TW202221132A - Production of a mixture of mammalian milk oligosaccharides 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 describes a cell metabolically engineered for production of a mixture of at least three different mammalian milk oligosaccharides. Furthermore, the present invention provides a method for the production of a mixture of at least three different mammalian milk oligosaccharides by a cell as well as the purification of at least one of said mammalian milk oligosaccharides from the cultivation.

Description

Production of a mixture of mammalian milk oligosaccharides by cells

The present invention is in the technical field of synthetic biology and metabolic engineering. More specifically, the present invention is in the technical field of fermentation of metabolically engineered cells. The present invention describes a cell metabolically engineered for the production of a mixture of at least three different mammalian milk oligosaccharides. Furthermore, the present invention provides a method for producing by cells a mixture of at least three different mammalian milk oligosaccharides and purifying at least one of these mammalian milk oligosaccharides from culture.

Mammalian milk oligosaccharide (MMO) is a type of complex carbohydrate present in mammalian milk. Oligosaccharide moieties are major components in mammalian milk. MMOs are involved in many biological phenomena, such as differentiation, development, biorecognition processes, and immune processes, and can, for example, act as probiotics, antiadhesives, and antimicrobials. Such multiple functionalities account for the great diversity of their structures (Boehm and Stahl, J. Nutrition 137(3) (2007) 847S-849S; Bode L., Early Human Devel. 91(11) (2015) 619-622) . Due to the broad functional spectrum of this type of oligosaccharide, mixtures of MMOs have great scientific and commercial value. However, the availability of mixtures of MMOs is limited because production relies on chemical or chemical enzymatic synthesis or on purification from natural sources. 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 from which biotechnologists synthesize MMOs 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 MMOs 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 a single MMO rather than a mixture of MMOs.

It is an object of the present invention to provide tools and methods by which mixtures of mammalian milk oligosaccharides comprising at least three different MMOs can be passed through cells in an efficient, time-efficient and cost-effective manner and if necessary in a continuous process , preferably produced by a single cell.

According to the present invention, this object and other objects are achieved by providing a cell and a method for producing a mammalian milk oligosaccharide mixture comprising at least three different MMOs, wherein the cell is genetically modified for the production of the MMOs.

Surprisingly, it has now been found that it is possible to generate MMO mixtures comprising at least three different MMOs from a single cell. The present invention provides a metabolically engineered cell and a method for producing a mammalian milk oligosaccharide mixture comprising at least three different MMOs. The method comprises the steps of: providing cells expressing at least two glycosyltransferases and capable of synthesizing one or more nucleotide-sugars as donors for one or more of the glycosyltransferases; and The cells were cultured in the MMO mixture. The present invention also provides methods for isolating at least one, preferably all, of these MMOs from a mammalian milk oligosaccharide mixture. Furthermore, the present invention provides a cell metabolically engineered for the production of a mixture of MMOs comprising at least three different MMOs. definition

Words used in this specification to describe the invention and various specific examples thereof are to be understood not only in the sense of their commonly defined meanings, but also to include structures specifically defined in this specification outside the scope of the commonly defined meanings , 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.

The various embodiments and aspects of the embodiments disclosed herein are to be construed not only in the order and context specifically described in this specification, but also in any order and 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, specific examples 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 specific examples described have been presented for purposes of illustration only, and should not be construed as limiting the invention. It will be apparent to those skilled in the art that changes, other specific examples, 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, the scope of 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. Throughout the application, the verb "to comprise" may be replaced by "to consist of" or "to consist essentially of", and vice versa. 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. Thus 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", the most The preferred substitution is "at least seven".

Specific examples as identified herein may be grouped 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 "genetically modified" are used interchangeably and indicate that the cell replicates a heterologous nucleic acid, or Expressed by a heterologous nucleic acid (that is, a sequence "foreign to said cell" or a sequence "foreign to said location or environment in said cell" sequence) encoded peptides or proteins. 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 that is foreign to a particular cell (eg, from a different species), or if from the same source, from 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 optional 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 DeoR in B. subtilis , 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 "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 (eg, including but not limited 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) Not limited to) apoptosis and autophagy.

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 "wild type" refers to the genetic or phenotypic condition commonly known as it occurs in nature.

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.

As used herein, the term "derivative" of a polypeptide is one that can contain deletions, additions or substitutions of amino acid residues within the amino acid sequence of the polypeptide, but which result in silent changes, thereby producing functionally equivalent Polypeptide of polypeptide. Amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphiphilic properties of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; planar neutral amines Base acids include glycine, serine, threonine, cysteine, tyrosine, aspartamine, and glutamic acid; positively charged (basic) amino acids include arginine, amino acids and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In the context of the present invention, a derivative polypeptide as used herein refers to a polypeptide capable of exhibiting substantially similar in vitro and/or in vivo activities as the original polypeptide, such as by a variety of criteria, including but not limited to enzymes activity), and which can be differentially modified during or after translation. In addition, non-classical amino acids or chemical amino acid analogs can be introduced into the original polypeptide sequence in substitution or addition form.

In some embodiments, the present invention contemplates making functional variants by modifying the structure of an enzyme as used in the present invention. Variants can be created by amino acid substitutions, deletions, additions, or combinations thereof. For example, it is reasonable to expect that independent substitution of leucine with isoleucine or valine, independent substitution of aspartic acid with glutamic acid, independent substitution of threonine with serine, or independent substitution of amine groups Similar substitution of an acid with a structurally related amino acid (eg, conservative mutation) does not have a significant effect on the biological activity of the resulting molecule. Conservative substitutions are substitutions that occur within a family of amino acids that are related in the side chains. Whether changes in the amino acid sequence of the polypeptides of the invention result in functional homologues can readily be determined by assessing the ability of the variant polypeptide to generate a response in cells in a manner similar to the wild-type polypeptide.

The term "functional homolog" as used herein describes those molecules that share sequence similarity (in other words, homology) and also share at least one functional characteristic (such as biochemical activity) (Altenhoff et al ., PLoS Comput. Biol. 8 (2012) e1002514). Functional homologues 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 proteins . Sequence analysis may involve BLAST, Reciprocal BLAST, or PSI using the amino acid sequences of biomass-modulating polypeptides, glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis, or membrane proteins as reference sequences to non-redundant databases, respectively -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 proteins, respectively. . 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.

In the context of polynucleotides, "Fragment" refers to any portion of a clone or polynucleotide molecule, especially a portion of a polynucleotide that retains the useful functional characteristics of a full-length polynucleotide molecule. Useful fragments include oligonucleotides and polynucleotides that can be used in hybridization or amplification techniques or to modulate replication, transcription or translation. "Polynucleotide fragment" refers to any subsequence of a polynucleotide of SEQ ID NO (or Genbank NO.), which typically comprises any of the polynucleotide sequences provided herein. At least about 9, 10, 11, 12 contiguous nucleotides or consist thereof, eg, at least about 30 nucleotides or at least about 50 nucleotides. Exemplary fragments may additionally or alternatively include a region comprising, consisting essentially of, or consisting of a conserved family domain encoding a polypeptide. Exemplary fragments may additionally or alternatively include fragments comprising conserved domains of polypeptides. Therefore, a fragment of a polynucleotide SEQ ID NO (or Genbank NO.) preferably means a nucleotide sequence comprising or consisting of the polynucleotide SEQ ID NO (or Genbank NO.), of which no more than 200 , 150, 100, 50 or 25 consecutive nucleotide deletions, preferably no more than 50 consecutive nucleotide deletions, and the nucleotide sequence retention can be assessed by those of ordinary skill in the art through routine experimentation Available functional characteristics (eg, activity) of full-length polynucleotide molecules. Alternatively, a fragment of a polynucleotide SEQ ID NO (or Genbank NO.) preferably means a nucleotide sequence comprising a quantified contiguous nucleus from the polynucleotide SEQ ID NO (or Genbank NO.) nucleotides or consisting of, and wherein the certain amount of contiguous nucleotides is at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0% of the full length of the polynucleotide SEQ ID NO (or Genbank NO.) , 82.0 %, 83.0 %, 84.0 %, 85.0 %, 86.0 %, 87.0 %, 88.0 %, 89.0 %, 90.0 %, 91.0 %, 92.0 %, 93.0 %, 94.0 %, 95.0 %, 95.5 %, 96.0 %, 96.5 %, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 100%, preferably at least 80%, more preferably at least 87%, even better at least 90%, even better at least 95%, most Preferably at least 97% and retain the useful functional characteristics (eg, activity) of the full-length polynucleotide molecule. Therefore, a fragment of a polynucleotide SEQ ID NO (or Genbank NO.) preferably means a nucleotide sequence comprising or consisting of the polynucleotide SEQ ID NO (or Genbank NO.), wherein a certain amount of Consecutive nucleotide deletions and wherein the amount does not exceed 50.0%, 40.0%, 30.0% of the full length of the polynucleotide SEQ ID NO (or Genbank NO.), preferably not more than the polynucleotide SEQ ID NO (or Genbank NO.) or Genbank NO.) of the full length of 20.0%, 15.0%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, better not more than 15%, even better not more than 10%, even better not more than 5%, best not more than 2.5%, and wherein the fragment retention can be determined by the relevant technical field Available functional characteristics (eg, activity) of full-length polynucleotide molecules routinely assessed by those of ordinary skill in

Throughout the application, polynucleotide sequences may be represented by SEQ ID NOs or, alternatively, by GenBank NOs. Accordingly, unless expressly stated otherwise, the terms "polynucleotide SEQ ID NO" and "polynucleotide GenBank NO." are used interchangeably.

Fragments may additionally or alternatively include subsequences of polypeptides and protein molecules, or subsequences of polypeptides. In some cases, a fragment or domain is a subsequence of a polypeptide that performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar extent, as the intact polypeptide. A "subsequence of the polypeptide" as defined herein refers to a sequence derived from contiguous amino acid residues of a polypeptide. For example, a polypeptide fragment can contain identifiable structural motifs or functional domains, such as DNA binding sites or domains that bind to DNA promoter regions, activation domains, or protein-protein interaction domains, and can initiate transcription. Fragments can vary in size from as few as 3 amino acid residues to the full length of a complete polypeptide, eg, at least about 20 amino acid residues in length, eg, at least about 30 amino acid residues in length. Thus, a fragment of a polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) preferably means a polypeptide sequence comprising or consisting of the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.), wherein no more than 80 , 60, 50, 40, 30, 20 or 15 consecutive amino acid residues are deleted, preferably no more than 40 consecutive amino acid residues are deleted, and it can be obtained by those having ordinary knowledge in the art according to Routinely assessed intact polypeptides preferably perform at least one biological function of the intact polypeptide to a similar or greater extent in substantially the same manner. Alternatively, a fragment of a polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) preferably means a polypeptide sequence comprising a quantification of contiguous amino acids from the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) residues or consisting of, and wherein the certain amount of contiguous amino acid residues is at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0 %, 82.0 %, 83.0 %, 84.0 %, 85.0 %, 86.0 %, 87.0 %, 88.0 %, 89.0 %, 90.0 %, 91.0 %, 92.0 %, 93.0 %, 94.0 %, 95.0 %, 95.5 %, 96.0 % , 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 100%, preferably at least 80%, more preferably at least 87%, even better at least 90%, even better at least 95% , preferably at least 97%, and which preferably perform at least one biological function of the intact polypeptide to a similar or greater extent in substantially the same manner as the intact polypeptide can be routinely assessed by those of ordinary skill in the art. Thus, a fragment of a polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) preferably means a polypeptide sequence comprising or consisting of the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.), wherein a certain amount of Consecutive amino acid residues are deleted and wherein the amount does not exceed 50.0%, 40.0%, 30.0% of the full length of the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.), preferably not more than the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) 20.0%, 15.0%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0 %, 1.5%, 1.0%, 0.5%, better not more than 15%, even better not more than 10%, even better not more than 5%, best not more than 2.5%, and it is in accordance with and can be obtained by the relevant technology Intact polypeptides routinely assessed by those of ordinary skill in the art preferably perform at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar or greater extent.

Throughout the application, polypeptide sequences may be represented by SEQ ID NO or alternatively by UniProt ID or GenBank NO. Accordingly, unless expressly stated otherwise, the terms "polypeptide SEQ ID NO" and "polypeptide UniProt ID" and "polypeptide GenBank NO. (polypeptide GenBank NO.)" are used interchangeably.

Preferably, a fragment of a polypeptide is a functional fragment derived from a polypeptide that preferably possesses at least one property or activity of the polypeptide to a similar or greater extent. Functional fragments may, for example, include functional or conserved domains of polypeptides. It is understood that a polypeptide or fragment thereof may have conservative amino acid substitutions that have substantially no effect on the activity of the polypeptide. By conservative substitution, it means that one hydrophobic amino acid is substituted with another hydrophobic amino acid or one polar amino acid is substituted with another polar amino acid or one acidic amino acid is substituted with another acidic amino acid Or one basic amino acid is substituted by 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. Domains can be identified, for example, by Pfam (El-Gebali et al., Nucleic Acids Res. 47 (2019) D427-D432) or the Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih. gov/cdd) (Lu et al, Nucleic Acids Res. 48 (2020) D265-D268) name qualification. The content of each database is fixed and does not change with each release. When the content of a particular database changes, this particular database receives a new release version with a new release date. All release versions of each database and their corresponding release dates and specific content as noted on such specific release dates are available and known to those skilled in the art. The PFAM database (https://pfam.xfam.org/) used in this article is Pfam version 33.1 released on June 11, 2020. Protein sequence information and functional information can be provided by comprehensive sources of protein sequence and annotation data, such as, for example, the Universal Protein Resource (UniProt) (www.uniprot.org) (Nucleic Acids Res. 2021, 49(D1), D480-D489). UniProt includes a specialized and well-managed protein database called the UniProt Knowledge Base (UniProtKB), as well as the UniProt Reference Sequence Set (UniRef) and the UniProt Archive (UniParc). UniProt identifiers (UniProt IDs) are unique to each protein present in the database. UniProt ID as used herein is the UniProt ID in the UniProt database version on May 05, 2021. The NIH Gene Sequence Database (https://www.ncbi.nlm.nih.gov/genbank/) (Nucleic Acids Res. 2013, 41(D1), D36) is used in this paper as it exists on May 05, 2021 -D42) The respective Genbank Accession Number (GenBank No.) in the version refers to proteins that do not have a UniProt ID.

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. Mammalian milk 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 (e.g. β-1,3-N-acetylglucosaminyl transferase, beta-1,6-N-acetylglucosaminyltransferase), N-acetylgalactosaminyltransferase (e.g. alpha-1,3-N-acetylgalactosaminotransferase) enzyme, β-1,3-N-acetylgalactosyltransferase), glucosyltransferase, mannosyltransferase, N-acetylmannosyltransferase, xylosyltransferase, Glucuronosyltransferase, galacturonyltransferase, glucosaminyltransferase, N-hydroxyacetylneuraminosyltransferase, rhamnosyltransferase, N-acetylrhamnosyltransferase Enzymes, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosaminotransferase, UDP-N-acetylglucosamine enolacetonyl transfer Enzymes and Fucosaminotransferases.

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-arabinose-4-hexulose glycosyltransferase for the biosynthesis of pseudoamines, which are sialic saccharides 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 terms "nucleotide-sugar", "nucleotide-activated sugar" or "activated sugar" are used interchangeably herein and refer to the activation of a monosaccharide form. 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-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 , CMP-N-hydroxyacetyl neuraminic acid (CMP-Neu5Gc), UDP-glucuronide, UDP-half Lacturonate, GDP-rhamnose or UDP-xylose. Nucleotide-sugars act as sugar donors in glycosylation reactions. Glycosylation reactions are reactions 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. Preferably, the oligosaccharide as described herein contains a monosaccharide selected from the list as used herein below. As used herein, "LNB (lacto-N-biose)-based oligosaccharide" refers to an oligosaccharide, as defined herein, which contains LNB at its reducing end . As used herein, "LacNAc (N-acetyllactosamine)-based oligosaccharide" refers to an oligosaccharide, as defined herein, which contains LacNAc at its reducing end.

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.

As used herein, the term "disaccharide" refers to a sugar composed of two monosaccharide units. 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).

As used herein, "mammalian milk oligosaccharide" (MMO) refers to oligosaccharides such as (but not limited to) the following: lacto-N-triose II, 3-fucosyllactose, 2' - Fucoyllactose, 6-fucoyllactose, 2',3-difucoyllactose, 2',2-difucoyllactose, 3,4-difucoyllactose, 6'-sialic acid Sylactose, 3'-Sialylactose, 3,6-Disialylactose, 6,6'-Disialylactose, 8,3-Disialylactose, 3,6-Disialylactose Lacto-N-tetraose, lactose difucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentose II, lacto-N-fucopentaose I, lacto-N-fucopentose -N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentose VI, sialyl lacto-N-neotetraose d, sialyl lacto-N-neotetraose Sugar c, sialyl lacto-N-tetrasaccharide b, sialyl lacto-N-tetrasaccharide a, lacto-N-difucose I, lacto-N-difucose II, lacto-N - Hexose, lacto-N-neohexaose, p-lacto-N-hexaose, monofucosylmonosialyllacto-N-neotetraose c, monofucosyl p-lacto-N- Hexaccharide, monofucosylated lacto-N-hexasaccharide III, isofucosylated lacto-N-hexasaccharide III, isofucosylated lacto-N-hexasaccharide I, sialyl lactose -N-Hexose, Sialyllacto-N-Neohexaose II, Difucosyl-p-lacto-N-hexaose, Difucosyllacto-N-hexaose, Difucosyl Lacto-N-hexasaccharide a, difucosyl lacto-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.

As used herein, "lactose-based mammalian milk oligosaccharide (MMO)" refers to an MMO, as defined herein, which contains lactose at its reducing end.

As used herein, "sialylated oligosaccharide" is to be understood as a charged sialic acid containing an oligosaccharide (ie an oligosaccharide having a sialic acid residue). It has acidic properties. Sialylated 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), sialylated lacto-N-triose, sialylated lacto-N-tetrasaccharide, sialylated lacto-N-neotetraose, monosialylated lacto-N- Hexose, disialolacto-N-hexasaccharide I, monosialolacto-N-neohexaose I, monosialolacto-N-neohexaose II, disialolacto-N-neo Hexose, disialolacto-N-tetrasaccharide, disialolacto-N-hexasaccharide II, sialyllacto-N-tetrasaccharide a, disialolacto-N-hexasaccharide I, saliva Sialyllacto-N-tetrasaccharide b, Sialyllacto-N-neotetrasaccharide c, Sialyllacto-N-neotetrasaccharide d, 3'-Sialyl-3-fucosyllactose, Disialyl Acid monofucosyl lacto-N-neohexaose, monofucosyl monosialyl lacto-N-octasaccharide (sialyl Lea), sialyl lacto-N-fucohexaose II, disialic acid lacto-N-fucopentaose II, monofucosyldisialolacto-N-tetrasaccharide; and oligosaccharides with one or several sialic acid residues, including (but not limited to): selected from GM3 The oligosaccharide moiety of the ganglioside (3' sialyllactose, Neu5Acα-2,3Galβ-4Glc); and oligosaccharides, including the 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, GD 1a 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(Neu5Acα-2,8Neu5Acα2 ,3) Galβ-1,4Glc, GQ1b Neu5Acα-2,8Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)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(Neu5Aα-2,8Neu5Aα-2,8Neu5Aα2,3)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 ; which can all 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- The glycosyltransferase of the acceptor molecule 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 "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 keys. Examples include 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL), lactodifucotetraose (LDFT), lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucoa Pentasaccharide 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), Monofuco-N-hexaose III (MFLNH III), Difuco-N-N - Hexose (DFLNHa), difucosyl-lacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, disialomonofucosyllacto-N-neohexaose, mono Fuco-monosialyllacto-N-octasaccharide (Sialyl-Lea), sialyl-lacto-N-fucohexaose II, disialo-lacto-N-fucopentose II, monofuco basedisialolacto-N-tetrasaccharide.

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 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 are 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.

The terms "LNB" and "Lacto-N-biose" are used interchangeably and refer to the disaccharide Gal-b1,3-GlcNAc.

The terms "LacNAc" and "N-acetyllactosamine" are used interchangeably and refer to the disaccharide Gal-b1,4-GlcNAc.

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 hexasaccharide" (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 reuse pathway L-fucose kinase/GDP-fucoidase Sugar pyrophosphorylase, and fucosyltransferase producing alpha 1,2, alpha 1,3, alpha 1,4 and/or alpha 1,6 fucosylated oligosaccharides.

The "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 transfer Enzymes, Glucosamine-1-Phosphate Acetyltransferase, Sialic Acid Synthase, N-Acetylneuraminic Acid Lysase, N-Acetylneuraminic Acid-9-Phosphate Synthase, N-Acylneuramine Acid-9-phosphate phosphatase and/or CMP-sialic acid synthase, and sialyltransferase producing alpha 2,3, alpha 2,6 and/or alpha 2,8 sialylated oligosaccharides.

"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, Galactosyltransferases that bind galactose at the 4 and/or 6 hydroxyl groups.

The "N-acetylglucosamine carbohydrate pathway" as used herein is a biochemical pathway consisting of the 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-ethyl Acetyltransferase, N-acetylglucosamine-1-phosphate uridine transferase, glucosamine-1-phosphate acetyltransferase, and/or glucosamine-1-phosphate acetyltransferase, and Glycosyltransferases that produce alpha or beta bound N-acetylglucosamine on the 3, 4 and/or 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-acetylhemifold 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 Enzymes, 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-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 (D-fructose-6-phosphate amidotransferase)", "fructose-6-phosphate aminotransferase (fructose-6-phosphate aminotransferase)", "phosphate glucose "glucosaminephosphate isomerase", "glucosamine 6-phosphate synthase", "GlcN6P synthase", "GFA", "glms", "glmS" and " glmS*54" is used interchangeably and refers to an enzyme that catalyzes the conversion of D-fructose-6-phosphate to D-glucosamine-6-phosphate using L-glutamic acid.

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.

Terms "N-acetylglucosamine-6-P deacetylase (N-acetylglucosamine-6-P deacetylase)", "N-acetylglucosamine-6-phosphate deacetylase (N-acetylglucosamine-6-phosphate deacetylase)" acetylglucosamine-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 produce 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) to convert N-acetylglucosamine 1-phosphate (GlcNAc-1-P) to UD P-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 the ability to dephosphorylate N-acylneuraminate-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 terms "identical" or "identical" or "identical" in the context of two or more nucleic acid or polypeptide sequences when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection "Percent identity" or "% identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides. For sequence comparison, one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Next, the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence according to the specified program parameters. The percent identity can be calculated globally within the full-length sequence of the reference sequence, resulting in an overall percent identity score. Alternatively, percent identity can be calculated within a partial sequence of a reference sequence, resulting in a percent local identity score. The use of the full length of the reference sequence in a local sequence alignment yields an overall percent identity score between the test and reference sequences. The percent identity can use different algorithms such as, for example, BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25: 17, 3389-402) , Clustal Omega method (Sievers et al, 2011, Mol. Syst. Biol. 7:539), MatGAT method (Campanella et al, 2003, BMC Bioinformatics, 4:29) or EMBOSS Needle assay.

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

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

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

The EMBOSS Needle (https://galaxy-iuc.github.io/emboss-5.0-docs/needle.html) uses the Needleman-Wunsch global alignment algorithm when its entire length is considered Find the best alignment of two sequences (including gaps). The best alignment is ensured by a dynamic programming method by exploring all possible alignments and selecting the best one. The Needleman-Wunsch algorithm is a member of a class of algorithms that can compute optimal scores and alignments in the order of mn steps (where 'n' and 'm' are the lengths of the two sequences). Gap opening penalty (default 10.0) is the score deducted when a gap is created. The default values assume you are using the EBLOSUM62 matrix for protein sequences. A gap extension (default 0.5) penalty is added to the standard gap penalty for each base or residue in the gap. This is the way to punish long gaps.

As used herein, a polypeptide having an amino acid sequence of at least 80% sequence identity to the full-length sequence of the reference polypeptide sequence is understood to be 80%, 81%, 82%, 83% to the full-length amino acid sequence of the reference polypeptide sequence , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50 100% sequence identity. Throughout the application, unless expressly specified otherwise, include amino acid sequences (or nucleotide sequence) polypeptide (or DNA sequence)/an amino acid sequence with at least 80% sequence identity to the full-length amino acid sequence (or nucleotide sequence) of the reference polypeptide (or nucleotide sequence) ( or nucleotide sequence) of the polypeptide (or DNA sequence) composition/has an amino acid with at least 80% sequence identity with the full-length amino acid sequence (or nucleotide sequence) of the reference polypeptide (or nucleotide sequence) The polypeptide (or DNA sequence) of the sequence (or nucleotide sequence), usually indicated by SEQ ID NO or UniProt ID or Genbank NO., preferably has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% sequence identity.

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

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 inside (intracellular) and outside (extracellular) cells.

The terms "membrane transporter protein" and "membrane protein" are used interchangeably and refer to proteins that are part of or interact with a cell membrane and control the flow of molecules and information across cells. Thus, membrane proteins are involved in transport, whether they are imported into or exported from cells.

Such membrane transporters may be transporters as defined by the Transporter Classification Database, P-P-bond hydrolysis driven transporters, β-Barrel Porins , paratransporters, putative transporters, and phosphotransfer-driven group translocation proteins, a database 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, known as the Transporter Classification (TC) system. The TCDB classification search described here is based on the TCDB as published on June 17, 2019 .org is defined.

Transporter is the collective name for uniporters, symporters, and antiporters that utilize vector-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 transporters are included in this category when using carrier-mediated processes to catalyze uniporters when a single species is transported by facilitated diffusion or in a membrane potential-dependent process (if the solute is charged) ; catalytic antiporters when two or more species are transported in opposite directions in a tightly coupled process, not coupled to a direct energy form other than chemoosmotic energy; and/or when two or more species are transported in opposite directions When multiple species are transported together in the same direction in a tightly coupled process and are not coupled to a direct energy form other than chemoosmotic energy, catalyzing cotransport proteins, which are all 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. The dynamic association of transport proteins and enzymes produces a functional membrane transport metabolite that imports substrates typically obtained from the extracellular compartment directly 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 glycolytic 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 the 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 temporarily 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 member is confirmed, or eliminated from the transport protein 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 as classified in this group under the TCDB system published on June 17, 2019 include, but are not limited to, copper transport proteins.

Phosphate transfer-driven group translocation protein is also known as bacterial phosphoenolpyruvate:sugar phosphotransferase system (phosphotransferase system; PTS) PEP-dependent phosphonium group transfer-driven group translocation protein. 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 A 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 uniport proteins, solute: cation (H+, but hardly Na+) symporters and/or solutes :H+ or solute:solute antiporter. 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 is a protein belonging to the eggNOGv4.5 family ENOG410XTE9. InterPro domains can be identified by using the online tool at https://www.ebi.ac.uk/interpro/ or 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 enzyme 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 chelatin precursor, biosynthesis depends on the hydroxylation of ornithine catalyzed by L-ornithine N5-monooxygenase. In the NIS pathway, an important step in chelatin biosynthesis is N(6)-hydroxylysine synthase.

Transport proteins are required to export chelatin to the outside of the cell. So far, four superfamilies of membrane proteins have been identified in the process: the major facilitator superfamily (MFS); the 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 to export 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 cells 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-acetyl-galactosamine, phosphorylated carbohydrates such as for example (but not limited to) glucose-1-phosphate salt, galactose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, mannose-6-phosphate, mannose-1-phosphate, Glycerol-3-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-acetyl-glucosamine-6-phosphate, N-acetylmannan Glycosamine-6-phosphate, N-acetylglucosamine-1-phosphate, N-acetyl-neuraminic acid-9-phosphate and/or nucleotide activated carbohydrates 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. 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, nuclear transporter Glycoside-activated sugar transporter, wherein the transporter is internalized into precursor-supplemented medium for oligosaccharide synthesis.

The term "acceptor" as used herein refers to a disaccharide or oligosaccharide that can be modified by a glycosyltransferase. Examples of such receptors include lactose, lacto-N-disaccharide (LNB), lacto-N-triose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), N-ethyl Acyl-lactosamine (LacNAc), lacto-N-pentasaccharide (LNP), lacto-N-neopentasaccharide, p-lacto-N-pentasaccharide, p-lacto-N-neopentasaccharide, lacto-N-neopentasaccharide Sugar I (lacto-N-novopentaose I), lacto-N-hexaose (lacto-N-hexaose; LNH), lacto-N-neohexaose (LNnH), lacto-N-new Hexose (para lacto-N-neohexaose; pLNnH), para lacto-N-hexaose (pLNH), lacto-N-heptaose, lacto-N-neoheptose, para-lacto-N -Lacto-N-octaose, lacto-N-octaose (LNO), lacto-N-octaose, isolacto-N-octaose, lacto-N-octaose Octose, isomilk-N-octaose, neonatal milk-N-octaose, paramilk-N-neoctaose, isomilk-N-nonaose, neonatal milk-N-nonaose, milk-N- Nona sugar, lacto-N-deca sugar, isolacto-N-deca sugar, neonatal milk-N-deca sugar, lacto-N-neodeca sugar; galactosyl lactose, extended with 1, 2, 3, 4, Lactose containing 5 or more N-acetyllactosamine units and/or 1, 2, 3, 4, 5 or more lacto-N-disaccharide units and containing 1 or more N-acetyllactosamine units and or oligosaccharides of 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 metabolically engineered cell for producing a mixture comprising at least three different mammalian milk oligosaccharides (MMOs), preferably lactose-based MMOs, more preferably human milk oligosaccharides Sugars (HMOs), ie, metabolically engineered for use in producing cells comprising a mixture of at least three different MMOs, preferably HMOs. Throughout this application, unless expressly stated otherwise, the features "a mammalian milk oligosaccharide" or "mammalian milk oligosaccharides" or "an MMO" or "MMOs", respectively, are preferably referred to as "a lactose-based Mammalian milk oligosaccharide" or "lactose-based mammalian milk oligosaccharides" or "a lactose-based MMO" or "lactose-based MMOs", more preferably "a human milk oligosaccharide" or "multiple lactose-based MMOs", respectively Human milk oligosaccharide" or "one HMO" or "multiple HMOs" replacement. Provided herein is a single metabolically engineered cell capable of expressing, preferably expressing at least two glycosyltransferases and capable of synthesizing one or more sugars as donors for one or more of the glycosyltransferases - Nucleotides. Throughout this application, unless expressly stated otherwise, "genetically modified cell" or "metabolically engineered cell" preferably means genetically modified or metabolically engineered, respectively, to produce a basis comprising at least three Cells of the mixture of the MMOs of the present invention. In the context of the present invention, preferably at least three different MMOs of the mixture as disclosed herein are not present in wild-type precursor cells of the metabolically engineered cell.

According to a second aspect, the present invention provides a method for producing a mixture comprising at least three different mammalian milk oligosaccharides. The method includes the following steps: i) Provide a cell, preferably a single cell, capable of expressing, preferably expressing at least two glycosyltransferases and capable of synthesizing one or more nucleotide-sugars, wherein the one or more nucleotide-sugars are used for the one or more donors such as glycosyltransferases, and ii) culturing the cell under conditions that allow expression of the glycosyltransferases and synthesis of the one or more nucleotide-sugars, iii) Preferably, at least one of the mammalian milk oligosaccharides is isolated from the culture, more preferably, all of the mammalian milk oligosaccharides are isolated from the culture.

Within the scope of the present invention, permissive conditions are understood to be conditions related to physical or chemical parameters including, but not limited to, temperature, pH, pressure, osmotic pressure and product/precursor/acceptor concentrations.

In a specific embodiment, allowable conditions may include a temperature range of 30 +/- 20 degrees Celsius, a pH range of 7 +/- 3.

In the context of the present invention, it is understood that the cells produce these oligosaccharides intracellularly. It will be further understood by those of ordinary skill in the art that some or substantially all of the oligosaccharides produced remain intracellular and/or are excreted outside the cell via passive or active transport.

According to the present invention, the method for producing a mixture comprising at least three different MMOs can utilize non-metabolically engineered cells or can utilize metabolically engineered cells, ie metabolically engineered for the production of a mixture comprising Cells of the mixture of at least three different MMOs.

According to a preferred embodiment of the methods and cells of the present invention, metabolically engineered cells are modified with gene expression modules, wherein expression from any of these expression modules is persistent or by natural Inducer production.

These expression modules are also referred to as transcriptional units and comprise polynucleotides for expression of recombinant genes including coding gene sequences and appropriate transcriptional and/or translational control signals operably linked to the coding genes. 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 recombinants Gene. 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 exist in plastid, cosmid, phage, liposome or viral form stably transformed/transfected into the metabolically engineered cell. Among them, such vectors include chromosomal, episomal and virus-derived vectors, for example, bacterial plastids, phages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viral vectors, and combinations thereof. Vectors, such as those derived from plastids 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. Generally, in this regard, any system or vector suitable for maintaining, amplifying or expressing polynucleotides and/or expressing polypeptides in a host can be used for expression. 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 the expression system or portion thereof or 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 polypeptides that function in the synthesis of the MMO 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 MMOs. The recombinant genes encode endogenous proteins with modified expression or activity, preferably the endogenous proteins are overexpressed; or the recombinant genes encode heterogenous introductions and are expressed 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 performance of each of these performance 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.

The present invention provides different types of cells for this production of mammalian milk oligosaccharide mixtures comprising three or more MMOs using a single metabolically engineered cell. For example, the invention provides a cell wherein the cell expresses two different glycosyltransferases and the cell synthesizes a single nucleotide-sugar that is the supply for both the expressed glycosyltransferases body. The invention also provides a cell wherein the cell expresses three different glycosyltransferases and the cell synthesizes a single nucleotide-sugar that is the donor of all three of the expressed glycosyltransferases. The present invention also provides a cell, wherein the cell expresses two different glycosyltransferases and the cell synthesizes two different nucleotide-sugars, wherein the first nucleotide-sugar is for the first glycosyltransferase body and the second nucleotide-sugar is the donor for the second glycosyltransferase. The invention also provides a cell, wherein the cell expresses three or more glycosyltransferases and the cell synthesizes one or more different nucleotide-sugars that are donors for all of the expressed glycosyltransferases .

In the methods and cells described herein, the cells preferably comprise multiple copies of the same coding DNA sequence encoding a protein. In the context of the present invention, the protein may be a glycosyltransferase, a membrane 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.

In one embodiment of the method and/or cell according to the present invention, the mixture comprises at least three, preferably at least four, more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight , at least nine, at least ten different mammalian milk oligosaccharides. In a preferred embodiment, any of the mammalian milk oligosaccharides is a human milk oligosaccharide, preferably all of the oligosaccharides are human milk oligosaccharides.

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 additional and/or alternative embodiments of the methods and/or cells according to the invention, the mixture comprises at least three different mammalian milk oligosaccharides that differ in degree of polymerization (DP). The degree of polymerization of an oligosaccharide refers to the number of monosaccharide units present in the oligosaccharide structure. As used herein, an oligosaccharide has a degree of polymerization of three (DP3) or higher, the latter comprising any of 4 (DP4), 5 (DP5), 6 (DP6) or longer. The mammalian milk oligosaccharide mixture as described herein preferably comprises at least three different mammalian milk oligosaccharides, wherein all the mammalian milk oligosaccharides present in the mixture have different degrees of polymerization from each other. For example, the oligosaccharide mixture is composed of three MMOs, wherein the first MMO is a trisaccharide with a degree of polymerization of 3 (DP3), the second MMO is a tetrasaccharide with a degree of polymerization of 4 (DP4), and the third MMO is Pentasaccharide with a degree of polymerization of 5 (DP5). According to one embodiment of the method and/or cell of the invention, the cell produces a mixture comprising four different MMOs or more than four different MMOs. In a specific example, such a mixture comprises at least four different MMOs, three of which have different degrees of polymerization of the oligosaccharides. In a specific example, all of the MMOs in the mixture have different degrees of polymerization as described herein.

In additional and/or alternative preferred embodiments of the methods and/or cells according to the invention, the MMO mixture as described herein consists of charged and neutral mammalian milk oligosaccharides. Accordingly, the present invention preferably provides a cell that is metabolically engineered for the production of a mixture comprising at least three different MMOs and wherein the mixture is composed of charged and neutral MMOs, wherein the cell is capable of expressing at least two glycosyl groups Transferase, preferably at least three, more preferably at least four, even more preferably at least five, most preferably at least six, at least seven, at least eight glycosyltransferases, and capable of synthesizing one or more nucleotide-sugars , preferably at least two, more preferably at least three, even more preferably at least four, and most preferably at least five nucleotide-sugars, wherein the nucleotide-sugar(s) are used for the glycosyltransferases donor. In other words, the present invention preferably provides a metabolically engineered cell for producing (preferably the cell produces) a mixture comprising at least three different MMOs and wherein the mixture consists of charged and neutral MMOs, wherein the cell Genetically modified for use in producing the mixture, the cells are capable of expressing at least two glycosyltransferases, preferably at least three, more preferably at least four, even more preferably at least five, most preferably at least six, at least seven, At least eight glycosyltransferases, and capable of synthesizing one or more nucleotide-sugars, wherein the nucleotide-sugar(s) are donors for the glycosyltransferases. Furthermore, the present invention preferably provides a method for producing a mixture comprising at least three different MMOs, and wherein the mixture is composed of charged and neutral MMOs, the method comprising steps as described herein (wherein preferably provided as herein Metabolically engineered cells disclosed). Charged oligosaccharides (MMOS) are oligosaccharide structures containing one or more negatively charged monosaccharide subunits including N-acetylneuraminic acid (Neu5Ac) (commonly referred to as sialic acid), N-hydroxyacetylneuraminic acid (Neu5Gc), Neu4Ac, _Neu5Ac9N3 , _Neu4,5Ac2 , _Neu5,7Ac2 , _Neu5,9Ac2 , Neu5,7(8,9)Ac2, glucuronate and galactose Aldehydes. Charged oligosaccharides (MMOs) are also known as acidic oligosaccharides (MMOs). Throughout this application, the charged MMOs are preferably sialylated MMOs. Sialic acid belongs to a family of derivatives of neuraminic acids (5-amino-3,5-dideoxy-D-glycerol-D-galactose-nonan-2-ketonic acid). Neu5Gc is a derivative of sialic acid formed by hydroxylation of the N-acetyl group at C5 of Neu5Ac. Neutral oligosaccharides (MMOs) are non-sialylated oligosaccharides (MMOs) and therefore do not contain acidic monosaccharide subunits. Neutral oligosaccharides (MMOs) include uncharged fucosylated oligosaccharides (MMOs) that contain one or more fucose subunits in their glycan structure and uncharged non-rocks that lack any fucose subunits Fluoroglycosylated oligosaccharides (MMOs).

