 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 (PTS sugar) of E. coli K-12. PtsI is one of the two sugar-nonspecific protein components of PTS sugars (PtsI and PtsH), which together with sugar-specific endomembrane permease enzymes achieve a phosphotransfer stage that results in coupled phosphorylation and transport of various carbohydrate substrates link. HPr (histidine-containing protein) is one of two sugar-nonspecific protein components of PTS sugars. It accepts the phosphooxyl group from phosphorylase I (PtsI-P) and then transfers it to the EIIA domain of any of the many carbohydrate-specific enzymes (collectively referred to as Enzyme II) of the PTS carbohydrate. In reactions requiring PtsH and PtsI, Crr or EIIAGlc are phosphorylated by PEP.

In another and/or additional preferred embodiment, the cells are further modified to compensate for the absence of the PTS system of the carbon source by the introduction and/or overexpression of the corresponding permease. etc. are, for example, permease or ABC transport proteins including, but not limited to, transport proteins that specifically import lactose, such as those encoded by the LacY gene from E. coli; transport proteins that import sucrose, such as those encoded by cscB from E. coli a gene-encoded transporter; a glucose-import transporter, such as the transporter encoded by the galP gene from E. coli; a fructose-import transporter, such as the transporter encoded by the fruI gene from Streptococcus mutans ; or are sorbitol/mannitol ABC transporters, such as those encoded by the cluster SmoEFGK of Rhodobacter sphaeroides; cocoon honey/sucrose/maltose transporters, such as those encoded by the genes of Sinorhizobium meliloti A transporter protein encoded by the cluster ThuEFGK; and an N-acetylglucosamine/galactose/glucose transporter protein, such as the transporter protein encoded by NagP of Shewanella oneidensis . Examples of combinations of PTS deletion and overexpression of alternative transporters are: 1) deletion of a glucose PTS system (eg ptsG gene) combined with introduction and/or overexpression of a glucose permease (eg galP of glcP), 2) fructose PTS Deletion of system (eg one or more of fruB, fruA, fruK genes) combined with introduction and/or overexpression of fructose permease (eg fruI), 3) deletion of lactose PTS system with lactose permease (eg LacY) and/or 4) deletion of the sucrose PTS system combined with the introduction and/or overexpression of a sucrose permeating enzyme such as cscB.

In another preferred embodiment, cells are modified 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) to compensate for the lack of carbon source PTS system. Examples of combinations of PTS deletion with overexpression of alternative transport proteins 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), and Introduction and/or combination of overexpression of glucokinase (eg glk), and/or 2) deletion of fructose PTS system (eg one or more of fruB, fruA, fruK genes), and fructose permease (eg fruI) Combination of introduction and/or overexpression, combined with introduction and/or overexpression of fructokinase (eg frk or mak).

In another and/or additional preferred embodiment and as a means for the enhanced production and/or supply of PEP, cells by introducing any one or more of a list comprising or in a Modification of any one or more aspects of the list to modify: phosphoenolpyruvate synthase activity (EC: 2.7.9.2, eg encoded by ppsA in E. coli), phosphoenolpyruvate carboxykinase activity (EC 4.1.1.32 or EC 4.1.1.49, for example encoded by PCK in Corynebacterium glutamicum or by pckA in E. coli, respectively); phosphoenolpyruvate carboxylase activity (EC 4.1 .1.31, for example in E. coli encoded by ppc); oxaloacetate decarboxylase activity (EC 4.1.1.112, for example in E. coli encoded by eda); pyruvate kinase activity (EC 2.7.1.40 , for example in E. coli by pykA and pykF); pyruvate carboxylase activity (EC 6.4.1.1, for example in Bacillus subtilis by pyc); and malate dehydrogenase activity ( EC 1.1.1.38 or EC 1.1.1.40, for example in E. coli encoded by maeA or maeB, respectively).

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

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

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

In an exemplary embodiment, cells are genetically modified by different adaptations such as overexpression of phosphoenolpyruvate synthase in combination with deletion of the pyruvate kinase gene, overexpression of phosphoenolpyruvate synthase Expression combined with deletion of phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase combined with deletion of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase and pyruvate kinase Deletion combination of genes, overexpression of phosphoenolpyruvate carboxykinase and deletion of phosphoenolpyruvate carboxylase gene, combination of overexpression of phosphoenolpyruvate carboxykinase and deletion of pyruvate carboxylase gene, Overexpression of oxaloacetate decarboxylase combined with deletion of pyruvate kinase gene, combination of overexpression of oxaloacetate decarboxylase and deletion of phosphoenolpyruvate carboxylase gene, overexpression of oxaloacetate decarboxylase and pyruvate Deletion combination of carboxylase gene, overexpression of malate dehydrogenase and deletion combination of pyruvate kinase gene, combination of deletion of malate dehydrogenase overexpression and phosphoenolpyruvate carboxylase gene and/or apple Overexpression of acid dehydrogenase combined with deletion of the pyruvate carboxylase gene.

In another exemplary embodiment, the cells are genetically modified by different adaptations such as overexpression of phosphoenolpyruvate synthase in combination with overexpression of phosphoenolpyruvate carboxykinase, phosphoenolacetone Overexpression of acid synthase combined with overexpression of oxaloacetate decarboxylase, overexpression of phosphoenolpyruvate synthase combined with overexpression of malate dehydrogenase, overexpression of phosphoenolpyruvate carboxykinase and grass Overexpression combination of oxalate decarboxylase, combination of overexpression of phosphoenolpyruvate carboxykinase and overexpression of malate dehydrogenase, combination of overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase, phosphate Overexpression of enolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase, overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase Overexpression and combination of overexpression of malate dehydrogenase, overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase Combination of overexpression, overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and combination of overexpression of malate dehydrogenase and/or overexpression of phosphoenolpyruvate synthase and oxaloacetate Overexpression of decarboxylase combined with overexpression of malate dehydrogenase.

In another exemplary embodiment, the cells are genetically modified by different adaptations such as overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and between pyruvate kinase genes Combination of deletions, overexpression of phosphoenolpyruvate synthase and overexpression of oxalate decarboxylase and combination of deletions of pyruvate kinase gene, overexpression of phosphoenolpyruvate synthase and overexpression of malate dehydrogenase And the combination of deletion of pyruvate kinase gene, overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and combination of deletion of pyruvate kinase gene, overexpression of phosphoenolpyruvate carboxykinase and malate Overexpression of dehydrogenase and combination of deletion of pyruvate kinase gene, overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and combination of deletion of pyruvate kinase gene, excess of phosphoenolpyruvate synthase Expression combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and deletion of the pyruvate kinase gene, overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase Overexpression and overexpression of malate dehydrogenase and deletion combinations of pyruvate kinase genes, overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and Overexpression of malate dehydrogenase and deletion combination of pyruvate kinase gene, overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and pyruvate kinase gene Combination of deletions, overexpression of phosphoenolpyruvate synthase and overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and deletion of the pyruvate kinase gene.

In another exemplary embodiment, cells are genetically modified by different adaptations such as overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate Deletion combination of carboxylase gene, overexpression of phosphoenolpyruvate synthase and overexpression of oxaloacetate decarboxylase and deletion combination of phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase Expression combined with overexpression of malate dehydrogenase and deletion of phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and phosphoenolpyruvate carboxylate Deletion combination of phosphoenolpyruvate gene, overexpression of phosphoenolpyruvate carboxykinase and overexpression of malate dehydrogenase and deletion combination of phosphoenolpyruvate carboxylase gene, overexpression of oxaloacetate decarboxylase and apple Overexpression of acid dehydrogenase and deletion combination of phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase Expression and combination of deletions in the phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and overexpression of malate dehydrogenase and phosphoenolacetone Deletion combination of acid carboxylase gene, overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and phosphorylation Deletion combination of enolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and phosphoenolpyruvate carboxylase gene A combination of deletions, overexpression of phosphoenolpyruvate synthase and overexpression of oxaloacetate decarboxylase and overexpression of malate dehydrogenase and a combination of deletions of the phosphoenolpyruvate carboxylase gene.

In another exemplary embodiment, the cells are genetically modified by different adaptations such as overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and pyruvate carboxylase Deletion combination of genes, overexpression of phosphoenolpyruvate synthase and overexpression of oxalate decarboxylase and deletion combination of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase and malate dehydrogenase Enzyme overexpression and combination of deletion of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and combination of deletion of pyruvate carboxylase gene, phosphoenolpyruvate Overexpression of carboxykinase and overexpression of malate dehydrogenase and deletion of pyruvate carboxylase gene combination, overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and combination of pyruvate carboxylase gene Deletion combination, overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and deletion combination of pyruvate carboxylase gene, phosphoenolpyruvate synthase Enzyme overexpression and phosphoenolpyruvate carboxykinase overexpression and malate dehydrogenase and pyruvate carboxylase gene deletion combinations, phosphoenolpyruvate synthase overexpression and phosphoenolacetone Overexpression of acid carboxykinase and overexpression of oxaloacetate decarboxylase and overexpression of malate dehydrogenase and deletion combination of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase and oxalate decarboxylation Enzyme overexpression and malate dehydrogenase and deletion combination of pyruvate carboxylase gene, phosphoenolpyruvate synthase and oxalate decarboxylase overexpression and malate dehydrogenase A combination of overexpression and deletion of the pyruvate carboxylase gene.

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

In another exemplary embodiment, cells are genetically modified by different adaptations such as overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and pyruvate kinase genes and Deletion combination of pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase and overexpression of oxaloacetate decarboxylase, and pyruvate kinase gene and pyruvate carboxylase Deletion combination of gene and phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase and overexpression of malate dehydrogenase and pyruvate kinase gene and pyruvate carboxylase gene and phosphoenol Deletion combination of pyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and pyruvate kinase gene and pyruvate carboxylase gene and phosphoenolpyruvate carboxylase Deletion combination of genes, overexpression of phosphoenolpyruvate carboxykinase and overexpression of malate dehydrogenase, and deletion combination of pyruvate kinase gene and pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, Overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and deletion combination of pyruvate kinase gene and pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, phosphoenolpyruvate synthase Overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and deletion combination of pyruvate kinase gene and pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, phosphate Overexpression of enolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and overexpression of malate dehydrogenase and pyruvate kinase gene and pyruvate carboxylase gene and phosphoenolpyruvate carboxylase Deletion combinations of genes, overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and pyruvate kinase genes and Deletion combination of pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase and acetone Deletion combination of acid kinase gene and pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, overexpression of phosphoenolpyruvate synthase and overexpression of oxalate decarboxylase and malate dehydrogenase Overexpression and combination of deletions of the pyruvate kinase gene and the pyruvate carboxylase gene and the phosphoenolpyruvate carboxylase gene.

According to another preferred embodiment of the method of the present invention, the cells comprise modifications for reducing acetate production compared to unmodified precursor cells. The modification may be any one or more selected from the group comprising: overexpression of acetyl-CoA synthase, complete or partial deletion or rendering of pyruvate dehydrogenase less functional and complete or partial deletion Lactate dehydrogenase may cause its function to be weak.

In another embodiment of the methods of the invention, the cells are modified in the expression or activity of at least one acetyl-CoA synthetase, such as, eg, acs from Escherichia coli, Saccharomyces cerevisiae, Homo sapiens, mouse. In a preferred embodiment, the acetyl-CoA synthetase is an endogenous protein of the cell with modified expression or activity, preferably the endogenous acetyl-CoA synthase is overexpressed; or , the acetyl-CoA synthetase is heterogeneously introduced and expressed in the cell, preferably a heterologous protein that is overexpressed. The endogenous acetyl-CoA synthetase can have a modified expression in cells that also express the heterologous acetyl-CoA synthase. In a more preferred embodiment, the cells are modified in the expression or activity of the acetyl-CoA synthase acs (UniProt ID P27550) from E. coli. In another and/or additional preferred embodiment, the cell has at least 80% overall sequence identity with the full length of the polypeptide from Escherichia coli (UniProt ID P27550) and has acetyl-CoA synthetase activity from a Functional homologues, variants or derivatives of E. coli (UniProt ID P27550) modified in performance or activity.

In alternative and/or additional other embodiments of the methods of the invention, the cells are modified in the expression or activity of at least one pyruvate dehydrogenase, such as, for example, from Escherichia coli, Saccharomyces cerevisiae, Homo sapiens, and brown mouse. In a preferred embodiment, the cell has been modified to have at least one partially or completely deleted or mutated gene encoding pyruvate dehydrogenase by means generally known to those of ordinary skill in the art to produce at least one acetone A protein with weak or incapacitated acid dehydrogenase activity. In a more preferred embodiment, the cells have a complete knockout of the gene encoding poxB, resulting in cells lacking pyruvate dehydrogenase activity.

In alternative and/or additional other embodiments of the methods of the invention, the cells are modified in the expression or activity of at least one lactate dehydrogenase, such as, for example, from Escherichia coli, Saccharomyces cerevisiae, Homo sapiens, and brown mouse. In a preferred embodiment, the cell has been modified to have at least one partially or fully deleted or mutated lactate dehydrogenase-encoding gene by means generally known to those of ordinary skill in the art to produce at least one functionally less or A protein incapacitated by lactate dehydrogenase activity. In a more preferred embodiment, the cell has a complete knockout of the gene encoding ldhA, resulting in a lack of lactate dehydrogenase activity in the cell.

According to another preferred embodiment of the method of the present invention, the cell comprises any of the following proteins comprising reduced or reduced expression and/or abrogated, attenuated, reduced or delayed activity compared to unmodified precursor cells One or more of: β-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N -Acetyl glucosamine arrestin, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecyl isopentenyl-phosphoglucose-1-phosphotransferase, L-fuculokinase ), L-fucose isomerase, N-acetylneuraminic acid lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epomerase , 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 control protein, transcriptional repressor protein IclR, lon protease, glucose-specific shifter Phosphophosphotransferase IIBC component ptsG, glucose-specific translocation phosphotransferase (PTS) IIBC component malX, enzyme IIA ^Glc , β-glucoside-specific PTS enzyme II, fructose-specific PTS polyphosphooxytransferase FruA And FruB, alcohol dehydrogenase aldehyde dehydrogenase, pyruvate-formate dissociation enzyme, acetate kinase, phosphoacyltransferase (phosphoacyltransferase), phosphoacetyltransferase, pyruvate decarboxylase.

According to another preferred embodiment of the method of the present invention, the cell comprises a catabolic pathway for the selected monosaccharide, disaccharide or oligosaccharide, the catabolic pathway being at least partially inactivated, the monosaccharide, disaccharide or oligosaccharide Participate in and/or be required for the production of this mixture of at least two oligosaccharides.

In another embodiment of the method of the invention, at least one of the oligosaccharides in the mixture produced by the cell is a mammalian milk oligosaccharide. The cell can produce a mammalian milk oligosaccharide in a mixture of the produced at least two oligosaccharides. The cells can produce more than one mammalian milk oligosaccharide in the mixture of the produced at least two oligosaccharides. In a preferred embodiment, all of the oligosaccharides in the produced mixture of at least two oligosaccharides are mammalian milk oligosaccharides. Throughout the application, unless expressly stated otherwise, the characteristics "oligosaccharide" or "oligosaccharides" are preferably represented by "mammalian milk oligosaccharide (MMO), preferably human milk oligosaccharide (HMO), respectively. (mammalian milk oligosaccharide (MMO), preferably human milk oligosaccharide (HMO))” or “mammalian milk oligosaccharide (MMO), preferably human milk oligosaccharide (HMO) (mammalian milk oligosaccharides (MMOs), preferably human milk oligosaccharides (HMOs)” ))"replace.

Therefore, a preferred method according to the present invention is a method for producing a mixture comprising at least two MMOs, preferably HMOs, the method comprising the steps as described herein. Preferably, the mixture comprises at least three, more preferably at least four, even better at least five, even better at least six, even better at least seven different MMOs (preferably HMOs).

In another embodiment of the method of the invention, any of the oligosaccharides produced by the cell is an antigen of the human ABO blood group system. In a more preferred embodiment, all of the oligosaccharides produced by the cell are antigens of the human ABO lineage system. Therefore, a preferred method according to the present invention is a method for producing a mixture comprising at least two antigens of the human ABO blood group system, the method comprising the steps as described herein. Preferably, the mixture comprises at least three different antigens of the human ABO blood group system.

Another embodiment of the present invention provides a method and a cell wherein a mixture comprising at least two different oligosaccharides is produced in a fungal, yeast, bacterial, insect, animal, plant or protozoal cell as described herein and/or or resulting from it. The cell line is selected from a list comprising bacteria, yeast, protozoa or fungi, or refers to plant or animal cells. The latter bacterium preferably belongs to the phylum Proteobacteria or Firmicutes or Cyanobacteria or Deinococcus-Thermus. A bacterium belonging to the latter phylum Proteobacteria preferably belongs to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacteria preferably refers to any strain belonging to the species Escherichia coli, such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term refers to a cultured E. coli strain (designated E. coli K12 strain) that is well suited to laboratory settings and that, unlike wild-type strains, has lost its ability to thrive in the gut . Well-known examples of E. coli K12 strains are K12 wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Therefore, the present invention specifically relates to a mutated and/or transformed E. coli cell or strain as indicated above, wherein the E. coli strain is the K12 strain. The Escherichia coli K12 strain is more preferably Escherichia coli MG1655. A bacterium belonging to the phylum Firmicutes is preferably of the class Bacilli, preferably of the order Lactobacilliales having a member such as Lactobacillus lactis , Leuconostoc mesenteroides , or having Such as Bacillales from members of the genus Bacillus, such as Bacillus subtilis or B. amyloliquefaciens . A bacterium belonging to the phylum Actinobacteria, preferably belonging to the Corynebacteriaceae family with members Corynebacterium glutamicum or C. afermentans , or belonging to the family Streptomyces griseus ( Streptomyces griseus ) or the Streptomycetaceae family of 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 (with members such as, for example, Saccharomyces cerevisiae, S. bayanus , S. boulardii ), Pichia (with members such as For example, Pichia pastoris ( Pichia pastoris ), Pichia anomala ( P. anomala ), Pichia kluyveri ( P. kluyveri members), Komagataella (Komagataella), Hansenula ( Hansenula), Kluyveromyces (with members such as eg Kluyveromyces lactis , K. marxianus , K. thermotolerans ), Debaromyces, Yarrowia (such as, for example, Yarrowia lipolytica) or Starmerella (such as, for example, Starmerella bombicola )). The latter yeast is preferably selected from Pichia pastoris , Yarrowia lipolytica , Saccharomyces cerevisiae and Kluyveromyces lactis . The latter fungus preferably belongs to the genus Rhizopus , Dictyostelium , Penicillium , Mucor or Aspergillus . Plant cells include cells of flowering and non-flowering plants, as well as algal cells such as Chlamydomonas, Chlorella, and the like. Preferably, the plant is a tobacco, alfalfa, rice, tomato, cotton, rapeseed, soybean, maize or corn plant. The latter animal cells are preferably derived from non-human mammals (eg cattle, buffalo, pigs, sheep, mice, rats), birds (eg chickens, ducks, ostriches, turkeys, pheasants), fish (eg swordfish, salmon, tuna, sea bass, trout, catfish), invertebrates (e.g. lobster, crab, shrimp, clams, oysters, mussels, sea urchins), reptiles (e.g. snakes, crocodiles, turtles), amphibians ( eg frogs) or insects (eg flies, nematodes) or are genetically modified cell lines derived from human cells devoid of embryonic stem cells. Both human and non-human mammalian cells are preferably selected from a list comprising: epithelial cells such as eg mammary epithelial cells, embryonic kidney cells (eg HEK293 or HEK 293T cells), fibroblasts, COS cells, Chinese hamster ovary ( Chinese hamster ovary; CHO) cells, murine myeloma cells such as eg N20, SP2/0 or YB2/0 cells, NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, such as described in WO21067641. The latter insect cells are preferably derived from: Spodoptera frugiperda such as eg Sf9 or Sf21 cells, Bombyx mori , Mamestra brassicae , Trichoplusia ni such as eg BTI- TN-5B1-4 cells or Drosophila melanogaster such as Drosophila S2 cells. The latter protozoal cell is preferably a Leishmania tarentolae cell.

In a preferred embodiment of the method of the invention, the cells are viable Gram-negative bacteria comprising reduced or eliminated synthetic poly-N-acetyl- Glucosamine (poly-N-acetyl-glucosamine; PNAG), Enterobacterial Common Antigen (ECA), cellulose, kolac acid, nuclear oligosaccharide, Osmoregulated Periplasmic Glucan; OPG), glycerol glucoside, polysaccharide and/or cocoon honey.