According to additional and/or alternative embodiments of the methods and/or cells of the invention, at least one of the oligosaccharides of the mixture is fucosylated, sialylated, galactosylated, glucosylated , Xylosylated, Mannosylated, with N-acetylglucosamine, with N-acetylneuraminic acid, with N-hydroxyacetylneuraminic acid, with N-acetylgalactosamine , containing rhamnose, containing glucuronic acid ester, containing galacturonic acid ester and/or containing N-acetylmannosamine.

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

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

Alternatively or additionally, the mixture of MMOs comprises at least one MMO having three or more monosaccharide subunits connected to each other via glycosidic bonds, wherein at least one of the monosaccharide residues is N-acetylglucosamine (GlcNAc) residues. The oligosaccharide may contain more than one GlcNAc residue, eg, two, three or more. The oligosaccharides may be neutral oligosaccharides or charged oligosaccharides, eg also comprising sialic acid structures. 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, glucose.

Alternatively or additionally, the MMO mixture comprises at least one galactosylated MMO and contains at least one galactose monosaccharide subunit. The galactosylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits connected 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.

In additional and/or alternative preferred embodiments of the methods and/or cells according to the invention, the MMO mixture as described herein consists of charged (preferably sialylated) and neutral MMOs, wherein the charged in the mixture The relative abundance of (preferably sialylated) MMOs is at least 5%, preferably at least 7%, more preferably at least 10%. Preferably, the relative abundance of the charged MMOs in the mixture is less than 20%, preferably less than 15%. Therefore, the relative abundance of the charged MMO in the mixture is preferably 5%-20%, preferably 5%-15%, more preferably 10%-15%, even more preferably 12%-14%, and most preferably Preferably, it reflects the relative abundance of charged MMOs in human breast milk and/or colostrum. Those of ordinary skill in the art will further understand that if the relative abundance of charged oligosaccharides in a mixture is defined, the remainder of the oligosaccharides in the mixture is inevitably neutral oligosaccharides.

In a preferred embodiment of the method and/or cell according to the present invention, the neutral MMOs do not comprise afucosylated MMOs. In an alternative preferred embodiment, the neutral MMOs do not comprise fucosylated MMOs. In a more preferred embodiment, the neutral MMOs comprise fucosylated MMOs and non-fucosylated MMOs. In an even more preferred embodiment, the relative abundance of fucosylated MMOs in the neutral MMO portion of the mixture is at least 10%, preferably at least 20%, more preferably at least 30%, most preferably at least 35% . Preferably, the relative abundance of fucosylated MMO in the neutral MMO fraction of the mixture is 10%-60%, preferably 20%-60%, better 30%-60%, even better 30 %-50%, optimally reflecting the relative abundance of fucosylated MMOs in the neutral MMO fraction in human breast milk and/or colostrum.

In additional and/or alternative embodiments of the methods and/or cells according to the invention, the MMO mixture as described herein comprises fucosylated MMOs in a relative abundance of at least 10% in the mixture, Preferably at least 20%, more preferably at least 30%, even better at least 40%, even better at least 50%, most preferably at least 55%. Preferably, the relative abundance of the fucosylated MMOs in the mixture is less than 90%, preferably less than 80%, more preferably less than 70%, even more preferably less than 60%. Therefore, the relative abundance of the fucosylated MMOs in the mixture is preferably 10%-90%, preferably 20%-80%, more preferably 30%-60%, even better 40% -55%, which best reflects the relative abundance of fucosylated MMOs in human breast milk and/or colostrum.

Thus, in additional and/or alternative embodiments of the methods and/or cells according to the present invention, the MMO mixture as described herein consists of charged (preferably sialylated) and neutral MMOs, wherein charged (preferably saliva) The relative abundance of acidified) MMOs and/or fucosylated MMOs are as described herein.

In additional and/or alternative embodiments of the methods and/or cells according to the invention, the relative abundance of each oligosaccharide in the mixture as described herein is at least 5%, preferably at least 10%.

In the context of the present invention, the oligosaccharide mixture as disclosed herein is preferably the direct result of metabolic engineering of cells as described herein. This means that preferably at least one, more preferably at least two, even more preferably at least three, and optimally all of the oligosaccharides in the mixture according to the invention are not by wild-type precursors of the metabolically engineered cell cell production.

In the context of the present invention, the mixture of at least three different mammalian milk oligosaccharides according to the present invention may comprise lactose-based MMOs as described herein, LNB-based MMOs, LacNAc-based MMOs and other oligosaccharides that are not MMOs . In a preferred embodiment of the method and/or cell according to the present invention, the mixture comprises at least three MMOs, preferably lactose-based MMOs, more preferably HMOs, and optionally at least one, preferably at least two, More preferably at least three, even more preferably at least four other oligosaccharides that are not MMOs. In alternative and/or additional preferred embodiments of the methods and/or cells according to the present invention, mammalian milk oligosaccharides constitute at least 50%, preferably at least 60%, more preferably at least 70% of the oligosaccharide mixture according to the present invention %, even better at least 80%, even better at least 90%. In a more preferred embodiment of the method and/or cell according to the present invention, all oligosaccharides in the mixture are MMOs, preferably lactose-based MMOs, more preferably HMOs. As already stated herein, mixtures as disclosed herein are preferably the direct result of metabolic engineering of cells as described herein.

The names of MMOs as described herein are based on as disclosed by Urashima et al. Probiotics in human milk. Origins and Functions of Milk-Borne Oligosaccharides and Bacteria", Chapters 2 and 3, eds. M. McGuire, M. McGuire, L. Bode, Elsevier, Academic Press, p. 506) Sugar name and formula.

Throughout this application, unless expressly stated otherwise, the feature "at least one" is preferably replaced with "a", likewise the feature "at least two" is preferably replaced with "two", etc.

In a more preferred embodiment of the method and/or cell according to the invention, the MMO mixture comprising at least three different MMOs as described herein consists of charged (preferably sialylated) and neutral MMOs, and wherein the mixture Consists of, consists of, or consists essentially of: a) at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine , at least ten different charged MMOs preferably selected from the following: a1) Lactose-based charged MMO, preferably any of the following: 3'SL, 6'SL, F-SL, LSTa, LSTb, LSTc, LSTd, F-LSTa, F-LSTb, F-LSTc, S-LNH I, S-LNH II, S-LNnH I, S-LNnH II, S-para-LNnH, FS-LNH, FS-LNH I, FS-LNH II, FS-LNH III, FS-LNH IV, FS-LNnH I, FS-LNnH II, FS-para-LNnH I, FS-para-LNnH II, DFS-LNH I, DFS-LNH III, DFS-LNH IV, DFS-LNnH, S-LNO, FS-LNO I, FS-LNO II, FS-Iso-LNO, DFS-Iso-LNO I, DFS-Iso-LNO II, DFS-LNO I, DFS-NO II, DFS-LNO III, TFS-LNO, TFS-Iso- LNO, DS-LNT, FDS-LNT I, FDS-LNT II, DS-LNH I, DS-LNH II, DS-LNnH, FDS-LNH I, FDS-LNH II, FDS-LNH III, FDS-LNnH, TS -LNH, SLNnD, FS-nascent-LNP I, DF-para-LNH sulfate I, DF-para-LNH sulfate II, TF-para-LNH sulfate, Neu5Ac-a2,3-Gal-b1,3- Gal-b1,4-Glc, Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,6-[GlcNAc-b1,3]-Gal-b1,4- Glc, Neu5Gc-a2,8-Neu5Ac-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,8-Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,8-Neu5Ac- a2,3-Gal-b1,4-Glc, Neu5Gc-a2,8-Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,3-[Neu5Ac-a2,6 ]-Gal-b1,4-Glc, Gal-b1,6-[Neu5Ac-a2,3]-Gal-b1,4-Glc, Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4- Glc, Neu5Gc-a2,3-Gal-b1,3-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Gal-b1,4- GlcNAc-b1,6-[Neu5Ac-a2,3-Gal-b1,3]-Gal-1,4-Glc, Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,6-[Gal-b1 ,3]-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,6-Gal-b1,4-Glc, Gal-b1,3-[Neu5Gc-a2 ,6]-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1, 4-GlcNAc-b1,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, FS Gal-LNnH, DFSGal-LNnH, Gal-MSLNnH, MSDF-para-LNnH, Neu5GcLNnT, GM2 tetrasaccharide, SLNOa , and/or a2) LNB-based charged MMO, preferably 3'-sialyllactose-N-biose (3'-sialyllacto-N-biose; 3'SLNB), 6'-sialyllactose-N-disaccharide ( 6'-sialyllacto-N-biose; 6'SLNB) any of; and/or a3) LacNAc-based charged oligosaccharides, preferably 3'-sialyllactosamine (3'SLacNAc), 6'-sialyllactosamine (6'SLacNAc), Neu5Gc-a2,3-Gal-b1,4 - any of GlcNAc; and Combine with: b) at least one, preferably at least two, more preferably at least three, even better at least four, even better at least five, even better at least six, preferably at least seven, at least eight, at least nine , at least ten different neutral fucosylated MMOs preferably selected from the following: b1) Lactose-based neutral fucosylated MMO, preferably any of the following: 2'FL, 3-FL, DiFL, LNFP-I, LNFP-II, LNFP-III, Gal-LNFP- III, LNFP-V, LNDFH I, LNDFH II, LNDFH III, F-LNH I, F-LNH II, F-LNH III, F-LNnH II, F-LNnH I, F-para-LNH I, F-pair -LNH II, F-para-LNnH, DF-LNH II, DF-LNH I, DF-LNnH, DF-para-LNH, DF-para-LNH II, DF-para-LNH III, DF-para-LNnH, TF -LNH I, TF-LNH II, TF-para-LNH I, TF-para-LNH II, TF-para-LNnH, F-LNO I, F-LNO II, F-LNO III, F-LNnO, F- LNnO II, F-iso-LNO, F-iso-LNnO I, F-nascent-LNnO, F-para-LNO, DF-iso-LNnO, DF-LNO I, DF-LNO II, DF-LNO III, DF -LNnO I, DF-LNnO II, DF-LNnO III, DF-iso-LNO I, DF-iso-LNO II, DF-iso-LNO III, DF-iso-LNO IV, DF-iso-LNO V, DF -iso-LNO VI, DF-iso-LNO VII, DF-para-LNnO, TF-LNO I, TF-LNO II, TF-LNnO, TF-iso-LNO I, TF-iso-LNO II, TF-iso -LNO III, TF-iso-LNO IV, TF-iso-LNnO, Tetra-F-iso-LNO, Tetra-F-para-LNO, Penta-F-iso-LNO, F-LND I, F-LND II , DF-LND I, DF-LND II, DF-LND III, DF-LND IV, DF-LND V, DF-LND VI, TriF-LND I, TriF-LND II, TriF-LND III, TriF-LND IV , TriF-LND V, TriF-LND VI, TriF-LND VII, TetraF-LND I, TetraF-LND II, TetraF-LND III, F-LNnD I, F-LNnD II, DF-LNnD, DF-Newborn-LND , A-tetrasaccharide, DF D Gal-LNnH (Gal-a1,3-Gal-b1,4-GlcNAc-b1,6-[Gal-a1,3-Gal-b1,4-[Fuc -a1,3]-GlcNAc-b1,3]-Gal-b1,4-Glc), 3-F-heteroglobular trisaccharide, B-tetrasaccharide, B-pentasaccharide, B-hexasaccharide, B-heptasaccharide , DF DGal-LNnT (Gal-a1,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3-Gal-b1,4-[Fuc-a1,3]-Glc), TF DGaI-LNnHa, TF DGaI-LNnHb, DFGal-para-LNnH; and/or b2) neutral fucosylated MMO based on LNB, preferably either 2'FLNB, 4'FLNB; and/or b3) Neutral fucosylated MMO based on LacNAc, preferably any one of 2'FLacNAc and 3-FLacNAc; and/or c) at least one, preferably at least two, more preferably at least three, even better at least four, even better at least five, even better at least six, preferably at least seven, at least eight, at least nine , at least ten different neutral afucosylated MMOs preferably selected from: c1) Lactose-based neutral afucosylated MMO, preferably any one of the following: Gal-a1,4-Gal-b1,4-Glc (Gal-a1,4-lactose or globulotrisaccharide) ), β3'-galactosylose, β6'-galactosylose, lacto-N-triose II (LN3), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), LNH, LNnH, iso-LNO, nascent-LNO, nascent-LNnO, LND, iso-LND, GalNAc-a1,3-Gal-b1,4-Glc, nascent-LNP I, Gal-nascent-LNP I, Gal- Nascent-LNP II, Gal-nascent-LNP III, Iso-LNT, DGalLNnH, galili pentasaccharide, Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-Gal- b1,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, nascent-LNT, and/or c2) neutral afucosylated MMO based on LNB; and/or c3) Neutral afucosylated MMOs based on LacNAc, such as eg poly-LacNAc, LacDiNAc, Optionally combined with any one or more of charged and/or neutral oligosaccharides that are not MMOs.

Preferred mixtures in this context of the invention comprise mixtures of at least three different charged and neutral MMOs, wherein the mixtures comprise, consist of, or consist essentially of at least one, preferably at least two more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten preferably selected from the following Different charged MMOs: 3'SL, 6'SL, F-SL, LSTa, LSTb, LSTc, LSTd, F-LSTa, F-LSTb, F-LSTc, S-LNH I, S-LNH II, S- LNnH I, S-LNnH II, S-para-LNnH, FS-LNH, FS-LNH I, FS-LNH II, FS-LNH III, FS-LNH IV, FS-LNnH I, FS-LNnH II, FS- para-LNnH I, FS-para-LNnH II, DFS-LNH I, DFS-LNH III, DFS-LNH IV, DFS-LNnH, S-LNO, FS-LNO I, FS-LNO II, FS-iso-LNO , DFS-iso-LNO I, DFS-iso-LNO II, DFS-LNO I, DFS-NO II, DFS-LNO III, TFS-LNO, TFS-iso-LNO, DS-LNT, FDS-LNT I, FDS -LNT II, DS-LNH I, DS-LNH II, DS-LNnH, FDS-LNH I, FDS-LNH II, FDS-LNH III, FDS-LNnH, TS-LNH, SLNnD, FS-Neo-LNP I, DF-para-LNH sulfate I, DF-para-LNH sulfate II, TF-para-LNH sulfate, Neu5Ac-a2,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1, 3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, Neu5Gc-a2,8-Neu5Ac-a2, 3-Gal-b1,4-Glc, Neu5Ac-a2,8-Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,8-Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Gal- b1,6-[Neu5Ac-a2,3]-Gal-b1,4-Glc, Neu5 Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1, 3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Gal-b1,4-GlcNAc-b1,6-[Neu5Ac-a2,3-Gal-b1,3]-Gal-1,4- Glc, Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,6-Gal-b1,4-Glc, Gal-b1,3-[Neu5Gc-a2,6]-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac -a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, FS Gal -LNnH, DFSGal-LNnH, Gal-MSLNnH, MSDF-para-LNnH, Neu5GcLNnT, GM2 Tetrasaccharide, SLNOa, 3'-Sialyllacto-N-Disaccharide (3'SLNB), 6'-Sialyllactoide -N-Disaccharide (6'SLNB), 3'-Sialyllactosamine (3'SLacNAc), 6'-Sialyllactosamine (6'SLacNAc), Neu5Gc-a2,3-Gal-b1,4 -GlcNAc, with at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least Nine, at least ten different combinations of neutral fucosylated MMOs preferably selected from 2'FL, 3-FL, DiFL, LNFP-I, LNFP-II, LNFP-III, Gal-LNFP- III, LNFP-V, LNDFH I, LNDFH II, LNDFH III, F-LNH I, F-LNH II, F-LNH III, F-LNnH II, F-LNnH I, F-para-LNH I, F-pair -LNH II, F-para-LNnH, DF-LNH II, DF-LNH I, DF-LNnH, DF-para-LNH, DF-para-LNH II, DF-para-LNH III, DF-para-LNnH, TF -LNH I, TF-LNH II, TF-para-LNH I, TF-para-LNH II, TF-para-LNnH, F-LNO I, F-LNO II, F-LNO III, F-LNnO, F-LNnO II, F-iso-LNO, F-iso-LNnO I, F-nascent-LNnO, F-para-LNO, DF-iso-LNnO, DF-LNO I, DF-LNO II, DF-LNO III, DF-LNnO I, DF-LNnO II, DF-LNnO III, DF-iso-LNO I, DF-iso-LNO II, DF-iso-LNO III, DF-iso- LNO IV, DF-iso-LNO V, DF-iso-LNO VI, DF-iso-LNO VII, DF-para-LNnO, TF-LNO I, TF-LNO II, TF-LNnO, TF-iso-LNO I , TF-iso-LNO II, TF-iso-LNO III, TF-iso-LNO IV, TF-iso-LNnO, tetra-F-iso-LNO, tetra-F-para-LNO, five-F-iso- LNO, F-LND I, F-LND II, DF-LND I, DF-LND II, DF-LND III, DF-LND IV, DF-LND V, DF-LND VI, TriF-LND I, TriF-LND II, TriF-LND III, TriF-LND IV, TriF-LND V, TriF-LND VI, TriF-LND VII, TetraF-LND I, TetraF-LND II, TetraF-LND III, F-LNnD I, F-LNnD II, DF-LNnD, DF-nascent-LND, A-tetrasaccharide, DF D Gal-LNnH (Gal-a1,3-Gal-b1,4-GlcNAc-b1,6-[Gal-a1,3-Gal- b1,4-[Fuc-a1,3]-GlcNAc-b1,3]-Gal-b1,4-Glc), 3-F-heteroglobular trisaccharide, B-tetrasaccharide, B-pentasaccharide, B-hexasaccharide Sugar, B-heptaose, DF DGal-LNnT (Gal-a1,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3-Gal-b1,4-[Fuc-a1,3 ]-Glc), TF DGal-LNnH a, TF DGal-LNnH b, DFGal-para-LNnH, 2'FLNB, 4'FLNB, 2'FLacNAc, 3-FlacNAc, and/or with at least one, preferably at least two more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten preferably selected from the following The different neutral afucosylated MMO combinations: Gal-a1, 4-Gal-b1, 4-Glc (Gal-a1, 4-lactose or globulotrisaccharide), β3'-galactosylose, β6'-galactosylose, lacto-N-triose II (LN3), lacto-N-neotetraose (LNnT), lacto-N -Tetrasaccharide (LNT), LNH, LNnH, iso-LNO, nascent-LNO, nascent-LNnO, LND, iso-LND, GalNAc-a1,3-Gal-b1,4-Glc, nascent-LNP I, Gal- Nascent-LNP I, Gal-nascent-LNP II, Gal-nascent-LNP III, iso-LNT, DGalLNnH, galili pentasaccharide, Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, Gal -b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal -b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, nascent-LNT, poly - LacNAc, LacDiNAc, optionally combined with any one or more of charged and/or neutral oligosaccharides that are not MMOs.

More preferred mixtures in this context of the invention comprise mixtures of at least three different charged and neutral MMOs, wherein the mixtures comprise, consist of, or consist essentially of: selected from a list comprising At least three charged and neutral MMOs of: 3'SL, 6'SL, F-SL, LSTa, LSTb, LSTc, LSTd, F-LSTa, F-LSTb, F-LSTc, S-LNH I, S-LNH II , S-LNnH I, S-LNnH II, S-para-LNnH, FS-LNH, FS-LNH I, FS-LNH II, FS-LNH III, FS-LNH IV, FS-LNnH I, FS-LNnH II , FS-para-LNnH I, FS-para-LNnH II, DFS-LNH I, DFS-LNH III, DFS-LNH IV, DFS-LNnH, S-LNO, FS-LNO I, FS-LNO II, FS- iso-LNO, DFS-iso-LNO I, DFS-iso-LNO II, DFS-LNO I, DFS-NO II, DFS-LNO III, TFS-LNO, TFS-iso-LNO, DS-LNT, FDS-LNT I, FDS-LNT II, DS-LNH I, DS-LNH II, DS-LNnH, FDS-LNH I, FDS-LNH II, FDS-LNH III, FDS-LNnH, TS-LNH, SLNnD, FS-Newborn- LNP I, DF-para-LNH sulfate I, DF-para-LNH sulfate II, TF-para-LNH sulfate, Neu5Ac-a2,3-Gal-b1,3-Gal-b1,4-Glc, Gal -b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, Neu5Gc-a2,8-Neu5Ac -a2,3-Gal-b1,4-Glc, Neu5Ac-a2,8-Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,4 -Glc, Neu5Gc-a2,8-Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc , Gal-b1,6-[Neu5Ac-a2,3]-Gal-b1,4-Glc, Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal -b1,3-Gal- b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Gal-b1,4-GlcNAc-b1,6-[Neu5Ac-a2 ,3-Gal-b1,3]-Gal-1,4-Glc, Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,4- Glc, Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,6-Gal-b1,4-Glc, Gal-b1,3-[Neu5Gc-a2,6]-Gal-b1,4- Glc, Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,6-[GlcNAc -b1,3]-Gal-b1,4-Glc, FS Gal-LNnH, DFSGal-LNnH, Gal-MSLNnH, MSDF-para-LNnH, Neu5GcLNnT, GM2 tetrasaccharide, SLNOa, 3'-sialylated lacto-N -Disaccharide (3'SLNB), 6'-Sialyllacto-N-disaccharide (6'SLNB), 3'-Sialyllactosamine (3'SLacNAc), 6'-Sialyllactosamine ( 6'SLacNAc), Neu5Gc-a2,3-Gal-b1,4-GlcNAc, 2'FL, 3-FL, DiFL, LNFP-I, LNFP-II, LNFP-III, Gal-LNFP-III, LNFP-V , LNDFH I, LNDFH II, LNDFH III, F-LNH I, F-LNH II, F-LNH III, F-LNnH II, F-LNnH I, F-para-LNH I, F-para-LNH II, F -para-LNnH, DF-LNH II, DF-LNH I, DF-LNnH, DF-para-LNH, DF-para-LNH II, DF-para-LNH III, DF-para-LNnH, TF-LNH I, TF -LNH II, TF-para-LNH I, TF-para-LNH II, TF-para-LNnH, F-LNO I, F-LNO II, F-LNO III, F-LNnO, F-LNnO II, F- iso-LNO, F-iso-LNnO I, F-nascent-LNnO, F-para-LNO, DF-iso-LNnO, DF-LNO I, DF-LNO II, DF-LNO III, DF-LNnO I, DF -LNnO II, DF-LNnO III, DF-iso-LNO I, D F-iso-LNO II, DF-iso-LNO III, DF-iso-LNO IV, DF-iso-LNO V, DF-iso-LNO VI, DF-iso-LNO VII, DF-para-LNnO, TF- LNO I, TF-LNO II, TF-LNnO, TF-iso-LNO I, TF-iso-LNO II, TF-iso-LNO III, TF-iso-LNO IV, TF-iso-LNnO, tetra-F- Iso-LNO, Tetra-F-para-LNO, Penta-F-Iso-LNO, F-LND I, F-LND II, DF-LND I, DF-LND II, DF-LND III, DF-LND IV, DF-LND V, DF-LND VI, TriF-LND I, TriF-LND II, TriF-LND III, TriF-LND IV, TriF-LND V, TriF-LND VI, TriF-LND VII, TetraF-LND I, TetraF-LND II, TetraF-LND III, F-LNnD I, F-LNnD II, DF-LNnD, DF-nascent-LND, A-tetrasaccharide, DF D Gal-LNnH (Gal-a1,3-Gal-b1 ,4-GlcNAc-b1,6-[Gal-a1,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3]-Gal-b1,4-Glc), 3-F- Isoglobular trisaccharide, B-tetrasaccharide, B-pentasaccharide, B-hexasaccharide, B-heptaose, DF DGal-LNnT (Gal-a1,3-Gal-b1,4-[Fuc-a1,3]- GlcNAc-b1,3-Gal-b1,4-[Fuc-a1,3]-Glc), TF DGal-LNnH a, TF DGal-LNnH b, DFGal-para-LNnH, 2'FLNB, 4'FLNB, 2 'FLacNAc, 3-FLacNAc, Gal-a1,4-Gal-b1,4-Glc (Gal-a1,4-lactose or globular trisaccharide), β3'-galactosylose, β6'-galactosylose, Lacto-N-triose II (LN3), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), LNH, LNnH, iso-LNO, neo-LNO, neo-LNnO, LND, iso- -LND, GalNAc-a1,3-Gal-b1,4-Glc, nascent-LNP I, Gal-nascent-LNP I, Gal-nascent-LNP II, Gal-nascent-LNP III, iso-LNT, DGalLNnH, galili Pentasaccharide, Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, Ga l-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3- Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, newborn-LNT, Poly-LacNAc and LacDiNAc.

Examples of such preferred mixtures include mixtures comprising, consisting of, or consisting essentially of: 3'-sialyllactose, 6'-sialyllactose, sialylated milk-N-propane Sugar, sialylated lacto-N-tetrasaccharide, 2'-fucosyllactose (2'FL), 3-fucosyllactose (3-FL), difucosyllactose, lacto-N-rock Phycopentaose I (LNFP-I; Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), lacto-N-fucopentose II (LNFP-II; Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-Gal-b1,4-Glc), lacto-N-fucopentaose V (LNFP-V; Gal-b1,3-GlcNAc -b1,3-Gal-b1,4-[Fuc-a1,3]-Glc), lacto-N-triose II (LN3) and lacto-N-tetraose (LNT).

Another example of such preferred mixtures includes mixtures comprising, consisting of, or consisting essentially of: 3'-sialyllactose, 6'-sialyllactose, sialylated milk-N - Triose, sialyl lacto-N-neotetraose, lacto-N-fucopentose III (LNFP III; Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3-Gal- b1,4-Glc), lacto-N-triose II (LN3), lacto-N-neotetraose (LNnT) and GalNAc-b1,3-LNnT.

Another example of such preferred mixtures includes mixtures comprising, consisting of, or consisting essentially of: 3'-sialyllactose, sialylated lactose-N-triose, sialylated lactose- N-tetrasaccharide, 3-fucosyllactose (3-FL), lacto-N-fucopentaose V (LNFP-V; Gal-b1,3-GlcNAc-b1,3-Gal-b1,4- [Fuc-a1,3]-Glc), lacto-N-triose II (LN3) and lacto-N-tetraose (LNT).

Another example of such preferred mixtures includes mixtures comprising, consisting of, or consisting essentially of: 2'-fucosyllactose, 3-fucosyllactose, 3'-sialic acid Syllactose, 6'-sialyllactose, LN3, LNT and LNnT.

Another example of such preferred mixtures includes mixtures comprising, consisting of, or consisting essentially of: 2'-fucosyllactose, 3-fucosyllactose, 3'-sialic acid Syllactose, 6'-sialyllactose, LN3, sialylated LN3, LNT, LNnT, sialylated LNT, fucosylated LNT, sialylated LNnT, fucosylated LNnT.

Exemplary mixtures are described in the Examples section in this context of the invention.

In this context of the present invention, other preferred mixtures comprise mixtures of at least three charged and neutral fucosylated MMOS and no neutral non-fucosylated MMO, wherein the mixtures comprise, from Consists of or consists essentially of at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, At least eight, at least nine, at least ten different charged MMOs, preferably selected from any of the following: 3'SL, 6'SL, F-SL, LSTa, LSTb, LSTc, LSTd, F-LSTa , F-LSTb, F-LSTc, S-LNH I, S-LNH II, S-LNnH I, S-LNnH II, S-para-LNnH, FS-LNH, FS-LNH I, FS-LNH II, FS -LNH III, FS-LNH IV, FS-LNnH I, FS-LNnH II, FS-para-LNnH I, FS-para-LNnH II, DFS-LNH I, DFS-LNH III, DFS-LNH IV, DFS- LNnH, S-LNO, FS-LNO I, FS-LNO II, FS-iso-LNO, DFS-iso-LNO I, DFS-iso-LNO II, DFS-LNO I, DFS-NO II, DFS-LNO III , TFS-LNO, TFS-iso-LNO, DS-LNT, FDS-LNT I, FDS-LNT II, DS-LNH I, DS-LNH II, DS-LNnH, FDS-LNH I, FDS-LNH II, FDS -LNH III, FDS-LNnH, TS-LNH, SLNnD, FS-Neo-LNP I, DF-para-LNH Sulfate I, DF-para-LNH Sulfate II, TF-para-LNH Sulfate, Neu5Ac-a2 ,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,6-[GlcNAc-b1, 3]-Gal-b1,4-Glc, Neu5Gc-a2,8-Neu5Ac-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,8-Neu5Gc-a2,3-Gal-b1,4-Glc , Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,8-Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1 ,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Gal-b1,6-[Neu5A c-a2,3]-Gal-b1,4-Glc, Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-Gal-b1 ,4-Glc, Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Gal-b1,4-GlcNAc-b1,6-[Neu5Ac-a2, 3-Gal-b1,3]-Gal-1,4-Glc, Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,4-Glc , Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,6-Gal-b1,4-Glc, Gal-b1,3-[Neu5Gc-a2,6]-Gal-b1,4-Glc , Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,6-[GlcNAc- b1,3]-Gal-b1,4-Glc, FS Gal-LNnH, DFSGal-LNnH, Gal-MSLNnH, MSDF-para-LNnH, Neu5GcLNnT, GM2 tetrasaccharide, SLNOa, 3'-Sialyl lacto-N- Disaccharide (3'SLNB), 6'-Sialyllacto-N-disaccharide (6'SLNB), 3'-Sialyllactosamine (3'SLacNAc), 6'-Sialyllactosamine (6 'SLacNAc), Neu5Gc-a2,3-Gal-b1,4-GlcNAc, with at least one, preferably at least two, more preferably at least three, even better at least four, even better at least five, even better at least six, preferably at least seven, at least eight, at least nine, at least ten different combinations of neutral fucosylated MMOs preferably selected from any of the following: 2'FL, 3- FL, DiFL, LNFP-I, LNFP-II, LNFP-III, Gal-LNFP-III, LNFP-V, LNDFH I, LNDFH II, LNDFH III, F-LNH I, F-LNH II, F-LNH III, F-LNnH II, F-LNnH I, F-para-LNH I, F-para-LNH II, F-para-LNnH, DF-LNH II, DF-LNH I, DF-LNnH, DF-para-LNH, DF-para-LNH II, DF-para-LNH III, DF-para-LNnH, TF-LNH I, TF-LNH II, TF-para-LNH I, TF-para-LNH II, TF-para-LNnH, F-LNO I, F-LNO II, F-LNO III, F-LNnO, F-LNnO II, F-iso-LNO, F-iso-LNnO I, F-nascent-LNnO , F-para-LNO, DF-iso-LNnO, DF-LNO I, DF-LNO II, DF-LNO III, DF-LNnO I, DF-LNnO II, DF-LNnO III, DF-iso-LNO I, DF-iso-LNO II, DF-iso-LNO III, DF-iso-LNO IV, DF-iso-LNO V, DF-iso-LNO VI, DF-iso-LNO VII, DF-para-LNnO, TF- LNO I, TF-LNO II, TF-LNnO, TF-iso-LNO I, TF-iso-LNO II, TF-iso-LNO III, TF-iso-LNO IV, TF-iso-LNnO, tetra-F- Iso-LNO, Tetra-F-para-LNO, Penta-F-Iso-LNO, F-LND I, F-LND II, DF-LND I, DF-LND II, DF-LND III, DF-LND IV, DF-LND V, DF-LND VI, TriF-LND I, TriF-LND II, TriF-LND III, TriF-LND IV, TriF-LND V, TriF-LND VI, TriF-LND VII, TetraF-LND I, TetraF-LND II, TetraF-LND III, F-LNnD I, F-LNnD II, DF-LNnD, DF-nascent-LND, A-tetrasaccharide, DF D Gal-LNnH (Gal-a1,3-Gal-b1 ,4-GlcNAc-b1,6-[Gal-a1,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3]-Gal-b1,4-Glc), 3-F- Isoglobular trisaccharide, B-tetrasaccharide, B-pentasaccharide, B-hexasaccharide, B-heptasaccharide, DF DGal-LNnT (Gal-a1,3-Gal-b1,4-[Fuc-a1,3]- GlcNAc-b1,3-Gal-b1,4-[Fuc-a1,3]-Glc), TF DGal-LNnH a, TF DGal-LNnH b, DFGal-para-LNnH, 2'FLNB, 4'FLNB, 2 'FLacNAc, 3-FlacNAc, optionally combined with any one or more of charged and/or neutral oligosaccharides that are not MMOs.

In this context of the invention, more preferred mixtures comprise mixtures of at least three different charged and neutral fucosylated MMOs and no neutral non-fucosylated MMOs, wherein the mixtures comprise, from Consists of or consists essentially of at least three charged and neutral fucosylated MMOs selected from the list comprising: 3'SL, 6'SL, F-SL, LSTa, LSTb, LSTc, LSTd, F-LSTa, F-LSTb, F-LSTc, S-LNH I, S-LNH II, S-LNnH I, S-LNnH II, S-para-LNnH, FS-LNH, FS-LNH I, FS-LNH II, FS-LNH III, FS-LNH IV, FS-LNnH I, FS-LNnH II, FS-para-LNnH I, FS-para-LNnH II, DFS-LNH I, DFS-LNH III, DFS-LNH IV , DFS-LNnH, S-LNO, FS-LNO I, FS-LNO II, FS-iso-LNO, DFS-iso-LNO I, DFS-iso-LNO II, DFS-LNO I, DFS-NO II, DFS -LNO III, TFS-LNO, TFS-iso-LNO, DS-LNT, FDS-LNT I, FDS-LNT II, DS-LNH I, DS-LNH II, DS-LNnH, FDS-LNH I, FDS-LNH II, FDS-LNH III, FDS-LNnH, TS-LNH, SLNnD, FS-nascent-LNP I, DF-para-LNH sulfate I, DF-para-LNH sulfate II, TF-para-LNH sulfate, Neu5Ac-a2,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,6-[GlcNAc -b1,3]-Gal-b1,4-Glc, Neu5Gc-a2,8-Neu5Ac-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,8-Neu5Gc-a2,3-Gal-b1, 4-Glc, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,8-Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,3- Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Gal-b1,6-[Neu5Ac-a2,3]-Gal-b1,4-Glc, Neu5Ac-a2,3- GlcNAc-b1,3-Gal-b1,4-Glc, Ne u5Gc-a2,3-Gal-b1,3-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Gal- b1,4-GlcNAc-b1,6-[Neu5Ac-a2,3-Gal-b1,3]-Gal-1,4-Glc, Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,6- [Gal-b1,3]-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,6-Gal-b1,4-Glc, Gal-b1,3- [Neu5Gc-a2,6]-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,3- Gal-b1,4-GlcNAc-b1,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, FS Gal-LNnH, DFSGal-LNnH, Gal-MSLNnH, MSDF-para-LNnH, Neu5GcLNnT, GM2 Tetrasaccharide, SLNOa, 3'-Sialyllacto-N-disaccharide (3'SLNB), 6'-Sialyllacto-N-disaccharide (6'SLNB), 3'-Sialyllactosamine ( 3'SLacNAc), 6'-Sialyllactosamine (6'SLacNAc), Neu5Gc-a2,3-Gal-b1,4-GlcNAc, 2'FL, 3-FL, DiFL, LNFP-I, LNFP-II , LNFP-III, Gal-LNFP-III, LNFP-V, LNDFH I, LNDFH II, LNDFH III, F-LNH I, F-LNH II, F-LNH III, F-LNnH II, F-LNnH I, F -para-LNH I, F-para-LNH II, F-para-LNnH, DF-LNH II, DF-LNH I, DF-LNnH, DF-para-LNH, DF-para-LNH II, DF-para-LNH III, DF-para-LNnH, TF-LNH I, TF-LNH II, TF-para-LNH I, TF-para-LNH II, TF-para-LNnH, F-LNO I, F-LNO II, F- LNO III, F-LNnO, F-LNnO II, F-iso-LNO, F-iso-LNnO I, F-nascent-LNnO, F-para-LNO, DF-iso-LNnO, DF-LNO I, DF- LNO II, DF-LNO III, DF-LNnO I, DF-LNnO II, D F-LNnO III, DF-iso-LNO I, DF-iso-LNO II, DF-iso-LNO III, DF-iso-LNO IV, DF-iso-LNO V, DF-iso-LNO VI, DF-iso -LNO VII, DF-para-LNnO, TF-LNO I, TF-LNO II, TF-LNnO, TF-iso-LNO I, TF-iso-LNO II, TF-iso-LNO III, TF-iso-LNO IV, TF-iso-LNnO, tetra-F-iso-LNO, tetra-F-para-LNO, penta-F-iso-LNO, F-LND I, F-LND II, DF-LND I, DF-LND II, DF-LND III, DF-LND IV, DF-LND V, DF-LND VI, TriF-LND I, TriF-LND II, TriF-LND III, TriF-LND IV, TriF-LND V, TriF-LND VI, TriF-LND VII, TetraF-LND I, TetraF-LND II, TetraF-LND III, F-LNnD I, F-LNnD II, DF-LNnD, DF-nascent-LND, A-tetrasaccharide, DF D Gal -LNnH (Gal-a1,3-Gal-b1,4-GlcNAc-b1,6-[Gal-a1,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3]-Gal -b1,4-Glc), 3-F-heteroglobular trisaccharide, B-tetrasaccharide, B-pentasaccharide, B-hexasaccharide, B-heptasaccharide, DF DGal-LNnT (Gal-a1,3-Gal- b1,4-[Fuc-a1,3]-GlcNAc-b1,3-Gal-b1,4-[Fuc-a1,3]-Glc), TF DGal-LNnH a, TF DGal-LNnH b, DFGal-pair -LNnH, 2'FLNB, 4'FLNB, 2'FLacNAc, 3-FlacNAc.

Exemplary mixtures are described in the Examples section in this context of the invention.

In this context of the present invention, other preferred mixtures comprise mixtures of at least three charged and neutral afucosylated MMOS and no neutral fucosylated MMOs, wherein the mixtures comprise, from Consists of or consists essentially of at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, At least eight, at least nine, at least ten different charged MMOs, preferably selected from any of the following: 3'SL, 6'SL, F-SL, LSTa, LSTb, LSTc, LSTd, F-LSTa , F-LSTb, F-LSTc, S-LNH I, S-LNH II, S-LNnH I, S-LNnH II, S-para-LNnH, FS-LNH, FS-LNH I, FS-LNH II, FS -LNH III, FS-LNH IV, FS-LNnH I, FS-LNnH II, FS-para-LNnH I, FS-para-LNnH II, DFS-LNH I, DFS-LNH III, DFS-LNH IV, DFS- LNnH, S-LNO, FS-LNO I, FS-LNO II, FS-iso-LNO, DFS-iso-LNO I, DFS-iso-LNO II, DFS-LNO I, DFS-NO II, DFS-LNO III , TFS-LNO, TFS-iso-LNO, DS-LNT, FDS-LNT I, FDS-LNT II, DS-LNH I, DS-LNH II, DS-LNnH, FDS-LNH I, FDS-LNH II, FDS -LNH III, FDS-LNnH, TS-LNH, SLNnD, FS-Neo-LNP I, DF-para-LNH Sulfate I, DF-para-LNH Sulfate II, TF-para-LNH Sulfate, Neu5Ac-a2 ,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,6-[GlcNAc-b1, 3]-Gal-b1,4-Glc, Neu5Gc-a2,8-Neu5Ac-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,8-Neu5Gc-a2,3-Gal-b1,4-Glc , Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,8-Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1 ,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Gal-b1,6-[Neu5A c-a2,3]-Gal-b1,4-Glc, Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-Gal-b1 ,4-Glc, Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Gal-b1,4-GlcNAc-b1,6-[Neu5Ac-a2, 3-Gal-b1,3]-Gal-1,4-Glc, Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,4-Glc , Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,6-Gal-b1,4-Glc, Gal-b1,3-[Neu5Gc-a2,6]-Gal-b1,4-Glc , Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,6-[GlcNAc- b1,3]-Gal-b1,4-Glc, FS Gal-LNnH, DFSGal-LNnH, Gal-MSLNnH, MSDF-para-LNnH, Neu5GcLNnT, GM2 tetrasaccharide, SLNOa, 3'-Sialyl lacto-N- Disaccharide (3'SLNB), 6'-Sialyllacto-N-disaccharide (6'SLNB), 3'-Sialyllactosamine (3'SLacNAc), 6'-Sialyllactosamine (6 'SLacNAc), Neu5Gc-a2,3-Gal-b1,4-GlcNAc, with at least one, preferably at least two, more preferably at least three, even better at least four, even better at least five, even better at least six, preferably at least seven, at least eight, at least nine, at least ten different combinations of neutral afucosylated MMOs preferably selected from any of the following: Gal-a1,4 -Gal-b1,4-Glc (Gal-a1,4-lactose or globulotrisaccharide), β3'-galactosylose, β6'-galactosylose, lacto-N-triose II (LN3), lactose -N-Neotetraose (LNnT), Lacto-N-tetraose (LNT), LNH, LNnH, iso-LNO, nascent-LNO, nascent-LNnO, LND, iso-LND, GalNAc-a1,3-Gal- b1,4-Glc, nascent-LNP I, Gal-nascent-LNP I, Gal-nascent-LNP II, Gal-nascent-LNP III, iso-LNT, DGalLNnH, galili pentasaccharide, Gal-b1,3-Gal- b1,3-Gal-b1,4-Gl c. Gal-b1, 3-Gal-b1, 3-Gal-b1, 3-Gal-b1, 4-Glc, Gal-b1, 3-Gal-b1, 3-Gal-b1, 3-Gal-b1, 3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, newborn- LNT, Poly-LacNAc, LacDiNAc.

In this context of the present invention, more preferred mixtures comprise mixtures of at least three different charged and neutral afucosylated MMOs and no neutral fucosylated MMOs, wherein the mixtures comprise, from Consists of or consists essentially of at least three charged and neutral afucosylated MMOs selected from the list comprising: 3'SL, 6'SL, F-SL, LSTa, LSTb, LSTc, LSTd , F-LSTa, F-LSTb, F-LSTc, S-LNH I, S-LNH II, S-LNnH I, S-LNnH II, S-para-LNnH, FS-LNH, FS-LNH I, FS- LNH II, FS-LNH III, FS-LNH IV, FS-LNnH I, FS-LNnH II, FS-para-LNnH I, FS-para-LNnH II, DFS-LNH I, DFS-LNH III, DFS-LNH IV, DFS-LNnH, S-LNO, FS-LNO I, FS-LNO II, FS-iso-LNO, DFS-iso-LNO I, DFS-iso-LNO II, DFS-LNO I, DFS-NO II, DFS-LNO III, TFS-LNO, TFS-iso-LNO, DS-LNT, FDS-LNT I, FDS-LNT II, DS-LNH I, DS-LNH II, DS-LNnH, FDS-LNH I, FDS- LNH II, FDS-LNH III, FDS-LNnH, TS-LNH, SLNnD, FS-nascent-LNP I, DF-para-LNH sulfate I, DF-para-LNH sulfate II, TF-para-LNH sulfate , Neu5Ac-a2,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,6-[ GlcNAc-b1,3]-Gal-b1,4-Glc, Neu5Gc-a2,8-Neu5Ac-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,8-Neu5Gc-a2,3-Gal-b1 ,4-Glc, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,8-Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,3 -Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Gal-b1,6-[Neu5Ac-a2,3]-Gal-b1,4-Glc, Neu5Ac-a2,3 -GlcNAc-b1,3-Gal-b1,4-Glc, N eu5Gc-a2,3-Gal-b1,3-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Gal- b1,4-GlcNAc-b1,6-[Neu5Ac-a2,3-Gal-b1,3]-Gal-1,4-Glc, Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,6- [Gal-b1,3]-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,6-Gal-b1,4-Glc, Gal-b1,3- [Neu5Gc-a2,6]-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Neu5Ac-a2,3- Gal-b1,4-GlcNAc-b1,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, FS Gal-LNnH, DFSGal-LNnH, Gal-MSLNnH, MSDF-para-LNnH, Neu5GcLNnT, GM2 Tetrasaccharide, SLNOa, 3'-Sialyllacto-N-disaccharide (3'SLNB), 6'-Sialyllacto-N-disaccharide (6'SLNB), 3'-Sialyllactosamine ( 3'SLacNAc), 6'-Sialyllactosamine (6'SLacNAc), Neu5Gc-a2,3-Gal-b1,4-GlcNAc, Gal-a1,4-Gal-b1,4-Glc (Gal-a1 ,4-lactose or globulotrisaccharide), β3'-galactosylose, β6'-galactosylose, lacto-N-triose II (LN3), lacto-N-neotetraose (LNnT), lacto- N-tetrasaccharide (LNT), LNH, LNnH, iso-LNO, nascent-LNO, nascent-LNnO, LND, iso-LND, GalNAc-a1,3-Gal-b1,4-Glc, nascent-LNP I, Gal - Nascent-LNP I, Gal-nascent-LNP II, Gal-nascent-LNP III, iso-LNT, DGalLNnH, galili pentasaccharide, Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3- Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, newborn-LNT, Poly-LacNAc, LacDiNAc.

Exemplary mixtures are described in the Examples section in this context of the invention.

In this context of the invention, other preferred mixtures comprise mixtures of at least three neutral fucosylated and neutral afucosylated MMOSs and uncharged MMOs, wherein these mixtures comprise, from Consists of or consists essentially of at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, At least eight, at least nine, at least ten different neutral fucosylated MMOs, preferably selected from any of the following: 2'FL, 3-FL, DiFL, LNFP-I, LNFP-II , LNFP-III, Gal-LNFP-III, LNFP-V, LNDFH I, LNDFH II, LNDFH III, F-LNH I, F-LNH II, F-LNH III, F-LNnH II, F-LNnH I, F -para-LNH I, F-para-LNH II, F-para-LNnH, DF-LNH II, DF-LNH I, DF-LNnH, DF-para-LNH, DF-para-LNH II, DF-para-LNH III, DF-para-LNnH, TF-LNH I, TF-LNH II, TF-para-LNH I, TF-para-LNH II, TF-para-LNnH, F-LNO I, F-LNO II, F- LNO III, F-LNnO, F-LNnO II, F-iso-LNO, F-iso-LNnO I, F-nascent-LNnO, F-para-LNO, DF-iso-LNnO, DF-LNO I, DF- LNO II, DF-LNO III, DF-LNnO I, DF-LNnO II, DF-LNnO III, DF-iso-LNO I, DF-iso-LNO II, DF-iso-LNO III, DF-iso-LNO IV , DF-iso-LNO V, DF-iso-LNO VI, DF-iso-LNO VII, DF-p-LNnO, TF-LNO I, TF-LNO II, TF-LNnO, TF-iso-LNO I, TF -iso-LNO II, TF-iso-LNO III, TF-iso-LNO IV, TF-iso-LNnO, tetra-F-iso-LNO, tetra-F-para-LNO, penta-F-iso-LNO, F-LND I, F-LND II, DF-LND I, DF-LND II, DF-LND III, DF-LND IV, DF-LND V, DF-LND VI, TriF-LND I, TriF-LND II, TriF-LND III, TriF-LND IV, TriF-LND V, TriF-LND VI, TriF-LND VII, TetraF-LND I, T etraF-LND II, TetraF-LND III, F-LNnD I, F-LNnD II, DF-LNnD, DF-nascent-LND, A-tetrasaccharide, DF D Gal-LNnH (Gal-a1,3-Gal-b1 ,4-GlcNAc-b1,6-[Gal-a1,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3]-Gal-b1,4-Glc), 3-F- Isoglobular trisaccharide, B-tetrasaccharide, B-pentasaccharide, B-hexasaccharide, B-heptasaccharide, DF DGal-LNnT (Gal-a1,3-Gal-b1,4-[Fuc-a1,3]- GlcNAc-b1,3-Gal-b1,4-[Fuc-a1,3]-Glc), TF DGal-LNnH a, TF DGal-LNnH b, DFGal-para-LNnH, 2'FLNB, 4'FLNB, 2 'FLacNAc, 3-FlacNAc, with at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least Eight, at least nine, at least ten different combinations of neutral afucosylated MMOs preferably selected from any of the following: Gal-a1,4-Gal-b1,4-Glc (Gal- a1,4-lactose or globulotrisaccharide), β3'-galactosylose, β6'-galactosylose, lacto-N-triose II (LN3), lacto-N-neotetraose (LNnT), lactose -N-tetrasaccharide (LNT), LNH, LNnH, iso-LNO, nascent-LNO, nascent-LNnO, LND, iso-LND, GalNAc-a1,3-Gal-b1,4-Glc, nascent-LNP I, Gal-nascent-LNP I, Gal-nascent-LNP II, Gal-nascent-LNP III, iso-LNT, DGalLNnH, galili pentasaccharide, Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc , Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3 -Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, newborn-LNT , Poly-LacNAc, LacDiNAc.

In this context of the invention, more preferred mixtures comprise mixtures of at least three different neutral fucosylated and neutral afucosylated MMOs and uncharged MMOs, wherein the mixtures comprise, from Consists of or consists essentially of at least three neutral fucosylated and neutral afucosylated MMOs selected from the list comprising: 2'FL, 3-FL, DiFL, LNFP-I , LNFP-II, LNFP-III, Gal-LNFP-III, LNFP-V, LNDFH I, LNDFH II, LNDFH III, F-LNH I, F-LNH II, F-LNH III, F-LNnH II, F- LNnH I, F-para-LNH I, F-para-LNH II, F-para-LNnH, DF-LNH II, DF-LNH I, DF-LNnH, DF-para-LNH, DF-para-LNH II, DF-para-LNH III, DF-para-LNnH, TF-LNH I, TF-LNH II, TF-para-LNH I, TF-para-LNH II, TF-para-LNnH, F-LNO I, F-LNO II, F-LNO III, F-LNnO, F-LNnO II, F-iso-LNO, F-iso-LNnO I, F-nascent-LNnO, F-para-LNO, DF-iso-LNnO, DF-LNO I, DF-LNO II, DF-LNO III, DF-LNnO I, DF-LNnO II, DF-LNnO III, DF-iso-LNO I, DF-iso-LNO II, DF-iso-LNO III, DF- iso-LNO IV, DF-iso-LNO V, DF-iso-LNO VI, DF-iso-LNO VII, DF-para-LNnO, TF-LNO I, TF-LNO II, TF-LNnO, TF-iso- LNO I, TF-iso-LNO II, TF-iso-LNO III, TF-iso-LNO IV, TF-iso-LNnO, tetra-F-iso-LNO, tetra-F-para-LNO, penta-F- Iso-LNO, F-LND I, F-LND II, DF-LND I, DF-LND II, DF-LND III, DF-LND IV, DF-LND V, DF-LND VI, TriF-LND I, TriF -LND II, TriF-LND III, TriF-LND IV, TriF-LND V, TriF-LND VI, TriF-LND VII, TetraF-LND I, TetraF-LND II, TetraF-LND III, F-LNnD I, F -LNnD II, DF-LNnD, DF-NEW Raw-LND, A-tetrasaccharide, DF D Gal-LNnH (Gal-a1,3-Gal-b1,4-GlcNAc-b1,6-[Gal-a1,3-Gal-b1,4-[Fuc-a1 ,3]-GlcNAc-b1,3]-Gal-b1,4-Glc), 3-F-heteroglobular trisaccharide, B-tetrasaccharide, B-pentasaccharide, B-hexasaccharide, B-heptasaccharide, DF DGal-LNnT (Gal-a1,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3-Gal-b1,4-[Fuc-a1,3]-Glc), TF DGal- LNnH a, TF DGal-LNnH b, DFGal-para-LNnH, 2'FLNB, 4'FLNB, 2'FLacNAc, 3-FLacNAc, Gal-a1,4-Gal-b1,4-Glc (Gal-a1,4 -lactose or globulotriose), β3'-galactosylose, β6'-galactosylose, lacto-N-triose II (LN3), lacto-N-neotetraose (LNnT), lacto-N- Tetrasaccharide (LNT), LNH, LNnH, iso-LNO, nascent-LNO, nascent-LNnO, LND, iso-LND, GalNAc-a1,3-Gal-b1,4-Glc, nascent-LNP I, Gal-nascent -LNP I, Gal-nascent-LNP II, Gal-nascent-LNP III, iso-LNT, DGalLNnH, galili pentasaccharide, Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, Gal- b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal- b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, nascent-LNT, poly- LacNAc, LacDiNAc.

Exemplary mixtures are described in the Examples section in this context of the invention.

In this context of the invention, other preferred mixtures comprise mixtures of at least three charged MMOS and no neutral MMOs, wherein the mixtures comprise, consist of or consist essentially of at least three, preferably At least four, more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different charged MMOs preferably selected from any of the following: 3'SL, 6'SL, F-SL, LSTa, LSTb, LSTc, LSTd, F-LSTa, F-LSTb, F-LSTc, S-LNH I, S-LNH II, S-LNnH I, S-LNnH II, S-para-LNnH, FS-LNH, FS-LNH I, FS-LNH II, FS-LNH III, FS-LNH IV, FS-LNnH I, FS-LNnH II, FS-para-LNnH I, FS - Para-LNnH II, DFS-LNH I, DFS-LNH III, DFS-LNH IV, DFS-LNnH, S-LNO, FS-LNO I, FS-LNO II, FS-iso-LNO, DFS-iso-LNO I, DFS-Iso-LNO II, DFS-LNO I, DFS-NO II, DFS-LNO III, TFS-LNO, TFS-Iso-LNO, DS-LNT, FDS-LNT I, FDS-LNT II, DS- LNH I, DS-LNH II, DS-LNnH, FDS-LNH I, FDS-LNH II, FDS-LNH III, FDS-LNnH, TS-LNH, SLNnD, FS-nascent-LNP I, DF-para-LNH sulfate Salt I, DF-p-LNH Sulfate II, TF-p-LNH Sulfate, Neu5Ac-a2,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-[Neu5Ac-a2 ,6]-Gal-b1,4-Glc, Neu5Ac-a2,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, Neu5Gc-a2,8-Neu5Ac-a2,3-Gal-b1, 4-Glc, Neu5Ac-a2,8-Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,8- Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc, Gal-b1,6-[Neu5Ac -a2,3]-Gal-b1,4-Glc, Neu5Ac-a2,3 -GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac- a2,6]-Gal-b1,4-Glc, Gal-b1,4-GlcNAc-b1,6-[Neu5Ac-a2,3-Gal-b1,3]-Gal-1,4-Glc, Neu5Ac-a2 ,6-Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,4-Glc, Neu5Gc-a2,6 -Gal-b1,4-Glc, Gal-b1,3-[Neu5Gc-a2,6]-Gal-b1,4-Glc, Neu5Gc-a2,3-Gal-b1,3-[Neu5Ac-a2,6] -Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, FS Gal-LNnH, DFSGal- LNnH, Gal-MSLNnH, MSDF-para-LNnH, Neu5GcLNnT, GM2 tetrasaccharide, SLNOa, 3'-Sialyllacto-N-disaccharide (3'SLNB), 6'-Sialyllacto-N-disaccharide (6'SLNB), 3'-Sialyllactosamine (3'SLacNAc), 6'-Sialyllactosamine (6'SLacNAc), Neu5Gc-a2,3-Gal-b1,4-GlcNAc.

Exemplary mixtures are described in the Examples section in this context of the invention.

In this context of the invention, other preferred mixtures comprise mixtures of at least three neutral MMOS and uncharged MMOs, wherein the mixtures comprise, consist of, or consist essentially of at least three, preferably At least four, more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different neutral MMOs preferably selected from any of the following : 2'FL, 3-FL, DiFL, LNFP-I, LNFP-II, LNFP-III, Gal-LNFP-III, LNFP-V, LNDFH I, LNDFH II, LNDFH III, F-LNH I, F-LNH II, F-LNH III, F-LNnH II, F-LNnH I, F-para-LNH I, F-para-LNH II, F-para-LNnH, DF-LNH II, DF-LNH I, DF-LNnH , DF-para-LNH, DF-para-LNH II, DF-para-LNH III, DF-para-LNnH, TF-LNH I, TF-LNH II, TF-para-LNH I, TF-para-LNH II, TF-para-LNnH, F-LNO I, F-LNO II, F-LNO III, F-LNnO, F-LNnO II, F-iso-LNO, F-iso-LNnO I, F-nascent-LNnO, F -para-LNO, DF-iso-LNnO, DF-LNO I, DF-LNO II, DF-LNO III, DF-LNnO I, DF-LNnO II, DF-LNnO III, DF-iso-LNO I, DF- iso-LNO II, DF-iso-LNO III, DF-iso-LNO IV, DF-iso-LNO V, DF-iso-LNO VI, DF-iso-LNO VII, DF-para-LNnO, TF-LNO I , TF-LNO II, TF-LNnO, TF-iso-LNO I, TF-iso-LNO II, TF-iso-LNO III, TF-iso-LNO IV, TF-iso-LNnO, tetra-F-iso- LNO, Tetra-F-para-LNO, Penta-F-iso-LNO, F-LND I, F-LND II, DF-LND I, DF-LND II, DF-LND III, DF-LND IV, DF- LND V, DF-LND VI, TriF-LND I, TriF-LND II, TriF-LND III, TriF-LND IV, TriF-LND V, TriF-LND VI, TriF-LND VII, TetraF-LND I, TetraF- LND II, TetraF-LND III, F-LNnD I, F-LNnD II, DF-LNnD, DF-nascent-LND, A-tetrasaccharide, DF D Gal-LNnH (Gal-a1,3-Gal-b1,4-GlcNAc-b1,6-(Gal-a1,3 -Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1,3)-Gal-b1,4-Glc), 3-F-isoglobular trisaccharide, B-tetrasaccharide, B-pentasaccharide, B-hexasaccharide, B-heptaose, DF DGal-LNnT (Gal-a1,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3-Gal-b1,4-[Fuc- a1,3]-Glc), TF DGal-LNnH a, TF DGal-LNnH b, DFGal-para-LNnH, 2'FLNB, 4'FLNB, 2'FLacNAc, 3-FLacNAc, Gal-a1,4-Gal- b1,4-Glc (Gal-a1,4-lactose or globulotrisaccharide), β3'-galactosylose, β6'-galactosylose, lacto-N-trisaccharide II (LN3), lacto-N- Neotetraose (LNnT), lacto-N-tetraose (LNT), LNH, LNnH, iso-LNO, neo-LNO, neo-LNnO, LND, iso-LND, GalNAc-a1,3-Gal-b1,4 -Glc, nascent-LNP I, Gal-nascent-LNP I, Gal-nascent-LNP II, Gal-nascent-LNP III, iso-LNT, DGalLNnH, galili pentasaccharide, Gal-b1,3-Gal-b1,3 -Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1 ,3-Gal-b1,3-Gal-b1,4-Glc,Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1 , 4-Glc, nascent-LNT, poly-LacNAc, LacDiNAc.

Examples of such preferred mixtures include mixtures comprising, consisting of, or consisting essentially of: 2'-fucosyllactose (2'FL), lacto-N-triose II (LN3 ), lacto-N-tetraose (LNT) and lacto-N-fucopentaose I (LNFP-I; Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4- Glc).

Another example of such preferred mixtures includes mixtures comprising, consisting of, or consisting essentially of: lacto-N-triose II (LN3), lacto-N-neotetraose (LNnT) , lacto-N-tetraose (LNT), p-lacto-N-neopentose, p-lacto-N-pentose, p-lacto-N-neohexaose and p-lacto-N-hexaose.

Exemplary mixtures are described in the Examples section in this context of the invention.

To produce lactose-based oligosaccharides as described herein, ie lactose-based mammalian milk oligosaccharides, and in one embodiment of the methods and/or cells according to the invention, lactose may be added to the culture such that the cells Absorption can be passive or via active transport; or lactose can be produced by cells (eg, after metabolic engineering of cells for this purpose as known to those of ordinary skill in the art), preferably intracellularly. Lactose can thus be used as a receptor in the synthesis of mammalian milk oligosaccharides or human milk oligosaccharides (preferably all lactose-based MMOs or HMOs), which lactose is preferably included in the oligosaccharide mixture according to the invention as described herein middle. Lactose-producing cells can be obtained by expressing N-acetylglucosamine β-1,4-galactosyltransferase and UDP-glucose 4-epimerase. More preferably, the cells are modified for enhanced lactose production. The modification may be any one or more selected from the group comprising: overexpression of N-acetylglucosamine β-1,4-galactosyltransferase, overexpression of UDP-glucose 4-epimerase Excessive performance. Alternatively, cells that use lactose as a receptor in glycosylation reactions preferably have transport proteins for uptake of lactose from culture. More preferably, the cells are optimized for lactose uptake. The optimization may be the overexpression of a lactose transporter such as a lactose permease from eg Escherichia coli, Kluyveromyces lactis or Lactobacillus casei BL23. The lactose permease is preferably expressed continuously. Lactose can be added at the beginning of the culture, or it can be added when sufficient biomass has formed during the growth phase of the culture, ie, the MMO production phase (started by adding lactose to the culture) is separate from the growth phase. In a preferred embodiment, lactose is added at the beginning and/or during the culture, ie the growth phase and the production phase are not separated.

In a preferred embodiment of the method and/or cell according to the present invention, the cell is resistant to lactose killing when 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 the growth of cells 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 includes expression and/or overexpression of exogenous and/or endogenous lactose transporter genes by a heterologous promoter that does not produce a lactose-killing phenotype, and/or modification of a gene that does not produce a lactose-killing phenotype The codon usage of the lactose transporter produces an altered representation of the lactose transporter. The contents of WO 2016/075243 are incorporated by reference in this regard.

To produce LNB-based oligosaccharides as described herein and in additional and/or alternative embodiments of the methods and/or cells of the invention, LNB (i.e. lacto-N-disaccharide, Gal- b1,3-GlcNAc) so that the cell can take up passively or via active transport; or LNB can be produced by the cell (eg after metabolically engineering the cell for this purpose as known to those of ordinary skill in the art ), preferably produced intracellularly. LNB can thus be used as acceptor in the synthesis of LNB-based oligosaccharides (preferably all LNB-based oligosaccharides), which LNB is preferably included in the oligosaccharide mixture according to the invention as described herein. LNB-producing cells can be obtained by expressing N-acetylglucosamine β-1,3-galactosyltransferases that can modify GlcNAc (produced in cells and/or taken up passively or via active transport) to form LNBs . Preferably, LNB-producing cells are capable of expressing, preferably expressing, enzymes required for the synthesis of GlcNAc, such as glucosamine 6-phosphate N-acetyltransferase, phosphatase, N-acetylglucosamine beta-1 ,3-galactosyltransferase, L-glutamic acid-D-fructose-6-phosphate aminotransferase and UDP-glucose 4-epimerase, preferably glucosamine 6-phosphate N-acetyl Transferases and phosphatases (preferably, HAD-type phosphatases). Preferably, the cell is metabolically engineered for LNB production. More preferably, the cell is metabolically engineered for enhanced production of LNB. The cells are preferably modified to express and/or overexpress any one or more of a polypeptide comprising: glucosamine 6-phosphate N-acetyltransferase, phosphatase, N-acetylglucosamine beta- 1,3-Galactosyltransferase, L-glutamic acid-D-fructose-6-phosphate aminotransferase and UDP-glucose 4-epimerase.

Cells that use LNB as a receptor in a glycosylation reaction preferably have transport proteins for uptake of LNB from culture. More preferably, cells are optimized for LNB uptake. The optimization may be the overexpression of LNB transporter proteins such as lactose permease from eg Escherichia coli, Kluyveromyces lactis or Lactobacillus casein BL23. The lactose permease is preferably expressed continuously. LNB can be added at the beginning of the culture, or it can be added when sufficient biomass has formed during the growth phase of the culture, ie, the oligosaccharide production phase (started by adding LNB to the culture) is separate from the growth phase. In a preferred embodiment, LNB is added at the beginning and/or during the culture, ie the growth phase and the production phase are not separated.

To produce LacNAc-based oligosaccharides as described herein and in additional and/or alternative embodiments of the methods and/or cells according to the invention, LacNAc (i.e. N-acetyllactosamine, Gal can be added to the culture) -b1,4-GlcNAc) so that the cell can take up passively or via active transport; or LacNAc can be produced by the cell (e.g., metabolically engineer the cell for this purpose as known to those of ordinary skill in the art ), preferably produced intracellularly. LacNac can thus be used as acceptor in the synthesis of LacNAc-based oligosaccharides (preferably all LacNAc-based oligosaccharides), which LacNAc is preferably included in the oligosaccharide mixture according to the invention as described herein. LacNAc-producing cells can be obtained by expressing an N-acetylglucosamine beta-1,4-galactosyltransferase that can modify GlcNAc (produced in cells and/or taken up passively or via active transport) to form LacNAc . Preferably, LacNAc-producing cells are capable of expressing, preferably expressing, enzymes required for the synthesis of GlcNAc, such as glucosamine 6-phosphate N-acetyltransferase, phosphatase, N-acetylglucosamine beta-1 ,4-galactosyltransferase, L-glutamic acid-D-fructose-6-phosphate aminotransferase and UDP-glucose 4-epimerase, preferably glucosamine 6-phosphate N-acetyl Transferases and phosphatases (preferably, HAD-type phosphatases). Preferably, the cell is metabolically engineered for the production of LacNAc. More preferably, the cell is metabolically engineered for enhanced production of LacNAc. The cells are preferably modified to express and/or overexpress any one or more of a polypeptide comprising: glucosamine 6-phosphate N-acetyltransferase, phosphatase, N-acetylglucosamine beta- 1,4-Galactosyltransferase, L-glutamic acid-D-fructose-6-phosphate aminotransferase and UDP-glucose 4-epimerase.

Cells using LacNAc as receptors in glycosylation reactions preferably have transport proteins for uptake of LacNAc from culture. More preferably, cells are optimized for LacNAc uptake. The optimization may be the overexpression of LNB transporter proteins such as lactose permease from eg Escherichia coli, Kluyveromyces lactis or Lactobacillus casein BL23. The lactose permease is preferably expressed continuously. LacNAc can be added at the beginning of the culture, or it can be added when sufficient biomass has formed during the growth phase of the culture, ie, the oligosaccharide production phase (started by adding LacNAc to the culture) is separate from the growth phase. In a preferred embodiment, LacNAc is added at the beginning and/or during the culture, ie the growth phase and the production phase are not separated.

In additional and/or alternative preferred embodiments of the methods and/or cells according to the present invention, the cells are capable of expressing at least two, preferably at least three, more preferably at least four, even better at least as described herein Five, even better at least six, most preferably at least seven glycosyltransferases.

In additional and/or alternative preferred embodiments of the methods and/or cells according to the invention, at least one, preferably at least two, more preferably all of the glycosyltransferases are involved in at least three different mammals Production of this mixture of lacto-oligosaccharides.

In additional and/or alternative preferred embodiments of the methods and/or cells according to the present invention, any one of the glycosyltransferases is preferably selected from the list comprising: fucosyltransferases, Sialyltransferase, Galactosyltransferase, Glucosyltransferase, Mannosyltransferase, N-Acetylglucosaminyltransferase, N-Acetylgalactosyltransferase, N-Acetyltransferase Mannosylaminotransferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosaminotransferase, N-hydroxyacetylneuramidotransferase, rhamnose Syltransferase, N-Acetyl rhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine aminotransferase, UDP -N-Acetylglucosamine enolacetonyltransferase and fucosaminotransferase, 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 selected from the list comprising: α-1,3-N-acetylgalactosaminyltransferase and β- 1,3-N-Acetylgalactosaminyltransferase.

In another embodiment of the methods and/or cells of the present invention, the cells are modified in the expression or activity of at least one, preferably at least two, more preferably all, of the glycosyltransferases. 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 A heterologous protein that is preferably overexpressed in the cell. The endogenous glycosyltransferase can have a modified expression in cells that also express the heterologous glycosyltransferase.

In another embodiment of the method and/or cell of the present invention, at least one, preferably at least both, of the glycosyltransferases 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 . In a preferred embodiment of the method and/or cell of the present invention, the cell is capable of expressing at least one, preferably at least two, fucosyltransferases selected from the group consisting of: α-1,2-fucosyl Glycosyltransferases, α-1,3/1,4-fucosyltransferases and α-1,6-fucosyltransferases. Preferably, the fucosyltransferases are selected from the following organisms: such as, for example, Helicobacter species, such as, for example, Helicobacter pylori, Helicobacter mustelae; Akkermansia species such as eg Akkermansia muciniphila; Bacteroides species such as eg Bacteroides fragilis, Bacteroides vulgatus, ovale Bacteroides ovatus; Escherichia coli species, such as eg Escherichia coli O126, Escherichia coli O55:H7; Lachnospiraceae species; Tannerella species; Clostridium species; Salmonella Salmonella species, such as eg Salmonella enterica, Methanosphaerula palustries; Butyrivibrio species; Prevotella species; Pyrromonas (Porphyromonas) species such as eg Porphyromonas catoniae; Arabidopsis thaliana; Homo sapiens; Mus musculus. In a more preferred embodiment of the method and/or cell of the present invention, the fucosyltransferases are selected from the group consisting of α-1,2-fucosyltransferase and α-1,3/1, List of 4-fucosyltransferases.

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. Throughout this application, unless expressly stated otherwise, features "enhanced" and/or "optimized" for production preferably mean the modifications introduced into cells as described herein and/or Metabolic engineering results in higher yields compared to wild-type precursor cells of the modified cells or metabolically engineered cells. For example, "enhanced GDP-fucose production" preferably means higher intracellular production of GDP-fucose in modified cells compared to wild-type precursor cells that do not contain these specific modifications .

Preferably, the cell in this context comprises a fucosylation pathway as described herein.

In another embodiment of the method and/or cell of the present invention, at least one, preferably at least both, of the glycosyltransferases 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 may be a CMP-sialic acid synthase, such as N-acylneuraminic acid cytidine syltransferase from several species including Homo sapiens, Neisseria meningitidis , and Pasteurella multocida . In a preferred embodiment of the method and/or cell of the present invention, the cell is capable of expressing the enzyme selected from the group consisting of α-2,3-sialyltransferase, α-2,6-sialyltransferase and α-2 , at least one, preferably at least two sialyltransferases of 8-sialyltransferase. Preferably, the sialyltransferases are selected from organisms such as, for example, Pasteurella species, such as, for example, Pasteurella septicemia, Pasteurella dagmatis ; Photobacterium species such as eg Photobacterium damselae, Photobacterium spp. JT-ISH-224, Photobacterium phosphoreum, Photobacterium leiognathi; Pyrromonas Genus species such as eg Pyrromonas catarrhalis; Streptococcus species such as eg Streptococcus suis, Streptococcus agalactiae, Streptococcus entericus; Neisseria meningitidis Bacteria; Campylobacter jejuni; Haemophilus species such as for example Haemophilus somnus, Haemophilus ducreyi, Haemophilus parahaemolyticus, Haemophilus parasuis; Vibrio species; Alistipes species, e.g. Alistipes CAG:268, Alistobacter AL-1, Alistipes (Alistipes shahii), Alistipes timonensis; Actinobacillus species such as eg Actinobacillus suis, Actinobacillus capsulatus; Homo sapiens; Mus musculus. In a more preferred embodiment of the method and/or cell of the present invention, the sialyltransferases are selected from the group consisting of α-2,3-sialyltransferase and α-2,6-sialyltransferase List of enzymes.

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 comprising: deletion of N-acetylglucosamine-6-phosphate deacetylase, deletion of glucosamine-6-phosphate deaminase, salivary Overexpression of the gene encoding acid synthase and overexpression of the gene encoding N-acetyl-D-glucosamine-2-epimerase.

Preferably, the cell in this context comprises a sialylation pathway as described herein.

In another embodiment of the method and/or cell of the present invention, at least one, preferably at least two of the glycosyltransferases 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 cells producing UDP-GlcNAc may express enzymes that convert, for example, GlcNAc added to the cell to UDP-GlcNAc. Such enzymes may be N-acetyl-D-glucosamine kinase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine metathesis from several species including Homo sapiens, Escherichia coli Enzymes and N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase. Alternatively, cells can (preferably metabolically engineered to) express enzymes required for synthesis of GlcNAc, such as glucosamine 6-phosphate N-acetyltransferase, phosphatases, glycosyltransferases, L-gluten Amino acid-D-fructose-6-phosphate aminotransferase and UDP-glucose 4-epimerase, preferably glucosamine 6-phosphate N-acetyltransferase and phosphatase (preferably, HAD-type phosphate enzymes). In a preferred embodiment of the method and/or cell of the present invention, the cell is capable of expressing the enzyme selected from the group consisting of β-1,3-N-acetylglucosaminyltransferase and β-1,6-N-acetylglucosamine At least one, preferably at least two N-acetylglucosaminyltransferases. Preferably, the N-acetylglucosaminyltransferases are selected from the following organisms: such as, for example, Neisseria species, such as, for example, Neisseria meningitidis, Neisseria lactis Neisseria lactamica, Neisseria polysaccharea, Neisseria elongata, Neisseria gonorrhoeae, Neisseria subflava; Pasteurella Species such as eg Pasteurella dacoma; Neorhizobium species such as eg Neorhizobium galegae; Haemophilus species such as eg Haemophilus parainfluenzae, Ducre Haemophilus; Homo sapiens; Mus musculus.

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- Overexpression of phosphoaminotransferase, overexpression of phosphoglucosamine mutase, and overexpression of N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase Performance.

Preferably, the cells in this context comprise the N-acetylglucosamine carbohydrate pathway as described herein.

In another embodiment of the method and/or cell of the present invention, at least one, preferably at least both, of the glycosyltransferases 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. In a preferred embodiment of the method and/or cell of the present invention, the cell is capable of expressing at least one selected from the group consisting of β-1,3-galactosyltransferase and β-1,4-galactosyltransferase, Preferably at least two galactosyltransferases, and/or the cell is capable of expressing at least one selected from the group consisting of α-1,3-galactosyltransferase and α-1,4-galactosyltransferase, preferably at least one Two galactosyltransferases. Preferably, the galactosyltransferases are selected from organisms such as, for example, Escherichia coli species, such as, for example, Escherichia coli O55:H7, Escherichia coli DEC1B, Escherichia coli DEC1D; Neisseria species, such as e.g. Neisseria meningitidis, Neisseria lactis, Neisseria polysaccharide, Neisseria longum, Neisseria gonorrhoeae, Neisseria flavus; Chinella species such as e.g. gold denitrification Kingella denitrificans; Brucella species such as eg Brucella canis, Brucella suis; Salmonella species such as eg Salmonella enterica, ferrous oxide Pseudomonas Pseudogulbenkiana ferrooxidan, Corynebacterium glutamicum; Streptococcus species; Arabidopsis; Homo sapiens; Mus musculus.