In a more preferred embodiment of the method, the reduction or elimination of synthetic poly-N-acetyl-glucosamine (PNAG), enterobacterial common antigen (ECA), cellulose, kolaric acid, ribo-oligosaccharide, Osmotic regulation of periplasmic glucan (OPG), glycerol glucoside, polysaccharide and/or cocoon sugar by participating in the poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA) , any one or more sugar groups in the synthesis of any one of cellulose, kolaric acid, ribo-oligosaccharide, osmoregulatory periplasmic glucan (OPG), glycerol glucoside, polysaccharide and/or cocoon Mutations of transferases provide for deletion or lower expression of any of the glycosyltransferases. These glycosyltransferases include the glycosyltransferase gene encoding the poly-N-acetyl-D-glucosamine synthase subunit, UDP-N-acetylglucosamine-undecyl isopentenyl-phosphate N -Acetylglucosamine phosphotransferase, Fuc4NAc (4-acetamido-4,6-dideoxy-D-galactose)transferase, UDP-N-acetyl-D-mannuronic acid transfer Enzymes, glycosyltransferase genes encoding catalytic subunits of cellulose synthase, cellulose biosynthesis proteins, colaric acid biosynthesis glucuronyltransferase, colaric acid biosynthesis galactosyltransferase, colaric acid biosynthesis Synthetic fucosyltransferase, UDP-glucose:undecopentenyl-phosphoglucose-1-phosphotransferase, putative Kola biosynthetic glycosyltransferase, UDP-glucuronic acid:LPS(HepIII) glycosyl Transferase, ADP-heptose-LPS heptosyltransferase 2, ADP-heptose:LPS heptosyltransferase 1, putative ADP-heptose:LPS heptosyltransferase 4, lipopolysaccharide core biosynthesis protein, UDP-glucose:(glucosyl)LPS α-1,2-glucosyltransferase, UDP-D-glucose:(glucosyl)LPS α-1,3-glucosyltransferase, UDP-D-galactose:( Glucosyl) lipopolysaccharide-1,6-D-galactosyltransferase, lipopolysaccharide glucosyltransferase I, lipopolysaccharide core heptosyltransferase 3,β-1,6-galactosylfuranosyltransferase, Undecyl isopentenyl-phosphate 4-deoxy-4-carbamoylamino-L-arabinosyltransferase, lipid IVA 4-amino-4-deoxy-L-arabinosyltransferase, bacterpene alcohol Glucosyltransferase, putative family 2 glycosyltransferase, osmoregulatory periplasmic glucan (OPG) biosynthesis protein G, OPG biosynthesis protein H, glucosylglycerate phosphorylase, hepatic glucose synthase, 1, 4-alpha-glucan branching enzyme, 4-alpha-glucanotransferase and cocoon-6-phosphate synthase. In an exemplary embodiment, the cell is mutated in any one or more of the glycosyltransferases comprising pgaC, pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC , wcaA, wcaC, wcaE, wcaI, wcaJ, wcaL, waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbl, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM , glgA, glgB, malQ, otsA, and yaiP, wherein the mutation provides deletion or lower expression of any of the glycosyltransferases.

In an alternative and/or additional preferred embodiment of the method, the reduction or elimination of poly-N-acetyl-glucosamine (PNAG) synthesis is by overexpression of carbon storage regulator-encoding genes, Na+/H+ inversion Deletion of the gene encoding the transporter protein regulator and/or deletion of the gene encoding the sensor histidine kinase is provided.

Microorganisms or cells as used herein can be grown on monosaccharides, disaccharides, oligosaccharides, polysaccharides, polyols, glycerol; complex media including molasses, corn infusion, meringue, pancreas, yeast extract, or mixtures thereof (eg, mixed Feedstocks, preferably mixed monosaccharide feedstocks such as, for example, hydrolyzed sucrose) are grown on as the main carbon source. The term "complex medium" means a medium in which the exact composition is not defined. The term primarily means the most important carbon source for the biological product of interest, biomass formation, carbon dioxide and/or by-product formation (such as acids and/or alcohols, such as acetate, lactate and/or ethanol), i.e. all required 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% of the carbon is derived from the carbon sources indicated above. In one 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 specified above. Common major carbon sources include (but are not limited to) glucose, glycerol, fructose, maltose, lactose, arabinose, malto-oligosaccharide, maltotriose, sorbitol, xylose, rhamnose, sucrose, galactose, mannose Sugar, methanol, ethanol, cocoon honey, starch, cellulose, hemicellulose, molasses, corn infusion, high fructose syrup, acetate, citrate, lactate and pyruvate. The term complex medium means a medium in which the exact composition is not defined. Examples are molasses, corn steep liquor, meringue, pancreas or yeast extract.

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

According to the present invention, the method as described herein preferably comprises the step of isolating at least one of the oligosaccharides from the culture.

The term "separating from said cultivation" means harvesting, collecting or extracting any of the oligosaccharides, preferably all of the oligosaccharides, from the cells and/or the medium in which they are grown .

Any of the oligosaccharides can be isolated from the aqueous medium in which the cells are grown in a known manner. Where the oligosaccharide is still present in the cells producing the oligosaccharide mixture, conventional means for releasing or extracting the oligosaccharide from the cells can be used, such as cell disruption using: high pH, heat shock, Sonication, French press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergents, hydrolysis... media and/or cell extracts together and respectively can then be further used to isolate the oligosaccharides. This preferably involves clarifying the oligosaccharide-containing mixture to remove suspended particles and contaminants, especially cells, cellular components, insoluble metabolites, and debris produced by culturing genetically modified cells. In this step, the oligosaccharide-containing mixture can be clarified in a conventional manner. Preferably, the oligosaccharide-containing mixture is clarified by centrifugation, flocculation, decantation and/or filtration. Another step in separating the oligosaccharide from the oligosaccharide-containing mixture preferably involves removing substantially all proteins as well as peptides, amino acids, RNA and DNA, as well as any endotoxins and glycolipids that might interfere with subsequent separation steps, from the oligosaccharide-containing mixture. The mixture of oligosaccharides is removed, preferably after it has been clarified. In this step, proteins and related impurities can be removed from the oligosaccharide-containing mixture in a conventional manner. Preferably, proteins, salts, by-products, dyes, endotoxins and other related impurities are removed from the oligosaccharide-containing mixture by ultrafiltration, nanofiltration, two-phase partition, reverse osmosis, microfiltration, activated carbon or carbon treatment, Treatment with nonionic surfactants, enzymatic digestion, tangential flow high performance filtration, tangential flow ultrafiltration, electrophoresis (e.g. using slab-polyacrylamide or sodium dodecyl sulfate-polyacrylamide coagulation) Gel electrophoresis (PAGE)), affinity chromatography (using affinity ligands including, for example, DEAE-agarose, poly-L-lysine and polymyxin-B, endotoxin-selective sorbent matrices) , ion exchange chromatography (such as (but not limited to) cation exchange, anion exchange, mixed bed ion exchange, inside-out ligand attachment), hydrophobic interaction chromatography and/or gel filtration (i.e. particle size size exclusion chromatography), specifically by chromatography, more specifically by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography. In addition to size exclusion chromatography, the oligosaccharide is retained in the oligosaccharide-containing mixture by retention of proteins and related impurities by the chromatography medium or membrane of choice.

In another preferred embodiment, the methods as described herein also provide for further purification of any one or more of the oligosaccharide(s) from the oligosaccharide mixture. Further purification of the oligosaccharide(s) can be achieved eg by using (activated) charcoal or carbon, nanofiltration, ultrafiltration or ion exchange to remove any residual DNA, protein, LPS, endotoxin or other impurities. Alcohols such as ethanol and hydroalcoholic mixtures can also be used. Another purification step is achieved by crystallization, evaporation or precipitation of the product. Another purification step is drying the produced oligosaccharides, e.g. spray drying, freeze drying, spray freeze drying, freeze spray drying, band dry, belt dry, vacuum belt drying Drying, vacuum belt drying, tumble drying, drum drying, vacuum tumble drying or vacuum tumble drying.

In an exemplary embodiment, isolation and purification of at least one, preferably all, of the oligosaccharides produced is performed in a process comprising the following steps in any order: a) Contacting the culture or its clarified form with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da, ensuring retention of the oligosaccharides produced and leaving proteins, salts, by-products, dyes and at least a portion of other related impurities passed, b) using the membrane, the retentate from step a) is subjected to a diafiltration process with an aqueous solution of inorganic electrolyte, followed by diafiltration with pure water as appropriate to remove excess electrolyte, c) and collecting the oligosaccharide-enriched retentate(s) in salt form from the cations of the electrolyte.

In an alternative exemplary embodiment, the isolation and purification of at least one, preferably all of the oligosaccharides produced is performed in a process comprising the following steps in any order: or its clarified form is subjected to two membrane filtration steps, one membrane having a molecular weight cut-off between about 300 and about 500 Daltons, and another membrane having a molecular weight cut-off between about 600 and about 800 Daltons between.

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

In an alternative exemplary embodiment, the isolation and purification of at least one, preferably all, of the oligosaccharides produced is performed in the following manner. The cultures containing the produced oligosaccharides, biomass, media components and contaminants were subjected to the following purification steps: i) separation of biomass from the culture, ii) cation exchanger treatment for removal of positively charged species, iii) anion exchanger treatment for removal of negatively charged species, iv) performing a nanofiltration step and/or an electrodialysis step, Therein is provided a purified solution comprising the produced oligosaccharides, the purity of which is greater than or equal to 80%. Optionally, the purified solution is dried by any one or more drying steps selected from the list comprising: spray drying, lyophilization, spray freeze drying, freeze spray drying, ribbon drying, ribbon drying, vacuum ribbon Drying, vacuum belt drying, drum drying, drum drying, vacuum drum drying and vacuum drum drying.

In an alternative exemplary embodiment, the isolation and purification of at least one, preferably all of the oligosaccharides produced is performed in a process comprising the following steps, in any order: Enzymatic treatment of the culture ; removing biomass from the culture; ultrafiltration; nanofiltration; and performing 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) a dissolving agent comprising water; and/or iv) an operating temperature of 15 to 60 degrees Celsius.

In a specific embodiment, the present invention provides the oligosaccharide or oligosaccharide mixture produced, which is dried to powder by any one or more drying steps selected from the list comprising: spray drying, lyophilization, spray freezing Drying, freeze spray drying, strip drying, belt drying, vacuum strip drying, vacuum belt drying, tumble drying, drum drying, vacuum tumble drying and vacuum drum drying, where the dry powder contains <15-wt. % water, preferably <10-wt.% water, more preferably <7-wt.% water, most preferably <5-wt.% water.

In a second aspect, the present invention provides the use of a method as described herein for the manufacture of a mixture comprising at least two different oligosaccharides.

To identify oligosaccharides in a mixture comprising at least two different oligosaccharides produced in cells as described herein, monomeric building blocks (e.g. monosaccharide or glycan unit composition), mutator groups of side chains State, presence and position of substituents, degree of polymerization/molecular weight, and linkage pattern can be identified by standard methods known in the art, such as, for example, methylation analysis, reductive cleavage, hydrolysis, gas chromatography -Mass chromatography (gas chromatography-mass spectrometry; GC-MS), matrix-assisted laser desorption/ionization-mass spectrometry (Matrix-assisted laser desorption/ionization-mass spectrometry; MALDI-MS), electrospray ionization-mass spectrometry method (Electrospray ionization-mass spectrometry; ESI-MS), high performance liquid chromatography (HPLC) with ultraviolet or refractive index detection, high performance anion exchange chromatography (High-Performance Anion-Exchange chromatography) with pulsed amperometric detection with Pulsed Amperometric Detection; HPAEC-PAD), capillary electrophoresis (CE), infrared (IR)/Raman spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. Crystal structures can be solved using, for example, solid-state NMR, Fourier transform infrared (FT-IR) spectroscopy, and wide-angle X-ray scattering (WAXS). 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 components of sugars, methods such as, for example, acid-catalyzed hydrolysis, high performance liquid chromatography (HPLC) or gas-liquid chromatography (GLC) (in conversion after aldol acetate). To determine the glycosidic bond, the sugars were methylated in DMSO with methyl iodide and a strong base, hydrolyzed to achieve reduction of partially methylated sugar alcohols, acetylated of methylated aldol acetates, and acetylated by GLC. /MS (gas-liquid chromatography combined with mass spectrometry) for analysis. To sequence oligosaccharides, partial depolymerization is performed using acids or enzymes to determine structure. To identify the mutator configuration, oligosaccharides are subjected to enzymatic analysis, for example, by contacting them with enzymes specific for a particular type of linkage, such as β-galactosidase or α-glucosidase, and NMR. Can be used to analyze products. Products containing mixtures of oligosaccharides

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

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

"Prebiotics" are substances that promote the growth of microorganisms that are beneficial to the host, especially those in the gastrointestinal tract. In some embodiments, the dietary supplement provides a variety of probiotic cobiotics, including oligosaccharide mixtures produced and/or purified by the processes disclosed in this specification, to promote the growth of one or more beneficial microorganisms. Examples of probiotic components of dietary supplements include other probiotic molecules (such as HMO) and plant polysaccharides (such as inulin, pectin, b-glucans and xylo-oligosaccharides). "Probiotic" products typically contain live microorganisms that replace or add to the microbiota of the gastrointestinal tract for the benefit of the recipient. Examples of such microorganisms include Lactobacillus species (eg, L. acidophilus and L. bulgaricus ), Bifidobacterium species (eg, B. animalis ), B. longum and B. infantis (eg Bi-26) and Saccharomyces boulardii . In some embodiments, mixtures of oligosaccharides produced and/or purified by the procedures of this specification are administered orally in combination with such microorganisms.

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

In some embodiments, the oligosaccharide mixture is incorporated into human infant food (eg, infant formula). Infant formula is generally a manufactured food intended for feeding infants as a complete or partial substitute for human breast milk. In some embodiments, the infant formula is sold in powder form and prepared by mixing with water for bottle feeding or cup feeding to infants. The composition of infant formula is typically designed to approximately mimic human breast milk. In some embodiments, the oligosaccharide mixture produced and/or purified by the process in this specification is included in an infant formula to provide nutritional benefits similar to those provided by oligosaccharides in human breast milk. In some embodiments, the oligosaccharide mixture is mixed with one or more ingredients of the infant formula. Examples of infant formula ingredients include skim milk, carbohydrate sources such as lactose, protein sources such as whey protein concentrate and casein, fat sources such as vegetable oils such as palm oil, high oleic safflower oil, canola oil, coconut oil and/or sunflower oil; and fish oil), vitamins (such as vitamins A, Bb, Bi2, C and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, citric acid) sodium and calcium phosphate) and possibly human milk oligosaccharide (HMO). Such HMOs may include, for example, DiFL, lacto-N-triose II, LNT, LNnT, lacto-N-fucopentaose I, lacto-N-neofucopentose, lacto-N-fucopentose 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 comprise skim milk, a carbohydrate source, a protein source, a fat source, and/or vitamins and minerals.

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

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

In some embodiments, the oligosaccharide mixture is incorporated into a feed formulation, wherein the feed is selected from the list comprising pet food, animal milk replacer, veterinary product, post-weaning feed, or nursery feed.

Each embodiment disclosed in the context of one aspect of the invention is also disclosed in the context of all other aspects of the invention, unless 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 nomenclature used herein and the experimental procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described above and below are those well known and commonly used in the art, in cell culture Experimental procedures, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization. Nucleic acid and peptide synthesis is performed using standard techniques. Generally, purification steps are carried out according to the manufacturer's instructions.

Other advantages follow specific implementations, examples and accompanying drawings. It goes without saying that the features mentioned above and those still to be explained below can be used not only in the respectively specified combination but also in other combinations or independently without departing from the scope of the present invention. The present invention relates to the following specific embodiments: 1. A method for producing a mixture of at least two different oligosaccharides by cells, the method comprising the steps of: i. providing a glycosyltransferase expressing a glycosyltransferase capable of synthesizing a nucleotide- A cell of sugar, wherein the nucleotide-sugar is a donor for the glycosyltransferase, and ii. culturing the cell under conditions that allow expression of the glycosyltransferase and synthesis of the nucleotide-sugar, and iii. adding at least two receptors to the culture so that the cells can produce at least two oligosaccharides, preferably, any one of these receptors is a disaccharide or an oligosaccharide, iv. preferably, At least one of the oligosaccharides is isolated from the culture, more preferably, all of the oligosaccharides are isolated from the culture. 2. The method of embodiment 1, wherein the cell is a metabolically engineered cell modified with at least one gene expression module, wherein the expression from any one of the at least one expression module is persistent or borrowed. Produced by natural inducers. 3. The method of any one of embodiments 1 or 2, wherein the cell produces a mixture of three or more different oligosaccharides. 4. The method of any one of embodiments 1 to 3, wherein the cell produces a mixture of different oligosaccharides, wherein at least two of the oligosaccharides differ in degree of polymerization. 5. The method of any one of embodiments 1 to 4, wherein the glycosyltransferase is selected from a list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase, glucose Syltransferase, Mannosyltransferase, N-Acetylglucosaminyltransferase, N-Acetylgalactosaminyltransferase, N-Acetylmannosyltransferase, Xylosyltransferase , glucuronidyl transferase, galacturonyl transferase, glucosamine transferase, N-hydroxyacetyl neuroaminotransferase, rhamnosyl transferase. 6. The method of any one of embodiments 1 to 5, wherein the cell is modified in the expression or activity of the glycosyltransferase. 7. The method of any one of embodiments 1 to 6, wherein the glycosyltransferase is a fucosyltransferase and the donor nucleotide-sugar is GDP-fucose (GDP-Fuc). 8. The method of any one of embodiments 1 to 7, wherein the glycosyltransferase is a sialyltransferase and the donor nucleotide-sugar is CMP-N-acetylneuraminic acid (CMP-Neu5Ac ). 9. The method of any one of embodiments 1 to 8, wherein the glycosyltransferase is N-acetylglucosaminyltransferase and the donor nucleotide-sugar is UDP-N-acetylglucosamine Amine (UDP-GlcNAc). 10. The method of any one of embodiments 1 to 9, wherein the glycosyltransferase is a galactosyltransferase and the donor nucleotide-sugar is UDP-galactose (UDP-Gal). 11. The method of any one of embodiments 1 to 10, wherein the nucleotide-sugar is selected from a list comprising GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N -Acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), CMP- N-hydroxyacetylneuraminic acid (CMP-Neu5Gc), UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose. 12. The method of any one of embodiments 1 to 11, wherein the degree of polymerization of any of the receptors is 3 or more, preferably all of the receptors have a degree of polymerization of 3 Or more. 13. The method of any one of embodiments 1 to 12, wherein all of the receptors have different degrees of polymerization. 14. The method of any one of embodiments 1 to 13, wherein the culture is supplemented with at least 3 receptors for making the oligosaccharide mixture. 15. The method of any one of embodiments 1 to 14, wherein the oligosaccharide mixture comprises at least one oligosaccharide that is fucosylated, sialylated, galactosylated, glucosyl Glucosylation, Xylosylation, Mannosylation, with N-acetylglucosamine, with N-acetylneuraminic acid, with N-hydroxyacetylneuraminic acid, with N-acetylgalactose Amine, containing rhamnose, containing glucuronide, containing galacturonate, and/or containing N-acetylmannosamine. 16. The method of any one of embodiments 1 to 15, wherein the oligosaccharide mixture comprises at least one fucosylated oligosaccharide. 17. The method of any one of embodiments 1 to 16, wherein the oligosaccharide mixture comprises at least one sialylated oligosaccharide. 18. The method of any one of embodiments 1 to 17, wherein the oligosaccharide mixture comprises at least one oligosaccharide comprising N-acetylglucosamine monosaccharide units. 19. The method of any one of embodiments 1 to 18, wherein the oligosaccharide mixture comprises at least one galactosylated oligosaccharide. 20. The method of any one of embodiments 1 to 16, wherein all of the oligosaccharides in the mixture are fucosylated oligosaccharides. 21. The method of embodiments 1 to 16 and 20, wherein the cell produces three fucosylated oligosaccharides. 22. The method of any one of embodiments 1-21, wherein the receptors are manufactured by a method comprising extraction from natural sources, biotechnological processes, physical processes, chemical processes, and combinations thereof. 23. The method of any one of embodiments 1-22, wherein any of the receptors is fully converted to any of the oligosaccharides. 24. The method of any one of embodiments 1 to 23, wherein the cell is further metabolically engineered for i) modified expression of endogenous membrane proteins, and/or ii) modified activity of endogenous membrane proteins , and/or iii) the expression of a homologous membrane protein, and/or iv) the expression of a heterologous membrane protein, wherein the membrane protein is involved in the secretion of any of the oligosaccharides from the cell, preferably wherein the membrane The protein is involved in the secretion of all of the oligosaccharides from the cell. 25. The method of any one of embodiments 1 to 24, wherein the cell is further metabolically engineered for i) modified expression of endogenous membrane proteins, and/or ii) modified activity of endogenous membrane proteins , and/or iii) the expression of a homologous membrane protein, and/or iv) the expression of a heterologous membrane protein, wherein the membrane protein is involved in the uptake of precursors for the synthesis of any of the oligosaccharides in the mixture , preferably wherein the membrane protein is involved in the absorption of all desired precursors, more preferably wherein the membrane protein is involved in the absorption of all the receptors. 26. The method of any one of embodiments 1 to 25, wherein any of the oligosaccharides is a mammalian milk oligosaccharide, preferably wherein all of the oligosaccharides are mammalian milk oligosaccharides. 27. The method of any one of embodiments 1 to 25, wherein any one of the oligosaccharides is an antigen of the human ABO blood group system, preferably wherein all of the oligosaccharides are antigens of the human ABO blood group system. 28. The method of any one of embodiments 1 to 27, wherein the cell line is selected from the group consisting of microorganisms, plants or animal cells, preferably the microorganisms are bacteria, fungi or yeasts, preferably the plants are rice, Cotton, rapeseed, soybean, maize or corn plants, preferably the animal is an insect, fish, bird or non-human mammal, preferably the animal cell is a mammalian cell line. 29. The method of any one of embodiments 1 to 28, wherein the cell is a bacterium, preferably a strain of Escherichia coli, more preferably a cell of a strain of Escherichia coli of strain K-12, even more preferably strain of Escherichia coli K-12 For Escherichia coli MG1655. 30. The method of any one of embodiments 1 to 28, wherein the cell is a yeast cell. 31. The method of any one of embodiments 1 to 30, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated carbon or carbon treatment, tangential flow High performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography. 32. The method of any one of embodiments 1 to 31, further comprising purifying any of the oligosaccharides from the cell. 33. The method of any one of embodiments 1 to 32, wherein the purification comprises at least one of the following steps: using activated carbon or carbon; using charcoal, nanofiltration, ultrafiltration or ion exchange; using alcohol; using water Alcohol mixture; crystallization; evaporation; precipitation; drying, spray drying or lyophilization. 34. Use of the method of any one of embodiments 1 to 33 for the manufacture of a mixture of at least two different oligosaccharides. Furthermore, the present invention relates to the following preferred specific embodiments: 1. A method for producing a mixture of at least two different oligosaccharides by a cell, preferably a single cell, the method comprising the steps of: i. providing a method capable of expressing transglycosylation Enzymes and cells capable of synthesizing a nucleotide-sugar, preferably a single cell, wherein the nucleotide-sugar is a donor for the glycosyltransferase, and ii. in allowing expression of the glycosyltransferase and synthesis of the culturing the cell under nucleotide-sugar conditions, and iii. adding at least two receptors to the culture such that the cell is capable of producing at least two oligosaccharides, preferably any of the receptors Is a disaccharide or an oligosaccharide, iv. Preferably, at least one of the oligosaccharides is isolated from the culture, more preferably, all of the oligosaccharides are isolated from the culture. 2. as the method of preferred embodiment 1, wherein any one of these receptors is a disaccharide or mammalian milk oligosaccharide (mammalian milk oligosaccharide; MMO), preferably, any one of these receptors The one is mammalian milk oligosaccharide. 3. The method of any one of preferred embodiments 1 or 2, wherein the cell is a cell that has been metabolically engineered to produce the mixture and/or is modified with at least one gene expression module from which the at least one expression model is derived. The performance of any of the groups is either persistent or produced by natural inducers. 4. The method of any one of preferred embodiments 1 to 3, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein. 5. The method of any one of preferred embodiments 1 to 4, wherein the cell produces a mixture of three or more different oligosaccharides. 6. The method of any one of preferred embodiments 1 to 5, wherein the cell produces a mixture of different oligosaccharides, wherein at least two oligosaccharides differ in degree of polymerization. 7. The method of any one of preferred embodiments 1 to 6, wherein the glycosyltransferase is selected from a list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase , Glucosyltransferase, Mannosyltransferase, N-Acetylglucosaminyltransferase, N-Acetylgalactosaminyltransferase, N-Acetylmannosyltransferase, Xylosyl Transferase, glucuronyltransferase, galacturonyltransferase, glucosaminyltransferase, N-hydroxyacetylneuramidotransferase, rhamnosyltransferase, N-acetyl rhamnosyl Glycosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP-N-acetylglucosamine Enolacetonyltransferase and fucosyltransferase, - preferably, the fucosyltransferase is selected from the list comprising: α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,4-fucosyltransferase and α-1,6-fucosyltransferase, - preferably, the sialyltransferase is selected from the list comprising α-2,3-sialyltransferase, α-2,6-sialyltransferase and α-2,8-sialyltransferase, - preferably, The galactosyltransferase is selected from the list comprising: β-1,3-galactosyltransferase, N-acetylglucosamine β-1,3-galactosyltransferase, β-1,3-galactosyltransferase 4-Galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, α-1,3-galactosyltransferase and α-1,4-galactosyltransferase , - preferably, the glucosyltransferase is selected from the list comprising: α-glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase and β-glucosyltransferase - 1,4-glucosyltransferase, - preferably, the mannosyltransferase is selected from the list comprising: α-1,2-mannosyltransferase, α-1,3-mannose Syltransferase and α-1,6-mannosyltransferase, - preferably, the N-acetylglucosaminyltransferase is selected from the list comprising: galactoside β-1,3- N-acetylglucosaminyltransferase and β-1,6-N-acetylglucosaminyltransferase, - preferably, the N-acetylgalactosaminyltransferase is α-1, 3-N-Acetylgalactosaminyltransferase. 8. The method of any one of preferred embodiments 1 to 7, wherein the cell is modified in the expression or activity of the glycosyltransferase. 9. The method of any one of preferred embodiments 1 to 8, wherein the glycosyltransferase is a fucosyltransferase and the donor nucleotide-sugar is GDP-fucose (GDP-Fuc) . 10. The method of any one of the preferred embodiments 1 to 9, wherein the glycosyltransferase is a sialyltransferase and the donor nucleotide-sugar is CMP-N-acetylneuraminic acid (CMP). -Neu5Ac). 11. The method of any one of preferred embodiments 1 to 10, wherein the glycosyltransferase is N-acetylglucosaminyltransferase and the donor nucleotide-sugar is UDP-N-acetyl Glucosamine (UDP-GlcNAc). 12. The method of any one of preferred embodiments 1 to 11, wherein the glycosyltransferase is a galactosyltransferase and the donor nucleotide-sugar is UDP-galactose (UDP-Gal). 13. The method of any one of preferred embodiments 1 to 12, wherein the glycosyltransferase is N-acetylgalactosamine transferase and the donor nucleotide-sugar is UDP-N-ethyl Acylgalactosamine (UDP-GalNAc). 14. The method of any one of preferred embodiments 1 to 13, wherein the glycosyltransferase is N-acetylmannosylaminotransferase and the donor nucleotide-sugar is UDP-N-ethyl Acylmannosamine (UDP-ManNAc). 15. The method of any one of preferred embodiments 1 to 14, wherein the nucleotide-sugar is selected from a list comprising the following: GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP -N-Acetylgalactosamine (UDP-GalNAc), UDP-N-Acetylmannosamine (UDP-ManNAc), GDP-Mannose (GDP-Man), UDP-Glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy--L-arabinose-4-hexulose, UDP-2-acetamido-2,6-dideoxy--L-to Throse-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetylamino-2,6-dideoxy-L-mannan sugar), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy- L-galactose), UDP-N-acetyl-L-neumosamine (UDP-L-PneNAC or UDP-2-acetylamino-2,6-dideoxy-L-talose) , UDP-N-Acetylmuramic acid, UDP-N-Acetyl-L-Isorhamnosamine (UDP-L-QuiNAc or UDP-2-Acetylamino-2,6-dideoxy -L-glucose), GDP-L-isorhamnose, CMP-N-hydroxyacetylneuraminic 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, UDP-xylose . 16. The method of any one of preferred embodiments 1 to 15, wherein the degree of polymerization of any one of these acceptors is 3 or more, preferably wherein the degree of polymerization of all of these acceptors for 3 or more. 17. The method of any one of preferred embodiments 1 to 16, wherein all the receptors have different degrees of polymerization. 18. The method of any one of preferred embodiments 1 to 17, wherein the culture is supplemented with at least 3 receptors for the manufacture of the oligosaccharide mixture, preferably with at least 4 receptors, more preferably with At least 5 receptors. 19. The method of any one of preferred embodiments 1 to 18, wherein the oligosaccharide mixture comprises at least one oligosaccharide that is fucosylated, sialylated, galactosylated, Glycosylation, Xylosylation, Mannosylation, with N-acetylglucosamine, with N-acetylneuraminic acid, with N-hydroxyacetylneuraminic acid, with N-acetylneuraminic acid Galactosamine, containing rhamnose, containing glucuronic acid ester, containing galacturonic acid ester, and/or containing N-acetylmannosamine. 20. The method of any one of preferred embodiments 1 to 19, wherein the oligosaccharide mixture comprises charged and/or neutral oligosaccharides, preferably wherein at least one of these charged oligosaccharides is a sialic acid group Oligosaccharides. 21. The method of any one of preferred embodiments 1 to 19, wherein the oligosaccharide mixture comprises fucosylated and/or unfucosylated neutral oligosaccharides. 22. The method of any one of preferred embodiments 1 to 21, wherein the oligosaccharide mixture comprises at least one fucosylated oligosaccharide. 23. The method of any one of preferred embodiments 1 to 20, wherein the oligosaccharide mixture comprises at least one sialylated oligosaccharide. 24. The method of any one of preferred embodiments 1 to 23, wherein the oligosaccharide mixture comprises at least one oligosaccharide comprising N-acetylglucosamine monosaccharide units. 25. The method of any one of preferred embodiments 1 to 24, wherein the oligosaccharide mixture comprises at least one galactosylated oligosaccharide. 26. The method of preferred embodiment 21, wherein all the oligosaccharides in the mixture are fucosylated oligosaccharides. 27. The method of preferred embodiments 1 to 26, wherein the cell produces three fucosylated oligosaccharides. 28. The method of any one of preferred embodiments 1 to 27, wherein the receptors are manufactured by a method comprising extraction from natural sources, biotechnological processes, physical processes, chemical processes, and combinations thereof. 29. The method of any one of preferred embodiments 1 to 28, wherein any of the receptors is fully converted to any of the oligosaccharides. 30. The method of any one of preferred embodiments 1 to 29, wherein the cell intracellularly manufactures the mixture of at least two oligosaccharides, and wherein a part or substantially all of the manufactured oligosaccharides remain in the cell. are excreted inside and/or outside the cell via passive or active transport. 31. The method of any one of preferred embodiments 1 to 30, wherein the cell is further metabolically engineered for i) modified expression of endogenous membrane proteins, and/or ii) modified expression of endogenous membrane proteins. modification activity, and/or iii) expression of homologous membrane proteins, and/or iv) expression of heterologous membrane proteins, wherein the membrane proteins are involved in the secretion of any of the oligosaccharides from the cell, preferably wherein The membrane protein is involved in the secretion of all of the oligosaccharides from the cell. 32. The method of any one of preferred embodiments 1 to 31, wherein the cell is further metabolically engineered for i) modified expression of endogenous membrane proteins, and/or ii) modified expression of endogenous membrane proteins. Modification activity, and/or iii) expression of homologous membrane proteins, and/or iv) expression of heterologous membrane proteins, wherein the membrane proteins are involved in uptake of any of the oligosaccharides used in the synthesis of the mixture Precursors, preferably wherein the membrane protein is involved in the absorption of all desired precursors, more preferably wherein the membrane protein is involved in the absorption of all the receptors. 33. The method of any one of preferred embodiments 31 or 32, wherein the membrane protein is selected from the list comprising: transporter protein, PP-bond hydrolysis-driven transporter protein, β-barrel porin, auxin Transport proteins, putative transport proteins and phosphate transfer driven group translocation proteins, preferably, these transport proteins include MFS transport proteins, sugar efflux transport proteins and chelatin export proteins, preferably, these PP-bonds Hydrolysis-driven transport proteins include ABC transport proteins and chelatin export proteins. 34. The method of any one of preferred embodiments 31 to 33, wherein the membrane protein provides an improved manufacture of the mixture of at least two oligosaccharides and/or can achieve and/or enhance this mixture of at least two oligosaccharides. outflow of the mixture. 35. The method of any one of preferred embodiments 1 to 34, wherein the cells are resistant to lactose killing when grown in an environment where lactose is combined with one or more other carbon sources. 36. The method of any one of preferred embodiments 1 to 35, wherein the cell comprises a modification for reducing acetate production compared to an unmodified precursor cell. 37. The method of preferred embodiment 36, wherein the cell comprises any one of the following proteins comprising reduced or reduced expression and/or eliminated, attenuated, reduced or delayed activity compared to unmodified precursor cells or more: β-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N- Acetyl glucosamine arrestin, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecyl isopentenyl-phosphoglucose-1-phosphotransferase, L-fucokinase, L- Fucose Isomerase, N-Acetyl Neuraminidase Lysase, N-Acetyl Mannosamine Kinase, N-Acetyl Mannosamine-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 fructose-6-phosphate Kinase isoenzyme 1, ATP-dependent 6-phosphofructokinase isoenzyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor protein IclR, lon protease, glucose-specific translocation phosphotransferase IIBC component ptsG, glucose-specific translocation phosphotransferase (PTS) IIBC component malX, enzyme IIA ^Glc , β-glucoside-specific PTS enzyme II, fructose-specific PTS polyphosphooxytransferase FruA and FruB, ethanol Dehydrogenase Aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacetyltransferase, phosphoacetyltransferase, pyruvate decarboxylase. 38. The method of any one of preferred embodiments 1 to 37, wherein the cell is capable of producing phosphoenolpyruvate (phosphoenolpyruvate; PEP). 39. The method of any one of preferred embodiments 1 to 38, wherein the cell is modified for enhanced production and/or supply of phosphoenolpyruvate (PEP) compared to unmodified precursor cells . 40. The method of any one of preferred embodiments 1 to 39, wherein any one of these oligosaccharides is a mammalian milk oligosaccharide, preferably, wherein all of these oligosaccharides are mammalian milk oligosaccharides sugar. 41. The method of any one of preferred embodiments 1 to 40, wherein any one of the oligosaccharides is an antigen of the human ABO blood group system, preferably wherein all of the oligosaccharides are of the human ABO blood group system. antigen. 42. The method of any one of preferred embodiments 1 to 41, wherein the cell is a bacterium, a fungus, a yeast, a plant cell, an animal cell or a protozoan cell, - preferably, the bacterium is Escherichia coli ( Escherichia coli ) strain, more preferably an E. coli strain of the K-12 strain, even more preferably the E. coli K-12 strain is E. coli MG1655, - preferably, the fungus belongs to a genus selected from the group comprising: Rhizopus Genus Rhizopus , Dictyostelium , Penicillium , Mucor or Aspergillus , - preferably, the yeast belongs to the group selected from the group consisting of Genera: Saccharomyces , Zygosaccharomyces , Pichia , Komagataella , Hansenula , Yarrowia , St. Starmerella ( Starmerella ), Kluyveromyces ( Kluyveromyces ) or Debaromyces ( Debaromyces ), - preferably, the plant cell is an algal cell or is derived from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soybean, maize or corn plants, - preferably, the animal cells are derived from non-human mammals, birds, fish, invertebrates, reptiles, amphibians or insects, or are derived from excluding embryonic stem cells Genetically modified cell lines of human cells, preferably the human and non-human mammalian cells are epithelial cells, embryonic kidney cells, fibroblasts, COS cells, Chinese hamster ovary (CHO) cells, murine Myeloma cells, NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, more preferably the insect cells are derived from Spodoptera frugiperda , Bombyx mori , Mamestra brassicae , Trichoplusia ni or Drosophila melanogaster , - preferably, the protozoal cell is a Leishmania tarentolae cell. 43. The method of preferred embodiment 42, wherein the cell is a viable Gram-negative bacterium comprising reduced or eliminated synthetic poly-N compared to unmodified precursor cells -Acetyl-glucosamine (poly-N-acetyl-glucosamine; PNAG), Enterobacterial Common Antigen (ECA), cellulose, kolaric acid, ribo-oligosaccharide, osmoregulatory periplasmic glucan (Osmoregulated Periplasmic Glucan; OPG), glycerol glucoside, polysaccharide and/or cocoon sugar. 44. The method of any one of preferred embodiments 1 to 43, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, two-phase partition, reverse osmosis, microfiltration, activated carbon or Carbon treatment, treatment with nonionic surfactants, enzymatic digestion, tangential flow high performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, Bit Exchange Chromatography. 45. The method of any one of preferred embodiments 1 to 44, further comprising purifying any of the oligosaccharides from the cell. 46. The method of preferred embodiment 45, wherein the purification comprises at least one of the following steps: using activated carbon or carbon; using charcoal, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange; using alcohol; using Hydroalcoholic Mixtures; Crystallization; Evaporation; Precipitation; Drying, Spray Drying, Freeze Drying, Spray Freeze Drying, Freeze Spray Drying, Band Drying, Belt Drying, Vacuum Belt Drying, Vacuum Belt drying, tumble drying, tumble drying, vacuum tumble drying or vacuum tumble drying. 47. Use of the method of any one of preferred embodiments 1 to 46 for the manufacture of a mixture of at least two different oligosaccharides.

The invention will be described in more detail in the examples. The following examples serve to further illustrate and illustrate the invention and are not intended to be limiting. EXAMPLES Example 1. Materials and Methods of Escherichia coli Culture Medium

Luria Broth (LB) medium was composed of 1% pancreas (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride ( VWR, Leuven, Belgium). The minimal medium used in 96-well plates or shake flasks in culture experiments contains 2.00 g/L NH4Cl, 5.00 g/L (NH4)2SO4, 2.993 g/L KH2PO4, 7.315 g/L K2HPO4, 8.372 g/L MOPS, 0.5 g/L NaCl, 0.5 g/L MgSO4.7H2O, 30 g/L sucrose or 30 g/L glycerol, 1 ml/L vitamin solution, 100 µl/L molybdate solution and 1 mL/L selenium solution. As specified in the respective examples, 20 g/L Lactose, 20 g/L 2'FL, 20 g/L 3-FL, 20 g/L 3'SL, 20 g/L 6'SL, 20 g/L /L LacNAc and/or 20 g/L LNB were additionally added to the medium as acceptors. Minimal medium was set to pH 7 using 1M KOH. The vitamin solution is composed of 3.6 g/L FeCl2.4H2O, 5 g/L CaCl2.2H2O, 1.3 g/L MnCl2.2H2O, 0.38 g/L CuCl2.2H2O, 0.5 g/L CoCl2.6H2O, 0.94 g/L ZnCl2, 0.0311 g/L H3BO4, 0.4 g/L Na2EDTA.2H2O and 1.01 g/L thiamine.HCl. The molybdate solution contained 0.967 g/L NaMoO4.2H2O. The selenium solution contained 42 g/L Seo2.

The basic medium for fermentation contains 6.75 g/L NH4Cl, 1.25 g/L (NH4)2SO4, 2.93 g/L KH2PO4 and 7.31 g/L KH2PO4, 0.5 g/L NaCl, 0.5 g/L MgSO4.7H2O, 30 g/L sucrose or 30 g/L glycerol, 1 mL/L vitamin solution, 100 µL/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above. 20 g/L Lactose, 20 g/L 2'FL, 20 g/L 3-FL, 20 g/L 3'SL, 20 g/L LacNAc and/or 20 g as specified in the respective Examples /L LNB was additionally added to the medium as acceptor.

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

pKD46 (Red helper plastid, ampicillin resistance), pKD3 (contains FRT flanked by chloramphenicol resistance (cat) gene), pKD4 (contains FRT flanked by kanamycin resistance (kan) gene) and pCP20 (expressed FLP recombinase activity) plastids were obtained from Prof. R. Cunin (Vrije Universiteit Brussel, Belgium, 2007). Plastids were maintained in host E. coli DH5α (F ⁻ , phi80d lacZΔM15 , Δ( lacZYA − argF ) U169, deoR , recA1 , endA1 , hsdR17(rk ⁻ , mk ⁺ ), phoA , supE44 , λ ⁻ , purchased from Invitrogen , thi -1, gyrA96 , rel A1). Strains and Mutations

Escherichia coli K12 MG1655 [λ ^- , F ^- , rph-1] was obtained from Escherichia coli Gene Reserve Center (USA) in March 2007, CGSC strain number: 7740. Gene disruption, gene introduction and gene replacement were performed using techniques disclosed by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). This technique is based on antibiotic selection following homologous recombination by λ Red recombinase. Subsequent catalysis by flippase recombinase ensures removal of the antibiotic selection cassette in the final producing strain. Transformants carrying the Red helper plastid pKD46 were grown at 30°C in 10 mL of LB medium containing ampicillin (100 mg/L) and L-arabinose (10 mM) to an OD ₆₀₀ nm of 0.6. Cells were made electrocompetent by washing with 50 mL of ice-cold water a first time and 1 mL of ice-cold water a second time. Next, cells were resuspended in 50 µL of ice-cold water. 50 µL of cells and 10-100 ng of linear double-stranded DNA product were electroporated by using a Gene Pulser™ (BioRad) (600 Ω, 25 µFD and 250 volts). After electroporation, cells were added to 1 mL of LB medium incubated at 37°C for 1 h and finally spread onto LB agar containing 25 mg/L chloramphenicol or 50 mg/L kanamycin for selection Antibiotic-resistant transformants. Selected mutants were verified by PCR with primers upstream and downstream of the modified region, and grown in LB agar at 42°C for loss of 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 a 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 body knockouts, homology regions were designed 50-nt upstream and 50-nt downstream of the start and stop codons of the gene of interest. For gene body embedding, the transcription start point (+1) must be considered. PCR products were PCR purified, digested with Dpnl, repurified from agarose gels, and suspended in elution buffer (5 mM Tris, pH 8.0). Selected mutants were transformed with pCP20 plastids, which are heat-induced ampicillin and chloramphenicol-resistant plastids showing temperature-sensitive replication and FLP synthesis. Ampicillin resistant transformants were selected at 30°C, after which some transformants were colonized in LB at 42°C and then tested for all antibiotic resistance and loss of FLP helper plastids. Gene knockout and gene insertion were checked with control primers.