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: deletion of a bifunctional 5'-nucleotidase/UDP-glycohydrolase encoding gene, galactose-1-phosphate uridine transfer Knockout of the enzyme encoding gene and overexpression of the UDP-glucose-4-epimerase encoding gene.

Preferably, the cells in this context comprise a galactosylation pathway as described herein.

In another embodiment of the method and/or cell of the present invention, at least one, preferably at least two of the glycosyltransferases 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 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. In a preferred embodiment of the method and/or cell of the present invention, the cell is capable of expressing an enzyme selected from the group consisting of α-1,3-N-acetylgalactosaminyltransferase and β-1,3-N-ethyl At least one, preferably at least two N-acetylgalactosaminyltransferases of acylgalactosaminyltransferases. Preferably, the N-acetylgalactosaminyltransferases are selected from organisms such as, for example, Helicobacter species, such as, for example, Helicobacter weasel; Haemophilus species, such as, for example, influenzae Haemophilus influenzae; Neisseria species, such as, eg, Neisseria meningitidis, Neisseria lactis, Neisseria polysaccharide, Neisseria longum, Neisseria gonorrhoeae, Microxanthella Serratia; Rickettsia species such as, for example, Rickettsia bellii, Rickettsia prowazekii, Rickettsia japonica, Connie Rickettsia conorii, Rickettsia felis, Rickettsia massiliae; Homo sapiens; Mus musculus.

Preferably, the cells are modified to produce UDP-GalNAc. More preferably, the cells are modified for enhanced UDP-GalNAc production. The modification may be any one or more selected from the group comprising: deletion of a bifunctional 5'-nucleotidase/UDP-glycohydrolase encoding gene, galactose-1-phosphate uridine transfer Knockout of the gene encoding the enzyme and overexpression of the gene encoding the UDP-glucose-4-epimerase.

Preferably, the cell in this context comprises an N-acetylgalactosaminylation pathway as described herein.

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

Throughout this application, whenever a protein is disclosed, for example, by reference to SEQ ID NO, i.e., a unique database number (eg, UNIPROT number), or by reference to the particular organism from which it originated, a specific instance of that protein may preferably be referred to by Any, preferably all substitutions of the following specific examples (and therefore the protein is considered to be disclosed according to all of the following specific examples): - proteins (e.g. by reference to SEQ ID NO i.e. unique database number (e.g. UNIPROT number) or by reference to the specific organism of origin), - a functional homologue, variant or derivative of the protein with at least 80% overall sequence identity to the full length of the protein, - a functional fragment of the protein with the same activity, or - Comprising a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity with the full-length amino acid sequence of the protein and having the same activity.

For example, when " H. pylori alpha-1,3- fucosyltransferase with SEQ ID NO: 05 is disclosed"05)", the specific example is preferably replaced by any one, preferably all of the following specific examples: - Helicobacter pylori alpha-1,3-fucosyltransferase with SEQ ID NO: 05, - An alpha-1,3-fucosyltransferase comprising the polypeptide sequence according to SEQ ID NO: 05, - having at least 80% overall sequence identity with the full length of SEQ ID NO: 05 and having an alpha-1,3-peptide a functional homologue, variant or derivative of SEQ ID NO: 05 of fucosyltransferase activity, - a functional fragment of SEQ ID NO: 05 and having alpha-1,3-fucosyltransferase activity, or - comprising a polypeptide comprising or an amino acid sequence having at least 80% sequence identity with the full-length amino acid sequence of SEQ ID NO: 05 and having alpha-1,3-fucosyltransferase activity composition.

In another embodiment of the method and/or cell 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 Base-2,6-dideoxy--L-lyxose-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetyl Amino-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2 -Acetamido-2,6-dideoxy-L-galactose), UDP-N-acetamido-L-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-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-glucuronide, UDP-galactose uronic acid ester, GDP-rhamnose, UDP-xylose. In a preferred embodiment, cells are capable of synthesizing two nucleotide-sugars. In a more preferred embodiment, the cell is capable of synthesizing at least three nucleotide-activating sugars. In an even more preferred embodiment, the cell is capable of synthesizing at least four nucleotide-activating sugars. In a preferred embodiment, the cell is capable of synthesizing at least five nucleotide-activating sugars. In another preferred embodiment, the cell is metabolically engineered for the production of nucleotide-sugars. In another preferred embodiment, cells are modified and/or engineered for optimal production of nucleotide-sugars, ie, enhanced production of nucleotide-sugars as described herein. In a more preferred embodiment, the cell is metabolically engineered for the production of two nucleotide-sugars. In an even more preferred embodiment, the cell is metabolically engineered for the production of three or more nucleotide-activated sugars.

In another embodiment of the method and/or cell of the invention, the cell expresses one or more polypeptides selected from the list comprising: mannose-6-phosphate isomerase, phosphomannose isomerase Otase, mannose-1-phosphate guanosyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, GDP- Fucose pyrophosphorylase, fucose-1-phosphate guanosyltransferase, L-glutamic acid-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase , phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acetylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-tablet Isomerase, N-acetylmannosamine-6-phosphate 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, N-Acetylneuraminic Acid Synthase, N-Acetylneuraminic Acid Lysase, N-Acetylneuraminic Acid-9-Phosphate Synthase , N-acyl neuraminic acid-9-phosphate phosphatase, N-acyl neuraminic acid cytidine syltransferase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1 - Uridine phosphate transferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridine syltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epiiso Constructing enzyme, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase.

In a preferred embodiment of the method and/or cell according to the present invention, a mixture of at least three different mammalian milk oligosaccharides according to the present invention can be used to produce a lactose-based neutral afucosyl by providing a mMMO is produced by cells 1) capable of absorbing lactose from a culture as described herein or capable of producing lactose after absorbing glucose by the action of b-1,4-galactosyltransferase as described herein; and 2) capable of expressing an N-acetylglucosaminyltransferase as described herein, preferably a galactoside β-1,3-N-acetylglucosaminyltransferase; 3) optionally capable of expressing at least One, preferably at least two galactosyltransferases as described herein, the galactosyltransferase(s) selected from the list comprising: N-acetylglucosamine beta-1,3-galactose syltransferase, N-acetylglucosamine beta-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase, alpha-1,4-galactosyltransferase; and 4) Optionally capable of expressing at least one, preferably at least two N-acetylgalactosaminyltransferases as described herein, the N-acetylgalactosaminyltransferase(s) selected from the group comprising alpha-1 ,3-N-Acetylgalactosaminyltransferase and β-1,3-N-Acetylgalactosaminyltransferase list and 5) can synthesize these glycosyltransferases (if present) The nucleotide-sugar of each.

In another and/or additional preferred embodiment of the method and/or cell according to the present invention, a mixture of at least three different mammalian milk oligosaccharides according to the present invention can be used to produce a lactose-based neutral by providing a fucosylated MMO cells 1) capable of absorbing lactose from a culture as described herein or capable of absorbing glucose by the action of b-1,4-galactosyltransferase as described herein and 2) capable of expressing at least one, preferably at least two, fucosyltransferases as described herein, the fucosyltransferase(s) selected from the group consisting of alpha-1,2-fucosyltransferases List of glycosyltransferases, α-1,3-fucosyltransferases, α-1,4-fucosyltransferases and α-1,6-fucosyltransferases; 3) as needed Can express N-acetylglucosaminyltransferase as described herein, preferably galactoside β-1,3-N-acetylglucosaminyltransferase; 4) can express at least one, more Preferably at least two galactosyltransferases as described herein, the galactosyltransferase(s) selected from the group comprising N-acetylglucosamine β-1,3-galactosyltransferase, N-acetylglucosamine List of glucosamine β-1,4-galactosyltransferase, α-1,3-galactosyltransferase, α-1,4-galactosyltransferase; 5) can express at least one, Preferably at least two N-acetylgalactosaminyltransferases as described herein, the N-acetylgalactosaminyltransferase(s) selected from the group comprising α-1,3-N-acetyl List of β-1,3-N-acetylgalactosaminyltransferases and β-1,3-N-acetylgalactosaminyltransferases; 6) Capable of synthesizing GDP-fucose, preferably the cell has a fucoid as defined herein glycosylation pathway, and 7) the nucleotide-sugar capable of synthesizing each of the glycosyltransferases (if present).

In another and/or additional preferred embodiment of the method and/or cell according to the present invention, a mixture of at least three different mammalian milk oligosaccharides according to the present invention can be used to produce lactose-based sialylation by providing a produced by cells that afucosylate MMOs that are 1) capable of taking up lactose from a culture as described herein or capable of taking up lactose by the action of b-1,4-galactosyltransferase as described herein and 2) capable of expressing at least one, preferably at least two, sialyltransferases as described herein, the sialyltransferase(s) being selected from groups comprising an alpha-2,3-sialyltransferase List of transferases, alpha-2,6-sialyltransferases and alpha-2,8-sialyltransferases; 3) Optionally capable of expressing an N-acetylglucosaminyltransferase as described herein , preferably galactoside β-1,3-N-acetylglucosaminyltransferase; 4) optionally capable of expressing at least one, preferably at least two, galactosyltransferases as described herein, the ( etc.) galactosyltransferase is selected from the group comprising N-acetylglucosamine β-1,3-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, α- List of 1,3-galactosyltransferase, α-1,4-galactosyltransferase; 5) optionally capable of expressing at least one, preferably at least two N-acetylgalactose as described herein Aminotransferases selected from the group consisting of α-1,3-N-acetylgalactosaminyltransferase and β-1,3-N-ethyl A list of acylgalactosaminyltransferases; 6) capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein, and 7) capable of synthesizing one of the glycosyltransferases (if present) Nucleotide-sugar of each.

In another and/or additional preferred embodiment of the method and/or cell according to the present invention, a mixture of at least three different mammalian milk oligosaccharides according to the present invention can be used to produce lactose-based sialylation by providing a fucosylated MMO cells 1) capable of absorbing lactose from a culture as described herein or capable of absorbing glucose by the action of b-1,4-galactosyltransferase as described herein and 2) capable of expressing at least one, preferably at least two, fucosyltransferases as described herein, the fucosyltransferase(s) selected from the group consisting of alpha-1,2-fucosyltransferases List of glycosyltransferases, α-1,3-fucosyltransferases, α-1,4-fucosyltransferases and α-1,6-fucosyltransferases; 3) capable of expressing At least one, preferably at least two sialyltransferases as described herein, the sialyltransferase(s) selected from the group consisting of alpha-2,3-sialyltransferase, List of acyltransferases and α-2,8-sialyltransferases; 4) optionally capable of expressing an N-acetylglucosaminyltransferase as described herein, preferably galactoside β-1, 3-N-Acetylglucosaminyltransferase; 5) optionally capable of expressing at least one, preferably at least two, galactosyltransferases as described herein, the (etc.) galactosyltransferases selected from the group consisting of N-acetylglucosamine β-1,3-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, α-1,3-galactosyltransferase, A list of alpha-1,4-galactosyltransferases; 6) optionally capable of expressing at least one, preferably at least two N-acetylgalactosyltransferases as described herein, the(s) N - Acetylgalactosaminyltransferase is selected from a list comprising α-1,3-N-acetylgalactosaminetransferase and β-1,3-N-acetylgalactosaminotransferase 7) capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein, 8) capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein and 9) a nucleotide-sugar capable of synthesizing each of the glycosyltransferases (if present).

In another and/or additional preferred embodiment of the method and/or cell according to the present invention, a mixture of at least three different mammalian milk oligosaccharides according to the present invention can be used for the production of LNB-based neutral by providing a from cells that afucosylate MMOs that are capable of producing LNB as described herein or capable of taking up LNB from culture; and capable of synthesizing UDP-Gal.

In another and/or additional preferred embodiment of the method and/or cell according to the present invention, a mixture of at least three different mammalian milk oligosaccharides according to the present invention can be used for the production of LNB-based neutral by providing a fucosylated MMO cells that are 1) capable of taking up LNB from a culture as described herein or capable of producing LNB as described herein; and 2) capable of expressing at least one, preferably at least two, as described herein The described fucosyltransferase, the (etc.) fucosyltransferase is selected from the group consisting of - List of 1,4-fucosyltransferases and α-1,6-fucosyltransferases; 3) Capable of synthesizing GDP-fucose, preferably the cells have fucose as defined herein sylation pathway, and 4) UDP-galactose can be produced on demand.

In another and/or additional preferred embodiment of the method and/or cell according to the present invention, a mixture of at least three different mammalian milk oligosaccharides according to the present invention can be used to generate LNB-based sialylation by providing a Afucosylated MMOs are produced from cells that are 1) capable of taking up LNB from a culture as described herein or capable of producing LNB as described herein; and 2) capable of expressing at least one, preferably at least two, such as The sialyltransferases described herein, the sialyltransferase(s) selected from the group consisting of α-2,3-sialyltransferase, α-2,6-sialyltransferase and α-2, A list of 8-sialyltransferases; 3) capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein, and 4) optionally capable of producing UDP-galactose.

In another and/or additional preferred embodiment of the method and/or cell according to the present invention, a mixture of at least three different mammalian milk oligosaccharides according to the present invention can be used to generate LNB-based sialylation by providing a fucosylated MMO cells that are 1) capable of taking up LNB from a culture as described herein or capable of producing LNB as described herein; and 2) capable of expressing at least one, preferably at least two, as described herein The described fucosyltransferase, the (etc.) fucosyltransferase is selected from the group consisting of - List of 1,4-fucosyltransferases and α-1,6-fucosyltransferases; 3) Capable of synthesizing GDP-fucose, preferably the cells have fucose as defined herein 4) capable of expressing at least one, preferably at least two sialyltransferases as described herein, the (etc.) sialyltransferases selected from the group comprising α-2,3-sialyltransferases , a list of α-2,6-sialyltransferase and α-2,8-sialyltransferase; 5) capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein; and 6) UDP-galactose can be produced as needed.

In another and/or additional preferred embodiment of the method and/or cell according to the present invention, a mixture of at least three different mammalian milk oligosaccharides according to the present invention can be used for the production of LacNAc-based neutral by providing a cells that afucosylate MMOs that are capable of producing LacNAc as described herein or capable of taking up LacNAc from culture as described herein; and capable of synthesizing UDP-Gal.

In another and/or additional preferred embodiment of the method and/or cell according to the present invention, a mixture of at least three different mammalian milk oligosaccharides according to the present invention can be used for the production of LacNAc-based neutral by providing a fucosylated MMO cells that are 1) capable of taking up LacNAc from a culture as described herein or capable of producing LacNAc as described herein; and 2) capable of expressing at least one, preferably at least two, as described herein The described fucosyltransferase, the (etc.) fucosyltransferase is selected from the group consisting of - List of 1,4-fucosyltransferases and α-1,6-fucosyltransferases; 3) Capable of synthesizing GDP-fucose, preferably the cells have fucose as defined herein sylation pathway, and 4) UDP-galactose can be produced on demand.

In another and/or additional preferred embodiment of the method and/or cell according to the present invention, a mixture of at least three different mammalian milk oligosaccharides according to the present invention can be used to generate LacNAc-based sialylation by providing a Afucosylated MMOs are produced from cells that are 1) capable of taking up LacNAc from a culture as described herein or capable of producing LacNAc as described herein; and 2) capable of expressing at least one, preferably at least two, such as The sialyltransferases described herein, the sialyltransferase(s) selected from the group consisting of α-2,3-sialyltransferase, α-2,6-sialyltransferase and α-2, A list of 8-sialyltransferases; 3) capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein, and 4) optionally capable of producing UDP-galactose.

In another and/or additional preferred embodiment of the method and/or cell according to the present invention, a mixture of at least three different oligosaccharides according to the present invention can be used for the production of LacNAc-based sialylated fucose by providing a oligosaccharides that are 1) capable of taking up LacNAc from a culture as described herein or capable of producing LacNAc as described herein; and 2) capable of expressing at least one, preferably at least two, as described herein The fucosyltransferase, the (etc.) fucosyltransferase is selected from the group consisting of α-1,2-fucosyltransferase, a list of ,4-fucosyltransferases and alpha-1,6-fucosyltransferases; 3) capable of synthesizing GDP-fucose, preferably the cells have fucosylation as defined herein pathway; 4) capable of expressing at least one, preferably at least two sialyltransferases as described herein, the (etc.) sialyltransferases selected from the group consisting of α-2,3-sialyltransferase, α- - a list of 2,6-sialyltransferases and alpha-2,8-sialyltransferases; 5) capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein; and 6 ) can produce UDP-galactose on demand.

In more preferred embodiments of the methods and/or cells according to the invention, lactose-based MMOs comprising neutral fucosylation (such as eg fucosyllactose) and fucosylated LNT and/or LNnT structures A mixture of at least three different MMOs (eg, LNFP-I, LNFP-II, LNFP-III, LNFP-V) and uncharged oligosaccharides can be produced by providing a cell 1) capable of Lactose is absorbed in culture or capable of producing lactose following absorption of glucose by the action of b-1,4-galactosyltransferase as described herein; and 2) capable of exhibiting at least one, preferably at least two, as described herein The fucosyltransferase, the (etc.) fucosyltransferase is selected from the group consisting of α-1,2-fucosyltransferase, a list of ,4-fucosyltransferases and alpha-1,6-fucosyltransferases; 3) capable of synthesizing GDP-fucose, preferably the cells have fucosylation as defined herein pathway; 4) capable of expressing an N-acetylglucosaminyltransferase as described herein, preferably a galactoside β-1,3-N-acetylglucosaminyltransferase; 5) capable of expressing at least one , preferably at least two galactosyltransferases as described herein, the (etc.) galactosyltransferases selected from the group consisting of N-acetylglucosamine β-1,3-galactosyltransferase, N- List of acetylglucosamine β-1,4-galactosyltransferase, α-1,3-galactosyltransferase, α-1,4-galactosyltransferase; 6) capable of synthesizing UDP-GlcNAc , preferably the cell has an N-acetylglucosaminylation pathway as defined herein; 7) is capable of synthesizing UDP-Gal, preferably the cell has a galactosylation pathway as defined herein.

In another more preferred embodiment of the method and/or cell according to the present invention, a charged, lactose-based MMO such as, for example, sialyllactose and sialylated lactose-N-triose and sialylated lactose-N-tetraose is included Mixtures of at least three different MMOs of sugars and/or sialylated lacto-N-neotetrasaccharides) and afucosylated oligosaccharides can be produced by providing a cell 1) capable of culturing from a culture as described herein and 2) capable of exhibiting at least one, preferably at least two, as described herein A sialyltransferase selected from the group consisting of α-2,3-sialyltransferase, α-2,6-sialyltransferase and α-2,8-sialic acid 3) capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein; 4) capable of expressing an N-acetylglucosaminyltransferase as described herein, preferably preferably galactoside β-1,3-N-acetylglucosaminyltransferase; 5) capable of expressing at least one, preferably at least two, galactosyltransferases as described herein, the(s) galactose The syltransferase is selected from the group consisting of N-acetylglucosamine β-1,3-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, α-1,3- List of galactosyltransferases, alpha-1,4-galactosyltransferases; 6) capable of synthesizing UDP-GlcNAc, preferably the cell has an N-acetylglucosaminylation pathway as defined herein; 7 ) is capable of synthesizing UDP-Gal, preferably the cell has a galactosylation pathway as defined herein.

In another more preferred embodiment of the method and/or cell according to the invention, a charged and neutral lactose-based MMO (such as eg fucosyllactose, sialyllactose, LN3, fucosylated LNT) is included and/or a mixture of at least three different MMOs of LNnT, sialylated lacto-N-triose and sialylated lacto-N-tetraose and/or sialylated lacto-N-neotetraose) can be produced by providing a cell , the cell 1) is capable of absorbing lactose from a culture as described herein or capable of producing lactose after absorbing glucose by the action of b-1,4-galactosyltransferase as described herein; and 2) capable of expressing at least One, preferably at least two sialyltransferases as described herein, the sialyltransferase(s) selected from the group consisting of α-2,3-sialyltransferase, α-2,6-sialic acid List of syltransferases and α-2,8-sialyltransferases; 3) capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein; 4) capable of expressing at least one, preferably At least two fucosyltransferases as described herein, the fucosyltransferase(s) selected from the group consisting of α-1,2-fucosyltransferase, α-1,3-fucose List of syltransferases, α-1,4-fucosyltransferases and α-1,6-fucosyltransferases; 5) Capable of synthesizing GDP-fucose, preferably the cells have as described herein Fucosylation pathway as defined; 6) capable of expressing an N-acetylglucosaminyltransferase as described herein, preferably a galactoside β-1,3-N-acetylglucosaminyltransferase 7) Capable of expressing at least one, preferably at least two, galactosyltransferases as described herein, the (etc.) galactosyltransferases selected from the group comprising N-acetylglucosamine β-1,3-galactosyltransferase List of lactosyltransferases, N-acetylglucosamine beta-1,4-galactosyltransferases, alpha-1,3-galactosyltransferases, alpha-1,4-galactosyltransferases; 8) capable of synthesizing UDP-GlcNAc, preferably the cell has an N-acetylglucosaminylation pathway as defined herein, 9) capable of synthesizing UDP-Gal, preferably the cell has a galactosyl as defined herein way of transformation.

Exemplary methods and cells according to the invention are described in the Examples section. It is emphasized that these examples show at least one way of producing a particular mixture. It will be understood by those of ordinary skill in the art that if any of the expressed enzymes have the same catalytic activity, preferably to a similar extent, which activity can be readily assessed by routine experimentation, then the Either can be replaced by another enzyme, wherein the activity of the enzyme is compared to the activity of a reference enzyme as disclosed herein (eg, in vitro conversion of substrates).

In a preferred embodiment of the methods and/or cells of the present invention, any of the MMOs, and more preferably all of the oligosaccharides, are consumed by passive transport, ie without resorting to active transport systems, from The energy of the cell is translocated to the outside of the cell.

In a preferred embodiment of the methods and/or cells of the present invention, the cells use at least one precursor for the production of any one or more of the oligosaccharides. As explained in the definitions disclosed herein, the term "precursor" should be understood. In a more preferred embodiment, the cell uses two or more precursors for the production of any one or more of the oligosaccharides.

In a preferred embodiment of the method of the present invention, the culture is fed with precursors and/or acceptors for the synthesis of any of the oligosaccharides in the mixture. As explained in the definitions disclosed herein, the term "acceptor" should be understood. In another preferred embodiment of the method, the culture is fed with at least two precursors and/or acceptors for use in synthesizing any or more, preferably all, of the oligosaccharides in the mixture. If two or more glycosyltransferases of the same classification are used (eg 2-fucosyltransferases), they have different affinities (eg one fucosyltransferase with affinity for lactose and the other for LacNAc Fucosyltransferases with affinity) are used to produce mixtures of oligosaccharides according to the invention, then this may be applicable.

In another embodiment of the method and/or cell as described herein, the cell produces a precursor for the production of any of the oligosaccharides. In a preferred embodiment, the cell produces one or more precursors for synthesizing the oligosaccharide mixture. In a more preferred embodiment, the cell is modified for optimal production of any of the precursors for the synthesis of any of the oligosaccharides.

In a preferred embodiment of the methods and/or cells of the invention, at least one precursor used to produce any of the oligosaccharides is fully converted to any of the oligosaccharides. In a more preferred embodiment, the cell completely converts any of the precursors to any of the oligosaccharides.

In another preferred embodiment of the method and/or cell of the present invention, the cell is 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) expression of heterologous membrane proteins, wherein the membrane protein is involved in the secretion of any of the MMOs outside the cell. The cell may express one of the membrane proteins involved in the secretion of any of the MMOs 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 MMOs to the outside of the cell. The cell producing a mixture of at least three MMOs can translocate any of the MMOs, the cell comprising passive diffusion, channel membrane proteins, membrane transport proteins, membrane carrier proteins.

In another preferred embodiment of the method and/or cell of the present invention, the cell is 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) expression of heterologous membrane proteins, wherein the membrane protein is involved in the uptake of precursors and/or receptors for the synthesis of any of the MMOs. In one embodiment, the cell expresses one of the membrane proteins involved in the uptake of any type of precursor and/or receptor used to synthesize any of the MMOs. In one embodiment, the cell expresses more than one of the membrane proteins involved in the uptake of at least one of the precursors and/or receptors. In one embodiment, cells are modified to take up more than one precursor and/or receptor for synthesis of any of the MMOs. In a preferred embodiment, cells are modified to take up all desired precursors. In another preferred embodiment, cells are modified for uptake of all receptors.

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

In another preferred embodiment of the methods and/or cells of the invention, the membrane protein provides improved production of any, preferably all, of the oligosaccharides. In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, the membrane protein enables efflux of any, preferably all, of the oligosaccharides. In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, the membrane protein provides enhanced efflux of any, preferably all, of the oligosaccharides.

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

In a preferred embodiment of the method and/or cell 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" shall 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 methods and/or cells of the present invention, the cells impart reduced viscosity. Reduction of 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, core oligosaccharides, osmoregulatory periplasmic glucans, and glycerol The synthesis of glucosylglycerol, polysaccharides and trehalose is reduced or eliminated.

According to another embodiment of the method and/or cell of the present invention, the cell is capable of producing phosphoenolpyruvate (PEP). In a preferred embodiment of the methods and/or cells 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, which takes up sucrose in the cytoplasm and forms sucrose-6-phosphate, 5) fructose-specific PTS transporter (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 the first step and fructose 1,6 diphosphate in the 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) trehalose-specific PTS enzymes, which take up trehalose and form trehalose-6-phosphate.

In another and/or additional preferred embodiment and as a means for enhanced production and/or supply of PEP, the intact 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 (PtsI and PtsH) carbohydrate-nonspecific protein components of PTS carbohydrates that, together with carbohydrate-specific inner membrane permease enzymes, affect the phosphotransfer cascade that results in coupled phosphorylation and transport of various carbohydrate substrates . HPr (histidine-containing protein) is one of two sugar-nonspecific protein components of PTS sugars. It accepts the phosphonium group from phosphorylase I (PtsI-P) and then transfers it to the EIIA domain of any of the many carbohydrate-specific enzymes of the PTS carbohydrate (collectively referred to as Enzyme II). 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 through the introduction and/or overexpression of the corresponding permease. These are, for example, permease or ABC transporters including, but not limited to, transporters that specifically import lactose, such as those encoded by the LacY gene from E. coli; transporters that import sucrose, such as those encoded by the LacY gene from E. coli The transporter protein encoded by the cscB gene; the transporter protein for glucose import, such as the transporter protein encoded by the galP gene from Escherichia coli; the transporter protein for fructose import, such as the transporter protein encoded by the fruI gene from Streptococcus mutans; Or sorbitol/mannitol ABC transporters, such as those encoded by the cluster SmoEFGK of Rhodobacter sphaeroides; trehalose/sucrose/maltose transporters, such as those encoded by the genes of Sinorhizobium meliloti Transport proteins encoded by the cluster ThuEFGK; and N-acetylglucosamine/galactose/glucose transport proteins, such as the transport 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, the 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 are treated 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 illustrative 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 illustrative embodiment, 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 illustrative embodiment, cells are genetically modified by different adaptations such as overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and 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 illustrative 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 illustrative embodiment, 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 illustrative 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 illustrative 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 methods and/or cells of the present invention, the cells comprise modifications for reducing acetate production as compared to unmodified precursor cells. The modification may be any one or more selected from the group comprising: overexpression, complete or partial deletion, or less functional pyruvate dehydrogenase, and complete or partial deletion of acetyl-CoA synthase Or show less functional lactate dehydrogenase.

In another embodiment of the method and/or cell of the invention, the cell is modified in the expression or activity of at least one acetyl-CoA synthetase, such as for example from Escherichia coli, Saccharomyces cerevisiae, The acs of man and 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 a heterologous protein that is heterogeneously introduced and expressed in the cell, preferably 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 is modified in the expression or activity of a functional homologue, variant or derivative of acs from E. coli (UniProt ID P27550), the functional homologue, The variant or derivative has at least 80% overall sequence identity to the full length of the polypeptide from E. coli (UniProt ID P27550) and has acetyl-CoA synthase activity.

In alternative and/or additional embodiments of the methods and/or cells of the present invention, the cells are 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 Modified. In a preferred embodiment, the cell has been modified to have at least one partially or completely deleted or mutated pyruvate dehydrogenase-encoding gene in a manner commonly known to those of ordinary skill in the art to produce at least one less functional Or a protein that disables pyruvate 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 embodiments of the methods and/or cells of the present 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. retouch. In a preferred embodiment, the cell has been modified to have at least one partially or completely deleted or mutated lactate dehydrogenase-encoding gene in a manner commonly known to those of ordinary skill in the art to produce at least one functionally lesser or A protein incapacitated by lactate dehydrogenase activity. In a more preferred embodiment, the cells have a complete deletion of the gene encoding ldhA, resulting in cells lacking lactate dehydrogenase activity.

According to another preferred embodiment of the method and/or cell of the present invention, the cell comprises a reduced or reduced expression and/or abrogated, attenuated, reduced or delayed activity comprising one of the following as compared to an unmodified precursor cell Any one or more of these proteins: beta-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6 - Phosphate deaminase, N-acetylglucosamine inhibitor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecopentenyl-phosphoglucosamine-1-phosphotransferase, L- Fucokinase, 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 transferase, glucose-1-phosphate adenylyltransferase (adenyyltransferase), glucose-1 - Phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IclR, lon protease, glucose-specific translocation phosphotransferase IIBC component ptsG, glucose-specific translocation phosphotransferase (PTS) IIBC component malX, enzyme IIA ^Glc , β-glucoside specific PTS enzyme II, fructose specific Sexual PTS polyphosphoryl transfer proteins FruA and FruB, alcohol dehydrogenase, aldehyde dehydrogenase, pyruvate-formate dissociation enzyme, acetate kinase, phosphoacyltransferase (phosphoacyltransferase), phosphoacetyltransferase, acetone acid decarboxylase.

According to another preferred embodiment of the method and/or cell of the present invention, the cell comprises a catabolic pathway for the selected monosaccharide, disaccharide or oligosaccharide, the catabolic pathway is at least partially inactivated, the monosaccharide, disaccharide or oligosaccharide The saccharides or oligosaccharides participate in and/or are required for the production of the oligosaccharides from the mixture.

Another embodiment of the present invention provides a method and a cell wherein a mixture comprising at least three different MMOs is in a fungal, yeast, bacterial, insect, animal, plant and protozoal cell and/or as described herein produced by it. The cell line is selected from a list comprising bacteria, yeast, protozoa or fungi, or refers to plant or animal cells. The latter bacteria preferably belong to the phylum Proteobacteria or Firmicutes or Cyanobacteria or Deinococcus-Thermus. Bacteria belonging to the later phylum Proteobacteria preferably belong to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacteria preferably refer to any strain belonging to the species Escherichia coli, such as, but not limited to, Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term refers to cultured E. coli strains designated as E. coli K12 strains - which are well adapted to the laboratory environment and which, unlike wild-type strains, have lost their presence in the intestinal tract. the ability to grow. Well-known examples of E. coli K12 strains are K12 wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Thus, the present invention 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. Bacteria belonging to the later Firmicutes phylum preferably belong to the Bacilli class, preferably the Lactobacilliales with members such as Lactobacillus lactis , Leuconostoc mesenteroides , or There are members such as Bacillales from the genus Bacillus, such as Bacillus subtilis or B. amyloliquefaciens . Bacteria belonging to the rear of the Actinobacteria phylum preferably belong to the Corynebacteriaceae family with members Corynebacterium glutamicum or C. afermentans , or belong to the family Streptomyces griseus ( Streptomyces griseus ) or the Streptomycetaceae family of S. fradiae . The latter yeast preferably belong to the phylum Ascomycota or Basidiomycota or Deuteromycota or Zygomycetes. The latter yeasts preferably belong 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 (eg, Yarrowia lipolytica) or Starmerella (eg, Starmerella bombicola ) )). The latter yeast is preferably selected from Pichia pastoris , Yarrowia lipolytica , Saccharomyces cerevisiae and Kluyveromyces lactis . The latter fungi preferably belong to the genera 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 cells are preferably Leishmania tarentolae cells.

In a preferred embodiment of the method and/or cell of the invention, the cell is a viable Gram-negative bacterium comprising reduced or eliminated synthetic poly-N- Acetyl-glucosamine (poly-N-acetyl-glucosamine; PNAG), Enterobacterial Common Antigen (ECA), cellulose, kolaric acid, core oligosaccharide, osmoregulatory periplasmic glucan ( Osmoregulated Periplasmic Glucan; OPG), glycerol glucosides, glycans and/or trehalose.

In a more preferred embodiment of the method and/or cell, the reduction or elimination of synthetic poly-N-acetyl-glucosamine (PNAG), enterobacterial common antigen (ECA), cellulose, kolac acid, Core oligosaccharides, osmoregulatory periplasmic glucans (OPG), glycerol glucosides, glycans and/or trehalose are produced by participating in the poly-N-acetyl-glucosamine (PNAG), enterobacterial common antigen Synthesis of any of (ECA), cellulose, kolac acid, core oligosaccharides, osmoregulatory periplasmic glucans (OPGs), glycerol glucosides, polysaccharides, and/or trehalose Mutations of glycosyltransferases provide for deletion or lower performance 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) sugar Syltransferase, 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-galactosylfuranosyl transferase Enzyme, Undecyl isopentenyl-phosphate 4-deoxy-4-carboxamido-L-arabinosyltransferase, Lipid IVA 4-amino-4-deoxy-L-arabinosyltransferase, Bacterial Terpene alcohol glucosyltransferase, putative family 2 glycosyltransferase, osmoregulatory periplasmic glucan (OPG) biosynthesis protein G, OPG biosynthesis protein H, glucosylglycerate phosphorylase, hepatic glucose synthase , 1,4-α-glucan branching enzyme, 4-α-glucan transferase and trehalose-6-phosphate synthase. In an illustrative 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 alternative and/or additional preferred embodiments of the method and/or cell, the reduction or elimination of poly-N-acetyl-glucosamine (PNAG) synthesis by overexpression of genes encoding carbon storage regulators, Na+ Deletion of the gene encoding the /H+ antiporter 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 for the organism, ie 100% of all required carbon is derived from the carbon sources indicated 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, trehalose, 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 production pathways and biomass pathways 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 another embodiment of the method of the present invention, the conditions allowing the production of the oligosaccharides in the mixture comprise culturing the cells of the present invention with a medium to produce the oligosaccharide mixture, wherein the medium is deficient in the production of the oligosaccharides any precursor and/or acceptor of any one, and in combination with the further addition of at least one precursor and/or acceptor feed to the culture medium for the production of any of the oligosaccharides, compared to It is preferably used to generate all of the oligosaccharides in the mixture.

In a preferred embodiment, the method for producing an oligosaccharide mixture as described herein comprises at least one of the following steps: i) using a culture medium comprising at least one precursor and/or acceptor; ii) reacting Add at least one precursor and/or acceptor feed to the medium in a reactor with a total reactor volume in the range of 250 mL (milliliters) to 10.000 ^m3 (cubic meters), preferably in a continuous manner, and more Preferably, the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium before adding the precursor and/or acceptor feed; iii) in the reactor Add at least one precursor and/or acceptor feed to the medium, wherein the total reactor volume is in the range of 250 mL (milliliters) to 10.000 m ³ (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium before addition of the precursor and/or acceptor feed, and wherein preferably, The pH of the precursor and/or acceptor feed is set between 3 and 7, and wherein preferably the temperature of the precursor and/or acceptor feed is maintained between 20°C and 80°C; iv) Add at least one precursor and/or acceptor feed to the medium in a continuous manner via the feed solution over a time course of 1 day, 2 days, 3 days, 4 days, 5 days; v) over a period of 1 day, 2 days , 3 days, 4 days, 5 days of this time course by adding at least one precursor and/or acceptor feed to the medium in a continuous manner by means of a feed solution, and wherein preferably the pH of the feed solution is set between 3 and 7, and wherein preferably, the temperature of the feed solution is maintained between 20°C and 80°C; the method produces a concentration in the final culture of at least 50 g/L, preferably at least 75 g/L L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L any of these oligosaccharides.

In another and/or additional preferred embodiment, the method for producing an oligosaccharide mixture as described herein comprises at least one of the following steps: i) using a method comprising at least 50, more preferably per liter of initial reactor volume At least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose culture medium, wherein the reactor volume is in the range of 250 mL to 10.000 ^m3 (cubic meters); ii) in one pulse or in At least one precursor and/or acceptor is added to the medium in a discontinuous (pulsed) manner, wherein the total reactor volume is in the range of 250 mL (milliliters) to 10.000 ^m3 (cubic meters), preferably such that the medium is The final volume does not exceed three times, preferably not more than twice, more preferably less than twice the volume of the medium before adding the precursor and/or acceptor feed pulse; iii) in one pulse or in discontinuous ( Add at least one precursor and/or acceptor feed to the culture medium in a pulsed mode, wherein the total reactor volume is in the range of 250 mL (milliliters) to 10.000 m ³ (cubic meters), preferably such that the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium before the addition of the precursor and/or acceptor feed pulse, and wherein preferably , the pH of the precursor and/or acceptor feed pulse is set between 3 and 7, and wherein preferably the temperature of the precursor and/or acceptor feed pulse is maintained between 20°C and 80°C ; iv) over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by feeding the solution Add at least one precursor and/or acceptor feed to the medium in a discontinuous (pulsed) manner; v) over 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hourly, 1 day, 2 days, 3 days, 4 days, 5 days time course to add at least one precursor and/or acceptor feed to the medium in a discontinuous (pulsed) manner by means of the feed solution, and wherein Preferably, the pH of the feed solution is set between 3 and 7, and wherein preferably, the temperature of the feed solution is maintained between 20°C and 80°C; the method yields in the final culture a concentration of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g /L, more preferably at least 200 g/L of any of these oligosaccharides.