In one example of GDP-fucose production, the mutant strain is derived from E. coli K12 MG1655 comprising knockout of the E. coli wcaJ and thyA genes and insertion of the gene body gene of the persistent transcription unit, the units containing, for example, from Sucrose transporter of CscB of Escherichia coli W (UniProt ID E0IXR1), fructokinase such as Frk derived from Zymomonas mobilis (UniProt ID Q03417), and fructokinase such as derived from Bifidobacterium adolescentis ) (UniProt ID A0ZZH6) the sucrose phosphorylase of BaSP. To produce fucosylated oligosaccharides, mutant GDP-fucose-producing strains were additionally modified with expressing plastids comprising persistent transcription units for alpha-1,2-fucosylation Enzymes, such as, for example, HpFutC from Helicobacter pylori (GenBank: AAD29863.1) and/or for alpha-1,3-fucosyltransferases, such as, for example, HpFucT from Helicobacter pylori (UniProt ID 030511), and Modified with a persistent transcription unit for a selection marker such as E. coli thyA (UniProt ID P0A884). The persistent transcription unit of the fucosyltransferase gene can also be present in mutant E. coli strains via gene body insertion. As described in WO2016075243 and WO2012007481, GDP-fucose production can be further knocked out in mutant E. coli strains by genomic deletion of the E. coli genes comprising glgC , agp , pfkA , pfkB , pgi , arcA , iclR , pgi and lon optimization. GDP-fucose production can additionally be optimized comprising mannose-6-phosphate isomerase (as eg, manA from E. coli (UniProt ID P00946)), phosphomannose mutase (as eg from E. coli manB (UniProt ID P24175)), mannose-1-phosphate guanosyltransferase (as e.g. manC from E. coli (UniProt ID P24174)), GDP-mannose 4,6-dehydratase (as e.g. from Gene body insertion of a persistent transcription unit of gmd from E. coli (UniProt ID P0AC88)) and GDP-L-fucose synthase such as eg fcl from E. coli (UniProt ID P32055). GDP-fucose production can also be obtained by gene body deletion of the E. coli fucK and fucI genes and gene body insertion of persistent transcription units containing a fucose permease, such as for example from E. coli fucP (UniProt ID P11551), and bifunctional fucosokinase/fucose-1-phosphate guanosyltransferase, such as eg fkp (UniProt ID SUV40286.1) from Bacteroides fragilis. If a mutant strain producing GDP-fucose is intended to produce a fucosylated lactose structure comprising 2'FL, 3-FL and DiFL, the strain is additionally gene-knocked out of the E. coli LacZ , LacY and LacA genes and Modified by gene body gene insertion of a persistent transcription unit for use in eg a lactose permease such as Escherichia coli LacY (UniProt ID P02920). Alternatively and/or additionally, the production of GDP-fucose and/or fucosylated structures can be further optimized in mutant E. coli strains by gene body insertion of a persistent transcription unit comprising a membrane Transport proteins such as, for example, MdfA from S. mogeni (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter johnsonii (UniProt ID D4BC23), MdfA from Escherichia coli (UniProt ID P0AEY8), from Regensburg MdfA from Yorkia (UniProt ID G9Z5F4), iceT from Escherichia coli (UniProt ID A0A024L207) or iceT from Citrobacter japonicus (UniProt ID D4B8A6).

In one embodiment of sialic acid production, the mutant strain is derived from E. coli K12 MG1655 comprising gene body insertions of persistent transcription units containing one or more copies of: glucosamine 6- Phosphate N-acetyltransferase, such as eg GNA1 from Saccharomyces cerevisiae (UniProt ID P43577); N-acetylglucosamine 2-epimerase, such as eg AGE from Bacteroides ovale (UniProt ID A7LVG6) and N-Acetylneuraminic acid synthase, as 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 body gene insertion of persistent transcription units containing UDP-N-acetylglucosamine 2-epimerase, such as for example from the jejunum NeuC of Campylobacter (UniProt ID Q93MP8), and N-acetylneuraminic acid synthase, such as eg Neisseria meningitidis (UniProt ID E0NCD4) or Campylobacter jejuni (UniProt ID Q93MP9).

Alternatively and/or additionally, sialic acid production can be obtained by gene body gene insertion of a persistent transcription unit containing, for example, phosphoglucosamine mutase, phosphoglucosamine mutase, glmM from Escherichia coli (UniProt ID P31120). N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase such as eg glmU (UniProt ID P0ACC7) from Escherichia coli, such as eg from Campylobacter jejuni UDP-N-acetylglucosamine 2-epimerase of NeuC (UniProt ID Q93MP8) and N-β-Epimerase such as eg from Neisseria meningitidis (UniProt ID E0NCD4) or Campylobacter jejuni (UniProt ID Q93MP9) Acyl neuraminic acid synthase.

Alternatively and/or additionally, sialic acid production can be obtained by gene body insertion of persistent transcription units containing, for example, bifunctional UDP-GlcNAc 2- from Mus musculus (strain C57BL/6J). Epimerase/N-Acetylmannosamine Kinase (UniProt ID Q91WG8), such as for example N-Acetylneuraminic acid-9-phosphate synthase (UniProt ID K9NPH9) from Pseudomonas sp. UW4 and such as For example N-acylneuraminic acid-9-phosphatase from candidate species Candidatus Magnetomorum 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 body insertion of a persistent transcription unit containing, for example, glmM phosphoglucosamine mutase from E. coli (UniProt ID P31120), such as N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase (UniProt ID P0ACC7) such as glmU from Escherichia coli, such as for example from Mus musculus (strain C57BL/6J) bifunctional UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase (UniProt ID Q91WG8), such as for example N-acetylneuraminic acid from Pseudomonas sp. UW4 -9-phosphate synthase (UniProt ID K9NPH9) and N-acyl neuraminic acid such as eg from the candidate species Magnetotropism HK-1 (UniProt ID KPA15328.1) or from Bacillus polymorpha (UniProt ID Q8A712) -9-phosphatase.

Sialic acid production can be further optimized in mutant E. coli strains with a large intestine comprising any one or more of nagA , nagB , nagC , nagD , nagE , nanA , nanE , nanK , manX , manY and manZ Genome knockout of Bacillus genes (as described in WO18122225); and/or E. coli genes comprising any one or more of nanT , poxB , ldhA , adhE , aldB , pflA , pflC , ybiY , ackA and/or pta gene body deletion; and gene body insertion of persistent transcription units comprising one or more copies of L-glutamic acid-D-fructose-6-phosphate aminotransferase, As eg mutant glmS*54 from E. coli (different from wild-type E. coli glmS by A39T, R250C and G472S mutations, with UniProt ID P17169, as by Deng et al. (Biochimie 88, 419-29 (2006)) ); preferred phosphatases, such as for example 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 or PsMupP from Pseudomonas sputum, ScDOG1 from Saccharomyces cerevisiae or BsAraL from Bacillus subtilis (as described in WO18122225) and acetyl-CoA synthase such as eg acs from Escherichia coli (UniProt ID P27550).

For sialylated oligosaccharide production, the sialic acid-producing strain is further modified to express: N-acylneuraminic acid cytidine acyltransferase, such as, for example, the NeuA enzyme from Campylobacter jejuni (UniProt ID Q93MP7), NeuA enzyme from Haemophilus influenzae (GenBank NO. AGV11798.1) or NeuA enzyme from Pasteurella septicemia (GenBank NO. AMK07891.1); and one or more copies of the following: Glycoside alpha-2,3-sialyltransferase, such as for example PmultST3 from Pasteurella septicemia (UniProt ID Q9CLP3) or a β-galactoside consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 PmultST3-like polypeptide with α-2,3-sialyltransferase activity, NmeniST3 from Neisseria meningitidis (GenBank NO. ARC07984.1) or PmultST2 from Pasteurella septicaemia subsp. septicemia strain Pm70 ( GenBank NO. AAK02592.1); β-galactoside α-2,6-sialyltransferase, such as, for example, PdST6 (UniProt ID 066375) from P. mermaidii or amino acid residue 108 from UniProt ID 066375 PdST6-like polypeptide with β-galactoside α-2,6-sialyltransferase activity composed of to 497 or P-JT-ISH-224-ST6 (UniProt ID A8QYL1 from Photophora JT-ISH-224) ) or a P-JT-ISH-224-ST6-like polypeptide with β-galactoside α-2,6-sialyltransferase activity consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 and/or As eg alpha-2,8-sialyltransferase from Mus musculus (UniProt ID Q64689). The continuous transcription units of N-acyl neuraminidyltransferase and sialyltransferase can be delivered to mutant strains via gene body insertion or via expression plastids. If mutant strains producing sialic acid and CMP-sialic acid are intended to produce sialylated lactose structures, the strains are additionally gene-knocked out of the E. coli LacZ , LacY and LacA genes and gene-inserted via the persistent transcription unit. Modified, this persistent transcription unit is used in lactose permease such as eg Escherichia coli LacY (UniProt ID P02920).

Alternatively and/or additionally, sialic acid and/or sialylated oligosaccharide production can be further optimized in mutant E. coli strains with gene body insertion of a persistent transcription unit comprising a membrane trafficking protein, Such as e.g. sialic acid transporters such as e.g. nanT from E. coli K-12 MG1655 (UniProt ID P41036), nanT from E. coli O6:H1 (UniProt ID Q8FD59), nanT from E. coli O157:H7 (UniProt ID Q8X9G8 ) or nanT (UniProt ID B1EFH1) from E. albertii ; or transporter proteins such as, for example, EntS (UniProt ID P24077) from E. coli, Kluyvera ascorbata ) EntS (UniProt ID A0A378GQ13) or EntS (UniProt ID A0A6Y2K4E8) from Salmonella enterica subsp. arizonae , MdfA (UniProt ID A0A2T7ANQ9) from S. MdfA from Escherichia coli (UniProt ID D4BC23), MdfA from Escherichia coli (UniProt ID P0AEY8), MdfA from Yorkia reginsburg (UniProt ID G9Z5F4), iceT from Escherichia coli (UniProt ID A0A024L207), from Young's iceT from Citrobacter (UniProt ID D4B8A6), SetA from Escherichia coli (UniProt ID P31675), SetB from Escherichia coli (UniProt ID P33026) or SetC from Escherichia coli (UniProt ID P31436); or ABC transporters such as For example oppF from Escherichia coli (UniProt ID P77737), lmrA from Lactococcus lactis subsp. lactis var. diacetoxylactate (UniProt ID A0A1V0NEL4) or Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4).

All mutant strains producing sialic acid, CMP-sialic acid and/or sialylated oligosaccharides are optionally suitable for growth on sucrose via gene body intercalation of persistent transcription units containing, for example, derived from Sucrose transport protein of CscB of E. coli W (UniProt ID E0IXR1 ), fructokinase (UniProt ID Q03417) such as Frk derived from Z. mobilis and sucrose phosphorylation as eg of BaSP derived from Bifidobacterium adolescentis Enzyme (UniProt ID A0ZZH6).

In one embodiment of the production of lacto-N-triose (LNT-II, LN3, GlcNAc-b1,3-Gal-b1,4-Glc), the mutant strain was derived from E. coli K12 MG1655 and subjected to E. coli LacZ , Knockout of LacY , LacA and nagB genes and gene body insertion modification with persistent transcription units for lactose permease such as LacY from E. coli (UniProt ID P02920) and as eg from meningitis Galactoside β-1,3-N-acetylglucosaminyltransferase of LgtA from Neisseria (GenBank: AAM33849.1). For the production of lacto- N -tetrasaccharides (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), the LN3-producing strains were further transported via gene body insertion or self-expressing plastids to Modification of the persistent transcription unit of the strain, for example, N-acetylglucosamine beta-1,3-galactosyltransferase (UniProt ID D3QY14) from WbgO of E. coli O55:H7. For the production of lacto- N -neotetrasaccharides (LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), the LN3-producing strain was further modified with a persistent transcription unit that N-Acetylglucosamine beta-1,4-galactosyltransferase (UniProt ID Q51116) for eg LgtB from Neisseria meningitidis. Optionally, multiple copies of the LgtA , wbgO and/or LgtB genes can be added to mutant E. coli strains. In addition, LNT and/or LNnT production can be enhanced by modifying the strain to improve UDP-GlcNAc production by inserting one or more gene bodies of a persistent transcription unit for mutations such as for example from E. coli L-glutamic acid-D-fructose-6-phosphate aminotransferase of type glmS*54 (different from wild-type E. coli glmS protein by A39T, R250C and G472S mutations, with UniProt ID P17169, as described by Deng et al. human (Biochimie 2006, 88: 419-429)). Additionally, the strain may optionally be modified to enhance UDP-galactose production by genomic deletion of the E. coli ushA , galT , ldhA and agp genes. Mutant E. coli strains may also optionally be adapted to have gene body insertions of a persistent transcription unit for use in, for example, UDP-glucose-4-epimerase (UniProt galE from E. coli). ID P09147), phosphoglucosamine mutase (UniProt ID P31120) as eg glmM from Escherichia coli and N-acetylglucosamine-1-phosphate uridine syltransferase as eg eg glmU from Escherichia coli/ Glucosamine-1-phosphate acetyltransferase (UniProt ID P0ACC7). Mutant strains are also optionally suitable for growth on sucrose via gene body intercalation of persistent transcription units containing, for example, the sucrose transport protein of CscB from E. coli W (UniProt ID E0IXR1 ), such as for example the source. The fructokinase from Frk of Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (UniProt ID A0ZZH6) as eg from BaSP of Bifidobacterium adolescentis. Alternatively and/or additionally, the production of LN3, LNT, LNnT and their derived oligosaccharides may be further optimized in mutant E. coli strains by gene body insertion of a persistent transcription unit comprising a membrane trafficking protein, Such as, for example, MdfA from S. mogeni (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter johnsonii (UniProt ID D4BC23), MdfA from Escherichia coli (UniProt ID P0AEY8), from Yorkia reginsburg MdfA from Escherichia coli (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, proteins involved in nucleotide-activated sugar synthesis, and/or membrane transport proteins are N-terminally and/or C-terminally fused to a soluble enhancer such as, for example, the following Tags: SUMO tag, MBP tag, His, FLAG, Strep-II, Halo-tag, NusA, thioredoxin, GST and/or Fh8 tag to enhance solubility (Cost et al., Front. Microbiol. 2014, https: //doi.org/10.3389/fmicb.2014.00063; Fox et al, Protein Sci. 2001, 10(3), 622-630; Jia and Jeaon, Open Biol. 2016, 6: 160196). Optionally, mutant E. coli strains are modified with gene body insertions encoding chaperone proteins, such as, for example, DnaK, DnaJ, GrpE or the persistent transcription unit of the GroEL/ES chaperone system (Baneyx F., Palumbo J.L. (2003) Improving Heterologous Protein Folding via Molecular Chaperone and Foldase Co-Expression. In: Vaillancourt P.E. (ed.) E. coliGene Expression Protocols. Methods in Molecular Biology™, Vol. 205. Humana Press).

Optionally, the mutant E. coli strain is modified to produce a glycosyl-minimized E. coli strain comprising a gene body deletion of any one or more of the non-essential glycosyltransferase genes, the non-essential glycosyltransferase genes Contains 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 promoter, UTR and terminator sequences were derived from Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148), De Mey et al. (BMC Biotechnol. 2007, 4(34), 1- 14), Dunn et al (Nucleic Acids Res. 1980, 8(10), 2119-2132), Kim and Lee (FEBS Letters 1997, 407(3), 353-356) and Mutalik et al (Nat. Methods 2013, 10, 354-360).

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

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

Pre-incubation of the 96-well microtiter plate experiments started in frozen vials in 150 µL 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 by diluting 400-fold with 400 µL of minimal 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 measure the sugar concentration at the end of the culture experiment, a sample of the whole culture fluid (= average of intracellular and extracellular sugar concentrations) was obtained from each well by boiling the culture fluid at 60°C for 15 min, after which the cells were briefly centrifuged .

Bioreactor pre-cultivation begins with an entire 1 mL frozen vial of a strain, inoculated in 250 mL or 500 mL of minimal medium in a 1 L or 2.5 L shake flask, and inoculated on an orbital shaker at 37°C. Incubate at 200 rpm for 24 hours. A 5 L bioreactor (250 mL inoculum in 2 L batch medium) was then inoculated; the process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). Cultivation conditions were set at 37°C and maximum agitation; pressure gas flow rates were strain and bioreactor dependent. The pH was controlled at 6.8 using 0.5 M H2S04 and 20% NH4OH. Cool the exhaust gas. When foaming occurs during fermentation, add 10% polysiloxane defoamer solution. Optical density

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

Standards such as (but not limited to) sucrose, lactose, LacNAc, lacto-N-disaccharide (LNB), fucosylated LacNAc (2'FLacNAc, 3-FLacNAc), sialylated LacNAc, (3' SLacNAc, 6'SLacNAc), fucosylated LNB (2'FLNB, 4'FLNB), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neo- Tetrasaccharides (LNnT), LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LSTa and LSTc were purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden) )). Other compounds were analyzed with in-house manufactured standards.

Neutral oligosaccharides were analyzed on a Waters Acquity H-stage UPLC with Evaporative Light Scattering Detector (ELSD) or refractive index (RI) detection. A volume of 0.7 µL was injected on a Waters Acquity UPLC BEH Amide column (2.1 × 100 mm; 130 Å; 1.7 µm) and an Acquity UPLC BEH Amide VanGuard column (130 Å, 2.1 × 5 mm). The column temperature was 50°C. The mobile phase consisted of a solution of 1/4 water and 2/4 acetonitrile to which 0.2% triethylamine was added. The method is isocratic at a flow rate of 0.130 mL/min. The drift tube temperature of the ELS detector was 50°C, and the N2 gas pressure was 50 psi, the gain was 200, and the data rate was 10 pps. The temperature of the RI detector was set to 35°C.

Sialyl oligosaccharides were analyzed on a Waters Acquity H-stage UPLC with refractive index (RI) detection. A volume of 0.5 µL was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm; 130 Å; 1.7 µm). The column temperature was 50°C. The mobile phase consisted of a mixture of 0.05% pyrrolidine in 70% acetonitrile, 26% ammonium acetate buffer (150 mM) and 4% methanol. The method is isocratic at a flow rate of 0.150 mL/min. The temperature of the RI detector was set to 35°C.

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

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

Neutral and sialylated sugars were analyzed at low concentrations (below 50 mg/L) using pulsed amperometric detection (PAD) on the Dionex HPAEC system. A volume of 5 µL was injected on 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 eluting agent A was deionized water, where eluting agent B was 200 mM sodium hydroxide and where eluting agent 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: 75% Eluent A initial isocratic step for 10 min, 0% to 4% Eluent C in 8 min Initial increase, second isocratic step with 71% Eluent A and 4% Eluent C maintained for 6 min, second increase in 2.6 min from 4% to 12% Eluent C, 63% Eluent A and 12% Eluent The third isocratic step of Reagent C was maintained for 3.4 min, and the third increase of 12% to 48% Eluent C in 5 min. As a washing step, 48% Solvent C was used for 3 min. For column equilibration, the initial conditions of 75% Eluent A and 0% Eluent C were restored within 1 min and maintained for 11 min. The flow rate applied was 0.5 mL/min. Example 2. Materials and methods of Saccharomyces cerevisiae

Grow strains on synthetic defined yeast medium with complete supplement mix (SD CSM) or CSM drop-out (SD CSM-Ura, SD CSM-Trp, SD CSM-His) containing 6.7 g/L of Yeast nitrogen base without amino acids (YNB without AA, Difco), 20 g/L agar (Difco) (solid culture), 22 g/L glucose monohydrate or 20 g/L lactose, 20 g/L 2'FL, 20 g/L3-FL, 20 g/L 3'SL, 20 g/L 6'SL, 20 g/L LacNAc and/or 20 g/L LNB and 0.79 g/L CSM or 0.77 g/L CSM-Ura, 0.77 g/L CSM-Trp, or 0.77 g/L CSM-His (MP Biomedicals). strain

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

In one example of the production of GDP-fucose, yeast expressed plastids such as eg p2a_2µ_Fuc (Chan 2013, Plasmid 70, 2-17) were used for the expression of 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, and a 2µ yeast ori and Ura3 selectable marker for selection and maintenance in yeast. This plastid additionally contains a persistent transcription unit for, eg, lactose permease from Kluyveromyces lactis LAC12 (UniProt ID P07921), eg, GDP-mannose from gmd of E. coli4,6 - Dehydratase (UniProt ID P0AC88) and GDP-L-fucose synthase such as eg fcl from E. coli (UniProt ID P32055). The yeast expression plastid p2a_2µ_Fuc2 was used as a surrogate expression plastid for the p2a_2µ_Fuc plastid containing a persistent transcription unit next to the ampicillin resistance gene, bacterial ori, 2µ yeast ori and the Ura3 selectable marker for: Bifunctional as eg LAC12 from Kluyveromyces lactis (UniProt ID P07921), as eg fucP from Escherichia coli (UniProt ID P11551) and bifunctional as eg from Bacillus fragilis fkp Sex-fucokinase/fucose-1-phosphate guanosyltransferase (UniProt ID SUV40286.1). For further production of fucosylated oligosaccharides, p2a_2µ_Fuc and its variant p2a_2µ_Fuc2 additionally contain alpha-1,2-fucosyltransferase enzymes such as HpFutC (GenBank: AAD29863.1) from Helicobacter pylori. persistent transcription unit.