In another more preferred embodiment, the method for producing an oligosaccharide mixture as described herein comprises at least one of the following steps: i) using a method comprising at least 50, more preferably at least 75, more per liter of initial reactor volume preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose medium, wherein the reactor volume is in the range of 250 mL to 10.000 m ³ (cubic meters); ii) adding to the medium a concentration of initial reaction per liter Lactose feed of at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose, wherein the total reactor volume is between 250 mL (milliliter) and 10.000 ^m3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium is no more than three times, preferably no more than twice, more preferably less than the volume of the medium before the addition of the lactose feed 2 times; iii) adding to the medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor The volume is in the range of 250 mL to 10.000 ^m3 (cubic meters), preferably added in a continuous manner, and preferably such that the final volume of the medium does not exceed three times the volume of the medium before the addition of the lactose feed , preferably not more than twice, more preferably less than 2 times, and wherein preferably the pH of the lactose feed is set between 3 and 7, and wherein preferably the temperature of the lactose feed is maintained at 20°C between 80°C and 80°C; iv) over the course of 1 day, 2 days, 3 days, 4 days, 5 days, the lactose feed was added to the medium by the feed solution in a continuous manner; v) over the course of 1 day, 2 days , 3 days, 4 days, 5 days of this time course by feeding solution to add lactose feed to the medium in a continuous manner, and wherein the concentration of the lactose feed solution is 50 g/L, preferably 75 g/L , better 100 g/L, better 125 g/L, better 150 g/L, better 175 g/L, better 200 g/L, better 225 g/L, better 250 g/L, Better 275 g/L, Better 300 g/L, Better 325 g/L, Better 350 g/L, Better 375 g/L, Better 400 g/L, Better 450 g/L, Better preferably 500 g/L, even better 550 g/L, optimally 600 g/L; and wherein preferably the pH of the feed solution is set between 3 and 7, and wherein preferably the feed The temperature of the solution is maintained between 20°C and 80°C; the method produces a concentration in the final culture of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L L, better at least 125 g/L, better at least 150 g/L, better at least 175 g/L, more preferably at least 200 g/L of any of these oligosaccharides.

Preferably, the lactose is fed by starting the culture at a concentration of at least 5 mM, preferably at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably at > This was achieved by adding lactose at a concentration of 300 mM.

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

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

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

Preferably, when carrying out the methods as described herein, the first stage of exponential cell growth is carried out by adding a carbon source, preferably lactose, to the culture prior to adding the precursor, preferably lactose, to the culture in the second stage. Glucose or sucrose was added to the medium to provide.

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

In another preferred embodiment of the method of the present invention, the first stage of exponential cell growth is carried out by adding a carbon-based substrate, preferably glucose or sucrose, to the medium comprising the precursor, preferably lactose Provided is followed by a second stage in which a carbon-based substrate, preferably glucose or sucrose, and a precursor, preferably lactose, are added to the culture.

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

In another preferred embodiment of the method, the culture medium contains at least one precursor selected from the group consisting of lactose, galactose, fucose, sialic acid, GlcNAc, GalNAc, lacto-N-disaccharide (LNB) ) and N-acetyllactosamine (LacNAc).

According to the present invention, the method as described herein preferably comprises the step of isolating any one or more of the oligosaccharides, preferably all of the oligosaccharides from the culture.

The term "separating from said cultivation" means harvesting, collecting or extracting any, 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 mammalian milk oligosaccharide(s) from the MMO mixture. Further purification of the MMO(s) can be for example by using (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange to remove any residual DNA, protein, LPS, endotoxin or other impurities to fulfill. 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 resulting 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, tumble drying, vacuum tumble drying or vacuum tumble drying.

In an illustrative embodiment, the isolation and purification of at least one, and preferably all, of the produced MMOs is performed in a process comprising the steps of the following, in any order: a) Contact the culture or its clarified form with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da, ensuring retention of the MMO produced and allowing 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 necessary to remove excess electrolyte, c) and collecting the MMO-enriched retentate(s) in salt form from the cations of the electrolyte.

In an alternative illustrative embodiment, the isolation and purification of at least one, preferably all, of the produced MMOs is performed in a process comprising the steps, in any order, of: The clarified form is subjected to two membrane filtration steps, where - a membrane having a molecular weight cut-off between about 300 and about 500 Daltons, and - Another membrane with a molecular weight cut-off between about 600 and about 800 Daltons.

In an alternative illustrative embodiment, the isolation and purification of at least one, preferably all, of the produced MMOs is performed in a process comprising the following steps in any order, the steps comprising using as H+ A step of treating the culture or its clarified form with a strong cation exchange resin in the form and a weak anion exchange resin in the free base form.

In an alternative illustrative embodiment, isolation and purification of at least one, preferably all, of the resulting oligosaccharides is performed in the following manner. The culture containing the produced oligosaccharides, biomass, media components and contaminants was subjected to the following purification steps: i) isolating 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, There is provided a purified solution comprising the produced MMO with a purity 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 Belt drying, vacuum belt drying, roller drying, roller drying, vacuum roller drying and vacuum roller drying.

In an alternative illustrative embodiment, the isolation and purification of at least one, preferably all of the produced MMOs is performed in a process comprising the steps of, in any order: enzymatically treating the culture; The cultures are removed from biomass; ultrafiltration; nanofiltration; and subjected to 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 resulting MMO or MMO mixture, which is dried to powder by any one or more drying steps selected from the list comprising: spray drying, lyophilization, spray freeze drying, Freeze spray drying, strip drying, belt drying, vacuum strip drying, vacuum belt drying, drum drying, roller drying, vacuum drum 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 third aspect, the present invention provides the use of a metabolically engineered cell as described herein for producing a mixture comprising at least three different mammalian milk oligosaccharides.

To identify oligosaccharides in mixtures comprising at least three different MMOs produced in cells as described herein, monomer building blocks (e.g. monosaccharide or glycan unit constituents), mutator configurations of side chains, The presence and location 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 spectrometry method (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 ( Electrospray ionization-mass spectrometry; ESI-MS), high performance liquid chromatography (HPLC) with UV or refractive index detection, high performance anion exchange chromatography with pulsed amperometric detection (High-Performance Anion-Exchange chromatography 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 includes 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, dietary supplements provide a variety of beneficial bacteria, 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 prebiotic components of dietary supplements include other prebiotic 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 gastrointestinal microbiota 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, 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 mixtures produced and/or purified by the process in this specification are included in infant formulas 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 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 typically 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 a list comprising pet food, animal milk replacer, veterinary product, post-weaning feed, or creep feed.

Each specific example 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 specific instances, instances 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 pertains to the following specific embodiments: 1. A metabolically engineered cell that produces a mixture of at least three different mammalian milk oligosaccharides, wherein the cell - Expresses at least two glycosyltransferases, and - Ability to synthesize one or more nucleotide-sugars, wherein the one or more nucleotide-sugars are one or more donors for the glycosyltransferase. 2. The cell of embodiment 1, wherein the cell is modified with a gene expression module, characterized in that the expression from any of the expression modules is persistent or produced by a natural inducer. 3. The cell of any one of embodiments 1 and 2, wherein the cell produces a mixture of charged and neutral mammalian milk oligosaccharides. 4. The cell of any one of embodiments 1 to 3, wherein the oligosaccharide mixture comprises at least three different oligosaccharides that differ in degree of polymerization. 5. The cell of any one of specific examples 1 to 4, wherein the cell produces four or more different mammalian milk oligosaccharides. 6. The cell of any one of specific examples 1 to 5, wherein any one of the glycosyltransferases is selected from the list comprising: fucosyltransferase, sialyltransferase, galactose Glycosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, N-acetylmannosyltransferase, Xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosaminyltransferase, N-hydroxyacetylneuraminosyltransferase, rhamnosyltransferase. 7. The cell of any one of embodiments 1 to 6, wherein the cell is modified in the expression or activity of at least one of the glycosyltransferases. 8. The cell of any one of embodiments 1 to 7, wherein one of the glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP- Fucose (GDP-Fuc). 9. The cell of any one of embodiments 1 to 8, wherein one of the glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-N - Acetyl neuraminic acid (CMP-Neu5Ac). 10. The cell of any one of embodiments 1 to 9, wherein one of these glycosyltransferases is N-acetylglucosaminyltransferase and one of these donor nucleotide-sugars The one is UDP-N-acetylglucosamine (UDP-GlcNAc). 11. The cell of any one of specific examples 1 to 10, wherein one of the glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is a UDP-half Lactose (UDP-Gal). 12. The cell of any one of specific examples 1 to 11, wherein any one of these nucleotide-sugars 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), CMP-N-glycolyl neuraminic acid (CMP-Neu5Gc), UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose. 13. The cell of any one of embodiments 1 to 12, wherein at least one of these mammalian milk oligosaccharides is fucosylated, sialylated, galactosylated, glycosylated, xylosylated , Mannosylated; with N-acetylglucosamine, with N-acetylneuraminic acid, with N-hydroxyacetylneuraminic acid, with N-acetylgalactosamine, with rhamnose , containing glucuronic acid ester, containing galacturonic acid ester and/or containing N-acetylmannosamine. 14. The cell of any one of embodiments 1 to 13, wherein the mammalian milk oligosaccharide mixture comprises at least one fucosylated mammalian milk oligosaccharide. 15. The cell of any one of embodiments 1 to 14, wherein the mammalian milk oligosaccharide mixture comprises at least one sialylated mammalian milk oligosaccharide. 16. The cell of any one of embodiments 1 to 15, wherein the mammalian milk oligosaccharide mixture comprises at least one mammalian milk oligosaccharide comprising N-acetylglucosamine monosaccharide units. 17. The cell of any one of embodiments 1 to 16, wherein the mammalian milk oligosaccharide mixture comprises at least one galactosylated mammalian milk oligosaccharide. 18. The cell of any one of specific examples 1 to 17, wherein the cell is further genetically modified for the following 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) expression of heterologous membrane proteins, wherein the membrane protein is involved in the secretion of any of the mammalian milk oligosaccharides from the mixture to the outside of the cell. 19. The cell of any one of specific examples 1 to 18, wherein the cell is further genetically modified for the following 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) expression of heterologous membrane proteins, wherein the membrane protein is involved in the uptake of precursors for the synthesis of any of the mammalian milk oligosaccharides. 20. The cell of any one of embodiments 1 to 19, wherein the cell produces a precursor for synthesizing any one of the mammalian milk oligosaccharides. 21. The cell of any one of embodiments 1 to 20, wherein any one of the mammalian milk oligosaccharides is a human milk oligosaccharide, preferably all of the oligosaccharides are human milk oligosaccharides. 22. A method of producing a mixture of at least three different mammalian milk oligosaccharides by cells, the method comprising the steps of: a. providing cells that express at least two glycosyltransferases and are capable of synthesizing one or more nucleotide-sugars, wherein the one or more nucleotide-sugars are used for one or more of the glycosyltransferases donor, and b. culturing the cell under conditions that allow expression of the glycosyltransferases and synthesis of the one or more nucleotide-sugars, c. Preferably, at least one of the mammalian milk oligosaccharides is isolated from the culture. 23. The method of embodiment 22, wherein the cell is a metabolically engineered cell of any one of embodiments 1-21. 24. The method of any one of embodiments 22 and 23, wherein the cell produces a mixture of charged and neutral mammalian milk oligosaccharides. 25. The method of any one of embodiments 22 to 24, wherein the oligosaccharide mixture comprises at least three different oligosaccharides that differ in degree of polymerization. 26. The method of any one of embodiments 22 to 25, wherein the cell produces four or more different mammalian milk oligosaccharides. 27. The method of any one of embodiments 22 to 26, wherein any one of the glycosyltransferases is selected from the list comprising: fucosyltransferase, sialyltransferase, half Lactosyltransferase, Glucosyltransferase, Mannosyltransferase, N-Acetylglucosaminyltransferase, N-Acetylgalactosaminyltransferase, N-Acetylmannosyltransferase , Xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosaminotransferase, N-hydroxyacetyl neuroaminotransferase, rhamnosyltransferase. 28. The method of any one of embodiments 22 to 27, wherein one of the glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP- Fucose (GDP-Fuc). 29. The method of any one of embodiments 22 to 28, wherein one of the glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-N - Acetyl neuraminic acid (CMP-Neu5Ac). 30. The method of any one of embodiments 22 to 29, wherein one of these glycosyltransferases is N-acetylglucosaminyltransferase and one of these donor nucleotide-sugars The one is UDP-N-acetylglucosamine (UDP-GlcNAc). 31. The method of any one of embodiments 22 to 30, wherein one of the glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is a UDP-half Lactose (UDP-Gal). 32. The method of any one of specific examples 22 to 31, wherein any one of these nucleotide-sugars 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), CMP-N-glycolyl neuraminic acid (CMP-Neu5Gc), UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose. 33. The method of any one of embodiments 22 to 32, wherein the mammalian milk oligosaccharide mixture comprises at least one fucosylated mammalian milk oligosaccharide. 34. The method of any one of embodiments 22 to 33, wherein the mammalian milk oligosaccharide mixture comprises at least one sialylated mammalian milk oligosaccharide. 35. The method of any one of embodiments 22 to 34, wherein the mammalian milk oligosaccharide mixture comprises at least one mammalian milk oligosaccharide comprising N-acetylglucosamine monosaccharide units. 36. The method of any one of embodiments 22 to 35, wherein the mammalian milk oligosaccharide mixture comprises at least one galactosylated mammalian milk oligosaccharide. 37. The method of any one of embodiments 22 to 36, wherein the mammalian milk oligosaccharide mixture comprises at least one mammalian milk oligosaccharide, the at least one mammalian milk oligosaccharide being fucosylated, sialylated, Galactosylation, glucosylation, xylosylation, mannosylation; with N-acetylglucosamine, with N-acetylneuraminic acid, with N-hydroxyacetylneuraminic acid, with N-Acetylgalactosamine, containing rhamnose, containing glucuronic acid ester, containing galacturonic acid ester, and/or containing N-acetylmannosamine. 38. The method of any one of specific examples 22 to 37, wherein the cell uses at least one precursor for synthesizing any one or more of these oligosaccharides, preferably, the cell uses two or more. A variety of precursors for the synthesis of any one or more of these oligosaccharides. 39. The method of any one of embodiments 22 to 38, wherein the cell produces a precursor for synthesizing any of the mammalian milk oligosaccharides. 40. The method of any one of specific examples 22 to 39, wherein the precursor for synthesizing any one of these mammalian milk oligosaccharides is completely converted into any one of these mammalian milk oligosaccharides By. 41. The method of any one of specific examples 22 to 40, wherein any one of these mammalian milk oligosaccharides is a human milk oligosaccharide, preferably, all of these mammalian milk oligosaccharides are human milk oligosaccharides sugar. 42. The method of any one of specific examples 22 to 41, 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. 43. The method of any one of embodiments 22 to 42, further comprising purifying any of the mammalian milk oligosaccharides from the cell. 44. The method of any one of embodiments 22 to 43, 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. 45. The cell of any one of specific examples 1 to 21 or the method of any one of specific examples 22 to 44, 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 animals are insects, fish, birds or non-human mammals, preferably the animal cells are mammalian cells strains. 46. The cell of any one of embodiments 1 to 21 and 45 or the method of any one of embodiments 22 to 45, wherein the cell is a bacterium, preferably a strain of E. coli, more preferably a strain of K-12 Cells of an E. coli strain, even more preferably the E. coli K-12 strain is E. coli MG1655. 47. The cell of any one of embodiments 1 to 21 and 45 or the method of any one of embodiments 22 to 45, wherein the cell is a yeast cell. 48. Use of a cell as in any one of Embodiments 1 to 21, 45 to 47 or a method as in any one of Embodiments 22 to 47 for producing a mixture of at least three different mammalian milk oligosaccharides.

Furthermore, the present invention relates to the following preferred specific embodiments: 1. A metabolically engineered cell producing a mixture of at least three different mammalian milk oligosaccharides, wherein the cell-metabolically engineered for producing The mixture, and - expresses at least two glycosyltransferases, and - is capable of synthesizing one or more nucleotide-sugars, wherein the one or more nucleotide-sugars are one or more donors for the glycosyltransferase body. 2. The cell of preferred embodiment 1, wherein the cell is capable of expressing, preferably at least five glycosyltransferases, preferably at least six, more preferably at least seven glycosyltransferases. 3. The cell of any one of preferred embodiments 1 or 2, wherein the mixture comprises, consists of or consists essentially of neutral fucosylated and non-fucosylated oligosaccharides and is uncharged Oligosaccharides. 4. The cell of any one of preferred embodiments 1 or 2, wherein the mixture comprises, consists of or consists essentially of neutral fucosylated oligosaccharides and is free of neutral afucosylated oligosaccharides or charged oligosaccharides. 5. The cell of any one of preferred embodiments 1 or 2, wherein the mixture comprises, consists of or consists essentially of neutral afucosylated oligosaccharides and is free of neutral fucosylated oligosaccharides or charged oligosaccharides. 6. The cell of any one of preferred embodiments 1 or 2, wherein the mixture comprises, consists of, or consists essentially of charged and neutral fucosylated and afucosylated oligosaccharides , preferably, at least one of the charged oligosaccharides is a sialylated oligosaccharide. 7. The cell of any one of preferred embodiments 1 or 2, wherein the mixture comprises, consists of, or consists essentially of and is free of neutral oligosaccharides, preferably, these charged oligosaccharides At least one of them is a sialylated oligosaccharide. 8. The cell of any one of preferred embodiments 1 to 7, wherein the cell is modified by a gene expression module, characterized in that the expression from any of these expression modules is persistent or by Produced by natural inducers. 9. The cell of any one of specific embodiments 1 to 8, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein. 10. The cell of any one of preferred embodiments 1 to 9, wherein the oligosaccharide mixture comprises at least three different oligosaccharides that differ in degree of polymerization. 11. The cell of any one of preferred embodiments 1 to 10, wherein the cell produces four or more different mammalian milk oligosaccharides. 12. The cell of any one of preferred embodiments 1 to 11, wherein any one of the glycosyltransferases is selected from the list comprising: fucosyltransferase, sialyltransferase, Galactosyltransferase, Glucosyltransferase, Mannosyltransferase, N-Acetylglucosaminyltransferase, N-Acetylgalactosaminyltransferase, N-Acetylmannosyltransferase Enzymes, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glycosaminotransferases, N-hydroxyacetylneuramidotransferases, rhamnosyltransferases, N- Acetyl rhamnosyltransferase, 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: alpha-1,2-fucosyl Glycosyltransferases, α-1,3-fucosyltransferases, α-1,4-fucosyltransferases and α-1,6-fucosyltransferases, - preferably, the 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-glucose Syltransferase and β-1,4-glucosyltransferase, - preferably, the mannosyltransferase is selected from the list comprising: α-1,2-mannosyltransferase, α-1 ,3-mannosyltransferase and alpha-1,6-mannosyltransferase, - preferably, the N-acetylglucosaminyltransferase is selected from the list comprising: galactoside beta -1,3-N-acetylglucosaminyltransferase and β-1,6-N-acetylglucosaminyltransferase, - preferably, the N-acetylgalactosaminyltransferase is selected from a list comprising alpha-1,3-N-acetylgalactosaminyltransferase and beta-1,3-N-acetylgalactosaminyltransferase. 13. The cell of any one of preferred embodiments 1 to 12, wherein the cell is modified in the expression or activity of at least one of the glycosyltransferases. 14. The cell of any one of preferred embodiments 1 to 13, wherein one of the glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-Fucose (GDP-Fuc). 15. The cell of any one of preferred embodiments 1 to 14, wherein one of the glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP -N-Acetyl neuraminic acid (CMP-Neu5Ac). 16. The cell of any one of preferred embodiments 1 to 15, wherein one of these glycosyltransferases is N-acetylglucosaminyltransferase and one of these donor nucleotide-sugars is One of them is UDP-N-acetylglucosamine (UDP-GlcNAc). 17. The cell of any one of preferred embodiments 1 to 16, wherein one of these glycosyltransferases is galactosyltransferase and one of these donor nucleotide-sugars is UDP -Galactose (UDP-Gal). 18. The cell of any one of the preferred embodiments 1 to 17, wherein the glycosyltransferase is N-acetylgalactosaminotransferase and the donor nucleotide-sugar is UDP-N-ethyl Acylgalactosamine (UDP-GalNAc). 19. The cell of any one of preferred embodiments 1 to 18, wherein the glycosyltransferase is N-acetylmannosylaminotransferase and the donor nucleotide-sugar is UDP-N-ethyl Acylmannosamine (UDP-ManNAc). 20. The cell of any one of preferred embodiments 1 to 19, wherein any one of these nucleotide-sugars 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), CMP-N-hydroxyacetyl neuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N ₃ , CMP-Neu4,5Ac ₂ , CMP-Neu5,7Ac ₂ , CMP-Neu5, 9Ac ₂ , CMP-Neu5,7(8,9)Ac ₂ , UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose, UDP-2-acetamido -2,6-dideoxy--L-arabino (arabino)-4-hexulose, UDP-2-acetamido-2,6-dideoxy--L-lyxose (lyxo) -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-neumosamine (UDP-L-PneNAC or UDP-2-acetylamino-2,6-dideoxy-L-talose) ), UDP-N-acetylmuramic acid, UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnosamine (quinovosamine) (UDP-L-QuiNAc or UDP-2-acetylamino-2,6 -dideoxy-L-glucose), GDP-L-isorhamnose. 21. The cell of any one of preferred embodiments 1 to 20, wherein the cell expresses one or more polypeptides selected from a list comprising: mannose-6-phosphate isomerase, phosphate Mannose mutase, mannose-1-phosphate guanosyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase , GDP-fucose pyrophosphorylase, fucose-1-phosphate guanosyltransferase, L-glutamic acid-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate Deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acetylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-Epimerase, N-Acetylmannosamine-6-Phosphate 2-Epimerase, Glucosamine 6-Phosphate N-Acetyltransferase, N-Acetylglucosamine-6-Phosphate Phosphatase, N-acetylmannosamine-6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate urine Glycosyltransferase, glucosamine-1-phosphate acetyltransferase, N-acetylneuraminic acid synthase, N-acetylneuraminic acid lyase, N-acetylneuraminic acid-9- Phosphate synthase, N-acyl neuraminic acid-9-phosphate phosphatase, N-acyl neuraminic acid cytidine syltransferase, galactose-1-epimerase, galactokinase, glucokinase, galactokinase Lactose-1-phosphate uridine transferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridine transferase, phosphoglucomutase, UDP-N-acetylglucosamine 4 - epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein the cell expresses any of these polypeptides or Modified in terms of activity. 22. The cell of any one of the preferred embodiments 1 to 21, wherein the cell is capable of synthesizing at least two nucleotide-sugars, preferably at least three nucleotide-sugars, more preferably at least four nucleotide-sugars. sugar, even better at least five nucleotide-sugars. 23. The cell of any one of preferred embodiments 1 to 22, wherein at least one of these mammalian milk oligosaccharides is fucosylated, sialylated, galactosylated, glucosylated, xylose N-acetylglucosamine, N-acetylneuraminic acid, N-hydroxyacetylneuraminic acid, N-acetylgalactosamine, murine Lisose, containing glucuronide, containing galacturonate and/or containing N-acetylmannosamine. 24. The cell of any one of preferred embodiments 1 to 23, wherein the mammalian milk oligosaccharide mixture comprises at least one fucosylated mammalian milk oligosaccharide. 25. The cell of any one of preferred embodiments 1 to 24, wherein the mammalian milk oligosaccharide mixture comprises at least one sialylated mammalian milk oligosaccharide. 26. The cell of any one of the preferred embodiments 1 to 25, wherein the mammalian milk oligosaccharide mixture comprises at least one mammalian milk oligosaccharide comprising N-acetylglucosamine monosaccharide units. 27. The cell of any one of preferred embodiments 1 to 26, wherein the mammalian milk oligosaccharide mixture comprises at least one galactosylated mammalian milk oligosaccharide. 28. The cell of any one of preferred embodiments 1 to 27, wherein the cell uses at least one precursor for producing any one or more of these oligosaccharides, preferably, the cell uses two or more precursors for the production of any one or more of the oligosaccharides, the precursor(s) being fed into the cell from the culture medium. 29. The cell of any one of preferred embodiments 1 to 28, wherein the cell produces at least one precursor for producing any one of the oligosaccharides. 30. The cell of any one of preferred embodiments 1 to 29, wherein the at least one precursor for producing any of the oligosaccharides is fully converted into any of the oligosaccharides. 31. The cell of any one of the preferred embodiments 1 to 30, wherein the cell intracellularly produces these oligosaccharides, and wherein part or substantially all of the oligosaccharides produced are maintained intracellularly and/or Excretion to the outside of the cell is via passive or active transport. 32. The cell of any one of preferred embodiments 1 to 31, wherein the cell is further genetically modified for the following i) modified expression of endogenous membrane proteins, and/or ii) modified endogenous membrane proteins activity, and/or iii) expression of a homologous membrane protein, and/or iv) expression of a heterologous membrane protein, wherein the membrane protein is involved in the secretion of any of the mammalian milk oligosaccharides from the mixture to the outside of the cell One, preferably, wherein the membrane protein is involved in the secretion of all of the oligosaccharides from the mixture from the cell. 33. The cell of any one of preferred embodiments 1 to 32, wherein the cell is further genetically modified for the following i) modified expression of endogenous membrane proteins, and/or ii) modified endogenous membrane proteins activity, and/or iii) expression of a homologous membrane protein, and/or iv) expression of a heterologous membrane protein, wherein the membrane protein is involved in the uptake of any of the mammalian milk oligosaccharides used in the synthesis of the mixture precursors and/or receptors, 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 such receptors. 34. The cell of any one of preferred embodiments 32 or 33, wherein the membrane protein is selected from a list comprising the following: transporter, PP-bond hydrolysis-driven transporter, β - Barrel porins, transport proteins, putative transport proteins and phosphate transfer driven group translocation proteins, preferably these transport proteins comprise MFS transport proteins, sugar efflux transport proteins and chelating iron Protein export proteins, preferably, the PP-bond hydrolysis-driven transport proteins include ABC transport proteins and chelatin export proteins. 35. The cell of any one of the preferred embodiments 32 to 34, wherein the membrane protein provides improved production of any one of these oligosaccharides and/or can achieve and/or enhance the production of any one of these oligosaccharides. outflow of either. 36. The cell of any one of preferred embodiments 1 to 35, wherein the cell is resistant to lactose killing when grown in an environment where lactose is combined with one or more other carbon sources. 37. The cell of any one of preferred embodiments 1 to 36, wherein the cell comprises a modification for reducing acetate production compared to an unmodified precursor cell. 38. The cell of preferred embodiment 37, wherein compared to an unmodified precursor cell, the cell comprises reduced or reduced expression and/or eliminated, attenuated, reduced or delayed activity comprising one of these proteins Any one or more of: β-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase , N-acetylglucosamine inhibitor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphoglucosamine-1-phosphotransferase, L-fucus 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 transferase, glucose-1-phosphate adenosyltransferase, glucose-1-phosphate Enzyme, ATP-dependent 6-phosphofructokinase isoenzyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor 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 Polyphosphoryl transfer proteins FruA and FruB, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate-formate dissociation enzyme, acetate kinase, phosphoryl transferase, phosphoacetyltransferase, pyruvate decarboxylase. 39. The cell of any one of preferred embodiments 1 to 38, wherein the cell is capable of producing phosphoenolpyruvate (phosphoenolpyruvate; PEP). 40. The cell of any one of preferred embodiments 1 to 39, wherein the cell is modified to enhance the production and/or supply of phosphoenolpyruvate (PEP) compared to unmodified precursor cells . 41. The cell of any one of preferred embodiments 1 to 40, wherein any one of these mammalian milk oligosaccharides is a human milk oligosaccharide, preferably, all these oligosaccharides are human milk oligosaccharides . 42. A method of producing a mixture of at least three different mammalian milk oligosaccharides by cells, the method comprising the steps of: a. providing a cell, preferably a single cell, expressing at least two glycosyltransferases and capable of synthesizing one or a Nucleotide-sugars, wherein the one or more nucleotide-sugars are one or more donors for the glycosyltransferases, and b. in allowing expression of the glycosyltransferases and synthesis of the one or more Culturing the cell under conditions of multiple nucleotide-sugars such that the cell produces at least three different mammalian milk oligosaccharides, c. preferably, at least one of the mammalian milk oligosaccharides is isolated from the culture, More preferably, all of the mammalian milk oligosaccharides are isolated from the culture. 43. The method of preferred embodiment 42, wherein the cell is capable of expressing, preferably at least five glycosyltransferases, preferably at least six, more preferably at least seven glycosyltransferases. 44. The method of any one of preferred embodiments 42 or 43, wherein the mixture comprises, consists of or consists essentially of neutral fucosylated and non-fucosylated oligosaccharides and is uncharged Oligosaccharides. 45. The method of any one of preferred embodiments 42 or 43, wherein the mixture comprises, consists of, or consists essentially of neutral fucosylated oligosaccharides and is free of neutral afucosylated oligosaccharides or charged oligosaccharides. 46. The method of any one of preferred embodiments 42 or 43, wherein the mixture comprises, consists of or consists essentially of neutral afucosylated oligosaccharides and is free of neutral fucosylation or charged oligosaccharides. 47. The method of any one of preferred embodiments 42 or 43, wherein the mixture comprises, consists of, or consists essentially of charged and neutral fucosylated and non-fucosylated oligosaccharides , preferably, at least one of the charged oligosaccharides is a sialylated oligosaccharide. 48. The method of any one of preferred embodiments 42 or 43, wherein the mixture comprises, consists of, or consists essentially of, and is free of neutral oligosaccharides, preferably, these charged oligosaccharides At least one of them is a sialylated oligosaccharide. 49. The method of any one of preferred embodiments 42 to 48, wherein the cell is a metabolically engineered cell of any one of embodiments 1 to 41. 50. The method of preferred embodiment 49, wherein the cell is modified with a gene expression module, characterized in that the expression from any of the expression modules is persistent or produced by a natural inducer. 51. The method of any one of preferred embodiments 49 or 50, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein. 52. The method of any one of preferred embodiments 42 to 51, wherein the oligosaccharide mixture comprises at least three different oligosaccharides that differ in degree of polymerization. 53. The method of any one of preferred embodiments 42 to 52, wherein the cell produces four or more different mammalian milk oligosaccharides. 54. The method of any one of preferred embodiments 42 to 53, wherein any one of the glycosyltransferases is selected from the list comprising the following: fucosyltransferase, sialyltransferase , Galactosyltransferase, Glucosyltransferase, Mannosyltransferase, N-Acetylglucosaminyltransferase, N-Acetylgalactosaminyltransferase, N-Acetylmannosylaminotransferase Transferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosaminotransferase, N-hydroxyacetylneuramidotransferase, rhamnosyltransferase, N -Acetyl rhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosaminotransferase, UDP-N-acetyl glucosamine enolacetonyltransferase and fucosyltransferase, - preferably, the fucosyltransferase is selected from the list comprising: α-1,2-fucosyl Transferases, α-1,3-fucosyltransferases, α-1,4-fucosyltransferases and α-1,6-fucosyltransferases, - preferably, the sialic acid The syltransferase 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-galactosyltransferase -1,4-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, α-1,3-galactosyltransferase and α-1,4-galactose Glycosyltransferase, - preferably, the glucosyltransferase is selected from the list comprising: α-glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase Enzymes and β-1,4-glucosyltransferase, - preferably, the mannosyltransferase is selected from the list comprising: α-1,2-mannosyltransferase, α-1,3 - mannosyltransferase and alpha-1,6-mannosyltransferase, - preferably, the N-acetylglucosaminyltransferase is selected from the list comprising: galactoside beta-1 ,3-N-acetylglucosamine transferase and β-1,6-N-acetylglucosamine transferase, - preferably, the N-acetylgalactosamine transferase is selected from From a list comprising the following: α-1,3-N-acetylgalactosaminyltransferase and β-1,3-N-acetylgalactosaminyltransferase, - preferably, the cell Modified in the expression or activity of at least one of the glycosyltransferases. 55. The method of any one of preferred embodiments 42 to 54, wherein one of the glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-Fucose (GDP-Fuc). 56. The method of any one of preferred embodiments 42 to 55, wherein one of the glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP -N-Acetyl neuraminic acid (CMP-Neu5Ac). 57. The method of any one of preferred embodiments 42 to 56, wherein one of the glycosyltransferases is N-acetylglucosaminyltransferase and one of the donor nucleotide-sugars is One of them is UDP-N-acetylglucosamine (UDP-GlcNAc). 58. The method of any one of preferred embodiments 42 to 57, wherein one of these glycosyltransferases is galactosyltransferase and one of these donor nucleotide-sugars is UDP -Galactose (UDP-Gal). 59. The method of any one of preferred embodiments 42 to 58, wherein the glycosyltransferase is N-acetylgalactosamine transferase and the donor nucleotide-sugar is UDP-N-ethyl Acylgalactosamine (UDP-GalNAc). 60. The method of any one of preferred embodiments 42 to 59, wherein the glycosyltransferase is N-acetylmannosylaminotransferase and the donor nucleotide-sugar is UDP-N-ethyl Acylmannosamine (UDP-ManNAc). 61. The method of any one of preferred embodiments 42 to 60, wherein any one of these nucleotide-sugars 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-dito Oxy--L-lyxose-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetylamino-2,6-di deoxy-L-mannose), 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-rhamnet Sugar, UDP-xylose. 62. The method of any one of preferred embodiments 42 to 61, wherein the cell expresses one or more polypeptides selected from a list comprising: mannose-6-phosphate isomerase, phosphate Mannose mutase, mannose-1-phosphate guanosyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase , GDP-fucose pyrophosphorylase, fucose-1-phosphate guanosyltransferase, L-glutamic acid-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate Deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acetylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-Epimerase, N-Acetylmannosamine-6-Phosphate 2-Epimerase, Glucosamine 6-Phosphate N-Acetyltransferase, N-Acetylglucosamine-6-Phosphate Phosphatase, N-acetylmannosamine-6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate urine Glycosyltransferase, glucosamine-1-phosphate acetyltransferase, N-acetylneuraminic acid synthase, N-acetylneuraminic acid lyase, N-acetylneuraminic acid-9- Phosphate synthase, N-acyl neuraminic acid-9-phosphate phosphatase, N-acyl neuraminic acid cytidine syltransferase, galactose-1-epimerase, galactokinase, glucokinase, galactokinase Lactose-1-phosphate uridine transferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridine transferase, phosphoglucomutase, UDP-N-acetylglucosamine 4 - epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein the cell expresses any of these polypeptides or Modified in terms of activity. 63. The method of any one of preferred embodiments 42 to 62, wherein the cell is capable of synthesizing at least two nucleotide-sugars, preferably at least three nucleotide-sugars, more preferably at least four nucleotide-sugars. sugar, even better at least five nucleotide-sugars. 64. The method of any one of preferred embodiments 42 to 63, wherein the mammalian milk oligosaccharide mixture comprises at least one fucosylated mammalian milk oligosaccharide. 65. The method of any one of preferred embodiments 42 to 64, wherein the mammalian milk oligosaccharide mixture comprises at least one sialylated mammalian milk oligosaccharide. 66. The method of any one of preferred embodiments 42 to 65, wherein the mammalian milk oligosaccharide mixture comprises at least one mammalian milk oligosaccharide comprising N-acetylglucosamine monosaccharide units. 67. The method of any one of preferred embodiments 42 to 66, wherein the mammalian milk oligosaccharide mixture comprises at least one galactosylated mammalian milk oligosaccharide. 68. The method of any one of preferred embodiments 42 to 67, wherein the mammalian milk oligosaccharide mixture comprises at least one mammalian milk oligosaccharide, the at least one mammalian milk oligosaccharide fucosylated, sialylated , galactosylation, glycosylation, xylosylation, mannosylation; the at least one mammalian milk oligosaccharide contains N-acetylglucosamine, contains N-acetylneuraminic acid, contains N- Hydroxyacetylneuraminic acid, containing N-acetylgalactosamine, containing rhamnose, containing glucuronide, containing galacturonic acid ester, and/or containing N-acetylmannosamine. 69. The method of any one of preferred embodiments 42 to 68, wherein the cell uses at least one precursor for producing any one or more of these oligosaccharides, preferably, the cell uses two or more precursors for the production of any one or more of the oligosaccharides, the precursor(s) being fed into the cell from the culture medium. 70. The method of any one of preferred embodiments 42 to 69, wherein the cell produces at least one precursor for producing any one of the mammalian milk oligosaccharides. 71. The method of any one of preferred embodiments 42 to 70, wherein the at least one precursor for producing any one of these mammalian milk oligosaccharides is completely converted into these mammalian milk oligosaccharides any of them. 72. The method of any one of preferred embodiments 42 to 71, wherein the cell intracellularly produces these oligosaccharides, and wherein part or substantially all of the oligosaccharides produced are maintained intracellularly and/or Excretion to the outside of the cell is via passive or active transport. 73. The method of any one of preferred embodiments 42 to 72, wherein the cell is further genetically modified for the following i) modified expression of endogenous membrane proteins, and/or ii) modified endogenous membrane proteins activity, and/or iii) expression of a homologous membrane protein, and/or iv) expression of a heterologous membrane protein, wherein the membrane protein is involved in the secretion of any of the mammalian milk oligosaccharides from the mixture to the outside of the cell One, preferably, wherein the membrane protein is involved in the secretion of all of the oligosaccharides from the mixture from the cell. 74. The method of any one of preferred embodiments 42 to 73, wherein the cell is further genetically modified for the following i) modified expression of endogenous membrane proteins, and/or ii) modified endogenous membrane proteins activity, and/or iii) expression of a homologous membrane protein, and/or iv) expression of a heterologous membrane protein, wherein the membrane protein is involved in the uptake of any of the mammalian milk oligosaccharides used in the synthesis of the mixture precursors and/or receptors, 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 such receptors. 75. The method of any one of preferred embodiments 73 or 74, wherein the membrane protein is selected from the list comprising: transport protein, PP-bond hydrolysis-driven transport protein, β-barrel porin, Auxiliary transport proteins, putative transport proteins and phosphate transfer driven group translocation proteins, preferably, these transport proteins comprise MFS transport proteins, sugar efflux transport proteins and chelating ferritin export proteins, preferably these Transport proteins driven by hydrolysis of PP-bonds include ABC transport proteins and chelatin export proteins. 76. The method of any one of preferred embodiments 73 to 75, wherein the membrane protein provides an improved production of any one of these oligosaccharides and/or can achieve and/or enhance the outflow of either. 77. The method of any one of preferred embodiments 42 to 76, wherein the cells are resistant to lactose killing when grown in an environment where lactose is combined with one or more other carbon sources. 78. The method of any one of preferred embodiments 42 to 77, wherein the cell comprises a modification for reducing acetate production compared to an unmodified precursor cell. 79. The method of preferred embodiment 78, wherein compared to an unmodified precursor cell, the cell comprises reduced or reduced expression and/or eliminated, attenuated, reduced or delayed activity of one of these proteins comprising the following Any one or more of: β-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase , N-acetylglucosamine inhibitor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecopentenyl-phosphoglucose-1-phosphotransferase, L-fucokinase , L-fucose isomerase, N-acetylneuraminic acid dissociation enzyme, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridine transferase, glucose-1-phosphate adenosyltransferase, glucose-1-phosphatase, ATP-dependent 6 - Phosphofructokinase isoenzyme 1, ATP-dependent 6-phosphofructokinase isoenzyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IclR, lon protease, glucose-specific Phosphophosphotransferase IIBC component ptsG, glucose-specific translocation phosphotransferase (PTS) IIBC component malX, enzyme IIA ^Glc , β-glucoside-specific PTS enzyme II, fructose-specific PTS polyphosphoryl transfer protein FruA and FruB, alcohol dehydrogenase, aldehyde dehydrogenase, pyruvate-formate dissociation enzyme, acetate kinase, phosphoacetyltransferase, phosphoacetyltransferase, pyruvate decarboxylase. 80. The method of any one of preferred embodiments 42 to 79, wherein the cell is capable of producing phosphoenolpyruvate (PEP). 81. The method of any one of preferred embodiments 42 to 80, wherein the cell is modified to enhance the production and/or supply of phosphoenolpyruvate (PEP) compared to unmodified precursor cells . 82. The method of any one of preferred embodiments 42 to 81, wherein any one of these mammalian milk oligosaccharides is a human milk oligosaccharide, preferably, all of these mammalian milk oligosaccharides are human Lacto-oligosaccharides. 83. The method of any one of preferred embodiments 42 to 82, wherein these conditions comprise: - use the culture medium comprising at least one precursor and/or acceptor for producing any one of these oligosaccharides , and/or - at least one precursor and/or acceptor feed is added to the medium for the production of any of the oligosaccharides. 84. The method of any one of preferred embodiments 42 to 83, comprising at least one of the following steps: i) using a culture medium comprising at least one precursor and/or acceptor; ii) in a reactor Add at least one precursor and/or acceptor feed to the medium, wherein the total reactor volume is in the range of 250 mL (milliliters) to 10.000 ^m3 (cubic meters), preferably in a continuous manner, and preferably such that The final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium before addition of the precursor and/or acceptor feed; iii) adding the The culture medium is supplemented with at least one precursor and/or acceptor feed, wherein the total reactor volume is in the range of 250 mL (milliliters) to 10.000 ^m3 (cubic meters), preferably in a continuous manner, and preferably such that the The final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium before adding the precursor and/or acceptor feed, and wherein preferably the precursor The pH of the bulk and/or acceptor feed is set between 3 and 7, and wherein preferably the temperature of the precursor and/or acceptor feed is maintained between 20°C and 80°C; iv) for 1 Add at least one precursor and/or acceptor feed to the medium in a continuous manner via the feed solution over a time course of days, 2 days, 3 days, 4 days, 5 days; v) over a period of 1 day, 2 days, 3 days The time course of days, 4 days, 5 days adds at least one precursor and/or acceptor feed to the medium in a continuous manner by means of a feed solution, and wherein preferably the pH of the feed solution is set at 3 between 7 and 7, and wherein preferably, the temperature of the feed solution is maintained between 20°C and 80°C; the method produces a concentration in the final culture of at least 50 g/L, preferably at least 75 g/L, More preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L of these any of the oligosaccharides. 85. The method of any one of preferred embodiments 42 to 83, comprising at least one of the following steps: i) using a method comprising at least 50, more preferably at least 75, more preferably at least 100 per liter of initial reactor volume , more preferably at least 120, more preferably at least 150 grams of lactose culture medium, wherein the reactor volume is in the range of 250 mL to 10.000 m ³ (cubic meters); ii) add to the culture medium a medium containing at least 1 per liter of initial reactor volume 50. Lactose feed of more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose, wherein the reactor volume is in the range of 250 mL to 10.000 ^m3 (cubic meters), more preferably preferably added in a continuous manner, and preferably such that the final volume of the medium does not exceed three times, preferably not more than twice, more preferably less than twice the volume of the medium before the addition of the lactose feed; iii) adding to the Medium supplementation Lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor volume is between 250 mL and 10.000 m ³ (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium does not exceed three times, preferably not more than twice the volume of the medium before the addition of the lactose feed , more preferably less than twice, and wherein preferably the pH of the lactose feed is set between 3 and 7, and wherein preferably the temperature of the lactose feed is maintained between 20°C and 80°C; iv ) feeds of lactose were added to the medium by the feed solution in a continuous manner over the time course of 1 day, 2 days, 3 days, 4 days, 5 days; v) over 1 day, 2 days, 3 days, 4 days, The time course of 5 days adds lactose feed to the medium in a continuous manner by means of a feed solution, and wherein the concentration of the lactose feed solution is 50 g/L, preferably 75 g/L, more preferably 100 g/L , better 125 g/L, better 150 g/L, better 175 g/L, better 200 g/L, better 225 g/L, better 250 g/L, better 275 g/L, Better 300 g/L, Better 325 g/L, Better 350 g/L, Better 375 g/L, Better 400 g/L, Better 450 g/L, Better 500 g/L, Even More preferably 550 g/L, most preferably 600 g/L, and wherein preferably the pH of the feed solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is maintained at 20°C between the temperature of /L, better at least 150 g/L, better at least 175 g/L, better to Any of these oligosaccharides at least 200 g/L. 86. The method of preferred embodiment 85, wherein the lactose feed is obtained by starting the culture at a concentration of at least 5 mM, preferably 30, 40, 50, 60, 70, 80, 90, 100, 150 mM concentrations, more preferably lactose is added at concentrations > 300 mM. 87. The method of any one of preferred embodiments 85 or 86, wherein the lactose feed is by adding lactose to the culture at a concentration such that at least 5 mM, more than 5 mM, is obtained throughout the production phase of the culture. Optimal lactose concentrations of 10 mM or 30 mM are used to achieve this. 88. The method of any one of preferred embodiments 42 to 87, wherein the host cells are cultured for at least about 60, 80, 100 or about 120 hours or in a continuous manner. 89. The method of any one of preferred embodiments 42 to 88, wherein the cell is cultured in a medium comprising a carbon source, the carbon source comprising monosaccharides, disaccharides, oligosaccharides, polysaccharides, polyols, glycerol; Culturing In a complex medium comprising molasses, corn infusion, protein extract, pancreas or yeast extract; preferably, wherein the carbon source is selected from the list comprising the following: glucose, glycerol, fructose, sucrose, maltose, lactose, Arabinose, malto-oligosaccharide, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn steep liquid, high fructose syrup, acetate, citrate, lactate and pyruvate. 90. The method of any one of preferred embodiments 42 to 89, wherein the substratum contains at least one precursor selected from the group consisting of lactose, galactose, fucose, sialic acid, GlcNAc, GalNAc, Lacto-N-biose (LNB), N-acetyllactosamine (LacNAc). 91. The method of any one of preferred embodiments 42 to 90, wherein the first stage of exponential cell growth is carried out by adding the precursor, preferably lactose, to the culture medium in the second stage based on A matrix of carbon, preferably glucose or sucrose is added to the medium to provide. 92. The method of any one of preferred embodiments 42 to 91, wherein the first stage of exponential cell growth is carried out by adding a carbon-based substrate, preferably glucose or sucrose, to a mixture comprising a precursor, preferably lactose. is provided in the medium, followed by a second stage in which only a carbon-based substrate, preferably glucose or sucrose, is added to the medium. 93. The method of any one of preferred embodiments 42 to 92, wherein the first stage of exponential cell growth is carried out by adding a carbon-based substrate, preferably glucose or sucrose to a precursor, preferably lactose, is provided in the medium, followed by a second stage in which a carbon-based substrate, preferably glucose or sucrose, and a precursor, preferably lactose, are added to the medium. 94. The method of any one of preferred embodiments 42 to 93, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, two-phase distribution, 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. 95. The method of any one of preferred embodiments 42 to 94, further comprising purifying any one of the mammalian milk oligosaccharides from the cell. 96. The method of any one of preferred embodiments 95, 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; use of alcohol; use of hydroalcoholic mixture; crystallization; evaporation; precipitation; drying, drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum belt drying Drying, vacuum belt drying, tumble drying, tumble drying, vacuum tumble drying or vacuum tumble drying. 97. The cell of any one of the preferred embodiments 1 to 41 or the method of any one of the preferred embodiments 42 to 96, wherein the cell is a bacterium, a fungus, a yeast, a plant cell, an animal cell or a progenitor. worm cells, - preferably, the bacteria are Escherichia coli ( Escherichia coli ) strains, more preferably Escherichia coli strains of K-12 strains, even more preferably the Escherichia coli K-12 strains are 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: Saccharomyces , Zygosaccharomyces , Pichia , Komagataella , Han Hansenula , Yarrowia , Starmerella , Kluyveromyces or Debaromyces , - preferably, the plant cell are algal cells or 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 Class, amphibian or insect, or a genetically modified cell line derived from human cells excluding embryonic stem cells, more preferably the human and non-human mammalian cells are epithelial cells, embryonic kidney cells, fibroblasts, COS cells , Chinese hamster ovary (Chinese hamster ovary; CHO) cells, murine myeloma cells, NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, preferably the insect cells are derived from Spodoptera frugiperda , Bombyx mori , Mamestra brassicae , Trichoplusia ni or Drosophila melanogaster , - preferably, the protozoal cell is Leishmania dow ( Leishmania tarentolae ) cells. 98. The cell of preferred embodiment 97 or the method of preferred embodiment 97, wherein the cell is a live Gram-negative bacterium compared to an unmodified precursor cell, the bacterium Contains reduced or eliminated synthetic poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, kolac acid, core oligo Sugars, Osmoregulated Periplasmic Glucan (OPG), glycerol glucosides, glycans and/or trehalose. 99. Use of the cell of any one of preferred embodiments 1 to 41, 97, 98 or the method of any one of preferred embodiments 42 to 98 for producing at least three different mammalian milk oligos. sugar mixture.