In an example of the production of UDP-galactose, the yeast showed that the plastid was derived from a continuous transcription unit containing a HIS3 selectable marker and a UDP-glucose-4-epimerase such as, for example, galE from E. coli (UniProt ID P09147). of the pRS420-plastid series (Christianson et al., 1992, Gene 110: 119-122). For the production of LN3 and LNT, this plastid is further modified with a persistent transcription unit for, for example, the lactose permease from LAC12 of Kluyveromyces lactis (UniProt ID P07921), as for example from Neisseria meningitidis Galactoside β-1,3-N-acetylglucosaminyltransferase of IgtA from bacteria (GenBank: AAM33849.1) and N-acetylglucosamine β such as, for example, WbgO from Escherichia coli O55:H7 -1,3-Galactosyltransferase (UniProt ID D3QY14). For the production of UDP-GalNAc, the plastids are additionally modified with a persistent transcription unit for 4-epimerase (UniProt ID Q8KN66) for e.g. WbpP from Pseudomonas aeruginosa, then optionally directed against e.g. Phosphoglucosamine mutase (UniProt P31120) of glmM of Bacillus and N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase such as glmU from Escherichia coli Gene insertion modification of a transferase (UniProt ID P0ACC7).

In one embodiment of 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 the TRP1 selection marker and persistent transcription units for one or more copies of L-glutamic acid-D-fructose-6-phosphate aminotransferase, such as, for example, mutants from Escherichia coli glmS*54 (different from wild-type E. coli glmS by A39T, R250C and G472S mutations, with UniProt ID P17169, as described by Deng et al. (Biochimie 88, 419-29 (2006))); Phosphatase, 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 E. coli genes or PsMupP from Pseudomonas sputum, ScDOG1 from Saccharomyces cerevisiae or BsAraL from Bacillus subtilis (as described in WO18122225); as eg AGE from Bacillus ovale N-acetylglucosamine 2-epimerase (UniProt ID A7LVG6), such as for example from Neisseria meningitidis (UniProt ID E0NCD4) or Campylobacter jejuni (UniProt ID Q93MP9) Amino acid synthase and eg NeuA from Campylobacter jejuni (UniProt ID Q93MP7), NeuA from Haemophilus influenzae (Genbank No. AGV11798.1) or NeuA from Pasteurella septicaemia (GenBank No. AMK07891.1 ) of N-acylneuraminic acid cytidine acyltransferase. Optionally, a persistent transcription unit comprising one or more copies of a glucosamine 6-phosphate N-acetyltransferase, such as eg GNA1 (UniProt ID P43577) from Saccharomyces cerevisiae, is also added. For the production of sialylated oligosaccharides, the plastids additionally comprise a continuous transcription unit for: such as, for example, the lactose permease from LAC12 of Kluyveromyces lactis (UniProt ID P07921); and one or more of the following A duplicate: as for example β-galactoside α-2,3-sialyltransferase from PmultST3 of Pasteurella septicemia (UniProt ID Q9CLP3) or consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity, NmeniST3 from Neisseria meningitidis (GenBank No. ARC07984.1) or from Pasteurella septicaemia PmultST2 of subsp. strain Pm70 (GenBank NO. AAK02592.1), β-galactoside α-2,6-sialyltransferase (UniProt ID 066375) such as, for example, PdST6 from P. mermaidii (UniProt ID 066375) A PdST6-like polypeptide with β-galactoside α-2,6-sialyltransferase activity consisting of amino acid residues 108 to 497 of P-JT-ISH- 224-ST6 (UniProt ID A8QYL1) or P-JT-ISH-224 with β-galactoside α-2,6-sialyltransferase activity consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 - ST6-like polypeptide and/or as eg alpha-2,8-sialyltransferase from Mus musculus (UniProt ID Q64689).

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

Optionally, mutant yeast strains are modified with gene body insertions encoding persistent transcription units for chaperone proteins such as, for example, Hsp31, Hsp32, Hsp33, Sno4, Kar2, Ssb1, Sse1, Sse2, Ssa1, Ssa2, Ssa3, Ssa4 , Ssb2, Ecm10, Ssc1, Ssq1, Ssz1, Lhs1, Hsp82, Hsc82, Hsp78, Hsp104, Tcp1, Cct4, Cct8, Cct2, Cct3, Cct5, Cct6 or Cct7 (Gong et al., 2009, Mol. Syst. Biol. 5 : 275).

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

Genes to be expressed, whether from plastids or gene bodies, 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 to that of the expression host. Genetic optimization using vendor tools. Culture conditions

Generally, yeast strains are initially grown on SD CSM disks to obtain single colonies. The plates were grown at 30°C for 2-3 days. Starting with a single colony, the preculture was grown overnight at 30 °C in 5 mL with shaking at 200 rpm. Subsequent 125 mL shake flask experiments were seeded with 2% of this preculture in 25 mL of medium. The flasks were incubated at 30°C with orbital shaking at 200 rpm. gene expression promoter

The gene was expressed using a synthetic persistent promoter, as described by Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012). Example 3. Production of an oligosaccharide mixture comprising 2'FL and DiFL with a modified E. coli host using lactose and 3-FL as acceptors

E. coli K12 strain modified for GDP-fucose production as described in Example 1 undergoes plastid transformation to express a persistent transcription unit for H. pylori alpha-1,2-rock Fluorosyltransferase HpFutC (GenBank: AAD29863.1). The novel strains were evaluated in growth experiments according to the culture conditions provided in Example 1, wherein the medium contained sucrose as carbon source and lactose and 3-FL as acceptors. Each strain was grown in four biological replicates in 96-well plates. After 72 h of incubation, the broth was collected and the sugar mixture was analyzed on UPLC. Experiments demonstrated that the novel strain produced an oligosaccharide mixture comprising 2'FL and 2',-3-fucosyllactose (DiFL) in whole broth samples. Example 4. Production of an oligosaccharide mixture comprising 2'FL , DiFL and 2'FLacNAc with a modified E. coli host using lactose and LacNAc as acceptors

E. coli K12 strain modified for GDP-fucose production as described in Example 1 undergoes plastid transformation to express a persistent transcription unit for H. pylori alpha-1,2-rock Fluorosyltransferase HpFutC (GenBank: AAD29863.1). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strain produced in a whole broth sample a mixture of 2'FL, DiFL and 2'-fucosylated N-acetyllactosamine ( 2'FLacNAc) in an oligosaccharide mixture containing sucrose as a carbon source and lactose and N-acetyllactosamine (Gal-b1,4-GlcNAc, LacNAc) as acceptors. Example 5. Production of an oligosaccharide mixture comprising 2'FL , 3-FL , DiFL , 2'FLacNAc , 3-FLacNAc and DiFLacNAc with a modified E. coli host using lactose and LacNAc as acceptors

E. coli K12 strain modified for GDP-fucose production as described in Example 1 undergoes plastid transformation expressing persistent transcription units for H. pylori alpha-1,2- Fucosyltransferase HpFutC (GenBank: AAD29863.1) and Helicobacter pylori α-1,3-fucosyltransferase HpFucT (UniProt ID O30511). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strains produced 2'FL, 3-FL and fucosylated LacNAc (i.e. 2'FLacNAc and 3'FLacNAc and 3-FL) in whole broth samples -FLacNAc) oligosaccharide mixture, wherein the medium contains sucrose as carbon source and lactose and LacNAc as acceptors. Since the enzyme HpFutC (GenBank: AAD29863.1) also showed fucosyltransferase activity on 2'FL and the enzyme HpFucT (UniProt ID O30511) also showed fucosyltransferase activity on 2'FLacNAc, the novel strain DiFL and Di-FLacNAc in the oligosaccharide mixture were also produced. Example 6. Production of an oligosaccharide mixture comprising 3-FL and 3-FLacNAc with a modified E. coli host using lactose and LacNAc as acceptors

E. coli K12 strain modified for GDP-fucose production as described in Example 1 undergoes plastid transformation expressing a persistent transcription unit for H. pylori alpha-1,3-rock Fluorosyltransferase HpFutC (UniProt ID O30511). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strain produced a sample of whole broth containing 3-FL and 3-fucosylated N-acetyllactosamine (3'FLacNAc ) in an oligosaccharide mixture containing sucrose as carbon source and lactose and LacNAc as acceptors. Example 7. Production of an oligosaccharide mixture comprising 2'FL , DiFL and 2'FLNB with a modified E. coli host using lactose and LNB as acceptors

E. coli K12 strain modified for GDP-fucose production as described in Example 1 undergoes plastid transformation to express a persistent transcription unit for H. pylori alpha-1,2-rock Fluorosyltransferase HpFutC (GenBank: AAD29863.1). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strain produced in a whole broth sample a sample containing 2'FL, DiFL and 2'-fucosylated lacto-N-disaccharide (2 'FLNB) oligosaccharide mixture in which the medium contains sucrose as a carbon source and lactose and lacto-N-disaccharide (Gal-b1,3-GlcNAc, LNB) as acceptors. Example 8. Production of an oligosaccharide mixture comprising 2'FL , 3-FL , DiFL , 2'FLNB , 4-FLNB and DiFLNB with a modified E. coli host using lactose and LNB as acceptors

E. coli K12 strain modified for GDP-fucose production as described in Example 1 undergoes plastid transformation expressing persistent transcription units for H. pylori alpha-1,2- Fucosyltransferase HpFutC (GenBank: AAD29863.1) and Helicobacter pylori α-1,3-fucosyltransferase HpFucT (UniProt ID O30511). Since lactose and LNB are suitable receptors for both H. pylori fucosyltransferases, the novel strain produced in a whole broth sample containing 2 when evaluated in growth experiments according to the culture conditions provided in Example 1 Oligosaccharide mixture of 'FL, 3-FL and fucosylated LNB (ie 2' FLNB and 4-FLNB) in which the medium contained sucrose as carbon source and lactose and LNB as acceptors. Since the enzyme HpFutC (GenBank: AAD29863.1) also showed fucosyltransferase activity on 2'FL and the enzyme HpFucT (UniProt ID O30511) also showed fucosyltransferase activity on 2'FLacNAc, the novel strain DiFL and difucosylated LNB (DiFLNB) in the oligosaccharide mixture were also produced. Example 9. Production of an oligosaccharide mixture comprising 3-FL and 4-FLNB with a modified E. coli host using lactose and LNB as acceptors

E. coli K12 strain modified for GDP-fucose production as described in Example 1 undergoes plastid transformation expressing a persistent transcription unit for H. pylori alpha-1,3-rock Fluorosyltransferase HpFutC (UniProt ID O30511). The novel strains produced 3-FL and 4-fucosylated lacto-N-disaccharide (4-FLNB) in whole broth samples when evaluated in growth experiments according to the culture conditions provided in Example 1 The oligosaccharide mixture in which the medium contains sucrose as carbon source and lactose and LNB as acceptors. Example 10. Production of an oligosaccharide mixture comprising 2'FLacNAc and 2'FLNB with a modified E. coli host using LacNAc and LNB as acceptors

E. coli K12 strain modified for GDP-fucose production as described in Example 1 undergoes plastid transformation to express a persistent transcription unit for H. pylori alpha-1,2-rock Fluorosyltransferase HpFutC (GenBank: AAD29863.1). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strain produced an oligosaccharide mixture comprising 2'FLacNAc and 2'FLNB in a whole broth sample, where the medium contained sucrose as a carbon source and LacNAc and LNB acts as a receptor. Example 11. Production of an oligosaccharide mixture comprising 3'FLacNAc and 4-FLNB with a modified E. coli host using LacNAc and LNB as acceptors

E. coli K12 strain modified for GDP-fucose production as described in Example 1 undergoes plastid transformation expressing a persistent transcription unit for H. pylori alpha-1,3-rock Fluorosyltransferase HpFutC (UniProt ID O30511). The novel strain produced an oligosaccharide mixture comprising 3-FLacNAc and 4-FLNB in a whole broth sample when assessed in growth experiments according to the culture conditions provided in Example 1, where the medium contained sucrose as a carbon source and LacNAc and LNB acts as a receptor. Example 12. Production of a mixture of oligosaccharides comprising 2'FLacNAc , 3-FLacNAc , DiFLacNAc , 2'FLNB , 4-FLNB and DiFLNB with modified E. coli hosts using LacNAc and LNB as acceptors

E. coli K12 strain modified for GDP-fucose production as described in Example 1 undergoes plastid transformation expressing persistent transcription units for H. pylori alpha-1,2- Fucosyltransferase HpFutC (GenBank: AAD29863.1) and Helicobacter pylori α-1,3-fucosyltransferase HpFucT (UniProt ID O30511). The novel strain produced oligosaccharides comprising 2'FLacNAc, 3-FLacNAc, DiFLacNAc, 2'FLNB, 4-FLNB and DiFLNB in whole broth samples when evaluated in growth experiments according to the culture conditions provided in Example 1 Mixtures in which the medium contained sucrose as carbon source and LacNAc and LNB as acceptors. Example 13. Production of an oligosaccharide mixture comprising 2'FL , DiFL , 2'FLacNAc and 2'FLNB with a modified E. coli host using lactose, LacNAc and LNB as acceptors

E. coli K12 strain modified for GDP-fucose production as described in Example 1 undergoes plastid transformation to express a persistent transcription unit for H. pylori alpha-1,2-rock Fluorosyltransferase HpFutC (GenBank: AAD29863.1). The novel strain produced an oligosaccharide mixture comprising 2'FL, DiFL, 2'FLacNAc, and 2'FLNB in a whole broth sample when assessed in growth experiments according to the culture conditions provided in Example 1, where the medium contained sucrose as carbon source and lactose, LacNAc and LNB as acceptors. Example 14. Production of an oligosaccharide mixture comprising 3-FL , 3'FLacNAc and 4-FLNB with a modified E. coli host using lactose, LacNAc and LNB as acceptors

E. coli K12 strain modified for GDP-fucose production as described in Example 1 undergoes plastid transformation expressing a persistent transcription unit for H. pylori alpha-1,3-rock Fluorosyltransferase HpFutC (UniProt ID O30511). The novel strain produced an oligosaccharide mixture comprising 3-FL, 3-FLacNAc and 4-FLNB in a whole broth sample when assessed in growth experiments according to the culture conditions provided in Example 1, where the medium contained sucrose as carbon source and lactose, LacNAc and LNB as acceptors. Example 15. Production of 2'FL , 3-FL , DiFL , 2'FLacNAc , 3-FLacNAc , DiFLacNAc , 2'FLNB , 4-FLNB with a modified E. coli host using lactose, LacNAc and LNB as acceptors and DiFLNB oligosaccharide mixture

E. coli K12 strain modified for GDP-fucose production as described in Example 1 undergoes plastid transformation expressing persistent transcription units for H. pylori alpha-1,2- Fucosyltransferase HpFutC (GenBank: AAD29863.1) and Helicobacter pylori α-1,3-fucosyltransferase HpFucT (UniProt ID O30511). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strains produced 2'FL, 3-FL, DiFL, 2'FLacNAc, 3-FLacNAc, DiFLacNAc, 2'FLacNAc, 2'FLacNAc, 2'FLacNAc, 2'FLacNAc, 2'FLacNAc, 2'FLacNAc Oligosaccharide mixture of FLNB, 4-FLNB and DiFLNB in which the medium contained sucrose as carbon source and lactose, LacNAc and LNB as acceptors. Example 16. Production of an Oligosaccharide Mixture Containing 3'SL and 3'SLacNAc with a Modified E. coli Host Using Lactose and LacNAc as Acceptors

E. coli K12 strains modified for sialic acid production as described in Example 1 undergo plastid transformation expressing persistent transcription units for amino acid residues 1 to 1 of UniProt ID Q9CLP3 268 PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity and N-acylneuraminic acid cytidine syltransferase (NeuA) from Pasteurella septicemia (NeuA) ( GenBank: AMK07891.1). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strain produced a mixture of oligosaccharides comprising 3'SL and sialylated LacNAc (3'SLacNAc) in samples of whole culture broth, wherein the medium contained Glycerol as carbon source and lactose and LacNAc as acceptors. Example 17. Production of an oligosaccharide mixture comprising 3'SL , 6'SL , 3'SLacNAc and 6'SLacNAc with modified E. coli hosts using lactose and LacNAc as acceptors

E. coli K12 strains modified for sialic acid production as described in Example 1 undergo plastid transformation expressing persistent transcription units for amino acid residues 1 to 1 of UniProt ID Q9CLP3 PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity composed of 268, with β-galactoside α-2 composed of amino acid residues 108 to 497 of UniProt ID 066375 , PdST6-like polypeptide with 6-sialyltransferase activity and N-acylneuraminic acid cytidine syltransferase (NeuA) from Pasteurella septicemia (GenBank: AMK07891.1). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strains produced 3'SL, 6'SL and sialylated LacNAc (i.e. 3'SLacNAc and 6'SLacNAc) in whole broth samples SLacNAc) oligosaccharide mixture in which the medium contains glycerol as carbon source and lactose and LacNAc as acceptors. Example 18. Production of an oligosaccharide mixture comprising 6'SL and 6'SLacNAc with a modified E. coli host using lactose and LacNAc as acceptors

E. coli K12 strains modified for sialic acid production as described in Example 1 undergo plastid transformation expressing persistent transcription units for amino acid residues 108 to 108 of UniProt ID 066375 PdST6-like polypeptide with β-galactoside α-2,6-sialyltransferase activity composed of 497 and NeuA from Pasteurella septicemia (GenBank: AMK07891.1). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strain produced a mixture of oligosaccharides comprising 6'SL and sialylated LacNAc (6'SLacNAc) in samples of whole culture broth, wherein the medium contained Glycerol as carbon source and lactose and LacNAc as acceptors. Example 19. Production of oligosaccharide mixtures comprising 3'SL and 3'SLNB with modified E. coli hosts using lactose and LNB as acceptors

E. coli K12 strains modified for sialic acid production as described in Example 1 undergo plastid transformation expressing persistent transcription units for amino acid residues 1 to 1 of UniProt ID Q9CLP3 268 PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity and NeuA from Pasteurella septicemia (GenBank: AMK07891.1). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strain produced an oligosaccharide mixture comprising 3'SL and sialylated LNB (3'SLNB) in a sample of whole broth, wherein the medium contained Glycerol as carbon source and lactose and LNB as acceptors. Example 20. Production of an oligosaccharide mixture comprising 3'SL , 6'SL , 3'SLNB and 6'SLNB with modified E. coli hosts using lactose and LNB as acceptors

E. coli K12 strains modified for sialic acid production as described in Example 1 undergo plastid transformation expressing persistent transcription units for amino acid residues 1 to 1 of UniProt ID Q9CLP3 PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity composed of 268, with β-galactoside α-2 composed of amino acid residues 108 to 497 of UniProt ID 066375 , PdST6-like polypeptide with 6-sialyltransferase activity and NeuA from Pasteurella septicemia (GenBank: AMK07891.1). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strain produced in whole broth samples a mixture of 3'SL, 6'SL, and sialylated LNB (ie, 3'SLNB and 6'SLNB and 6'SLNB) SLNB) oligosaccharide mixture, wherein the medium contains glycerol as carbon source and lactose and LNB as acceptors. Example 21. Production of oligosaccharide mixtures comprising 6'SL and 6'SLNB with modified E. coli hosts using lactose and LNB as acceptors

E. coli K12 strains modified for sialic acid production as described in Example 1 undergo plastid transformation expressing persistent transcription units for amino acid residues 108 to 108 of UniProt ID 066375 497 PdST6-like polypeptide with β-galactoside α-2,6-sialyltransferase activity and NeuA from Pasteurella septicemia (GenBank: AMK07891.1). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strain produced an oligosaccharide mixture comprising 6'SL and sialylated LNB (6'SLNB) in a sample of whole broth, wherein the medium contained Glycerol as carbon source and lactose and LNB as acceptors. Example 22. Production of an oligosaccharide mixture comprising 3'SLacNAc and 3'SLNB with a modified E. coli host using LacNAc and LNB as acceptors