Example Example 1. Materials and Methods for 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). Minimal medium in 96-well dishes or shake flasks used in culture experiments contained 2.00 g/L NH4Cl, 5.00 g/L (NH4)2SO4, 2.993 g/L KH2PO4, 7.315 g/L K2HPO4, 8.372 g/L MOPS, 0.5 g/L NaCl, 0.5 g/L MgSO4.7H2O, 30 g/L sucrose or 30 g/L glycerol, 1 ml/L vitamin solution, 100 µl/L molybdate solution, and 1 mL/L selenium solution. 0.30 g/L sialic acid, 20 g/L lactose, 20 g/L LacNAc and/or 20 g/L LNB were additionally added to the medium as precursors as specified in the respective examples. 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. 0.30 g/L sialic acid, 20 g/L lactose, 20 g/L LacNAc and/or 20 g/L LNB were additionally added to the medium as precursors as specified in the respective examples.

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), carbenicillin (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 The pCP20 (expressing FLP recombinase activity) plasmid system was obtained from Prof. R. Cunin (Vrije Universiteit Brussel, Belgium, in 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 lambda 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 with 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 ampicillin- and chloramphenicol-resistant plastids, showing temperature-sensitive replication and thermal induction of FLP synthesis. Ampicillin resistant transformants were selected at 30°C, followed by purification of a few colonies 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, which contains deletions of the E. coli wcaJ and thyA genes and gene body insertion of a persistent transcription unit that The unit contains a sucrose transporter such as CscB from Escherichia coli W with SEQ ID NO 01, a fructokinase with SEQ ID NO 02 as for example from Zymomonas mobilis and a fructokinase as for example from Zymomonas mobilis with Sucrose phosphorylase of Bifidobacterium adolescentis of SEQ ID NO 03. For the production of fucosylated oligosaccharides, mutant GDP-fucose-producing strains were additionally modified with expressing plastids comprising a persistent transcription unit of α-1,2-fucosyltransferase, As e.g. HpFutC from H. pylori with SEQ ID NO 04; and/or a continuous transcription unit of alpha-1,3-fucosyltransferase, as e.g. from HpFucT of H. pylori, and modified with a persistent transcription unit for selectable marker, such as, for example, E. coli thyA with SEQ ID NO 07. 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 optimize. GDP-fucose production can additionally be optimized, comprising mannose-6-phosphate isomerase (as eg, manA from E. coli having SEQ ID NO 08), phosphomannose mutase (as eg from Escherichia coli having SEQ ID NO 08) manB of Escherichia coli of SEQ ID NO 09), mannose-1-phosphate guanosyltransferase (as e.g. manC from Escherichia coli having SEQ ID NO 10), GDP-mannose 4,6-dehydratase ( As eg gmd from Escherichia coli with SEQ ID NO 11) and GDP-L-fucose synthase (as eg fcl from Escherichia coli with SEQ ID NO 12) gene body insertion. 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 fucose permeases, such as for example from those with SEQ ID fucP of E. coli NO 13, and bifunctional fucokinase/fucose-1-phosphate guanosyltransferase, such as eg fkp from Bacteroides fragilis with SEQ NO ID 14. If a GDP-fucose-producing mutant strain is intended to produce a fucosylated lactose structure, the strain is additionally genetically knocked out of the E. coli LacZ , LacY , and LacA genes and subjected to a lactose permease (eg, having SEQ ID NO 15) Gene body insertion modification of the persistent transcription unit of Escherichia coli LacY).

Alternatively, and/or additionally, the production of GDP-fucose and/or fucosylated structures can be further optimized in mutant E. coli strains with genomic insertion of a persistent transcription unit that Membrane transport proteins are included, 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 Regin MdfA from Y. burgdorferi (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207) or iceT from Citrobacter johnsonii (UniProt ID D4B8A6).

In one embodiment of sialic acid production, the mutant strain is derived from E. coli K12 MG1655, the E. coli comprising deletions of the E. coli nagA and nagB genes and gene body insertion of persistent transcription units containing, for example, For example glucosamine 6-phosphate N-acetyltransferase from GNA1 of Saccharomyces cerevisiae with SEQ ID NO 16, such as for example N-acetyltransferase from AGE of Bacteroides ovatus with SEQ ID NO 17 Glucosamine 2-epimerase and N-acetylneuraminic acid (Neu5Ac) synthase such as eg NeuB from Neisseria meningitidis having SEQ ID NO 18. Sialic acid production can be further optimized in mutant E. coli strains with genes comprising any one or more of the E. coli genes of nagC , nagD , nagE , nanA , nanE , nanK , manX , manY and manZ Body knockout (as described in WO18122225); and/or gene body knockout of E. coli genes comprising any one or more of nanT , poxB , ldhA , adhE , aldB , pflA , pflC , ybiY , ack and/or pta and gene body insertion of persistent transcription units comprising L-glutamic acid-D-fructose-6-phosphate aminotransferase, such as differing from, for example, A39T, R250C and G472S mutations A mutant glmS*54 from E. coli having SEQ ID NO 19 of wild type E. coli; and a phosphatase such as any one or more of the following: for example from E. coli having SEQ ID NO 20 or comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU yqaB of E. coli genes or PsMupP from Pseudomonas putida, ScDOG1 from Saccharomyces cerevisiae and BsAraL from Bacillus subtilis (as described in WO18122225); and as eg acs from E. coli (UniProt ID P27550) Acetyl-CoA synthase. Sialic acid production can also be obtained by deletion of the E. coli nagA and nagB genes and gene body insertion of persistent transcription units containing glucosamine phosphate such as, for example, glmM from E. coli with SEQ ID NO 42 Mutase, such as, for example, N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase, such as, for example, glmU from Escherichia coli with SEQ ID NO 43 UDP-N-acetylglucosamine 2-epimerase from NeuC of Campylobacter jejuni with SEQ ID NO 21 and N-ethyl glucosamine as for example from NeuB of Neisseria meningitidis with SEQ ID NO 18 Acyl neuraminic acid synthase. Also in this mutant strain, sialic acid production can be further optimized with gene body insertion of persistent transcription units comprising, for example, mutant glmS* from E. coli with SEQ ID NO 19 L-glutamic acid-D-fructose-6-phosphate aminotransferase of 54; and a phosphatase such as, for example, from Escherichia coli having SEQ ID NO 20 or comprising aphA, Cof, HisB, OtsB, SurE, Yaed, The yqaB of the E. coli genes of YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU may be derived from Escherichia coli PsMupP from Monascus, ScDOG1 from Saccharomyces cerevisiae and BsAraL from Bacillus subtilis (as described in WO18122225); and acetyl-CoA synthases such as eg acs from Escherichia coli (UniProt ID P27550).

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 persistent transcription units 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 candidate species Magnetotropism HK-1 (UniProt ID KPA15328.1) or from Bacillus polymorpha (UniProt ID Q8A712) -9-phosphatase.

For sialylated oligosaccharide production, the sialic acid producing strain further needs to express an N-acyl neuraminocytidyl transferase such as, for example, NeuA from Pasteurella septicaemia with SEQ ID NO 22 and as for example from a septicemic The β-galactoside α-2,3-sialyltransferase of PmultST3 of Pasteurella (UniProt ID Q9CLP3) or the PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 with transferase activity, NmeniST3 from Neisseria meningitidis (SEQ ID NO 24) or from Pasteurella septicaemia subsp. Pasteurella strain PmultST2 of Pm70 (GenBank NO. AAK02592.1), β-galactoside α-2,6-sialyltransferase (UniProt ID 066375) such as PdST6 from Pseudomonas mermaidii, or PmultST2 with as SEQ ID NO 25 PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID O66375 with β-galactoside α-2,6-sialyltransferase activity, P- JT-ISH-224-ST6 (UniProt ID A8QYL1) or from amino acid residues 18 to 18 of UniProt ID A8QYL1 having β-galactoside α-2,6-sialyltransferase activity as in SEQ ID NO 26 A P-JT-ISH-224-ST6-like polypeptide consisting of 514 and/or eg, α-2,8-sialyltransferase of Mus musculus (UniProt ID Q64689). The persistent transcription units of PmNeuA 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 genetically knocked out of the E. coli LacZ , LacY and LacA genes and subjected to a lactose permease (such as for example having SEQ ID NO 15). Gene body insertion modification of the persistent transcription unit of Escherichia coli LacY).

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 for example A sialic acid transporter such as, for example, 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 transport proteins, such as eg EntS from E. coli (UniProt ID P24077), 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), Lemonade Young's Acidobacillus iceT (UniProt ID D4B8A6), SetA from E. coli (UniProt ID P31675), SetB from E. coli (UniProt ID P33026) or SetC from E. coli (UniProt ID P31436); or ABC transport proteins, such as e.g. 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 can optionally be adapted for growth on sucrose via gene body intercalation of persistent transcription units containing, for example, derived from those with SEQ The sucrose transporter of CscB of E. coli W of ID NO 01, the fructokinase of Frk derived from Z. mobilis with SEQ ID NO 02 for example and the fructokinase of Bifidobacterium adolescentis as for example derived from Bifidobacterium adolescentis with SEQ ID NO 03 The sucrose phosphorylase of BaSP.

One implementation in the production of LN3 (GlcNAc-b1,3-Gal-b1,4-Glc) and oligosaccharides derived therefrom comprising lacto- N -tetrasaccharide (LNT) and lacto- N -neotetraose (LNnT) In an example, the mutant strain is derived from E. coli K12 MG1655 and modified by deletion of the E. coli LacZ and nagB genes, and modified with galactoside beta-1 such as LgtA from Neisseria meningitidis having SEQ ID NO 27 , Gene body insertion modification of the continuous transcription unit of 3-N-acetylglucosaminyltransferase. For LNT or LNnT production, mutant strains are further modified with persistent transcription units that can be delivered to the strain via gene body insertion or from expressing plastids, respectively, as for example from E. coli O55:H7 with SEQ ID NO 28, respectively. N-acetylglucosamine beta-1,3-galactosyltransferase of WbgO or N-acetylglucosamine beta-1,4 from LgtB of Neisseria meningitidis having SEQ ID NO 29 - Galactosyltransferase. Galactoside β-1,3-N-acetylglucosaminyltransferase, N-acetylglucosamine β-1,3-galactosyltransferase, and/or N-acetylglucosamine, as needed Multiple copies of the amine beta-1,4-galactosyltransferase gene can be added to mutant E. coli strains. In addition, LNT and/or LNnT production can be achieved by continuous transcription with L-glutamate-D-fructose-6-phosphate aminotransferase such as, for example, glmS*54 from Escherichia coli having SEQ ID NO 19 One or more gene bodies of the unit were inserted into the modified strain to improve UDP-GlcNAc production for enhancement. In addition, the strain can be optionally modified to enhance UDP-galactose production by genomic deletion of the E. coli ushA , galT , ldhA and agp genes. Mutant E. coli strains can also optionally be adapted to have gene body insertions with a persistent transcription unit for use in such as, for example, UDP-glucose-4-table from galE of E. coli having SEQ ID NO 30 Isomerases such as, for example, phosphoglucosamine mutase such as glmM from E. coli with SEQ ID NO 42 and N-acetylglucosamine-1-phosphate such as, for example, glmU from E. coli with SEQ ID NO 43 Uridine acetyltransferase/glucosamine-1-phosphate acetyltransferase. Mutant strains may also be adapted for growth on sucrose via gene body intercalation of a persistent transcription unit containing a sucrose transporter such as, for example, CscB from E. coli W having SEQ ID NO 01 , as eg fructokinase derived from Frk of Zymomonas mobilis with SEQ ID NO 02 and sucrose phosphorylase as eg from BaSP of Bifidobacterium adolescentis with SEQ ID NO 03.

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, 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, glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis and/or membrane transporters are N-terminally and/or C-terminally fused to soluble enhancer tags such as for example: SUMO tag, MBP tag , His, FLAG, Strep-II, Halo-tag, NusA, thioredoxin, GST and/or Fh8 tags to enhance their 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, mutant E. coli strains are modified to produce glycosyl-minimized E. coli strains comprising gene body deletions 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 Mutalik et al. (Nat. Methods 2013, No. 10, 354-360) and Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148) Libraries described: The gene lines were expressed using the promoters MutalikP5 ("PROM0005_MutalikP5") and apFAB82 ("PROM0050_apFAB82") as described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360), the UTRs used included GalE_BCD12 ("UTR0010_GalE_BCD12") and GalE_LeuAB ("UTR0014_GalE_LeuAB") as described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360) and terminator sequences used were as described by Cambray et al. ( ilvGEDA ("TER0007_ilvGEDA") described in Nucleic Acids Res. 2013, 41(9), 5139-5148). All genes were sequenced synthetically on Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com) and codon usage was adapted using the supplier's tools. The SEQ ID NOs described in the present invention are summarized in Table 1.

All strains were stored in cryovials (overnight LB cultures mixed with 70% glycerol at a 1:1 ratio) at -80°C. Table 1 : Summary of SEQ ID NOs described in the present invention SEQ ID NO name / identifier organism source Country of Origin of Digital Serial Information 01 CscB Escherichia coli W synthesis USA 02 Frk Z. mobilis synthesis U.K. 03 BaSP Bifidobacterium adolescentis synthesis Germany 04 HpFutC Helicobacter pylori UA1234 synthesis U.K. 05 HpFucT Helicobacter pylori UA1234 synthesis U.K. 06 mutHpFucT Helicobacter pylori UA1234 synthesis U.K. 07 thyA Escherichia coli K-12 MG1655 synthesis U.S. 08 manA Escherichia coli K-12 MG1655 synthesis U.S. 09 manB Escherichia coli K-12 MG1655 synthesis U.S. 10 manC Escherichia coli K-12 MG1655 synthesis U.S. 11 gmd Escherichia coli K-12 MG1655 synthesis U.S. 12 fcl Escherichia coli K-12 MG1655 synthesis U.S. 13 fucP Escherichia coli K-12 MG1655 synthesis U.S. 14 fkp Bacteroides fragilis synthesis U.K. 15 LacY Escherichia coli K-12 MG1655 synthesis U.S. 16 GNA1 Saccharomyces cerevisiae synthesis U.S. 17 AGE Bacteroides ovale synthesis U.S. 18 neuB Neisseria meningitidis synthesis U.K. 19 glmS*54 Escherichia coli K-12 MG1655 synthesis U.S. 20 Phosphatase Escherichia coli K-12 MG1655 synthesis U.S. twenty one neuC Campylobacter jejuni synthesis U.S. twenty two neuA Pasteurella septicaemia synthesis U.S. twenty three α-2,3-Sialyltransferase Pasteurella septicaemia synthesis U.S. twenty four α-2,3-Sialyltransferase Neisseria meningitidis synthesis U.K. 25 α-2,6-Sialyltransferase Mermaid Light Fungus synthesis Japan 26 α-2,6-Sialyltransferase Photobacterium sp. JT-ISH-224 synthesis Japan 27 LgtA Neisseria meningitidis synthesis U.K. 28 WbgO Escherichia coli O55:H7 synthesis Germany 29 LgtB Neisseria meningitidis MC58 synthesis U.K. 30 gale Escherichia coli K-12 MG1655 synthesis U.S. 31 Lac12 Kluyveromyces lactis synthesis U.S. 32 GCNT2 Homo sapiens synthesis unknown 33 α-1,3-Galactosyltransferase European cattle (Bos taurus) synthesis unknown 34 WbpP Pseudomonas aeruginosa synthesis U.S. 35 lgtD Haemophilus influenzae synthesis Ireland 36 lgtC Neisseria gonorrhoeae synthesis Japan 37 WbnI Escherichia coli K-12 MG1655 synthesis U.S. 38 BgtA Helicobacter weasel synthesis unknown 39 Mutant a1,3/4 fucosidase Bifidobacterium longum infantum synthesis U.S. 40 fucT54 Sideroxydans lithotrophicus ES-1 synthesis U.S. 41 WbdO Salmonella enterica subsp. salamae serovar Greenside synthesis U.K. 42 glmM Escherichia coli K-12 MG1655 synthesis U.S. 43 glmU Escherichia coli K-12 MG1655 synthesis U.S. 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 .

Pre-cultivation of bioreactors begins with an entire 1 mL frozen vial with a strain, inoculated in 250 mL or 500 mL of minimal medium in a 1 L or 2.5 L shake flask and incubated at 37°C on an orbital shaker. Incubate at 200 rpm for 24 h. 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 rate was dependent on strain and bioreactor. The pH was controlled at 6.8 using 0.5 M H2S04 and 20% NH4OH. Cool the exhaust gas. When foaming increases 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-tetraose (LNnT), LNFP-I, LNFP-II, LNFP-III, LNFP-V, LSTa, LSTc and LSTd were purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analyzed with in-house manufactured standards.

Neutral mammalian milk oligosaccharides were analyzed on a Waters Acquity Class H 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 ELS detector has an offset tube temperature of 50°C and a N2 gas pressure of 50 psi, a gain of 200 and a data transfer rate of 10 pps. The temperature of the RI detector was set to 35°C.

Siallylated mammalian milk 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 a mass spectrometer, a Waters with Electron Spray Ionisation (ESI) was used at a desolvation temperature of 450°C, a nitrogen desolvation gas flow rate of 650 L/h and a cone voltage of 20 V Xevo TQ-MS. For all mammalian milk 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. Mammalian milk 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 The third increase in eluate B from 40% to 100% within 5 min. 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 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 production of GDP-fucose, yeast expressed the plastid p2a_2µ_Fuc (Chan 2013, Plasmid 70, 2-17) 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, as well as 2µ yeast ori and Ura3 selectable markers for selection and maintenance in yeast. This plastid additionally contains a persistent transcription unit for, eg, lactose permease from LAC12 of Kluyveromyces lactis with SEQ ID NO 31, as eg, gmd from E. coli with SEQ ID NO 11 GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase as eg fcl from E. coli with SEQ ID NO 12. In another example, the yeast expression plastid p2a_2µ_Fuc2 can be used as a surrogate expression plastid for the p2a_2µ_Fuc plastid comprising the ampicillin resistance gene, bacterial ori, 2µ yeast ori and Ura3 selectable markers for Persistent transcription units such as lactose permease such as LAC12 from Kluyveromyces lactis with SEQ ID NO 31, fucose permease such as fucP from Escherichia coli with SEQ ID NO 13, and For example a bifunctional fucosokinase/fucose-1-phosphate guanosyltransferase from fkp of Bacteroides fragilis having SEQ NO ID 14. For further production of fucosylated oligosaccharides, p2a_2µ_Fuc and its variant p2a_2µ_Fuc2 additionally contain a persistent transcription unit for, for example, α-1,2- from HpFutC of Helicobacter pylori with SEQ ID NO 04 Fucosyltransferase, and/or an alpha-1,3-fucosyltransferase such as, for example, HpFucT from Helicobacter pylori having SEQ ID NO 05.

In one embodiment of the production of sialic acid and CMP-sialic acid, the yeast expressed plastids were derived from the pRS420 plastid series (Christianson et al., 1992, Gene 110: 119-122) containing the TRP1 selectable marker and A persistent transcription unit for use in: L-glutamate-D-fructose-6-phosphate aminotransferase, such as, for example, mutant glmS*54 from E. coli having SEQ ID NO 19; phosphatase, As e.g. from E. coli having SEQ ID NO 20 or comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YrbL, AppA, Gph, SerB, YbhA, YbiV , YqaB of the E. coli genes of YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonas sputum, ScDOG1 from Saccharomyces cerevisiae and BsAraL from Bacillus subtilis (as described in WO18122225) ; as for example N-acetylglucosamine 2-epimerase from AGE of Bacillus ovale with SEQ ID NO 17; as for example N from NeuB of Neisseria meningitidis with SEQ ID NO 18 - Acetylneuraminic acid synthase; and N-acetylneuraminic acid cytidine acyltransferase as eg NeuA from Pasteurella septicemia having SEQ ID NO 22. If desired, a continuous transcription unit for glucosamine 6-phosphate N-acetyltransferase such as eg GNA1 of Saccharomyces cerevisiae having SEQ ID NO 16 is also added. For the production of sialylated oligosaccharides, the plastids additionally comprise a continuous transcription unit for: a lactose permease such as, for example, LAC12 from Kluyveromyces lactis with SEQ ID NO 31; and, as for example, from Pasteurella septicaemia The β-galactoside α-2,3-sialyltransferase of PmultST3 of Bacillus (UniProt ID Q9CLP3) or the β-galactoside α-2,3-sialyltransferase with SEQ ID NO 23 A PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of active UniProt ID Q9CLP3, NmeniST3 from Neisseria meningitidis (SEQ ID NO 24) or from Pasteurella septicaemia strain Pm70 ( PmultST2 of GenBank NO. AAK02592.1), β-galactoside α-2,6-sialyltransferase (UniProt ID 066375) such as PdST6 from Pseudomonas mermaidii, or β-galactoside α-2,6-sialyltransferase such as SEQ ID NO 25 - PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID O66375 with galactoside α-2,6-sialyltransferase activity, P-JT- ISH-224-ST6 (UniProt ID A8QYL1) or consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 having β-galactoside α-2,6-sialyltransferase activity as in SEQ ID NO 26 P-JT-ISH-224-ST6-like polypeptide and/or such as eg Mus musculus α-2,8-sialyltransferase (UniProt ID Q64689).

In one example of the production of UDP-galactose, the yeast expressed plastids were derived from the pRS420-plastid series (Christianson et al., 1992, Gene 110: 119-122), which contained the HIS3 selectable marker and were used as e.g. The continuous transcription unit of the UDP-glucose-4-epimerase of galE of E. coli of SEQ ID NO 30. To produce LN3, this plastid is further modified with a persistent transcription unit for: as for example lactose permease from LAC12 of Kluyveromyces lactis with SEQ ID NO 31, from meningitis with SEQ ID NO 27 Galactoside β-1,3-N-acetylglucosaminyltransferase (lgtA) of Neisseria. To produce LN3-derived oligosaccharides like LNT or LNnT, the plastids are further modified with a persistent transcription unit for, for example, N-acetylide from WbgO of E. coli O55:H7 with SEQ ID NO 28, respectively Glucosamine β-1,3-galactosyltransferase or N-acetylglucosamine β-1,4-galactosyltransferase as for example from IgtB of Neisseria meningitidis having SEQ ID NO 29 enzymes.

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 2'FL - containing modified E. coli hosts expressing α -1,2- fucosyltransferases and α - 1,3 - fucosyltransferases from two compatibilizers Mammalian milk oligosaccharide mixture of , 3-FL and DiFL

E. coli K12 strain modified for GDP-fucose production as described in Example 1 was sequentially expressed with H. pylori alpha-1,2-fucosyltransferase having SEQ ID NO 04 The first plastid of the continuous transcription unit and the expression for the second compatible plastid transformation of the continuous transcription unit with the H. pylori alpha-1,3-fucosyltransferase of SEQ ID NO 05. The novel strains were evaluated in growth experiments according to the culture conditions provided in Example 1, wherein the medium contained sucrose and lactose. 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 a mixture of oligosaccharides comprising 2'FL, 3-FL and difucosylated lactose (DiFL) in whole broth samples. Example 4. Production of 2'FL , 3- _ _ _ _ _ Mammalian milk oligosaccharide mixture of FL and DiFL

E. coli strains modified for GDP-fucose production as described in Example 1 were further transformed with one of the two persistent transcription units, including those for A persistent transcription unit of Helicobacter pylori alpha-1,2-fucosyltransferase having SEQ ID NO 04 and use in Helicobacter pylori alpha-1,3-fucosyltransferase having SEQ ID NO 05 Another continuous transcription unit of an enzyme. The novel strains were evaluated in growth experiments according to the culture conditions provided in Example 1, wherein the medium contained sucrose and lactose. 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 a mixture of oligosaccharides comprising 2'FL, 3-FL and difucosylated lactose (DiFL) in whole broth samples. Example 5. Production of Mammalian Milk Oligosaccharide Mixtures Comprising 2'FL , 3-FL and DiFL Using Modified E. coli Hosts in Fed-Batch Fermentation

Mutant E. coli strains as described in Examples 3 and 4 were further evaluated in a fed-batch fermentation process. Fed-batch fermentation was performed as described in Example 1 at the bioreactor scale. In these examples, sucrose was used as the carbon source and lactose was added as a precursor to the batch medium. Regular broth samples were obtained and UPLC as described in Example 1 was used to measure the production of 2'FL, 3-FL and DiFL. The experiments demonstrated that the broth samples obtained at the end of the batch phase contained oligosaccharide mixtures of 2'FL and 3-FL and unmodified lactose, while the broth samples obtained at the end of the fed-batch phase contained 2' Oligosaccharide mixture of FL, 3-FL and DiFL. Since the ratio of lactose, 2'FL, 3-FL and DiFL changes over time during fed-batch, it can be done during the fermentation process by interrupting the fermentation at desired times during the fed-batch stage process to control. Example 6. Production of Mammalian Lactoligosaccharide Mixtures Containing 2'FL , 3 - FL , DiFL , 2'FLacNAc , 3 -FLacNAc , and Di - FLacNAc Using Modified E. coli Hosts

E. coli strains modified for GDP-fucose production as described in Example 1 were further transformed into plastids with one of the two persistent transcription units that expressed A persistent transcription unit of Helicobacter pylori alpha-1,2-fucosyltransferase having SEQ ID NO 04 and expression of Helicobacter pylori alpha-1,3-fucosyltransferase having SEQ ID NO 05 Another persistent transcription unit. Since lactose and N-acetyllactosamine (LacNAc, Gal-b1,4-GlcNAc) are suitable receptors for both H. pylori fucosyltransferases, growth experiments were performed according to the Culture conditions, novel strains were evaluated for the production of a mixture of oligosaccharides comprising 2'FL, 3-FL and fucosylated LacNAc (i.e. 2'FLacNAc and 3-FLacNAc) in the whole broth sample, wherein the medium contained sucrose as The carbon source as well as lactose and LacNAc were used as precursors. Since the enzyme with SEQ ID NO 04 also showed fucosyltransferase activity on 2'FL and the enzyme with SEQ ID NO 05 also showed fucosyltransferase activity on 2'FLacNAc, it was also useful to generate this Novel strains were evaluated for DiFL and bis-FLacNAc in oligosaccharide mixture. Example 7. Production of Mammalian Milk Oligosaccharide Mixtures Containing 2'FLacNAc , 3-FLacNAc and Di -FLacNAc Using Modified E. coli Hosts Expressing Different Fucosyltransferases

E. coli K-12 MG1655 strain optimized for GDP-fucose production as described in Example 1 with the E. coli N-acetylglucosamine-6-phosphate deacetylase (nagA) gene and deletion of the E. coli glucosamine-6-phosphate deaminase (nagB) gene and gene body insertion of the persistent transcription unit further modified for the production of GlcNAc and LacNAc for the following: having SEQ N-acetylglucosamine beta 1,4-galactosyltransferase (lgtB) of Neisseria meningitidis ID NO 29, mutant L-glutamic acid- from Escherichia coli having SEQ ID NO 19 D-fructose-6-phosphate aminotransferase (glmS*54) and glucosamine 6-phosphate N-acetyltransferase (GNA1) of Saccharomyces cerevisiae having SEQ ID NO 16. In the next step, the novel strain was additionally transformed with two compatible expressing plastids, wherein the first plastid contained a continuation of the H. pylori alpha-1,2-fucosyltransferase having SEQ ID NO 04 type transcription unit, and the second compatible plastid contains a persistent transcription unit for the H. pylori alpha-1,3-fucosyltransferase having SEQ ID NO 05. Alternatively, the novel strain can be transformed with an expressive plastid containing a continuous transcription unit for both the H. pylori fucosyltransferase having SEQ ID NOs 04 and 05. The growth experiments were performed according to the culture conditions provided in Example 1 for the production of 2'-fucosylated LacNAc (2'FLacNAc), 3-fucosylated LacNAc (3-FLacNAc) in the whole broth sample and a mixture of difucosylated LacNAc (di-FLacNAc), where the cultures contained sucrose as carbon source, were evaluated for all novel strains. Example 8. Production of Mammalian Milk Oligosaccharide Mixtures Containing 2'FL , 3-FL , DiFL , 2'FLNB , 4-FLNB and Di - FLNB Using Modified E. coli Hosts

E. coli strains modified for GDP-fucose production as described in Example 1 were further transformed into plastids with one of the two persistent transcription units that expressed A persistent transcription unit of Helicobacter pylori alpha-1,2-fucosyltransferase having SEQ ID NO 04 and expression of Helicobacter pylori alpha-1,3-fucosyltransferase having SEQ ID NO 05 A persistent transcription unit. Since lactose and lacto-N-disaccharide (LNB, Gal-b1,3-GlcNAc) are suitable receptors for both H. pylori fucosyltransferases, growth experiments were performed according to the culture provided in Example 1 Conditions, novel strains were evaluated for the production of a mixture of oligosaccharides comprising 2'FL, 3-FL and fucosylated LNB (ie 2'FLNB and 4-FLNB) in a whole broth sample in which the medium contained sucrose as carbon source and lactose and LNB as precursors. Since the enzyme with SEQ ID NO 04 also showed fucosyltransferase activity on 2'FL and the enzyme with SEQ ID NO 05 also showed fucosyltransferase activity on 2'FLNB, it was also useful to generate this Novel strains were evaluated for DiFL and di-FLNB in oligosaccharide mixture. Example 9. Production of Mammalian Milk Oligosaccharide Mixtures Containing 2'FL , 3-FL , DiFL , 2'FLNB , 4-FLNB and Di - FLNB Using Modified E. coli Hosts

E. coli strains modified for GDP-fucose production as described in Example 1 are further suitable for intracellular lactose synthesis by gene body knockout of lacZ , glk and galETKM operons and Gene body insertion of IgtB of Neisseria meningitidis of SEQ ID NO 29 and a continuous transcription unit from E. coli of SEQ ID NO 30 of UDP-glucose 4-epimerase (galE). In the next step, the mutant E. coli strain was transformed with an expression plastid having two persistent transcription units, namely an expression of the H. pylori alpha-1,2-fucosyltransferase having SEQ ID NO 04 A persistent transcription unit and a persistent transcription unit expressing the Helicobacter pylori alpha-1,3-fucosyltransferase having SEQ ID NO 05. In growth experiments according to the culture conditions provided in Example 1, for the production of 2'FL, 3-FL, DiFL and fucosylated LNB (ie 2'FLNB, 4-FLNB) in the whole culture broth sample and DiFLNB) in an oligosaccharide mixture containing sucrose as a carbon source and LNB as a precursor to evaluate novel strains. Example 10. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2'FLNB , 4-FLNB and Difucosylated LNB Using a Modified E. coli Host

The E. coli K-12 MG1655 strain optimized for GDP-fucose production as exemplified in Example 1 was further modified with deletion of the E. coli nagA and nagB genes and gene body insertion of the persistent transcription unit for Produces GlcNAc and LNB, these persistent transcription units for N-acetylglucosamine beta 1,3-galactosyltransferase (WbgO) from E. coli O55:H7 with SEQ ID NO 28, from Mutant glmS*54 of E. coli of NO 19 and GNA1 from Saccharomyces cerevisiae with SEQ ID NO 16. In the next step, the novel strain was additionally transformed with two compatible expressing plastids, the first plastid containing the persistent form for the H. pylori alpha-1,2-fucosyltransferase having SEQ ID NO 04 Transcription unit, and other plastids contain a persistent transcription unit for the H. pylori alpha-1,3-fucosyltransferase having SEQ ID NO 05. In growth experiments according to the culture conditions provided in Example 1, for the production of 2'-fucosylated LNB (2' FLNB), 4-fucosylated LNB (4-fucosylated LNB (4- FLNB) and oligosaccharide mixtures of difucosylated LNB (di-FLNB) were evaluated for novel strains, where the medium contained sucrose as a carbon source. Example 11. Production of Mammalian Milk Oligosaccharide Mixtures Containing 2'FLNB , 4-FLNB and Difucosylated LNB Using a Modified E. coli Host

The E. coli K-12 MG1655 strain optimized for GDP-fucose production as exemplified in Example 1 was further modified with deletion of the E. coli nagA and nagB genes and gene body insertion of the persistent transcription unit for GlcNAc and LNB were generated for WbgO from E. coli O55:H7 with SEQ ID NO 28, mutant glmS*54 from E. coli with SEQ ID NO 19 and from E. coli with SEQ ID NO 16 GNA1 of Saccharomyces cerevisiae. In the next step, the novel strain is additionally transformed with an expression plastid containing a persistent transcription unit for the H. pylori alpha-1,2-fucosyltransferase having SEQ ID NO 04 and Continuous Transcription Unit for Helicobacter pylori alpha-1,3-fucosyltransferase having SEQ ID NO 05. In the growth experiments according to the culture conditions provided in Example 1, for the production of 2'-fucosylated LNB (2' FLNB), 4-fucosylated LNB (4-fucosylated LNB (4- FLNB) and oligosaccharide mixtures of difucosylated LNB (di-FLNB) were evaluated for novel strains, where the medium contained sucrose as a carbon source. Example 12. Production of Mammalian Lactoligosaccharides Comprising 2'FL, DiFL , 2'FLNB and Gal - a1,3- ( Fuc - a1,2)-Gal-b1,3-GlcNAc Using Modified E. coli Hosts mixture

E. coli strains modified for GDP-fucose production as described in Example 1 exhibited plastid transformation with one with persistent transcription units α-1,2-fucosyltransferase from Helicobacter pylori and α-1,3-galactosyltransferase WbnI from Escherichia coli with SEQ ID NO 37. Since lactose and LNB are suitable receptors for H. pylori α-1,2-fucosyltransferase, in the growth experiments, according to the culture conditions provided in Example 1, for the production of 2'FL, DiFL and 2'FL, DiFL and Oligosaccharide mixtures of '-fucosylated LNB (2'FLNB) were evaluated for novel strains in which the medium contained sucrose as carbon source and lactose and LNB as precursors. In this experiment, since 2'FLNB is the acceptor of Escherichia coli α-1,3-galactosyltransferase, it also targets Gal-a1,3-(Fuc-a1,2)-Gal- Novel strains were evaluated for b1,3-GlcNAc production. Example 13. Production of Mammalian Milk Oligosaccharides Comprising 2'FL, DiFL , 2'FLNB and GalNAc - a1,3- ( Fuc -a1,2)-Gal-b1,3-GlcNAc Using Modified E. coli Hosts mixture

E. coli strains modified for GDP-fucose production as described in Example 1 exhibited plastid transformation with one with persistent transcription units α-1,2-fucosyltransferase of Helicobacter pylori and α-1,3-N-acetylgalactosaminyltransferase BgtA from Helicobacter weasels having SEQ ID NO 38. Since lactose and LNB are suitable receptors for H. pylori α-1,2-fucosyltransferase, in the growth experiments, according to the culture conditions provided in Example 1, for the production of 2'FL, DiFL and 2'FL, DiFL and Oligosaccharide mixtures of '-fucosylated LNB (2'FLNB) were evaluated for novel strains in which the medium contained sucrose as carbon source and lactose and LNB as precursors. In this experiment, since 2'FLNB is the receptor for the α-1,3-N-acetylgalactosaminyltransferase of Helicobacter weasel, it also targets GalNAc-a1,3-(Fuc in the oligosaccharide mixture. -a1,2)-Gal-b1,3-GlcNAc production evaluation of novel strains. Example 14. Production of Mammalian Lactoligosaccharides Comprising 2'FL, DiFL , 2'FLNB and Gal - a1,3- ( Fuc - a1,2)-Gal-b1,3-GlcNAc Using Modified E. coli Hosts mixture

E. coli strains modified for GDP-fucose production as described in Example 1 were further modified with deletion of the E. coli nagA and nagB genes and gene body insertion of a persistent transcription unit for the production of GlcNAc and LNB, which Isocontinuous transcription units for N-acetylglucosamine beta 1,3-galactosyltransferase (WbgO) from E. coli O55:H7 with SEQ ID NO 28, from E. coli with SEQ ID NO 19 Mutant glmS*54 and GNA1 from Saccharomyces cerevisiae with SEQ ID NO 16. In the next step, the novel strain was transformed with an expression plastid containing the alpha-1,2-fucosyltransferase from H. pylori having SEQ ID NO 04 and the The continuous transcription unit of the α-1,3-galactosyltransferase WbnI of Escherichia coli of NO 37. In growth experiments according to the culture conditions provided in Example 1, for the production of 2'FL, DiFL, 2'-fucosylated LNB and Gal-a1,3-(Fuc-a1,2)-Gal- Oligosaccharide mixtures of b1,3-GlcNAc were evaluated for novel strains in which the medium contained sucrose as carbon source and lactose as precursor. Example 15. Production of mammalian milk oligosaccharides comprising 2'FL, DiFL , 2'FLNB and GalNAc - a1,3- ( Fuc -a1,2)-Gal-b1,3-GlcNAc using a modified E. coli host mixture

E. coli strains modified for GDP-fucose production as described in Example 1 were further modified with deletion of the E. coli nagA and nagB genes and gene body insertion of a persistent transcription unit for the production of GlcNAc and LNB, which Iso-persistent transcription units were used for WbgO from E. coli O55:H7 with SEQ ID NO 28, mutant glmS*54 from E. coli with SEQ ID NO 19 and GNA1 from Saccharomyces cerevisiae with SEQ ID NO 16. In the next step, the novel strain was transformed with an expression plastid containing the alpha-1,2-fucosyltransferase from H. pylori having SEQ ID NO 04 and the The continuous transcription unit of the α-1,3-N-acetylgalactosaminyltransferase BgtA of Helicobacter weaselis NO 38. In growth experiments according to the culture conditions provided in Example 1, for the production of 2'FL, DiFL, 2'-fucosylated LNB and GalNAc-a1,3-(Fuc-a1,2)-Gal- Oligosaccharide mixtures of b1,3-GlcNAc were evaluated for novel strains in which the medium contained sucrose as a carbon source and lactose as a precursor. Example 16. Production of a Mammalian Milk Oligosaccharide Mixture Containing 3'SL , 6'SL , 3'- sialylated LacNAc and 6' - sialylated LacNAc Using a Modified E. coli Host

Escherichia coli K-12 MG1655 strain with gene body insertion for a persistent transcription unit from Pasteurella septicemia with SEQ ID NO 22 Modified and contained a knockout of the E. coli lacZ gene, further transformed with an expression plastid containing the alpha-2,3-sialyltransferase for use from Pasteurella septicemia having SEQ ID NO 23 and A persistent transcription unit from the alpha-2,6-sialyltransferase of P. mermaidii having SEQ ID NO 25. In growth experiments according to the culture conditions provided in Example 1, for the production of 3'SL, 6'SL, 3'-sialylated LacNAc (3'SLacNAc) and 6'-sialylated LacNAc (3'SLacNAc) and 6'-sialylated ( 6'SLacNAc) in an oligosaccharide mixture to evaluate the novel strain, in which the medium contained glycerol as carbon source and sialic acid, lactose and LacNAc as precursors. Example 17. Production of a Mammalian Milk Oligosaccharide Mixture Containing 3'SL , 6'SL , 3'- sialylated LacNAc and 6' - sialylated LacNAc Using a Modified E. coli Host

Escherichia coli K-12 MG1655 strain with gene body insertion for a persistent transcription unit from Pasteurella septicemia with SEQ ID NO 22 Modification, further with gene body deletion of E. coli nagA , nagB and lacZ genes and gene body insertion mutation of the persistent transcription unit for mutant glmS*54 with SEQ ID NO 19 from E. coli , GNA1 with SEQ ID NO 16 from Saccharomyces cerevisiae, phosphatase yqaB from E. coli with SEQ ID NO 20, and LgtB with SEQ ID NO 29 from Neisseria meningitidis. In the next step, the novel strain was transformed with an expression plastid containing the alpha-2,3-sialyltransferase from Pasteurella septicemia having SEQ ID NO 23 and the Persistent transcription unit of α-2,6-sialyltransferase of P. mermaidii ID NO 25. In growth experiments according to the culture conditions provided in Example 1, for the production of 3'SL, 6'SL, 3'-sialylated LacNAc (3'SLacNAc) and 6'-sialylated LacNAc in the whole culture fluid sample A mixture of oligosaccharides (6'SLacNAc) was evaluated for novel strains in which the medium contained glycerol as a carbon source and sialic acid and lactose as precursors. Example 18. Production of a Mammalian Milk Oligosaccharide Mixture Containing 3'SL , 6'SL , 3'- sialylated LacNAc and 6' - sialylated LacNAc Using a Modified E. coli Host

The E. coli K-12 MG1655 strain modified to produce sialic acid as described in Example 1 was further modified with deletion of the E. coli lacZ gene and transformed with an expression plastid derived from a plasmid with SEQ ID NO. neuA of Pasteurella septicemia of 22, α-2,3-sialyltransferase from Pasteurella septicemia with SEQ ID NO 23, and α-2 from Photobacterium mermaidii with SEQ ID NO 25, Composition of the continuous transcription unit of 6-sialyltransferase. In growth experiments according to the culture conditions provided in Example 1, for the production of 3'SL, 6'SL, 3'-sialylated LacNAc (3'SLacNAc) and 6'-sialylated LacNAc in the whole culture fluid sample A mixture of oligosaccharides (6'SLacNAc) was evaluated for novel strains in which the medium contained glycerol as carbon source and lactose and LacNAc as precursors. Example 19. Production of a Mammalian Milk Oligosaccharide Mixture Containing 3'SL , 6'SL , 3'- sialylated LacNAc and 6' - sialylated LacNAc Using a Modified E. coli Host

The E. coli K-12 MG1655 strain modified to produce sialic acid as described in Example 1 was further genetically knocked out of the E. coli lacZ gene and used for LgtB from Neisseria meningitidis with SEQ ID NO 29 The gene body of the persistent transcription unit was mutated to generate LacNAc and transformed with an expression plastid containing neuA for Pasteurella septicemia with SEQ ID NO 22, Alpha-2,3-sialyltransferase from Pasteurella septicemia and a continuous transcription unit from the alpha-2,6-sialyltransferase from Photoluminum mermaidii having SEQ ID NO 25. In growth experiments according to the culture conditions provided in Example 1, for the production of 3'SL, 6'SL, 3'-sialylated LacNAc (3'SLacNAc) and 6'-sialylated LacNAc in the whole culture fluid sample A mixture of oligosaccharides (6'SLacNAc) was used to evaluate novel strains in which the medium contained glycerol as a carbon source and lactose as a precursor. Example 20. Production of sialylated LacNAc and polyLacNAc structures using modified E. coli hosts

The E. coli K-12 MG1655 strain modified to produce sialic acid as described in Example 1 was further mutated by gene body insertion for the persistent transcription unit with LgtB of SEQ ID NO 29 from Neisseria meningitidis to generate LacNAc and transformed with an expression plastid containing neuA for neuA from Pasteurella septicemia having SEQ ID NO 22, alpha-2 from Pasteurella septicemia having SEQ ID NO 23 , 3-sialyltransferase and a continuous transcription unit from the alpha-2,6-sialyltransferase of Photolumines mermaidii having SEQ ID NO 25. In the next step, the mutant strain was further transformed with a compatible expression plastid containing galactoside β-1,3-N from Neisseria meningitidis with SEQ ID NO 27 - The continuous transcription unit of acetylglucosaminyltransferase (LgtA). In growth experiments according to the culture conditions provided in Example 1, the novel strain was evaluated for the production of LacNAc and an oligosaccharide mixture comprising a polyLacNAc structure, namely (Gal-b1,4-GlcNAc)n, the structure Constructed from repeating N-acetyllactosamine units, β1,3 linked to each other by the alternative activities of LgtB transferase having SEQ ID NO 29 and LgtA transferase having SEQ ID NO 27; and 3'-sialylated LacNAc and 6'-sialylated LacNAc; and a sialylated polyLacNAc structure in which the Gal residues are sialylated, wherein the medium contains glycerol as a carbon source and wherein no precursor is required to supply the culture. Example 21. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3'SL , 6'SL , 3'- sialylated LNB and 6' - sialylated LNB Using Modified E. coli Hosts

Escherichia coli K-12 MG1655 strain modified by gene body insertion for the persistent transcription unit of neuA from Pasteurella septicemia with SEQ ID NO 22 and containing deletion of the Escherichia coli lacZ gene, further expressed in plastids Transformed, this expression plastid contains for α-2,3-sialyltransferase from Pasteurella septicemia with SEQ ID NO 23 and α-2 from Photoluminum mermaidii with SEQ ID NO 25, The continuous transcription unit of 6-sialyltransferase. In growth experiments according to the culture conditions provided in Example 1, for the production of 3'SL, 6'SL, 3'-sialylated LNB (3'SLNB) and 6'-sialylated LNB in the whole culture fluid sample A mixture of oligosaccharides (6'SLNB) was used to evaluate novel strains in which the medium contained glycerol as carbon source and sialic acid, lactose and LNB as precursors. Example 22. Production of Mammalian Milk Oligosaccharide Mixture Comprising 3'SL , 6'SL , 3'- sialylated LNB and 6' - sialylated LNB Using Modified E. coli Hosts

Escherichia coli K-12 MG1655 strain modified with gene body insertion for the persistent transcription unit of neuA from Pasteurella septicemia having SEQ ID NO 22, further modified with gene bodies of Escherichia coli nagA , nagB and lacZ genes Knockout and gene body insertion mutation of persistent transcription units for mutant glmS*54 with SEQ ID NO 19 from E. coli, GNA1 with SEQ ID NO 16 from Saccharomyces cerevisiae, Phosphatase yqaB of E. coli of SEQ ID NO 20 and WbgO of SEQ ID NO 28 from E. coli O55:H7 to produce LNB. In the next step, the novel strain is transformed with an expression plastid containing the alpha-2,3-sialyltransferase from Pasteurella septicemia having SEQ ID NO 23 and the Persistent transcription unit of α-2,6-sialyltransferase of P. mermaidii ID NO 25. In growth experiments according to the culture conditions provided in Example 1, for the production of 3'SL, 6'SL, 3'-sialylated LNB (3'SLNB) and 6'-sialylated LNB in the whole culture fluid sample (6'SLNB) oligosaccharide mixture to evaluate novel strains, in which the medium contains glycerol as carbon source and sialic acid and lactose as precursors. Example 23. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3'SL , 6'SL , 3'- sialylated LNB and 6' - sialylated LNB Using Modified E. coli Hosts

The E. coli K-12 MG1655 strain modified to produce sialic acid as described in Example 1 was further modified with deletion of the E. coli lacZ gene and transformed with an expression plastid derived from a plasmid with SEQ ID NO. neuA of Pasteurella septicemia of 22, α-2,3-sialyltransferase from Pasteurella septicemia with SEQ ID NO 23, and α-2 from Photobacterium mermaidii with SEQ ID NO 25, Composition of the continuous transcription unit of 6-sialyltransferase. In growth experiments according to the culture conditions provided in Example 1, for the production of 3'SL, 6'SL, 3'-sialylated LNB (3'SLNB) and 6'-sialylated LNB in the whole culture fluid sample A mixture of oligosaccharides (6'SLNB) was used to evaluate novel strains in which the medium contained glycerol as carbon source and lactose and LNB as precursors. Example 24. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3'SL , 6'SL , 3'- sialylated LNB and 6' - sialylated LNB Using Modified E. coli Hosts

The E. coli K-12 MG1655 strain modified to produce sialic acid as described in Example 1 was further genetically knocked out of the E. coli lacZ gene and used for persistence of WbgO with SEQ ID NO 28 from E. coli O55:H7 The gene body of the type transcription unit was mutated to generate LNB and transformed with an expression plastid containing neuA from Pasteurella septicemia with SEQ ID NO 22, Alpha-2,3-sialyltransferase from Pasteurella haemorrhagic and a continuous transcription unit from the alpha-2,6-sialyltransferase from Photoluminum mermaidii having SEQ ID NO 25. In growth experiments according to the culture conditions provided in Example 1, for the production of 3'SL, 6'SL, 3'-sialylated LNB (3'SLNB) and 6'-sialylated LNB in the whole culture fluid sample (6'SLNB) mixture of oligosaccharides to evaluate novel strains in which the medium contained glycerol as carbon source and lactose as precursor. Example 25. Production of sialylated LNB using a modified E. coli host

The E. coli K-12 MG1655 strain modified to produce sialic acid as described in Example 1 was further subjected to gene body insertion mutations for the persistent transcription unit with WbgO of SEQ ID NO 28 from E. coli O55:H7 to LNB was produced and transformed with an expression plastid containing neuA for neuA from Pasteurella septicemia having SEQ ID NO 22, alpha-2 from Pasteurella septicemia having SEQ ID NO 23, 3-Sialyltransferase and a persistent transcription unit from the alpha-2,6-sialyltransferase of Photophora mermaidii having SEQ ID NO 25. Novel strains were evaluated for production of LNB as well as 3'-sialylated LNB and 6'-sialylated LNB in growth experiments according to the culture conditions provided in Example 1, where the medium contained glycerol as a carbon source and where no addition to the culture was required Supply precursors. Example 26. Production of Mammalian Milk Oligosaccharide Mixtures Containing GlcNAc-bl,3-Gal-bl,4-GlcNAc and Poly- LacNAc Structures by Modified E. coli Hosts

The E. coli K-12 MG1655 strain was knocked out of E. coli nagA and nagB genes and used for mutant glmS*54 with SEQ ID NO 19 from E. coli, GNA1 with SEQ ID NO 16 from S. cerevisiae and from meningitis Gene body insertion modification of the continuous transcription unit of Neisseria with LgtB of SEQ ID NO 29 to produce LacNAc. In the next step, the novel strain was additionally transformed with an expression plastid containing beta-1,3-N-acetyl galactoside for galactoside from Neisseria meningitidis with SEQ ID NO 27 The continuous transcription unit of glucosaminotransferase (LgtA). By subsequent action of the glmS*54 enzyme and the homologous EcGlmM and EcGlmU enzymes, the mutant strain is able to produce UDP-GlcNAc, which is used by the heterologous LgtA protein to modify LacNAc into GlcNAc-b1,3-Gal-b1,4- GlcNAc. The novel strains were evaluated for production of the GlcNAc-b1,3-Gal-b1,4-GlcNAc in whole culture broth samples in growth experiments according to the culture conditions provided in Example 1, wherein the medium contained glycerol as a carbon source. The novel strain was also evaluated for the production of a poly-LacNAc structure, namely (Gal-b1,4-GlcNAc)n, which is constructed from repeating N-acetyllactosamine units, by the heterologous LgtB expressed in the strain and The alternative activities of LgtA are β1,3 linked to each other. Example 27. Production of GlcNAc-b1,3-Gal-b1,4-GlcNAc , β -Gal-(1,4) -β- GlcNAc-(1,3)-[β - GalNAc-(1,4) -β- GlcNAc-(1,3)-[ β- Mammalian milk oligosaccharide mixture of GlcNAc- (1,6)]- β- Gal-(1,4)-GlcNAc and poly- LacNAc structures

In the next example, mutations suitable to generate LacNAc, UDP-GlcNAc, GlcNAc-b1,3-Gal-b1,4-GlcNAc and (Gal-b1,4-GlcNAc)n structures as described in Example 26 The somatic E. coli strain was further modified with a continuous transcription unit for the human N-acetyllactosamine beta-1,6-N-acetylglucosaminyltransferase GCNT2 having SEQ ID NO 32. The second performance plastid modification. In the growth experiments according to the culture conditions provided in Example 1, for the production of LacNAc, GlcNAc-b1,3-Gal-b1,4-GlcNAc, GlcNAc-b1,6-Gal-b1, Structure of 4-GlcNAc, (Gal-b1,4-GlcNAc)n and β-Gal-(1,4)-β-GlcNAc-(1,3)-[β-GlcNAc-(1,6)]-β- Oligosaccharide mixtures of Gal-(1,4)-GlcNAc were evaluated for novel strains in which the medium contained glycerol as a carbon source. Example 28. Production of Mammalian Milk Oligosaccharide Mixture Comprising Gal-a1,3-LacNAc , LN3 , LNnT and Gal-a1,3-LNnT by Modified E. coli Host

E. coli strains modified to produce LacNAc as described in Example 26 were further modified with genomic deletion of the E. coli lacZ gene and transformed with a compatible expressing plastid containing a persistent transcription unit for C-terminal catalytic domain of bovine alpha-1,3-galactosyltransferase (a3FTcd) having SEQ ID NO 33. In the growth experiments according to the culture conditions provided in Example 1, for the production of Gal-a1,3-LacNAc (Gal-a1,3-Gal-b1,4-GlcNAc), LN3, LNnT in the whole culture fluid sample and Gal-a1,3-LNnT (Gal-a1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc) oligosaccharide mixture evaluation of novel strains, wherein the medium contains glycerol as carbon source and lactose as precursors. Example 29. Production of LacNAc , GalNAc- b1,3- lactose, Gal-b1,3-GalNAc-b1,3- lactose, LN3 , LNnT , GalNAc-b1,3-LacNAc , Gal using a modified E. coli host Mammalian milk oligosaccharide mixture of -b1,3-GalNAc-b1,3-LacNAc , poly -LacNAc (Gal-b1,4-GlcNAc)n and GalNAc -ylated poly - LacNAc structures

The E. coli strains modified to produce LacNAc as described in Example 26 were further modified with gene body deletion of the E. coli lacZ gene and gene body insertion of persistent expression units for those having SEQ ID NO 4-Epimerase (WbpP) of Pseudomonas aeruginosa of 34, galactoside β-1,3-N-acetylglucosamine group from Neisseria meningitidis with SEQ ID NO 27 Transferase (LgtA) and β1,3-N-acetylgalactosaminyltransferase (LgtD) from Haemophilus influenzae having SEQ ID NO 35. In growth experiments according to the culture conditions provided in Example 1, the novel strain was evaluated for the production of an oligosaccharide mixture comprising LacNAc, LN3, LNnT, GalNAc-b1,3-lactose, Gal-b1,3-GalNAc -b1,3-lactose, GalNAc-b1,3-LacNAc, Gal-b1,3-GalNAc-b1,3-LacNAc, poly-LacNAc structure, namely (Gal-b1,4-GlcNAc)n, which consists of Repeated N-acetylglucosamine units constructed by N-acetylglucosamine β-1,4-galactosyltransferase and galactoside β-1,3-N-acetylglucosyl The alternative activities of glucosaminotransferase and GalNAc-ylated poly-LacNAc structures are β1,3 linked to each other, with the medium containing glycerol as carbon source and lactose as precursor. Example 30. Production of LNB , LN3 , LNT , GalNAc- b1,3- lactose, Gal-b1,3-GalNAc-b1,3- lactose, GalNAc-b1,3-LNB and Mammalian milk oligosaccharide mixture of Gal-b1,3-GalNAc-b1,3-LNB

E. coli strains were modified with gene body deletion of the E. coli nagB gene and gene body insertion of a persistent expression cassette for mutant glmS with SEQ ID NO 19 from E. coli to generate LNB 54. GNA1 having SEQ ID NO 16 from Saccharomyces cerevisiae and WbgO having SEQ ID NO 28 from E. coli O55:H7. In the next step, the LNB-producing E. coli strain was further modified with gene body deletion of the E. coli lacZ gene and gene body insertion modification of the persistent expression unit for Pseudomonas aeruginosa having SEQ ID NO 34 4-Epimerase of Monomonas (WbpP), Galactoside β-1,3-N-Acetylglucosaminyltransferase (LgtA) from Neisseria meningitidis having SEQ ID NO 27 and β1,3-N-acetylgalactosaminyltransferase (LgtD) from Haemophilus influenzae having SEQ ID NO 35. In the growth experiments according to the culture conditions provided in Example 1, for the production of LNB, LN3, LNT, GalNAc-b1,3-lactose, Gal-b1,3-GalNAc-b1,3-lactose, GalNAc-b1, Oligosaccharide mixture of 3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, GalNAc-b1,3-LNB and Gal-b1,3-GalNAc-b1,3-LNB evaluation of novel strains , wherein the medium contains glycerol as a carbon source and lactose as a precursor. Example 31. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Lactose Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as exemplified in Example 1 were further transformed with two compatible expressing plastids, wherein the first plastid contained the base for one or two selected fucose groups Persistent expression unit of transferase and wherein the second plastid contains for one or two selected sialyltransferases and N-acylneuraminic acid cytidine from Pasteurella septicemia having SEQ ID NO 22 Persistent expression unit of basal transferase (NeuA). Table 2 presents a summary of the six plastids used. Novel strains were evaluated in growth experiments according to the culture conditions provided in Example 1 for the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose structures as shown in Table 3 in whole broth samples, wherein The cultures contained sucrose as a carbon source and sialic acid and lactose as precursors. Table 2 : Summary of plastids with persistent transcription units for one or two fucosyltransferase genes or for one or two sialyltransferase genes plastid Fucosyltransferase expressed a1,2 -bonded a1,3 -bonded pMF_1A SEQ ID NO 04 none pMF_1B none SEQ ID NO 05 pMF_2 SEQ ID NO 04 SEQ ID NO 05 plastid sialyltransferase a2,3 -bonded a2,6 -bonded pMS_1A SEQ ID NO 23 none pMS_1B none SEQ ID NO 25 pMS_2 SEQ ID NO 23 SEQ ID NO 25 Table 3 : Evaluation of oligosaccharide production in mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contained sucrose as carbon source and sialic acid and lactose as precursors. strain plastid that exists * Oligosaccharide SF1 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL SF2 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL SF3 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL SF4 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL SF5 pMF_1A, pMS_2 2'FL, 3'SL, 3'S-2'FL, 6'SL, 6'S-2'FL SF6 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL SF7 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL SF8 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL SF9 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL *See Table 2 for plastid information . Example 32. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Lactose Structures Using Modified E. coli Hosts

The E. coli strain adapted for GDP-fucose production as exemplified in Example 1 was further modified with gene body deletion of the E. coli genes nagA , nagB , nanA , nanE and nanK and gene body insertion of the persistent transcription unit to For sialic acid production, these persistent transcription units are used for mutant glmS*54 from E. coli with SEQ ID NO 19, GNA1 from Saccharomyces cerevisiae with SEQ ID NO 16, oval with SEQ ID NO 17 Bacillus N-acetylglucosamine 2-epimerase (AGE) and Neisseria meningitidis N-acetylneuraminic acid synthase (neuB) having SEQ ID NO 18. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. Novel strains were evaluated in growth experiments according to the culture conditions provided in Example 1 for the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose structures as shown in Table 4 in whole broth samples, wherein The cultures contained sucrose as a carbon source and lactose as a precursor. Table 4 : Evaluation of oligosaccharide production in mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contained sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF10 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL SF11 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL SF12 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL SF13 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL SF14 pMF_1A, pMS_2 2'FL, 3'SL, 3'S-2'FL, 6'SL, 6'S-2'FL SF15 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL SF16 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL SF17 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL SF18 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL *See Table 2 for plastid information . Example 33. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Lactose Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as exemplified in Example 1 were further modified with gene body deletion of the E. coli genes nagA , nagB , nanA , nanE and nanK and gene body insertion of the persistent transcription unit to For sialic acid production, these persistent transcription units are used in mutant glmS*54 from E. coli with SEQ ID NO 19, UDP-N-acetylglucosamine 2 from Campylobacter jejuni with SEQ ID NO 21 - Epimerase (neuC) and N-acetylneuraminic acid synthase (neuB) of Neisseria meningitidis having SEQ ID NO 18. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. Novel strains were evaluated in growth experiments according to the culture conditions provided in Example 1 for the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose structures as shown in Table 5 in whole broth samples, wherein The cultures contained sucrose as a carbon source and lactose as a precursor. Table 5 : Evaluation of oligosaccharide production in mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contained sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF19 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL SF20 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL SF21 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL SF22 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL SF23 pMF_1A, pMS_2 2'FL, 3'SL, 3'S-2'FL, 6'SL, 6'S-2'FL SF24 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL SF25 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL SF26 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL SF27 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL *See Table 2 for plastid information . Example 34. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Lactose Structures Using Modified E. coli Hosts

E. coli strains adapted for sialic acid production as exemplified in Example 1 were further modified by genomic knockout of the E. coli wcaJ gene to increase the intracellular pool of GDP-fucose. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. Novel strains were evaluated in growth experiments according to the culture conditions provided in Example 1 for the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose structures as shown in Table 6 in whole broth samples, wherein The cultures contained sucrose as a carbon source and lactose as a precursor. Table 6 : Evaluation of oligosaccharide production in mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contained sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF28 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL SF29 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL SF30 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL SF31 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL SF32 pMF_1A, pMS_2 2'FL, 3'SL, 3'S-2'FL, 6'SL, 6'S-2'FL SF33 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL SF34 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL SF35 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL SF36 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL *See Table 2 for plastid information . Example 35. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Lactose Structures Using Modified E. coli Hosts

E. coli strains adapted for sialic acid production as exemplified in Example 1 were further modified by gene body deletion of E. coli wcaJ , fucK and fucI genes and gene body insertion of persistent expression units to increase GDP-fucose Intracellular pools of these persistent expression units for fucose permease (fucP) from Escherichia coli with SEQ ID NO 13 and bifunctional fucose kinase/ Fucose-1-phosphate guanosyltransferase (fkp). In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. Novel strains were evaluated in growth experiments according to the culture conditions provided in Example 1 for the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose structures as shown in Table 7 in whole broth samples, wherein The cultures contained sucrose as a carbon source and lactose as a precursor. Table 7 : Evaluation of oligosaccharide production in mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contained sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF37 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL SF38 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL SF39 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL SF40 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL SF41 pMF_1A, pMS_2 2'FL, 3'SL, 3'S-2'FL, 6'SL, 6'S-2'FL SF42 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL SF43 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL SF44 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL SF45 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL *See Table 2 for plastid information , Example 36. Production of a Mammalian Milk Oligosaccharide Mixture Containing LN3 , Siallylated LN3 , LNT , 3'SL and LSTa Using a Modified E. coli Host

E. coli strains modified to produce LNT as described in Example 1 were further modified with genomic deletion of the E. coli lacZ gene and transformed with expression plastids containing persistent expression cassettes using In NeuA from Pasteurella septicemia with SEQ ID NO 22 and alpha-2,3-sialyltransferase from Pasteurella septicemia with SEQ ID NO 23. In growth experiments according to the culture conditions in the 96-well plates provided in Example 1, for the production of LN3, 3'-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4- Glc), LNT, 3'SL and LSTa (Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) oligosaccharide mixture to evaluate novel strains in which the medium contained glycerol as A carbon source and both sialic acid and lactose serve as precursors. After 72 h of incubation, the broth was collected and the sugar mixture was analyzed as described in Example 1. Example 37. Production of a mammalian milk oligosaccharide mixture comprising 6'SL , LN3 , sialylated LN3 , LNnT and LSTc using a modified E. coli host