E. coli K12 strains modified for sialic acid production as described in Example 1 undergo plastid transformation expressing persistent transcription units for amino acid residues 1 to 1 of UniProt ID Q9CLP3 268 PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity and NeuA from Pasteurella septicemia (GenBank: AMK07891.1). The novel strain produced an oligosaccharide mixture comprising 3'SLacNAc and 3'SLNB in a whole broth sample when assessed in growth experiments according to the culture conditions provided in Example 1, where the medium contained glycerol as a carbon source and LacNAc and LNB acts as a receptor. Example 23. Production of an oligosaccharide mixture comprising 6'SLacNAc and 6'SLNB with a modified E. coli host using LacNAc and LNB as acceptors

E. coli K12 strains modified for sialic acid production as described in Example 1 undergo plastid transformation expressing persistent transcription units for amino acid residues 108 to 108 of UniProt ID 066375 PdST6-like polypeptide with β-galactoside α-2,6-sialyltransferase activity composed of 497 and NeuA from Pasteurella septicemia (GenBank: AMK07891.1). The novel strain produced an oligosaccharide mixture comprising 6'SLacNAc and 6'SLNB in whole broth samples when assessed in growth experiments according to the culture conditions provided in Example 1, where the medium contained glycerol as a carbon source and LacNAc and LNB acts as a receptor. Example 24. Production of an oligosaccharide mixture comprising 3'SLacNAc , 6'SLacNAc , 3'SLNB and 6'SLNB using a modified E. coli host using LacNAc and LNB as acceptors

E. coli K12 strains modified for sialic acid production as described in Example 1 undergo plastid transformation expressing persistent transcription units for amino acid residues 1 to 1 of UniProt ID Q9CLP3 PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity composed of 268, with β-galactoside α-2 composed of amino acid residues 108 to 497 of UniProt ID 066375 , PdST6-like polypeptide with 6-sialyltransferase activity and NeuA from Pasteurella septicemia (GenBank: AMK07891.1). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strain produced an oligosaccharide mixture comprising 3'SLacNAc, 6'SLacNAc, 3'SLNB and 6'SLNB in a whole broth sample, wherein the medium Contains glycerol as carbon source and LacNAc and LNB as acceptors. Example 25. Production of an oligosaccharide mixture comprising 3'SL , 3'SLacNAc and 3'SLNB using a modified E. coli host using lactose, LacNAc and LNB as acceptors

E. coli K12 strains modified for sialic acid production as described in Example 1 undergo plastid transformation expressing persistent transcription units for amino acid residues 1 to 1 of UniProt ID Q9CLP3 268 PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity and NeuA from Pasteurella septicemia (GenBank: AMK07891.1). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strain produced an oligosaccharide mixture comprising 3'SL, 3'SLacNAc and 3'SLNB in a whole broth sample, wherein the medium contained glycerol as carbon source and lactose, LacNAc and LNB as acceptors. Example 26. Production of an oligosaccharide mixture comprising 6'SL , 6'SLacNAc and 6'SLNB with a modified E. coli host using lactose, LacNAc and LNB as acceptors

E. coli K12 strains modified for sialic acid production as described in Example 1 undergo plastid transformation expressing persistent transcription units for amino acid residues 108 to 108 of UniProt ID 066375 497 PdST6-like polypeptide with β-galactoside α-2,6-sialyltransferase activity and NeuA from Pasteurella septicemia (GenBank: AMK07891.1). The novel strain produced an oligosaccharide mixture comprising 6'SL, 6'SLacNAc and 6'SLNB in a whole broth sample when assessed in growth experiments according to the culture conditions provided in Example 1, where the medium contained glycerol as carbon source and lactose, LacNAc and LNB as acceptors. Example 27. Production of oligosaccharide mixture comprising 3'SL , 6'SL , 3'SLacNAc , 6'SLacNAc , 3'SLNB and 6'SLNB with modified E. coli hosts using lactose , LacNAc and LNB as acceptors _ _

E. coli K12 strains modified for sialic acid production as described in Example 1 undergo plastid transformation expressing persistent transcription units for amino acid residues 1 to 1 of UniProt ID Q9CLP3 PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity composed of 268, with β-galactoside α-2 composed of amino acid residues 108 to 497 of UniProt ID 066375 , PdST6-like polypeptide with 6-sialyltransferase activity and NeuA from Pasteurella septicemia (GenBank: AMK07891.1). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strains produced 3'SL, 6'SL, 3'SLacNAc, 6'SLacNAc, 3'SLNB and 6' in whole broth samples Oligosaccharide mixture of SLNB in which the medium contains glycerol as carbon source and lactose, LacNAc and LNB as acceptors. Example 28. Production of an oligosaccharide mixture comprising 2'FL, DiFL , 2'FLacNAc , LN3 , LNT and LNFP - I using a modified E. coli host

E. coli strains modified for GDP-fucose production as described in Example 1 were further adapted by gene body insertion of persistent transcription units for lacto-N-triose (LN3, LNT-II, GlcNAc-b1,3-Gal-b1,4-Glc) and LNT (Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) production, these persistent transcription units are used in E. coli The mutant glmS*54 (different from wild-type E. coli glmS by A39T, R250C and G472S mutations, with UniProt ID P17169), galactoside β-1,3-N-β-galactoside from Neisseria meningitidis Acetylglucosaminyltransferase (LgtA) (UniProt ID Q9JXQ6) and N-acetylglucosamine beta-1,3-galactosyltransferase (WbgO) from Escherichia coli O55:H7 (UniProt ID D3QY14). In the next step, the novel strain was further transformed with an expressive plastid containing a persistent transcription unit for the α-1,2-fucosyltransferase (GenBank: AAD29863. 1). The novel strains produced 2'FL, DiFL, 2'FLacNAc, LN3, LNT and lacto-N-fucopentose in whole broth samples when evaluated in growth experiments according to the culture conditions provided in Example 1 Oligosaccharide mixture of I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), wherein the medium contains sucrose as carbon source and lactose and LacNAc as receptors body. Example 29. Production of oligosaccharide mixtures comprising 2'FL, DiFL , 2'FLacNAc , LN3 , LNT , LNFP - I and LNFP - II using modified E. coli hosts

Adapted as described in Example 28 for LN3 (GlcNAc-b1,3-Gal-b1,4-Glc) and LNT (Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) The produced E. coli strain was further transformed with an expressing plastid containing a persistent transcription unit for a mutant al,3/4 fucosidase from Bifidobacterium longum infantum subsp. ATCC 15697 (US8361756 in of SEQ ID: 18). When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strains produced 2'FL, DiFL, 2'FLacNAc, LN3, LNT, LNFP-I and milk-N- Oligosaccharide mixture of Fucopentaose II (LNFP-II, Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc), wherein the medium contains sucrose as a carbon source As well as lactose and LacNAc as receptors. Example 30. Production of Oligosaccharide Mixtures Comprising 3-FL , 3-FLacNAc , LN3 , LNnT , LNFP-III , and LNFP-VI Using Modified E. coli Hosts

E. coli strains modified for GDP-fucose production as described in Example 1 were further adapted for LN3 and LNnT production by gene body insertion of persistent transcription units for Mutant glmS*54 from E. coli (different from wild-type E. coli glmS by A39T, R250C and G472S mutations, with UniProt ID P17169), LgtA from Neisseria meningitidis (UniProt ID Q9JXQ6) and from meninges LgtB of Neisseria spp. (UniProt ID Q51116). In the next step, the novel strain was further transformed with expressing plastids containing persistent transcription units for the H. pylori alpha-1,3-fucosyltransferase HpFucT (UniProt ID O30511) and Its truncated variant with 66 amino acid residues deleted at its C-terminus is described by Bai et al. (2019, Carb. Res. 480, 1-6). The novel strains produced 3-FL, 3-FLacNAc, LN3 and LNnT, lacto-N-fucopentaose III (LNFP) in whole broth samples when assessed in growth experiments according to the culture conditions provided in Example 1 -III, Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1,3-Gal-b1,4-Glc) and lacto-N-fucopentaose VI (LNFP-VI, Gal-b1, 4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc) oligosaccharide mixture, wherein the medium contains sucrose as carbon source and lactose and LacNAc as acceptors. Example 31. Production of an oligosaccharide mixture comprising LN3 , sialylated LN3 , LNT , 3'SL , 3'SLacNAc and LSTa using a modified E. coli host

E. coli strains modified to produce sialic acid as described in Example 1 were further modified with gene body insertion of the persistent transcription unit to allow lacto-N-tetrasaccharide (LNT, Gal-b1,3-GlcNAc-b1,3 - production of Gal-b1,4-Glc), these persistent transcription units for galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from Neisseria meningitidis (UniProt ID Q9JXQ6) and for N-acetylglucosamine beta-1,3-galactosyltransferase (WbgO) (UniProt ID D3QY14) from E. coli O55:H7. In the next step, the novel strain was further modified with gene body deletion of the E. coli lacZ gene and transformed with an expressive plastid with persistent transcription units for NeuA from Pasteurella septicemia (GenBank: AMK07891.1) and a PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3. The novel strain produced LN3, 3'-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4) when evaluated in growth experiments according to the culture conditions provided in Example 1 -Glc), LNT, 3'SL, 3'SLacNAc and LSTa (Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) oligosaccharide mixture, wherein the medium contains Glycerol as carbon source and lactose and LacNAc as acceptors. Example 32. Production of an oligosaccharide mixture comprising LN3 , sialylated LN3 , LNnT , 6'SL , 6'SLacNAc and LSTc using a modified E. coli host

E. coli strains modified to produce sialic acid as described in Example 1 were further modified by gene body insertion of the persistent transcription unit to allow lacto- N -neotetrasaccharides (LNnT, Gal-b1, 4-GlcNAc-b1, 3-Gal-b1,4-Glc), the persistent transcription units for LgtA from Neisseria meningitidis (UniProt ID Q9JXQ6) and for LgtB from Neisseria meningitidis (UniProt ID Q51116). In the next step, the novel strain was further modified with gene body deletion of the E. coli lacZ gene and transformed with an expressive plastid with persistent transcription units for NeuA from Pasteurella septicemia (GenBank: AMK07891.1) and a PdST6-like polypeptide with β-galactoside α-2,6-sialyltransferase activity consisting of amino acid residues 108 to 497 of UniProt ID 066375. The novel strain produced LN3, 6'-sialylated LN3 (Neu5Ac-a2,6-(GlcNAc-b1,3)-Gal-b1 ) when evaluated in growth experiments according to the culture conditions provided in Example 1 ,4-Glc), 6'SL, 6'SLacNAc, LNnT and LSTc (Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc) oligosaccharide mixture, wherein The medium contained glycerol as carbon source and lactose and LacNAc as acceptors. Example 33. Production of Oligosaccharide Mixtures Containing 2'FL and DiFL with a Modified Saccharomyces cerevisiae Host Using Lactose and 3-FL as Acceptors

Saccharomyces cerevisiae strains were adapted for GDP-fucose production and fucosyltransferase expression with a yeast expression plastid (a variant of p2a_2µ_Fuc) as described in Example 2, the expression plastid comprising: Persistent transcription units: lactose permease (LAC12) from Kluyveromyces lactis (UniProt ID P07921), GDP-mannose 4,6-dehydratase (gmd) from Escherichia coli (UniProt ID P0AC88), from Escherichia coli GDP-L-fucose synthase (fcl) (UniProt ID P32055) and α-1,2-fucosyltransferase (GenBank: AAD29863.1) from Helicobacter pylori HpFutC. The mutant yeast strains produced oligosaccharide mixtures containing 2'FL and DiFL when evaluated on SD CSM-Ura omission medium containing lactose and 3-FL as acceptors. Example 34. Production of a mixture of oligosaccharides comprising 2'FL , 3-FL , DiFL , 2'FLacNAc , 3-FLacNAc and DiFLacNAc with a modified S. cerevisiae host using lactose and LacNAc as acceptors

Saccharomyces cerevisiae strains were adapted for GDP-fucose production and fucosyltransferase expression with a yeast expression plastid (a variant of p2a_2µ_Fuc) as described in Example 2, the expression plastid comprising: Persistent transcription units: lactose permease (LAC12) from Kluyveromyces lactis (UniProt ID P07921), GDP-mannose 4,6-dehydratase (gmd) from Escherichia coli (UniProt ID P0AC88), from Escherichia coli GDP-L-fucose synthase (fcl) (UniProt ID P32055), α-1,2-fucosyltransferase (GenBank: AAD29863.1) from Helicobacter pylori HpFutC and HpFucT from Helicobacter pylori α-1,3-fucosyltransferase (UniProt ID O30511). The mutant yeast strain produced a mixture of oligosaccharides containing 2'FL, 3-FL, DiFL, 2'FLacNAc, 3-FLacNAc, and DiFLacNAc when evaluated on SD CSM-Ura omission medium containing lactose and LacNAc as acceptors . Example 35. Production of an oligosaccharide mixture comprising 3'SL , 6'SL , 3'SLacNAc and 6'SLacNAc with a modified S. cerevisiae host using lactose and LacNAc as acceptors

S. cerevisiae strains were adapted for CMP-sialic acid production and expression of two sialyltransferases as described in Example 2 with a yeast expression plastid (pRS420 plastid variant) containing Persistent transcription units of: mutant glmS*54 from E. coli (different from wild-type E. coli glmS by A39T, R250C and G472S mutations, with UniProt ID P17169), phosphatase yqaB from E. coli (GenBank NO. NP_417175.1), AGE from Bacteroides ovale (UniProt ID A7LVG6), neuB from Neisseria meningitidis (UniProt ID E0NCD4), neuA from Pasteurella septicemia (GenBank: AMK07891.1) , a PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 and an amino acid residue with UniProt ID 066375 A PdST6-like polypeptide with β-galactoside α-2,6-sialyltransferase activity consisting of 108 to 497. The mutant yeast strains produced a mixture of oligosaccharides comprising 3'SL, 6'SL, 3'SLacNAc and 6'SLacNAc when evaluated on SD CSM-Trp omitted medium containing lactose and LacNAc as acceptors. Example 36. Evaluation of mutant E. coli strains in fed-batch fermentation

Mutant E. coli strains as described in Examples 4 and 5 were evaluated in a fed-batch fermentation process. Fed-batch fermentation was performed as described in Example 1 at the bioreactor scale. Sucrose was used as carbon source and lactose and LacNAc were added to the batch medium as acceptors. Conventional broth samples were obtained at several time points during the fermenting process and for the Examples respectively, compared to the culture experiments described herein and in which only the final samples were obtained at the end of the culture (ie, 72 hours as described herein). The strains described in 4 or 5 were evaluated for the production of oligosaccharide mixtures comprising 2'FL, DiFL and 2'FLacNAc or oligosaccharide mixtures comprising 2'FL, 3-FL, DiFL, 2'FLacNAc, 3-FLacNAc and DiFLacNAc . Example 37. Evaluation of mutant E. coli strains in fed-batch fermentation

Mutant E. coli strains as described in Example 27 were evaluated in a fed-batch fermentation process. Fed-batch fermentation was performed as described in Example 1 at the bioreactor scale. Sucrose was used as the carbon source and lactose, LacNAc and LNB were added to the batch medium as acceptors. Conventional broth samples were taken at several time points during the fermentation process and assessed for production comprising Oligosaccharide mixture of 3'SL, 6'SL, 3'SLacNAc, 6'SLacNAc, 3'SLNB and 6'SLNB. Example 38. Materials and Methods for Bacillus subtilis Medium

Two different media were used, namely enriched Luria broth (LB) and minimal medium for shake flasks (MMsf). Minimal medium uses a trace element mixture.

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 It is composed of 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 was composed of 1% pancreas (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 supplemented with 12 g/L agar (Difco, Erembodegem, Belgium).

Minimal medium for shake flask (MMsf) experiments containing 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 L tryptophan, 10 to 30 g/L glucose or another carbon source (including (but not limited to) fructose, maltose, sucrose, glycerol and maltotriose as specified in the examples), 10 ml/L trace Elemental mixture and 10 ml/L ferric citrate solution. The medium was set to pH 7 with 1M KOH. Depending on the experiment, lactose, 2'FL, 3-FL, 3'SL, LNB or LacNAc can be added as acceptors.

Sterilize complex media (eg LB) by autoclaving (121°C, 21') and sterilize 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, available from the Bacillus Gene Reserve (Ohio, USA).

Genetically deleted plastids via Cre/lox were constructed as described by Yan et al. (Appl. & Environm. Microbial., September 2008, pp. 5556-5562). Gene disruption occurs via homologous recombination with linear DNA and transformation via electroporation, as described by Xue et al. (J. Microb. Meth. 34 (1999) 183-191). The method of gene knockout is described by Liu et al. (Metab. Engine. 24 (2014) 61-69). This method uses 1000 bp of homology upstream and downstream of the gene of interest.

Integration vectors as described by Popp et al. (Sci. Rep., 2017, 7, 15158) are used as expression vectors and can be further used for genome integration if necessary. Promoters suitable for expression can be derived from partial repositories (iGems): sequence id: Bba_K143012, Bba_K823000, Bba_K823002 or Bba_K823003. Colonization can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

In one embodiment of the production of lactose-based oligosaccharides, mutant strains of B. subtilis are generated to contain a gene encoding a lactose importer such as, for example, E. coli lacY with UniProt ID P02920. For 2'FL, 3FL and diFL production, α-1,2- and/or α-1,3-fucosyltransferase expression constructs were additionally added to the strains.

In one embodiment of the production of lactose-N-triose (LNT-II, LN3, GlcNAc-b1,3-Gal-b1,4-Glc), the Bacillus subtilis strain is via a lactose importer (such as, for example, with a UniProt ID) Persistent expression of E. coli lacY from P02920 and galactoside β-1,3-N-acetylglucosaminyltransferase such as, for example, LgtA from Neisseria meningitidis (GenBank: AAM33849.1) Gene body insertion modification of the unit. For LNT production, the LN3-producing strain was further subjected to a persistent form of N-acetylglucosamine β-1,3-galactosyltransferase such as, for example, WbgO from E. coli O55:H7 (UniProt ID D3QY14) Transcription unit modification. In order to produce lacto- N -neotetrasaccharides (LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), the LN3-producing strain was further modified with a persistent transcription unit that N-Acetylglucosamine beta-1,4-galactosyltransferase (UniProt ID Q51116) for eg LgtB from Neisseria meningitidis. Both N-acetylglucosamine β-1,3-galactosyltransferase and N-acetylglucosamine β-1,4-galactosyltransferase can be delivered via gene body insertion or self-expressing plastids to the strain. To produce LNFP-I and other fucosylated derivatives of LNT and/or LNnT, the LNT and LNnT-producing strains can be further treated with α-1,2-fucosyltransferase and/or α-1,3 - Fucosyltransferase expresses construct modification.

In one example of sialic acid production, mutant B. subtilis strains were produced by overexpressing fructose-6-P-aminotransferase (UniProt ID P0CI73) to enhance the intracellular glucosamine-6-phosphate pool. In addition, the enzymatic activities of genes nagA, nagB and gamA were disrupted by gene knockout and glucosamine-6-P-aminotransferase (UniProt ID P43577) from Saccharomyces cerevisiae, N-acetylglucose from Bacillus ovale Amine-2-epimerase (UniProt ID A7LVG6) and N-acetylneuraminic acid synthase (UniProt ID Q93MP9) from Campylobacter jejuni were overexpressed on the gene body. To allow sialylated oligosaccharide production, the sialic acid-producing strain was further modified with an expression construct comprising: N-acylneuraminic acid cytidine acyltransferase NeuA (UniProt ID Q93MP7) from Campylobacter jejuni ; and one or more copies of: such as, for example, β-galactoside α-2,3-sialyltransferase from PmultST3 of Pasteurella septicemia (UniProt ID Q9CLP3) or an amine from UniProt ID Q9CLP3 A PmultST3-like polypeptide with β-galactoside α-2,3-sialyltransferase activity consisting of amino acid residues 1 to 268, NmeniST3 from Neisseria meningitidis (GenBank No. ARC07984.1) or PmultST2 from Pasteurella septicemia subsp. septicemia strain Pm70 (GenBank NO. AAK02592.1), such as, for example, β-galactoside α-2,6-sialyltransferase (UniProt ID O66375) or a PdST6-like polypeptide with β-galactoside α-2,6-sialyltransferase activity consisting of amino acid residues 108 to 497 of UniProt ID O66375 or from Photophora JT-ISH- 224 of P-JT-ISH-224-ST6 (UniProt ID A8QYL1) or composed of amino acid residues 18 to 514 of UniProt ID A8QYL1 with β-galactoside α-2,6-sialyltransferase activity P-JT-ISH-224-ST6-like polypeptide and/or α-2,8-sialyltransferase such as eg from Mus musculus (UniProt ID Q64689). Heterologous and Homologous Expression

Genes to be expressed, whether from plastids or gene bodies, 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 to that of the expression host. Genetic optimization using vendor tools. Culture conditions

Pre-incubation of 96-well microtiter plate experiments was performed starting from a frozen vial or from a single colony in an 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 by diluting 400-fold with 400 µL of MMsf medium. Each strain was grown in multiple wells of a 96-well plate as biological replicates. 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 briefly centrifuging cells for 5 min), or by boiling the culture at 90°C for 15 min before briefly centrifuging cells Or measure by boiling at 60°C for 60 min (= concentration of whole culture medium, concentration of intracellular and extracellular sugars, as defined herein).

In addition, cultures were diluted to measure optical density at 600 nm. The Cell Potency Index or CPI was determined by dividing the oligosaccharide concentration by the biomass (as a relative percentage compared to the reference strain). It is empirically determined that biomass is approximately 1/3 the optical density measured at 600 nm. Example 39. Production of an oligosaccharide mixture comprising 2'FL , LN3 , LNT , LNFP-I and 2'FLNB using a modified Bacillus subtilis host

Bacillus subtilis strains were first modified for LN3 production and growth on sucrose by gene body deletion of the nagB , glmS and gamA genes, and gene body insertion comprising the persistent transcription unit of the genes encoding the Lactose permease (LacY) (UniProt ID P02920), native fructose-6-P-aminotransferase (UniProt ID P0CI73), β-1,3-N-acetyl galactoside from Neisseria meningitidis Glucosaminotransferase LgtA (GenBank: AAM33849.1), sucrose transporter protein (CscB) from Escherichia coli W (UniProt ID E0IXR1), fructokinase (Frk) from Zymomonas mobilis (UniProt ID Q03417) and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (UniProt ID A0ZZH6). In the next step, the mutant strain was further transformed with a gene body comprising a continuous transcription unit of N-acetylglucosamine β-1,3-galactosyltransferase WbgO (UniProt ID D3QY14) from E. coli O55:H7 Embedding modifications to generate LNTs. In a subsequent step, the LNT-producing strain was transformed with an expressive plastid comprising a persistent transcription unit for the α-1,2-fucosyltransferase HpFutC (GenBank: AAD29863.1) from H. pylori. In growth experiments on MMsf medium containing lactose and LNB as acceptors according to the culture conditions provided in Example 38, for the production of 2'FL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal- A mixture of oligosaccharides of b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and 2'FLNB (Fuc-a1,2-Gal-b1,3-GlcNAc) was evaluated for novel strains. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 40. Production of oligos comprising 2'FL , LN3 , LNT , sialylated LN3 , LSTa , LNFP - I , 2'FLNB , 3' -SL and sialylated LNB using a modified Bacillus subtilis host sugar mixture

2'FL , LN3 , LNT , LNFP-I ( Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal- b1,4 - Glc ) and 2 ' can be produced as described in Example 39 A mutant B. subtilis strain of FLNB ( Fuc-a1,2-Gal-b1,3-GlcNAc ) was further genetically knocked out of the nagA gene and modified with a second compatible expression plastid containing a TRP1 selectable marker and a persistent transcription unit , these persistent transcription units are used in two copies of: mutant L-glutamic acid-D-fructose-6-phosphate aminotransferase (glmS*54) from E. coli (via A39T, R250C and G472S mutated to differ from wild-type E. coli glmS, with UniProt ID P17169); phosphatase, selected from the group consisting of 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 genes or PsMupP from Pseudomonas putida, ScDOG1 from Saccharomyces cerevisiae or from BsAraL from Bacillus subtilis (as described in WO18122225); N-Acetylglucosamine 2-Epimerase (AGE) from Bacillus ovale (UniProt ID A7LVG6), N from Neisseria meningitidis - Acetylneuraminic acid synthase (NeuB) (UniProt ID E0NCD4), N-acetylneuraminic acid cytidine syltransferase NeuA (Genbank No. AGV11798.1) from Haemophilus influenzae; and Three copies of the PmultST3 polypeptide (UniProt ID Q9CLP3) of Pasteurella haemorrhagic.

In growth experiments on MMsf medium containing lactose and LNB as acceptors according to the culture conditions provided in Example 38, for the production of 2'FL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal- b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), 2'FLNB (Fuc-a1,2-Gal-b1,3-GlcNAc), 3'-SL, sialylated LN3, saliva A mixture of acidylated LNB and LSTa (Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) was evaluated for novel strains. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 41. Materials and methods of Corynebacterium glutamicum culture medium

Two different media were used, namely enriched tryptone-yeast (TY) medium and minimal medium for shake flask (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 g/L biotin (pH 7.0) and 0.03 g/L protocatechuic acid.

Minimal medium for shake flask (MMsf) experiments containing 20 g/L (NH4)2SO4, 5 g/L urea, 1 g/L KH2PO4, 1 g/L K2HPO4, 0.25 g/L MgSO4.7H2O, 42 g/L MOPS , 10 to 30 g/L glucose or another carbon source (including (but not limited to) fructose, maltose, sucrose, glycerol and maltotriose as specified in the examples) and 1 ml/L trace element mixture. Depending on the experiment, lactose, LNB and/or LacNAc can be added as acceptors.

TY medium consisted of 1.6% pancreas (Difco, Ellen Bodheim, Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR, Leuven, Belgium). The TY agar (TYA) plate system consisted of TY medium supplemented with 12 g/L agar (Difco, Ellen Bodheim, Belgium).

Sterilize complex media (eg TY) by autoclaving (121°C, 21') and sterilize minimal media by filtration (0.22 µm Sartorius). When necessary, the medium is made selective by the addition of antibiotics (eg kanamycin, ampicillin). Strains and Mutations

Corynebacterium glutamicum ATCC 13032 is available from the American Type Culture Collection.

Integrating plastid vectors based on Cre/loxP technology as described by Suzuki et al. (Appl. Microbiol. Biotechnol., April 2005, 67(2):225-33) and as described by Okibe et al. (Journal of The temperature-sensitive shuttle vectors described in Microbiological Methods 85, 2011, 155-163) were constructed for gene deletion, mutation and insertion. Suitable promoters for (heterologous) gene expression can be derived from Yim et al. (Biotechnol. Bioeng., 2013 Nov, 110(11):2959-69). Colonization can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

In one embodiment of the production of lactose-based oligosaccharides, a mutant strain of Corynebacterium glutamicum is produced to contain a gene encoding a lactose importer such as, for example, E. coli lacY with UniProt ID P02920. For 2'FL, 3FL and diFL production, α-1,2- and/or α-1,3-fucosyltransferase expression constructs were additionally added to the strains.

In one embodiment of the production of lacto-N-triose (LNT-II, LN3, GlcNAc-b1,3-Gal-b1,4-Glc), the Corynebacterium glutamicum strain is via a lactose importer (such as, for example, Escherichia coli lacY with UniProt ID P02920) and galactoside β-1,3-N-acetylglucosaminyltransferase (eg, LgtA from Neisseria meningitidis) (GenBank: AAM33849.1) Gene body insertion modification of persistent expression units. For LNT production, the LN3-producing strain was further subjected to a persistent form of N-acetylglucosamine β-1,3-galactosyltransferase such as, for example, WbgO from E. coli O55:H7 (UniProt ID D3QY14) Transcription unit modification. In order to produce lacto-N-neotetraose (LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), the LN3-producing strain was further modified with a persistent transcription unit that N-Acetylglucosamine β-1,4-galactosyltransferase (UniProt ID Q51116) for eg LgtB from Neisseria meningitidis. Both N-acetylglucosamine β-1,3-galactosyltransferase and N-acetylglucosamine β-1,4-galactosyltransferase can be delivered via gene body insertion or self-expressing plastids to the strain. For the production of LNFP-I, the LNT-producing strain can be further modified with an alpha-1,2-fucosyltransferase expressing construct.

In one example of sialic acid production, mutant Corynebacterium glutamicum strains enhance intracellular glucosamine-6-phosphate pools by overexpressing fructose-6-P-aminotransferase (UniProt ID Q8NND3) produce. In addition, the enzymatic activities of genes nagA, nagB and gamA were disrupted by gene knockout and glucosamine-6-P-aminotransferase (UniProt ID P43577) from Saccharomyces cerevisiae, N-acetylglucose from Bacillus ovale Amine-2-epimerase (UniProt ID A7LVG6) and N-acetylneuraminic acid synthase (UniProt ID Q93MP9) from Campylobacter jejuni were overexpressed on the gene body. To allow sialylated oligosaccharide production, the sialic acid-producing strain is further modified with an expression construct comprising an N-acylneuraminic acid cytidine saccharide transferase, such as, for example, the NeuA enzyme from Campylobacter jejuni ( UniProt ID Q93MP7), NeuA enzyme from Haemophilus influenzae (GenBank NO. AGV11798.1) or NeuA enzyme from Pasteurella septicemia (GenBank NO. AMK07891.1); and one or more copies of: β-Galactoside α-2,3-sialyltransferase, such as for example PmultST3 from Pasteurella septicemia (UniProt ID Q9CLP3) or one consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 with β - PmultST3-like polypeptide with galactoside alpha-2,3-sialyltransferase activity, NmeniST3 from Neisseria meningitidis (GenBank NO. ARC07984.1) or from a strain of Pasteurella septicaemia subsp. PmultST2 of Pm70 (GenBank NO. AAK02592.1); β-galactoside α-2,6-sialyltransferase, such as PdST6 (UniProt ID 066375) from P. mermaidii or the amine group from UniProt ID 066375 A PdST6-like polypeptide with β-galactoside α-2,6-sialyltransferase activity consisting of acid residues 108 to 497 or P-JT-ISH-224-ST6 from Photophora JT-ISH-224 (UniProt ID A8QYL1) or P-JT-ISH-224-ST6-like with β-galactoside α-2,6-sialyltransferase activity consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 Polypeptides and/or alpha-2,8-sialyltransferase such as eg from Mus musculus (UniProt ID Q64689). Heterologous and Homologous Expression

Genes to be expressed, whether from plastids or gene bodies, 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 to that of the expression host. Genetic optimization using vendor tools. Culture conditions

Pre-incubation of 96-well microtiter plate experiments was performed starting from a frozen vial or from a single colony in a 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 by diluting 400-fold with 400 µL of MMsf medium. Each strain was grown in multiple wells of a 96-well plate as biological replicates. 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 incubation experiment, samples were taken from each well to measure the supernatant concentration (extracellular sugar concentration, after briefly centrifuging cells for 5 min), or by boiling the culture at 60°C for 15 min before briefly centrifuging cells Measurements (= concentration of whole broth, concentration of intracellular and extracellular sugars, as defined herein).

In addition, cultures were diluted to measure optical density at 600 nm. The Cell Potency Index or CPI is determined by dividing the measured oligosaccharide concentration, eg, sialyllactose concentration, in the complete broth by the biomass (as a relative percentage compared to a reference strain). It is empirically determined that biomass is approximately 1/3 the optical density measured at 600 nm. Example 42. Production of an oligosaccharide mixture comprising LN3 , LNT , LNFP-I , 2'-FL and 2'FLNB using a modified Corynebacterium glutamicum host

Corynebacterium glutamicum strains were first modified for LN3 production and growth on sucrose by gene body deletion of the ldh , cgl2645 and nagB genes and gene body insertion comprising the persistent transcription unit of the genes encoding from Lactose permease (LacY) from Escherichia coli (UniProt ID P02920), native fructose-6-P-aminotransferase (UniProt ID Q8NND3), β-1,3-N galactoside from Neisseria meningitidis - Acetylglucosamine transferase LgtA (GenBank: AAM33849.1), sucrose transporter protein (CscB) from Escherichia coli W (UniProt ID E0IXR1), fructokinase (Frk) from Zymomonas mobilis (UniProt ID Q03417) and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (UniProt ID A0ZZH6). In the next step, the mutant strain was further transformed with a gene body comprising a continuous transcription unit of N-acetylglucosamine β-1,3-galactosyltransferase WbgO (UniProt ID D3QY14) from E. coli O55:H7 Embedding modifications to generate LNTs. In a subsequent step, the LNT-producing strain was transformed with an expressive plastid comprising a persistent transcription unit for the α-1,2-fucosyltransferase HpFutC (GenBank: AAD29863.1) from H. pylori. In growth experiments on MMsf medium containing lactose and LNB as acceptors according to the culture conditions provided in Example 41 for the production of oligosaccharide mixtures comprising LN3, LNT, LNFP-I, 2'-FL and 2'FLNB Evaluate novel strains. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 43. Production of oligosaccharide mixture comprising LN3 , sialylated LN3 , LNnT , 6'SL , 6'SLacNAc and LSTc using a modified Corynebacterium glutamicum host

The strain of corynebacterium glutamicum was first modified to produce sialic acid by gene body deletion of ldh , cgl2645 , nagA and nagB genes and gene body insertion of persistent transcription units containing native fructose-6-P - aminotransferase (UniProt ID Q8NND3), glucosamine-6-P-aminotransferase from Saccharomyces cerevisiae (UniProt ID P43577), N-acetylglucosamine-2-episolone from Bacillus ovale Constructing enzyme (UniProt ID A7LVG6) and N-acetylneuraminic acid synthase (UniProt ID Q93MP9) from Campylobacter jejuni. The Corynebacterium glutamicum strain was further modified by gene body insertion of a persistent transcription unit to allow the production of LN3 and lacto-N-neotetraose (LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4 -Glc), these persistent transcription units are used for lactose permease LacY from E. coli (UniProt ID P02920), LgtA from Neisseria meningitidis (UniProt ID Q9JXQ6) and for Neisseria meningitidis LgtB (UniProt ID Q51116). In the next step, mutant strains were transformed with expressing plastids with persistent transcription units for NeuA from Pasteurella septicemia (GenBank: AMK07891.1) and amine from UniProt ID 066375 A PdST6-like polypeptide with β-galactoside α-2,6-sialyltransferase activity consisting of amino acid residues 108 to 497. In growth experiments on MMsf medium containing lactose and LacNAc as acceptors according to the culture conditions provided in Example 41, for the production of LN3, 6'-sialylated LN3 (Neu5Ac-a2,6-(GlcNAc- b1,3)-Gal-b1,4-Glc), 6'SL, 6'SLacNAc, LNnT and LSTc (Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4- Glc) oligosaccharide mixture to evaluate novel strains. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 44. Materials and Methods of Chlamydomonas reinhardtii Medium

Chlamydomonas reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7.0). TAP medium used 1000X stock Hutner's trace element mix. Hertner's trace element mixture consisted of 50 g/L Na2EDTA.H2O (Titriplex III), 22 g/L ZnSO4.7H2O, 11.4 g/L H3BO3, 5 g/L MnCl2.4H2O, 5 g/L FeSO4.7H2O , 1.6 g/L CoCl2.6H2O, 1.6 g/L CuSO4.5H2O and 1.1 g/L (NH4)6MoO3.

TAP medium contains 2.42 g/L Tris (para(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 and/or acceptors for sugar synthesis, precursors and/or acceptors such as, for example, galactose, glucose, fructose, fucose, GlcNAc, LNB and/or LacNAc can be added. The medium was sterilized by autoclaving (121°C, 21'). For stock cultures on slanted agar, use TAP medium containing 1% agar (with 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−) can be purchased from the Chlamydomonas Resource Center (https://www.chlamycollection.org) at the University of Minnesota, U.S.A., USA.

Expression plastids were derived from pSI103, eg, available from the Chlamydomonas Resource Center. Colonization can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation. Suitable promoters for (heterologous) gene expression can be derived, for example, from Scranton et al. (Algal Res. 2016, 15: 135-142). Targeted genetic modification, such as gene knockout or gene replacement, can be performed using the Crispr-Cas technology as described, for example, by Jiang et al. (Eukaryotic Cell 2014, 13(11): 1465-1469).

Transformation via electroporation was performed as described by Wang et al. (Biosci. Rep. 2019, 39: BSR2018210). Cells were grown in liquid TAP medium under constant aeration and continuous light at a light intensity of 8000 Lx until the cell density reached 1.0-2.0 x 107 cells/ml. Next, cells were seeded into freshly prepared liquid TAP medium at a concentration of 1.0×10 6 cells/ml and grown under continuous light for 18-20 h until the cell density reached 4.0×10 6 cells/ml. Next, cells were harvested by centrifugation at 1250 g for 5 min at room temperature, washed and resuspended with pre-cooled liquid TAP medium (Sigma, USA) containing 60 mM sorbitol and frozen for 10 min. Next, 250 µL of the cell suspension (corresponding to 5.0 x 107 cells) was placed in a pre-chilled 0.4 cm electroporation cuvette (400 ng/mL) with 100 ng of plastid DNA. 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, the cuvette was placed on ice for 10 minutes. Finally, the cell suspension was transferred to a 50 ml conical centrifuge tube containing 10 mL of fresh liquid TAP medium containing 60 mM sorbitol for overnight recovery with slow shaking in the dark. After overnight recovery, cells were re-harvested and seeded by starch embedding onto selective 1.5% (w/v) agar-TAP dishes containing ampicillin (100 mg/L) or chloramphenicol (100 mg/L). superior. The disks 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 one example of producing GDP-fucose, C. reinhardtii cells were modified with a transcription unit for GDP-fucose synthase from Arabidopsis (GER1, UniProt ID 049213).

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

In one embodiment of the production of UDP-N-acetylgalactosamine, the Chlamydomonas reinhardtii cells are treated with UDP-N-acetylglucosamine 4-epimerase from Pseudomonas aeruginosa serotype O6 Transcription unit modification of wbpP (UniProt ID Q8KN66).

In one embodiment of the production of LNB, the C. reinhardtii cells modified for UDP-galactose production are further modified with an expression plastid comprising a transcription unit for N-acetyl from E. coli O55:H7 Glucosamine beta-1,3-galactosyltransferase WbgO (UniProt ID D3QY14). In one embodiment of the production of LacNAc, the C. reinhardtii cells modified for UDP-galactose production are further modified with an expression plastid comprising a transcription unit for N-beta Acylglucosamine beta-1,4-galactosyltransferase LgtB (UniProt ID Q51116).

In one embodiment of CMP-sialic acid synthesis, C. reinhardtii cells are modified with persistent transcription units for use in eg UDP-N-acetylglucosamine-2 from GNE of Homo sapiens - Epimerase/N-Acetylmannosamine Kinase (UniProt ID Q9Y223) or a mutant form of a human GNE polypeptide comprising the R263L mutation, such as eg N-Acetylneuraminic acid-9 from NANS of Homo sapiens - Phosphate synthase (UniProt ID Q9NR45) and N-acylneuraminic acid cytidine syltransferase (UniProt ID Q8NFW8) as eg from CMAS of Homo sapiens. In one embodiment of the production of sialylated oligosaccharides, C. reinhardtii cells are modified with CMP-sialic acid transporter proteins such as eg CST from Mus musculus (UniProt ID Q61420) and selected from species such as eg Chi Golgi-localized sialyltransferase in human, Mus musculus, and brown mouse. Heterologous and Homologous Expression

Genes to be expressed, whether from plastids or gene bodies, 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 to that of the expression host. Genetic optimization using vendor tools. Culture conditions

Chlamydomonas reinhardtii cells were cultured in selective TAP-agar dishes at 23 +/- 0.5°C with a light intensity of 8000 Lx on a 14/10 h light/dark cycle. Cells were analyzed after 5 to 7 days in culture.

For high-density culture, cells can be cultured in closed systems such as, for example, Chen et al. (Bioresour. Technol. 2011, 102: 71-81) and Johnson et al. (Biotechnol. Prog. 2018, 34: 811-827) Vertical or horizontal tube photobioreactors, stirred tank photobioreactors or flat plate photobioreactors as described. Example 45. Production of an oligosaccharide mixture comprising 2'FLNB and 2'FLacNAc in mutant Chlamydomonas reinhardtii cells

Chlamydomonas reinhardtii cells were engineered for GDP-fucose production with gene body insertion of a persistent transcription unit comprising a GDP-fucose synthase from Arabidopsis as described in Example 44 Enzyme gene (GER1, UniProt ID O49213). In the next step, the mutant cells were transformed with an expressing plastid containing a persistent transcription unit for the alpha-1,2-fucosyltransferase HpFutC (GenBank: AAD29863. 1). According to the culture conditions provided in Example 44, novel strains were evaluated in growth experiments on TAP agar plates containing LNB and LacNAc as receptors. After 5 days of incubation, cells were harvested and analyzed on UPLC for 2'FLNB and 2'FLacNAc production. Example 46. Materials and Methods for Animal Cells Isolation of Mesenchymal Stem Cells from Adipose Tissue of Different Mammals

Fresh adipose tissue is obtained from slaughterhouses (eg, cattle, swine, sheep, chickens, ducks, catfish, snakes, frogs) or liposuction (eg, in the case of humans, after signed informed consent) and maintained at in phosphate buffered saline supplemented with antibiotics. Enzymatic digestion of adipose tissue followed by centrifugation to isolate mesenchymal stem cells. The isolated interleaf stem cells are transferred to cell culture flasks and grown under standard growth conditions (eg, 37°C, 5% CO2). Initial media included DMEM-F12, RPMI and Alpha-MEM media (supplemented with 15% fetal bovine serum) and 1% antibiotics. Then after the first pass, the medium was replaced with medium supplemented with 10% FBS (fetal bovine serum). For example, Ahmad and Shakoori (2013, Stem Cell Regen Med. 9(2): 29-36), herein incorporated by reference in its entirety for all purposes, describe the methods described in this example some variations of it. Isolation of mesenchymal stem cells from milk

This example illustrates the isolation of mesenchymal stem cells from milk collected under sterile conditions from a human or any other mammal, such as those described herein. An equal volume of phosphate buffered saline was added to the diluted milk, followed by centrifugation for 20 min. Cell pellets were washed three times with phosphate buffered saline, and cells were seeded in cell culture flasks in DMEM-F12, RPMI and Alpha-MEM medium supplemented with 10% fetal bovine serum and 1% antibiotics under standard culture conditions . For example, Hassiotou et al. (2012, Stem Cells. 30(10): 2164-2174), which is incorporated herein by reference in its entirety for all purposes, describe some of the methods described in this example. some variations. Differentiate stem cells using 2D and 3D culture systems

The isolated mesenchymal cells can be differentiated into mammary-like epithelial cells and luminal cells in 2D and 3D culture systems. See, eg, 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. 1999; Animal Cell Technology ': Basic & Applied Aspects, Springer, Dordrecht. 141-145; Williams et al. 2009, Breast Cancer Res 11(3): 26-43; and Arevalo et al. 2015, Am J Physiol Cell Physiol. 310(5) : C348-C356; each of which is incorporated herein by reference in its entirety for all purposes.

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

For 3D cultures, detached cells were trypsinized and cultured for six days in Matrigel, hyaluronic acid, or ultra-low attachment surface culture dishes, and by adding 10 ng/ml epithelial growth factor and 5 pg/ml insulin supplemented with Growth medium induction for differentiation and lactation. At confluence, cells were fed growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U/ml penicillin, 100 μg/ml streptomycin) and 5 pg/ml insulin for 48 hours . To induce differentiation, cells were fed complete growth medium containing 5 pg/ml insulin, 1 pg/ml cortisol, 0.65 ng/ml triiodothyronine, 100 nM dexamethasone and 1 pg/ml prolactin. After 24 h, serum was removed from the complete induction medium. Method of making mammary gland-like cells

Mammalian cells were induced to pluripotency by reprogramming with viral vectors encoding Oct4, Sox2, Klf4 and c-Myc. The resulting reprogrammed cells were then cultured in Mammocult medium (available from Stem Cell Technologies) or breast cell enrichment medium (DMEM, 3% FBS, estrogen, progesterone, heparin, cortisol, insulin, EGF) to allow It is mammary gland-like, from which the performance of selected milk components can be induced. Alternatively, epigenetic remodeling is performed using a remodeling system such as CRISPR/Cas9 to activate selected genes of interest, such as casein, alpha-lactalbumin to be persisted, to allow expression of their respective proteins, and/or Selected endogenous genes are down-regulated and/or knocked out as described, for example, in WO21067641, which is incorporated herein by reference in its entirety for all purposes. nourish

Complete growth medium including high glucose DMEM/F12, 10% FBS, 1% NEAA, 1% Penicillin-Streptomycin, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, and 5 pg/ml Cortisol . Complete lactation medium includes high glucose DMEM/F12, 1% NEAA, 1% penicillin-streptomycin, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, 5 pg/ml cortisol and 1 pg/ml ml prolactin (5 μg/ml, in Hyunh 1991). Cells were seeded on collagen-coated flasks in complete growth medium at a density of 20,000 cells/cm 2 and left to adhere and expand in complete growth medium for 48 hours, after which the medium was changed 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 lipids, lactose, casein, and whey, into the medium. The lactation medium of the desired concentration can be achieved by concentration or dilution by ultrafiltration. The desired salt balance of the lactation medium can be achieved by dialysis, eg, to remove undesired metabolites from the medium. The selective extraction of hormones and other growth factors used can be performed by resin purification, such as the use of nickel resins to remove HIS-tagged growth factors, to further reduce contaminant levels in the lactation product. Example 47. Production of an oligosaccharide mixture comprising 2'FLNB, 2'FLacNAc , 2' - FL , sialylated LNB , sialylated LacNAc and 3' - SL in non - mammary adult stem cells

Isolated mesenchymal cells and reprogrammed into mammary gland-like cells as described in Example 46 were modified by CRISPR-CAS to overexpress GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), human galacto- Glycoside alpha-1,2-fucosyltransferase FUT1 (UniProt ID P19526), N-acylneuraminic acid cytidine acyltransferase (UniProt ID Q99KK2) from Mus musculus and CMP- N-Acetylneuraminic acid-β-1,4-galactoside α-2,3-sialyltransferase ST3GAL3 (UniProt ID Q11203). Cells were seeded on collagen-coated flasks in complete growth medium at a density of 20,000 cells/cm 2 and left to adhere and expand in complete growth medium for 48 hours, after which the medium was changed to Complete lactation medium containing lactose, LacNAc and LNB as precursors for about 7 days. After culturing as described in Example 46, cells were subjected to UPLC to analyze the production of 2'-FL, 3'-SL, 2'FLNB, 2'FLacNAc, sialylated LNB and sialylated LacNAc. Example 48. Assessment of LacNAc , Sialyl- LacNAc , Sialyl- Lewisx , Fucosylated LacNAc , 3 -FL and 3'-SL Production in Non-Breast Adult Stem Cells

Isolated mesenchymal cells and cells reprogrammed into mammary-like cells as described in Example 46 were modified by CRISPR-CAS to overexpress β-1,4-galactosyltransferase 4 B4GalT4 from Homo sapiens (UniProt ID O60513), GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), Galactoside alpha-1,3-fucosyltransferase FUT3 from Homo sapiens (UniProt ID P21217), N-Acetylneuraminic acid cytidine syltransferase (UniProt ID Q99KK2) from Mus musculus and CMP-N-acetylneuraminic acid-β-1,4-galactoside α- from Homo sapiens 2,3-Sialyltransferase ST3GAL3 (UniProt ID Q11203). All genes introduced into cells are codon-optimized for the host. Cells were seeded on collagen-coated flasks in complete growth medium at a density of 20,000 cells/cm 2 and left to adhere and expand in complete growth medium for 48 hours, after which the medium was changed to Complete lactation medium containing LacNAc and lactose as acceptors for about 7 days. After culturing as described in Example 46, cells were subjected to UPLC to analyze the production of LacNAc, sialyl-LacNAc, sialyl-Lewis x, fucosylated LacNAc, 3-FL and 3'-SL. Example 49. Evaluation of membrane transport protein performance in modified E. coli hosts

The modified E. coli hosts described in Examples 29, 30, 31 and 32 were further modified by gene body insertion of a persistent transcription unit for a membrane transporter selected from a list comprising : MdfA from S. mogeni (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter johnsonii (UniProt ID D4BC23), MdfA from Escherichia coli (UniProt ID P0AEY8), from Yorkia reginsburg MdfA (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207), iceT from Citrobacter johnsonii (UniProt ID D4B8A6), nanT from E. coli O6:H1 (UniProt ID Q8FD59), from E. coli O157 : nanT from H7 (UniProt ID Q8X9G8), nanT from Escherichia coli (UniProt ID B1EFH1), EntS from Escherichia coli (UniProt ID P24077), EntS from Kluyveer ascorbate (UniProt ID A0A378GQ13), EntS from Salmonella enterica subsp. arizona (UniProt ID A0A6Y2K4E8), SetA from E. coli (UniProt ID P31675), SetB from E. coli (UniProt ID P33026), SetC from E. coli (UniProt ID P31436) , oppF (UniProt ID P77737) from Escherichia coli, lmrA (UniProt ID A0A1V0NEL4) from Lactococcus lactis subsp. lactis var. lactis and Blon_2475 (UniProt ID B7GPD4) from Bifidobacterium longum infantum. Novel strains each expressing one of these membrane transport proteins were evaluated in growth experiments according to the culture conditions provided in Example 1, wherein the medium contained sucrose as carbon source and lactose and LacNAc as acceptors. Each strain was grown in four biological replicates in 96-well plates. After 72 h of incubation, the broth (ie, the extracellular and intracellular fractions) and the extracellular and intracellular fractions individually were collected and analyzed for sugars on UPLC.

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Claims (47)

A method for producing a mixture of at least two different oligosaccharides by cells, preferably a single cell, the method comprising the steps of: i. Provide a cell, preferably a single cell, capable of expressing a glycosyltransferase and capable of synthesizing a nucleotide-sugar, wherein the nucleotide-sugar is a donor for the glycosyltransferase, and ii. culturing the cell under conditions that allow expression of the glycosyltransferase and synthesis of the nucleotide-sugar, and iii. adding at least two receptors to the culture such that the cells are capable of producing at least two oligosaccharides, preferably any one of these receptors is a disaccharide or an oligosaccharide, iv. Preferably, at least one of the oligosaccharides is isolated from the culture, more preferably, all of the oligosaccharides are isolated from the culture. The method of claim 1, wherein any of the receptors is a disaccharide or mammalian milk oligosaccharide (MMO), preferably, any of the receptors is a mammal Lacto-oligosaccharides. The method of any one of claims 1 or 2, wherein the cell is metabolically engineered to make the mixture and/or modified with at least one gene expression module from any of the at least one expression module One appears to be persistent or produced by natural inducers. The method of any one of claims 1 to 3, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein. The method of any one of claims 1 to 4, wherein the cell produces a mixture of three or more different oligosaccharides. The method of any one of claims 1 to 5, wherein the cell produces a mixture of different oligosaccharides, wherein at least two of the oligosaccharides differ in degree of polymerization. The method of any one of claims 1 to 6, wherein the glycosyltransferase is selected from the list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase, transglucosyltransferase Enzymes, Mannosyltransferase, N-Acetylglucosaminyltransferase, N-Acetylgalactosaminyltransferase, N-Acetylmannosyltransferase, Xylosyltransferase, Glucose Aldolase, galacturonyltransferase, glucosaminyltransferase, N-hydroxyacetylneuraminosyltransferase, rhamnosyltransferase, N-acetylrhamnosyltransferase , UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine (altrosamine) transaminase, UDP-N-acetylglucosamine enol acetone Enolpyruvyl transferase and fucosaminotransferase, - preferably, the fucosyltransferase is selected from the list comprising: α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1 ,4-fucosyltransferase and α-1,6-fucosyltransferase, - preferably, the sialyltransferase is selected from the list comprising: α-2,3-sialyltransferase, α-2,6-sialyltransferase and α-2,8- sialyltransferase, - preferably, the galactosyltransferase is selected from the list comprising: 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 list comprising α-glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase and β-glucosyltransferase 1,4-glucosyltransferase, - preferably, the mannosyltransferase is selected from the list comprising α-1,2-mannosyltransferase, α-1,3-mannosyltransferase and α-1,6- Mannosyltransferase, - preferably, the N-acetylglucosaminyltransferase is selected from the list comprising: galactoside β-1,3-N-acetylglucosaminyltransferase and β-1,6 -N-acetylglucosaminyltransferase, - Preferably, the N-acetylgalactosaminyltransferase is α-1,3-N-acetylgalactosaminyltransferase. The method of any one of claims 1 to 7, wherein the cell is modified in the expression or activity of the glycosyltransferase. The method of any one of claims 1 to 8, wherein the glycosyltransferase is a fucosyltransferase and the donor nucleotide-sugar is GDP-fucose (GDP-Fuc). The method of any one of claims 1 to 9, wherein the glycosyltransferase is a sialyltransferase and the donor nucleotide-sugar is CMP-N-acetylneuraminic acid (CMP-Neu5Ac). The method of any one of claims 1 to 10, wherein the glycosyltransferase is N-acetylglucosaminyltransferase and the donor nucleotide-sugar is UDP-N-acetylglucosamine ( UDP-GlcNAc). The method of any one of claims 1 to 11, wherein the glycosyltransferase is a galactosyltransferase and the donor nucleotide-sugar is UDP-galactose (UDP-Gal). The method of any one of claims 1 to 12, wherein the glycosyltransferase is N-acetylgalactosaminyltransferase and the donor nucleotide-sugar is UDP-N-acetylgalactose Amine (UDP-GalNAc). The method of any one of claims 1 to 13, wherein the glycosyltransferase is N-acetylmannosylaminotransferase and the donor nucleotide-sugar is UDP-N-acetylmannose Amine (UDP-ManNAc). The method of any one of claims 1 to 14, wherein the nucleotide-sugar is selected from the list comprising GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-B Acylgalactosamine (UDP-GalNAc), UDP-N-Acetylmannosamine (UDP-ManNAc), GDP-Mannose (GDP-Man), UDP-Glucose (UDP-Glc), UDP-2- Acetamido-2,6-dideoxy--L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy--L-lysole Sugar-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-Pneumosamine (UDP-L-PneNAC or UDP-2-Acetylamino-2,6-dideoxy-L-Tarot) sugar), UDP-N-Acetylmuramic acid, UDP-N-Acetyl-L-isorhamnosamine (UDP-L-QuiNAc or UDP-2-Acetylamino-2,6-di deoxy-L-glucose), GDP-L-isorhamnose, CMP-N-hydroxyacetylneuraminic 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, UDP- Xylose. The method of any one of claims 1 to 15, wherein any of the acceptors has a degree of polymerization of 3 or more, preferably wherein all of the acceptors have a degree of polymerization of 3 or more many. The method of any one of claims 1 to 16, wherein all the receptors have different degrees of polymerization. The method of any one of claims 1 to 17, wherein the culture is supplemented with at least 3 receptors for making the oligosaccharide mixture, preferably at least 4 receptors, more preferably at least 5 receptors body. The method of any one of claims 1 to 18, wherein the oligosaccharide mixture comprises at least one oligosaccharide that is fucosylated, sialylated, galactosylated, glycosylated, Xylosylation, Mannosylation, with N-acetylglucosamine, with N-acetylneuraminic acid, with N-hydroxyacetylneuraminic acid, with N-acetylgalactosamine, Contains rhamnose, contains glucuronide, contains galacturonate, and/or contains N-acetylmannosamine. The method of any one of claims 1 to 19, wherein the oligosaccharide mixture comprises charged and/or neutral oligosaccharides, preferably wherein at least one of the charged oligosaccharides is a sialylated oligosaccharide. The method of any one of claims 1 to 19, wherein the oligosaccharide mixture comprises fucosylated and/or unfucosylated neutral oligosaccharides. The method of any one of claims 1 to 21, wherein the oligosaccharide mixture comprises at least one fucosylated oligosaccharide. The method of any one of claims 1 to 20, wherein the oligosaccharide mixture comprises at least one sialylated oligosaccharide. The method of any one of claims 1 to 23, wherein the oligosaccharide mixture comprises at least one oligosaccharide comprising N-acetylglucosamine monosaccharide units. The method of any one of claims 1 to 24, wherein the oligosaccharide mixture comprises at least one galactosylated oligosaccharide. The method of claim 21, wherein all of the oligosaccharides in the mixture are fucosylated oligosaccharides. The method of claims 1 to 26, wherein the cell produces three fucosylated oligosaccharides. The method of any one of claims 1 to 27, wherein the receptors are manufactured by a method comprising extraction from natural sources, biotechnological processes, physical processes, chemical processes, and combinations thereof. The method of any one of claims 1 to 28, wherein any of the acceptors is fully converted to any of the oligosaccharides. The method of any one of claims 1 to 29, wherein the cell manufactures the mixture of at least two oligosaccharides intracellularly, and wherein a portion or substantially all of the manufactured oligosaccharides remain intracellularly and/or Excreted from the cell via passive or active transport. The method of any one of claims 1 to 30, wherein the cell is further metabolically engineered for use in i) Modified expression of endogenous membrane proteins, and/or ii) the modified activity of endogenous membrane proteins, and/or iii) Representation of homologous membrane proteins, and/or iv) performance of heterologous membrane proteins, wherein the membrane protein is involved in the secretion of any of the oligosaccharides from the cell, preferably wherein the membrane protein is involved in the secretion of all of the oligosaccharides from the cell. The method of any one of claims 1 to 31, wherein the cell is further metabolically engineered for use in i) Modified expression of endogenous membrane proteins, and/or ii) the modified activity of endogenous membrane proteins, and/or iii) Representation of homologous membrane proteins, and/or iv) performance of heterologous membrane proteins, wherein the membrane protein is involved in the uptake of precursors for the synthesis of any of the oligosaccharides in the mixture, preferably wherein the membrane protein is involved in the uptake of all desired precursors, more preferably wherein the membrane protein is involved in uptake all such receptors. The method of any one of claims 31 or 32, wherein the membrane protein is selected from the list comprising: porter, P-P-bond hydrolysis-driven transporter, β-barrel porin (β-barrel porin) Barrel Porin), accessory transporters, putative transporters, and phosphotransfer-driven group translocation proteins, Preferably, these transport proteins comprise MFS transport proteins, carbohydrate efflux transport proteins and chelatin export proteins, Preferably, the P-P-bond hydrolysis-driven transport proteins comprise ABC transport proteins and chelatin export proteins. The method of any one of claims 31 to 33, wherein the membrane protein provides for improved manufacture of the mixture of at least two oligosaccharides and/or enables and/or enhances the efflux of the mixture of at least two oligosaccharides. The method of any one of claims 1 to 34, wherein the cells are resistant to lactose killing when grown in an environment where lactose is combined with one or more other carbon sources. The method of any one of claims 1 to 35, wherein the cell comprises a modification for reducing acetate production compared to an unmodified precursor cell. The method of claim 36, wherein the cell comprises any one or more of proteins comprising reduced or reduced expression and/or abrogated, attenuated, reduced or delayed activity compared to an unmodified precursor cell: β-Galactosidase, Galactoside O-Acetyl Transferase, N-Acetyl Glucosamine-6-Phosphate Deacetylase, Glucosamine-6-Phosphate Deaminase, N-Acetyl Glucose Amine arrestin, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphoglucose-1-phosphotransferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminic acid lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB -Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridine syltransferase, 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 control protein, transcriptional repressor protein IclR, lon protease, glucose-specific translocation phosphate Transferase IIBC component ptsG, glucose specific translocation phosphotransferase (PTS) IIBC component malX, enzyme IIA ^Glc , β-glucoside specific PTS enzyme II, fructose specific PTS polyphosphooxytransferase FruA and FruB , alcohol dehydrogenase, aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase (phosphoacyltransferase), phosphoacetyltransferase, pyruvate decarboxylase. The method of any one of claims 1 to 37, wherein the cell is capable of producing phosphoenolpyruvate (phosphoenolpyruvate; PEP). The method of any one of claims 1 to 38, wherein the cells are modified for enhanced production and/or supply of phosphoenolpyruvate (PEP) compared to unmodified precursor cells. The method of any one of claims 1 to 39, wherein any of the oligosaccharides is a mammalian milk oligosaccharide, preferably, wherein all of the oligosaccharides are mammalian milk oligosaccharides. The method of any one of claims 1 to 40, wherein any of the oligosaccharides is an antigen of the human ABO blood group system, preferably wherein all of the oligosaccharides are antigens of the human ABO blood group system. The method of any one of claims 1 to 41, wherein the cells are bacteria, fungi, yeast, plant cells, animal cells or protozoal cells, - preferably, the bacteria are Escherichia coli strains, More preferably an Escherichia coli strain of the K-12 strain, even more preferably the Escherichia coli K-12 strain is Escherichia coli MG1655, - preferably, the fungus belongs to a genus selected from the group comprising: Rhizopus , Dictyostelium , Penicillium , Mucor or Aspergillus , - preferably, the yeast belongs to a genus selected from the group consisting of: Yeast Saccharomyces , Zygosaccharomyces , Pichia , Komagataella , Hansenula , Yarrowia , Stamos ( Starmerella ), Kluyveromyces ( Kluyveromyces ) or Debaromyces ( Debaromyces ), - preferably, the plant cells are algal cells or derived from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soybean , maize or maize plants, - preferably, the animal cells are derived from non-human mammals, birds, fish, invertebrates, reptiles, amphibians or insects, or are derived from human cells excluding embryonic stem cells Genetically modified cell lines, more preferably the human and non-human mammalian cells are epithelial cells, embryonic kidney cells, fibroblasts, COS cells, Chinese hamster ovary (CHO) cells, murine myeloma cells, NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, more preferably the insect cells are derived from Spodoptera frugiperda , Bombyx mori , Mamestra brassicae , Spodoptera frugiperda ( Trichoplusia ni ) or Drosophila melanogaster , - preferably, the protozoal cell is a Leishmania tarentolae cell. The method of claim 42, wherein the cell is a viable Gram-negative bacterium comprising a reduced or eliminated synthetic poly-N-acetyl group compared to an unmodified precursor cell - Glucosamine (poly-N-acetyl-glucosamine; PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, ribo-oligosaccharide, osmoregulatory periplasmic glucan (Osmoregulated Periplasmic Glucan; OPG), glycerol glucoside, polysaccharide and/or cocoon sugar. The method of any one of claims 1 to 43, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, two-phase partition, reverse osmosis, microfiltration, activated carbon or carbon treatment, treatment with 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 layer analysis. The method of any one of claims 1 to 44, further comprising purifying any of the oligosaccharides from the cell. The method of claim 45, wherein the purification comprises at least one of the following steps: using activated charcoal or carbon, using charcoal, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange, using alcohol, using a hydroalcoholic mixture, Crystallization, evaporation, precipitation, drying, spray drying, freeze drying, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum belt drying, vacuum belt drying, Tumble drying, tumble drying, vacuum tumble drying or vacuum tumble drying. Use of a method as claimed in any one of claims 1 to 46 for the manufacture of a mixture of at least two different oligosaccharides.