E. coli strains modified to produce LNnT as described in Example 1 were further modified with genomic deletion of the E. coli lacZ gene and transformed with expression plastids containing persistent expression cassettes using In NeuA from Pasteurella septicemia having SEQ ID NO 22 and one selected alpha-2,6-sialyltransferase. In this experiment, the α-2,6-sialyltransferase from Photoluminum mermaidii with SEQ ID NO 25 and α-2 from Photoluminum species JT-ISH-224 with SEQ ID NO 26 were tested, A 6-sialyltransferase wherein the alpha-2,6-sialyltransferase having SEQ ID NO 26 is expressed by two distinct transcription units. Therefore, three different strains were generated, each expressing a single alpha-2,6-sialyltransferase in a specific transcription unit. Table 8 shows a summary of the transcription units used for the selected alpha-2,6-sialyltransferase proteins. Novel strains were evaluated in growth experiments in 96-well plates according to the culture conditions provided in Example 1, where the medium contained glycerol as a carbon source and both sialic acid and lactose as precursors. After 72 h of incubation, the broth was collected and the sugar mixture was analyzed as described in Example 1. All novel strains produced oligosaccharide mixtures comprising 6'SL, LN3, LNnT and LSTc (Neu5Ac-a2,6-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc). FIG. 1 shows a chromatogram obtained from strain S2 analyzed by the Dionex method as described in Example 1. FIG. The compound 6'-sialylated LN3 (Neu5Ac-a2,6-(GlcNAc-bl,3)-Gal-bl, 4-Glc). The ratio of 6'SL, LN3, LNnT and LSTc produced in a mixture of new strains can be determined by selecting the α-2,6-sialyltransferases expressed and the transcription units used to express these sialyltransferases for precise adjustment. Table 8 : Summary of mutant E. coli strains expressing different a-2,6-sialyltransferases in specific transcription units. strain transcription unit Promoter UTR SEQ ID NO CDS terminator S1 "PROM0005_MutalikP5" "UTR0010_GalE_BCD12" 25 "TER0007_ilvGEDA" S2 "PROM0005_MutalikP5" "UTR0010_GalE_BCD12" 26 "TER0007_ilvGEDA" S3 "PROM0050_apFAB82" "UTR0014_GalE_LeuAB" 26 "TER0007_ilvGEDA" Example 38. Production of a mammalian milk oligosaccharide mixture comprising LN3 , LNnT , sialylated LN3 , 3'SL and LSTd using a modified E. coli host

E. coli strains modified to produce LNnT as described in Example 1 were further modified with genomic deletion of the E. coli lacZ gene and transformed with expression plastids containing persistent expression cassettes using In NeuA from Pasteurella septicemia having SEQ ID NO 22 and one selected alpha-2,3-sialyltransferase. In this experiment, alpha-2,3-sialyltransferase from Pasteurella septicemia with SEQ ID NO 23 and alpha-2,3 from Neisseria meningitidis with SEQ ID NO 324 were tested - Sialyltransferases, and alpha-2,3-sialyltransferases each colonize in two different transcription units. Thus, four different strains were generated, each expressing a single alpha-2,3-sialyltransferase in a specific transcription unit. Table 9 shows a summary of the transcription units used for the selected alpha-2,3-sialyltransferase proteins. Novel strains were evaluated in growth experiments in 96-well plates according to the culture conditions provided in Example 1, where the medium contained glycerol as a carbon source and both sialic acid and lactose as precursors. After 72 h of incubation, the broth was collected and the sugar mixture was analyzed as described in Example 1. All novel strains produced oligosaccharide mixtures comprising 3'SL, LN3, LNnT and LSTd (Neu5Ac-a2,3-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc). FIG. 2 shows a chromatogram obtained from strain S5 analyzed by the Dionex method as described in Example 1. FIG. The compound 3'-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4- ) can also be detected in these samples using UPLC with RI detection as described in Example 1 Glc). The ratio of 3'SL, LN3, LNnT and LSTd produced in a mixture of new strains can be determined by selecting the α-2,3-sialyltransferases expressed and the transcription units used to express these sialyltransferases for precise adjustment. Table 9 : Summary of mutant E. coli strains expressing different alpha-2,3-sialyltransferases in specific transcription units. strain transcription unit Promoter UTR SEQ ID NO CDS terminator S4 "PROM0050_apFAB82" "UTR0014_GalE_LeuAB" twenty three "TER0007_ilvGEDA" S5 "PROM0005_MutalikP5" "UTR0010_GalE_BCD12" twenty three "TER0007_ilvGEDA" S6 "PROM0050_apFAB82" "UTR0014_GalE_LeuAB" twenty four "TER0007_ilvGEDA" S7 "PROM0005_MutalikP5" "UTR0010_GalE_BCD12" twenty four "TER0007_ilvGEDA" Example 39. Production of a mammalian milk oligosaccharide mixture comprising LN3 , sialylated LN3 , LNT , 3'SL 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 units for use in the meninges derived from the meninges having SEQ ID NO 27 to allow the production of LNTs Galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) of Neisseria spp. and N-acetylglucosamine beta from Escherichia coli O55:H7 with SEQ ID NO 28 -1,3-Galactosyltransferase (WbgO). 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 septicaemia derived from septicaemia with SEQ ID NO 22 NeuA from Bacillus and alpha-2,3-sialyltransferase from Pasteurella septicemia having SEQ ID NO 23. In growth experiments according to the culture conditions provided in Example 1, for the production of LN3, 3'-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc), LNT, Oligosaccharide mixtures of 3'SL and LSTa evaluated novel strains in which the medium contained sucrose as carbon source and lactose as precursor. Example 40. Production of a mammalian milk oligosaccharide mixture comprising LN3 , sialylated LN3 , LNnT , 6'SL and LSTc 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 units for the meninges from the meninges having SEQ ID NO 27 to allow the production of LNnT LgtA from Neisseria meningitidis and LgtB from Neisseria meningitidis with SEQ ID NO 29. 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 septicaemia derived from septicaemia with SEQ ID NO 22 NeuA from Bacillus sp. and alpha-2,6-sialyltransferase from Photophora sp. JT-ISH-224 with SEQ ID NO 26. Novel strains were evaluated in growth experiments in 96-well plates according to the culture conditions provided in Example 1, where the medium contained sucrose as carbon source and lactose as precursor. After 72 h of incubation, the broth was collected and the sugar mixture was analyzed on UPLC. Experiments show that the novel strain produces an oligosaccharide mixture comprising LN3, 6'-sialylated LN3 (Neu5Ac-a2,6-(GlcNAc-b1,3)-Gal-b1,4-Glc), 6'SL, LNnT and LSTc . Example 41. Production of a mammalian milk oligosaccharide mixture comprising LN3 , sialylated LN3 , LNnT , 3'SL and LSTd 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 units for the meninges from the meninges having SEQ ID NO 27 to allow the production of LNnT LgtA from Neisseria meningitidis and LgtB from Neisseria meningitidis with SEQ ID NO 29. 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 septicaemia derived from septicaemia with SEQ ID NO 22 NeuA from Bacillus and alpha-2,3-sialyltransferase from Pasteurella septicemia having SEQ ID NO 23. In growth experiments according to the culture conditions provided in Example 1, for the production of LN3, 3'-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc), LNnT, Oligosaccharide mixtures of 3'SL and LSTd were evaluated for novel strains in which the medium contained sucrose as carbon source and lactose as precursor. Example 42. Production of fermentation broths comprising LN3 , sialylated LN3 , LNT , LSTa and 3' in fermentation broth of mutant E. coli strains when evaluated in a fed-batch fermentation process with glycerol, sialic acid and lactose SL Mammalian Milk Oligosaccharide Mixture

Mutant E. coli strains capable of producing LN3, LNT, 3'SL and LSTa as described in Example 36 were selected for further evaluation in a fed-batch fermentation process in a 5L bioreactor. Fed-batch fermentation was performed as described in Example 1 at the bioreactor scale. In these examples, glycerol was used as the carbon source and lactose was added as a precursor to the batch medium. During fed-batch, sialic acid was also added via an additional feed. Conventional broth samples were obtained at several time points during the fermentation process and evaluated for An oligosaccharide mixture comprising LN3, 3'-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-bl,3-Gal-bl,4-Glc), LNT, LSTa and 3'SL was produced. Example 43. Production of lactation comprising LN3 , sialylated LN3 , LNT , LSTa and 3'SL in fermentation broth of mutant E. coli strains when evaluated during fed-batch fermentation with sucrose and lactose animal milk oligosaccharide mixture

Mutant E. coli strains capable of producing LN3, LNT, 3'SL and LSTa as described in Example 39 were selected for further evaluation in a fed-batch fermentation process in a 5L bioreactor. Fed-batch fermentation was performed as described in Example 1 at the bioreactor scale. In these examples, sucrose was used as the carbon source and lactose was added as a precursor to the batch medium. Conventional broth samples were obtained at several time points during the fermentation process and evaluated for An oligosaccharide mixture comprising LN3, 3'-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-bl,3-Gal-bl,4-Glc), LNT, LSTa and 3'SL was produced. Example 44. Production of fermented broth containing LN3 , sialylated LN3 , LNnT , para - milk- , in fermentation broth of mutant E. coli strains when evaluated in a fed - batch fermentation process with glycerol, sialic acid, and lactose Mammalian milk oligosaccharide mixture of N- neohexaose , disialyl LNnT , LSTc and 6'SL

Mutant E. coli strains with a persistent transcription unit for α-2,6-sialyltransferase from Luminox sp. JT-ISH-224 with SEQ ID NO 26 as described in Example 37 were tested Selected for further evaluation during fed-batch fermentation in a 5L bioreactor. Fed-batch fermentation was performed as described in Example 1 at the bioreactor scale. In these examples, glycerol was used as the carbon source and lactose was added as a precursor to the batch medium. During fed-batch, sialic acid was also added via an additional feed. Regular broth samples were taken and the sugar produced was measured as described in Example 1. UPLC analysis showed that the fermented broth of the selected strains obtained after the batch phase contained lactose, LN3 and LNnT, while the fermented broth of the selected strains obtained after the fed-batch phase contained LN3, 6 Oligosaccharide mixture of '-sialylated LN3 (Neu5Ac-a2,6-(GlcNAc-b1,3)-Gal-b1,4-Glc), LNnT, LSTc and 6'SL. At the end of the fed-batch, the mixture also contained p-lacto-N-neohexaose (pLNnH), sialylated p-lacto-N-neohexaose and disialo-LNnT, i.e. due to limited detection levels and total Structures produced at small levels and not detected in growth assay analysis. Example 45. Production of LN3 , sialylated LN3 , LNnT , p - milk -N- in fermentation broth of mutant E. coli strains when evaluated during fed-batch fermentation with sucrose and lactose Oligosaccharide mixture of neohexasaccharide , disialyl LNnT , LSTc and 6'SL

Mutant E. coli strains capable of producing LN3, sialylated LN3, LNnT, 6'SL and LSTc as described in Example 40 were selected for further evaluation in a fed-batch fermentation process in a 5L bioreactor . Fed-batch fermentation was performed as described in Example 1 at the bioreactor scale. In these examples, sucrose was used as the carbon source and lactose was added as a precursor to the batch medium. Regular broth samples were taken and the sugar produced was measured as described in Example 1. UPLC analysis showed that the fermented broth of selected strains obtained after the batch phase contained lactose, LN3, 6'SL and LNnT, while the fermented broth of selected strains obtained after the fed-batch phase contained Oligosaccharide mixture comprising LN3, 6'-sialylated LN3 (Neu5Ac-a-2,6-(GlcNAc-b-1,3)-Gal-b-1,4-Glc), LNnT, LSTc and 6'SL . At the end of the fed-batch, the mixture also contained p-lacto-N-neohexaose, sialylated p-lacto-N-neohexaose, and disialylated LNnT, i.e. two due to limited detection levels and total production Smaller levels of structures not detected in growth assay analysis. Example 46. Production of mammalian milk oligosaccharide mixtures comprising LNT , LNnT and poly - galactosylated structures in modified E. coli hosts when evaluated in fed- batch fermentation

The E. coli strains modified to produce LNnT as described in Example 1 were further modified with gene body insertion for the persistent transcription unit of WbgO from E. coli O55:H7 having SEQ ID NO 28. In the next step, as described in Example 1, novel strains were evaluated in a fed-batch fermentation process in a 5L bioreactor. In this example, sucrose was used as the carbon source and lactose was added as a precursor to the batch medium. Conventional broth samples were obtained at several time points during the fermentation process and evaluated for Production contains lacto-N-triose II (LN3), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), p-lacto-N-neopentasaccharide, p-lacto-N- Pentasaccharide, p-lacto-N-neohexaose, p-lacto-N-hexaose, β-(1,3)galactoside-p-lacto-N-neopentose and β-(1,4) Oligosaccharide mixture of galactoside-p-lacto-N-pentasaccharide. Example 47. Production of Mammalian Milk Oligosaccharide Mixtures Containing Galactosylated and GalNAcylated Lactose Structures Using Modified E. coli Hosts

The E. coli strains optimized for UDP-galactose as described in Example 1 were further modified with deletion of the E. coli lacZ gene and insertion of a persistent expression unit for use in a persistent expression unit derived from a gene having SEQ ID NO. 36 Neisseria gonorrhoeae a1,4-galactosyltransferase (LgtC) to produce α-1,4-galactosylated lactose (Gal-a1,4-Gal- b1,4-Glc). In the next step, the mutant strain was further modified by gene body deletion of E. coli nagB gene and gene body insertion modification of the persistent expression unit for mutant glmS*54 of E. coli having SEQ ID NO 19 , and transformed with expression plastids comprising persistent expression units for 4-epimerase (WbpP) of Pseudomonas aeruginosa having SEQ ID NO 34 and from Pseudomonas aeruginosa having SEQ ID NO 34 β1,3-N-Acetylgalactosaminyltransferase (LgtD) of Haemophilus influenzae NO 35. In addition to its β1,3-N-acetyl-galactosaminyltransferase activity, the LgtD enzyme also has β1,3-galactosyltransferase activity and can add galactose to the non-reducing terminal GalNAc molecule, resulting in Terminal Gal-b1,3-GalNAc is generated at the non-reducing end of the glycan. In the growth experiments according to the culture conditions provided in Example 1, for the production of Gal-a1,4-Gal-b1,4-Glc (Gal-a1,4-lactose), Gal-a1,4-Gal-a1, 4-Gal-b1,4-Glc, Gal-a1,4-Gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, GalNAc-b1,3-Gal-b1,4-Glc ( GalNAc-b1,3-lactose), Gal-b1,3-GalNAc-b1,3-lactose, GalNAc-b1,3-Gal-a1,4-Gal-b1,4-Glc (ball-N-tetrasaccharide) and an oligosaccharide mixture of Gal-b1,3-GalNAc-b1,3-Gal-a1,4-Gal-b1,4-Glc, in which the medium contained glycerol as a carbon source and lactose as a precursor, the novel strains were evaluated. Example 48. Production comprising LN3 , LNT , GalNAc-b1,3-LNT , Gal-b1,3-GalNAc-b1,3-LNT , GalNAc -b1,3- lactose and Gal-b1 using a modified E. coli host ,3-GalNAc-b1,3- Lactose Oligosaccharide Mixture

The E. coli strain modified to produce LNT as described in Example 1 was further modified with insertion of persistent expression units for the 4-table of Pseudomonas aeruginosa having SEQ ID NO 34 Isomerase (WbpP) and β1,3-N-acetylgalactosaminyltransferase (LgtD) from Haemophilus influenzae having SEQ ID NO 35. In the growth experiments according to the culture conditions in Example 1, for the production of LN3, LNT, GalNAc-b1,3-LNT, Gal-b1,3-GalNAc-b1,3-LNT, GalNAc- Oligosaccharide mixtures of b1,3-lactose and Gal-b1,3-GalNAc-b1,3-lactose were evaluated for novel strains in which the medium contained glycerol as carbon source and lactose as precursor. Example 49. Production of LN3 , LNnT , GalNAc-b1,3-LNnT , Gal-b1,3-GalNAc-b1,3-LNnT , GalNAc -b1,3- lactose, Gal-b1 using a modified E. coli host ,3-GalNAc-b1,3- lactose and ( GalNAc- ) poly- LNnT structure oligosaccharide mixture

E. coli strains modified to produce LNnT as described in Example 1 were further modified with insertion of persistent expression units for WbpP from Pseudomonas aeruginosa having SEQ ID NO 34 and LgtD from Haemophilus influenzae with SEQ ID NO 35. In the growth experiment according to the culture conditions in Example 1, for the production of LN3, LNnT, GalNAc-b1,3-LNnT, Gal-b1,3-GalNAc-b1,3-LNnT, GalNAc- b1,3-lactose and Gal-b1,3-GalNAc-b1,3-lactose and poly-LNnT structures (by substitution activity of NmlgtA and NmLgtB) and GalNAc-ylated poly-LNnT structures (by substitution activity by HiLgtD) Novel strains were evaluated using a mixture of oligosaccharides in which the medium contained glycerol as a carbon source and lactose as a precursor. Example 50. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2'FL , DiFL , LN3 , LNT and LNFP-I Using a Modified E. coli Host

E. coli strains modified to produce GDP-fucose as described in Example 1 were further adapted for LN3 and LNT production by gene body insertion of persistent transcription units for Galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) of Neisseria meningitidis of SEQ ID NO 27 and N- from Escherichia coli O55:H7 of SEQ ID NO 28 Acetylglucosamine beta-1,3-galactosyltransferase (WbgO). In the next step, the novel strain was further transformed with an expression plastid containing a persistent transcription unit for alpha-1,2-fucosylation from H. pylori having SEQ ID NO 04 An enzyme, the expressing plastid accepts both LNT and lactose as receptors for its fucosyltransferase activity. When evaluated in growth experiments according to the culture conditions provided in Example 1, the novel strain produced 2'FL, DiFL, LN3, LNT and lacto-N-fucopentose I (LNFP- I. Oligosaccharide mixture of Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), wherein the medium contains sucrose as carbon source and lactose as precursor. Example 51. Production of Oligosaccharide Mixtures Comprising LN3 , LNT and LNFP-I Using Modified E. coli Hosts

The E. coli K-12 MG1655 strain modified for GDP-fucose production as described in Example 1 was further modified by genomic deletion of E. coli nagB and arabinose isomerase ( araA ) genes and a persistent transcription unit The gene body insertion was adapted for LN3 and LNT production, the persistent transcription units for galE from E. coli with SEQ ID NO 30, galactosides from Neisseria meningitidis with SEQ ID NO 27 β-1,3-N-Acetylglucosaminyltransferase (LgtA) and N-Acetylglucosamine β from Salmonella enterica subsp. salami serovar Grayside having SEQ ID NO 41 -1,3-Galactosyltransferase (WbdO). In the next step, the novel strain was further transformed with an expressing plastid containing a persistent transcription unit for a-1 from the ore-phagous iron-oxidizing bacteria ES-1 with SEQ ID NO 40, 2-fucosyltransferase (fucT54). In growth experiments according to the culture conditions provided in Example 1, for the production of LN3, LNT and lacto-N-fucopentose I (LNFP-I, Fuc-a1,2-Gal- The novel strains were evaluated with an oligosaccharide mixture of b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in which the medium contained sucrose as a carbon source and lactose as a precursor. Example 52. Production of 2'FL, DiFL , GalNAc - b1,3 - lactose, LN3 , LNT , GalNAc-b1,3-LNT , LNFP-1 and GalNAc-b1,3-LNFP using a modified E. coli host Mammalian milk oligosaccharide mixture of -I

E. coli strains modified for GDP-fucose production and for LNFP-I production as described in Example 50 were further transformed with compatible expressing plastids containing a persistent transcription unit using In bl,3-N-acetylgalactosaminyltransferase (LgtD) from Haemophilus influenzae having SEQ ID NO 35. In the growth experiments according to the culture conditions provided in Example 1, for the production of 2'FL, DiFL, GalNAc-b1,3-lactose (GalNAc-b1,3-Gal-b1,4- Glc), LN3, LNT, GalNAc-b1, 3-LNT (GalNAc-b1, 3-Gal-b1, 3-GlcNAc-b1, 3-Gal-b1, 4-Glc), LNFP-I (Fuc-a1, 2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and GalNAc-b1,3-LNFP-I (GalNAc-b1,3-(Fuc-a1,2)-Gal-b1 , 3-GlcNAc-b1,3-Gal-b1,4-Glc) oligosaccharide mixture to evaluate novel strains, in which the medium contains sucrose as a carbon source and lactose as a precursor. Example 53. Production of 2'FL, DiFL , LN3 , LNT , LNFP - I and Gal - a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1 using a modified E. coli host ,3-Gal-b1,4-Glc Oligosaccharide Mixture

E. coli strains modified for GDP-fucose production and for LNFP-I production as described in Example 50 were further transformed with compatible expressing plastids containing a persistent transcription unit using in α-1,3-galactosyltransferase WbnI from E. coli with SEQ ID NO 37. In growth experiments according to the culture conditions provided in Example 1, for the production of 2'FL, DiFL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal-b1,3- GlcNAc-b1,3-Gal-b1,4-Glc) and Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3 -Gal-b1,4-Glc) oligosaccharide mixture to evaluate novel strains, in which the medium contains sucrose as carbon source and lactose as precursor. Example 54. Production of 2'FL, DiFL , LN3 , LNT , LNFP - I and GalNAc - a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1 using a modified E. coli host ,3-Gal-b1,4-Glc Oligosaccharide Mixture

E. coli strains modified for GDP-fucose production and for LNFP-I production as described in Example 50 were further transformed with compatible expressing plastids containing a persistent transcription unit using In alpha-1,3-N-acetylgalactosaminyltransferase BgtA from Helicobacter weasels having SEQ ID NO 38. In growth experiments according to the culture conditions provided in Example 1, for the production of 2'FL, DiFL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal-b1,3- GlcNAc-b1,3-Gal-b1,4-Glc) and GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3 -Gal-b1,4-Glc) oligosaccharide mixture to evaluate novel strains, in which the medium contains sucrose as carbon source and lactose as precursor. Example 55. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2'FL , 3-FL , DiFL , LN3 , LNT and LNFP-I Using a Modified E. coli Host

Comprising glmS*54 from E. coli with SEQ ID NO 19, LgtA from Neisseria meningitidis with SEQ ID NO 27, E. coli O55:H7 with SEQ ID NO 28 as described in Example 50 Expression of WbgO and alpha-1,2-fucosyltransferase from Helicobacter pylori with SEQ ID NO 04 E. coli strains modified for GDP-fucose and LNFP-I production were further modified to contain Compatibility of the persistent transcription unit for alpha-1,3-fucosyltransferase (HpFucT) from Helicobacter pylori having SEQ ID NO 05 represents plastid transformation. In growth experiments according to the culture conditions provided in Example 1, for the production of 2'FL, 3-FL, DiFL, LN3, LNT and LNFP-I (Fuc-a1,2-Gal- The novel strains were evaluated with an oligosaccharide mixture of b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in which the medium contained sucrose as a carbon source and lactose as a precursor. Example 56. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LN3 , LNT and LNFP-II Using a Modified E. coli Host

The E. coli strain modified for GDP-fucose production as described in Example 1 was further adapted for LN3 and LNT production by gene body deletion of the E. coli nagB gene and gene body insertion of the persistent transcription unit, These persistent transcription units were used for LgtA from N. meningitidis with SEQ ID NO 27 and WbgO from E. coli O55:H7 with SEQ ID NO 28. In the next step, the novel strain was further transformed with an expressing plastid containing a persistent transcription unit for mutant al,3 from Bifidobacterium longum infantum subsp. infantis ATCC 15697 with SEQ ID NO 39 /4 Fucosidase. In growth experiments according to the culture conditions provided in Example 1, for the production of LN3, LNT and lacto-N-fucopentose II (LNFP-II, Gal-b1,3-(Fuc) in all culture broth samples Novel strains were evaluated with an oligosaccharide mixture of -a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc) in which the medium contained sucrose as a carbon source and lactose as a precursor. Example 57. Production of Mammalian Milk Oligosaccharide Mixtures Comprising 2'FL , DiFL , LN3 , LNT , LNFP-I and LNFP-II Using Modified E. coli Hosts

E. coli strains modified for GDP-fucose production and for LNFP-II production as described in Example 56 were further transformed with compatible expressing plastids containing a persistent transcription unit using In alpha-1,2-fucosyltransferase (HpFutC) from Helicobacter pylori having SEQ ID NO 04. In growth experiments according to the culture conditions provided in Example 1, for the production of 2'FL, DiFL, LN3, LNT, LNFP-I and lacto-N-fucopentose II (LNFP- II, the oligosaccharide mixture of Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc) evaluation of novel strains, in which the medium contains sucrose as carbon source and lactose as precursor . Example 58. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3-FL , LN3 , LNT and LNFP-II Using a Modified E. coli Host

E. coli strains modified for GDP-fucose production and for LNFP-II production as described in Example 56 were further transformed with compatible expressing plastids containing a persistent transcription unit using In alpha-1,3-fucosyltransferase (HpFucT) from Helicobacter pylori having SEQ ID NO 05. In growth experiments according to the culture conditions provided in Example 1, for the production of 3-FL, LN3, LNT and lacto-N-fucopentose II (LNFP-II, Gal-b1, 3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc) oligosaccharide mixture to evaluate novel strains, where the medium contains sucrose as carbon source and lactose as precursor. Example 59. Production of Mammalian Milk Oligosaccharide Mixtures Comprising 2'FL , 3-FL , DiFL , LN3 , LNT , LNFP-I and LNFP-II Using Modified E. coli Hosts

E. coli strains modified for GDP-fucose production and for LNFP-II production as described in Example 56 were further transformed with compatible expressing plastids containing persistent transcription units that For alpha-1,2-fucosyltransferase (HpFutC) from Helicobacter pylori with SEQ ID NO 04 and alpha-1,3-fucosyl from Helicobacter pylori with SEQ ID NO 05 Transferase (HpFucT). In growth experiments according to the culture conditions provided in Example 1, for the production of 2'FL, 3-FL, DiFL, LN3, LNT, LNFP-I and lacto-N-fucopentose in all culture broth samples II (LNFP-II, Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc) oligosaccharide mixture to evaluate novel strains, in which the medium contains sucrose as carbon source and Lactose as a precursor. Example 60. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3-FL , LN3 , LNT and LNFP-V Using a Modified E. coli Host

The E. coli strain modified for GDP-fucose production as described in Example 1 was further adapted for LN3 and LNT production by gene body deletion of the E. coli nagB gene and gene body insertion of the persistent transcription unit, These persistent transcription units were used for LgtA from N. meningitidis with SEQ ID NO 27 and WbgO from E. coli O55:H7 with SEQ ID NO 28. In the next step, the novel strain was further transformed with an expression plastid containing a persistent transcription unit for truncated al,3-fucosyl from H. pylori having SEQ ID NO 06 transferase. In the growth experiments according to the culture conditions provided in Example 1, for the production of 3-FL, LN3, LNT and lacto-N-fucopentose V (LNFP-V, Gal-b1, The oligosaccharide mixture of 3-GlcNAc-b1,3-Gal-b1,4-(Fuca1,3)-Glc) was evaluated for novel strains, wherein the medium contained sucrose as carbon source and lactose as precursor. Example 61. Production of Mammalian Milk Oligosaccharide Mixtures Comprising 3-FL , LN3 , LNnT , LNFP-III and LNFP-VI Using Modified E. coli Hosts

The E. coli strain modified for GDP-fucose production as described in Example 1 was further adapted for LN3 and LNnT production by gene body deletion of the E. coli nagB gene and gene body insertion of the persistent transcription unit, These persistent transcription units were used for LgtA from N. meningitidis with SEQ ID NO 27 and LgtB from N. meningitidis with SEQ ID NO 29. In the next step, the novel strain was further transformed with an expression plasmid containing a persistent transcription unit for a truncated alpha-1,3-fucobacterium from H. pylori having SEQ ID NO 06 Glycosyltransferase. In the growth experiments according to the culture conditions provided in Example 1, for the production of 3-FL, LN3 and LNnT, lacto-N-fucopentose III (LNFP-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 - Oligosaccharide mixture of Gal-b1,4-(Fuc-a1,3-)Glc) to evaluate novel strains, where the medium contains sucrose as carbon source and lactose as precursor. Example 62. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as described in Example 1 were further modified with gene body deletion of the E. coli nagB gene and gene body insertion of the persistent expression cassettes. For LgtA from Neisseria meningitidis with SEQ ID NO 27 and WbgO from E. coli O55:H7 with SEQ ID NO 28. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In the growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LN3, sialylated LN3, LNT and fucosylated and sialylated lactose, LN3, sialylated LN3, LNT and fucosylated and sialylated in all broth samples Oligosaccharide mixtures of LNT structures (Table 10) evaluated novel strains in which cultures contained sucrose as carbon source and sialic acid and lactose as precursors. Table 10 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and sialic acid and lactose as precursors. strain plastid that exists * Oligosaccharide SF46 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF47 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LN3, LNT, 3'S-LN3, LSTa SF48 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LN3, 6'S-LN3, LNT, LNFP-I SF49 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LN3, 6'S-LN3, LNT SF50 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa SF51 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, LN3, 3'S-LN3, 6'S-LN3, LNT, LSTa SF52 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF53 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LN3, 6'S-LN3, LNT, LNFP-I SF54 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LN3, 3'S-LN3, 6'S- LN3, LNT, LNFP-I, LSTa *See Table 2 for plastid information , Example 63. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as described in Example 1 were further modified with gene body deletion of the E. coli nagB gene and gene body insertion of the persistent expression cassettes. For LgtA from Neisseria meningitidis with SEQ ID NO 27 and LgtB from Neisseria meningitidis with SEQ ID NO 29. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LN3, sialylated LN3, LNnT and fucosylated and sialylated lactose, LN3, sialylated LN3, LNnT and fucosylated and sialylated in all broth samples Oligosaccharide mixtures of LNnT structures (Table 11) evaluated novel strains in which cultures contained sucrose as carbon source and sialic acid and lactose as precursors. Table 11 : Evaluation of production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and sialic acid and lactose as precursors. strain plastid that exists * Oligosaccharide SF55 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LN3, 3'S-LN3, LNnT, LSTd SF56 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF57 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LN3, 6'S-LN3, LNnT, LSTc SF58 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF59 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, LN3, 3'S-LN3, 6'S-LN3, LNnT, LSTc, LSTd SF60 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, LN3, 3'S-LN3, 6'S-LN3, LNnT, LNFP-III, LSTc, LSTd SF61 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF62 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF63 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LN3, 3'S-LN3, 6'S- LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plastid information , Example 64. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as described in Example 1 were further modified with gene body deletion of E. coli nagA , nagB , nanA , nanE and nanK genes and gene body insertion of a persistent expression cassette , these persistent expression cassettes were used for mutant glmS*54 from E. coli with SEQ ID NO 19, GNA1 from Saccharomyces cerevisiae with SEQ ID NO 16, AGE for Bacillus ovale with SEQ ID NO 17 , neuB from Neisseria meningitidis with SEQ ID NO 18, LgtA from Neisseria meningitidis with SEQ ID NO 27 and WbgO from Escherichia coli O55:H7 with SEQ ID NO 28. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LNB, LN3, sialylated lactose, LNB, fucosylated and sialylated LNB in all culture broth samples LN3, LNT, and an oligosaccharide mixture of fucosylated and sialylated LNT structures (Table 12) were evaluated for novel strains with cultures containing sucrose as carbon source and lactose as precursor. Table 12 : Evaluation of production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF64 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LNB, 2'FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF65 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LNB, 4-FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LSTa SF66 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LNB, 2'FLNB, 6'SLNB, LN3, 6'S-LN3, LNT, LNFP-I SF67 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LNB, 4-FLNB, 6'SLNB, LN3, 6'S-LN3, LNT SF68 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LNB, 2'FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa SF69 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LNB, 4-FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S- LN3, LNT, LSTa SF70 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF71 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 6'SLNB, LN3, 6'S-LN3, LNT, LNFP-I SF72 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LNB, 2' FLNB, 4-FLNB, Di-FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa *See Table 2 for plastid information , Example 65. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as described in Example 1 were further modified with gene body deletion of E. coli nagA , nagB , nanA , nanE and nanK genes and gene body insertion of a persistent expression cassette , these persistent expression cassettes were used for mutant glmS*54 from E. coli with SEQ ID NO 19, GNA1 from Saccharomyces cerevisiae with SEQ ID NO 16, AGE for Bacillus ovale with SEQ ID NO 17 , neuB from Neisseria meningitidis with SEQ ID NO 18, LgtA from Neisseria meningitidis with SEQ ID NO 27, and LgtB from Neisseria meningitidis with SEQ ID NO 29. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LacNAc, LN3, sialylated LN3, LNnT, and an oligosaccharide mixture of fucosylated and sialylated LNT structures (Table 13) were evaluated for novel strains in which cultures contained sucrose as carbon source and lactose as precursor. Table 13 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF73 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LSTd SF74 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LacNAc, 3'FlacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF75 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LSTc SF76 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LacNAc, 3'FlacNAc, 6'SlacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF77 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LacNAc, 2'FLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LSTc, LSTd SF78 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LacNAc, 3'FlacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S- LN3, LNnT, LNFP-III, LSTc, LSTd SF79 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF80 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF81 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2' FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plastid information , Example 66. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as described in Example 1 were further modified with gene body deletion of E. coli nagA , nagB , nanA , nanE and nanK genes and gene body insertion of a persistent expression cassette , these persistent expression cassettes were used for mutant glmS*54 from E. coli with SEQ ID NO 19, UDP-N-acetylglucosamine 2-epiiso from Campylobacter jejuni with SEQ ID NO 21 Construct (neuC), neuB from Neisseria meningitidis with SEQ ID NO 18, LgtA from Neisseria meningitidis with SEQ ID NO 27, and WbgO from E. coli O55:H7 with SEQ ID NO 28 . In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In the growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LN3, sialylated LN3, LNT and rock as shown in Table 14 in all broth samples Oligosaccharide mixtures of fucosylated and sialylated LNT structures evaluated novel strains in which cultures contained sucrose as carbon source and lactose as precursor. Table 14 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF82 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF83 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LN3, 3'S-LN3, LNT, LSTa SF84 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LN3, 6'S-LN3, LNT, LNFP-I SF85 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LN3, 6'S-LN3, LNT SF86 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa SF87 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, LN3, 3'S-LN3, 6'S-LN3, LNT, LSTa SF88 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF89 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LN3, 6'S-LN3, LNT, LNFP-I SF90 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LN3, LNT, 3'S-LN3, 6'S-LN3, LNFP-I, LSTa *See Table 2 for plastid information , Example 67. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as described in Example 1 were further modified with gene body deletion of E. coli nagA , nagB , nanA , nanE and nanK genes and gene body insertion of a persistent expression cassette , these persistent expression cassettes were used for mutant glmS*54 from E. coli with SEQ ID NO 19, neuC from Campylobacter jejuni with SEQ ID NO 21, Neisseria meningitidis with SEQ ID NO 18 neuB from bacteria, LgtA from Neisseria meningitidis with SEQ ID NO 27, and LgtB from Neisseria meningitidis with SEQ ID NO 29. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LN3, sialylated LN3, LNnT and fucosylated and sialylated lactose, LN3, sialylated LN3, LNnT and fucosylated and sialylated in all broth samples Oligosaccharide mixtures of LNnT structures (Table 15) evaluated novel strains in which cultures contained sucrose as carbon source and lactose as precursor. Table 15 : Evaluation of production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF91 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LN3, 3'S-LN3, LNnT, LSTd SF92 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF93 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LN3, 6'S-LN3, LNnT, LSTc SF94 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF95 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, LN3, 3'S-LN3, 6'S-LN3, LNnT, LSTc, LSTd SF96 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, LN3, 3'S-LN3, 6'S-LN3, LNnT, LNFP-III, LSTc, LSTd SF97 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF98 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF99 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LN3, 3'S-LN3, 6'S- LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plastid information , Example 68. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for sialic acid production as described in Example 1 were further genetically knocked out of the E. coli wcaJ gene to increase the intracellular pool of GDP-fucose and the gene-body insertion modification of the persistent expression cassette , these persistent expression cassettes were used for LgtA from Neisseria meningitidis with SEQ ID NO 27 and WbgO from E. coli O55:H7 with SEQ ID NO 28. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LNB, LN3, sialylated lactose, LNB, fucosylated and sialylated LNB in all culture broth samples LN3, LNT, and an oligosaccharide mixture of fucosylated and sialylated LNT structures (Table 16) were evaluated for novel strains with cultures containing sucrose as carbon source and lactose as precursor. Table 16 : Evaluation of production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF100 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LNB, 2'FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF101 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LNB, 4-FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LSTa SF102 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LNB, 2'FLNB, 6'SLNB, LN3, LNT, 6'S-LN3, LNFP-I SF103 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LNB, 4-FLNB, 6'SLNB, LN3, 6'S-LN3, LNT SF104 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LNB, 2'FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa SF105 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LNB, 4-FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S- LN3, LNT, LSTa SF106 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 3'SLNB, LN3, LNT, 3'S- LN3, LNFP-I, LSTa SF107 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 6'SLNB, LN3, 6'S-LN3, LNT, LNFP-I SF108 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LNB, 2' FLNB, 4-FLNB, Di-FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa *See Table 2 for plastid information . Example 69. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Siallylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for sialic acid production as described in Example 1 were further genetically knocked out of the E. coli wcaJ gene to increase the intracellular pool of GDP-fucose and the gene-body insertion modification of the persistent expression cassette , these persistent expression cassettes were used for LgtA from Neisseria meningitidis with SEQ ID NO 27 and LgtB from Neisseria meningitidis with SEQ ID NO 29. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LacNAc, LN3, sialylated lactose in all culture broth samples LN3, LNnT, and an oligosaccharide mixture of fucosylated and sialylated LNnT structures (Table 17) were evaluated for novel strains in which cultures contained sucrose as carbon source and lactose as precursor. Table 17 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF109 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LSTd SF110 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LacNAc, 3'FlacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF111 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LSTc SF112 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LacNAc, 3'FlacNAc, 6'SlacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF113 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LacNAc, 2'FLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LSTc, LSTd SF114 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LacNAc, 3'FlacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S- LN3, LNnT, LNFP-III, LSTc, LSTd SF115 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF116 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF117 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2' FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plastid information . Example 70. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for sialic acid production as described in Example 1 were further modified with gene body deletion of E. coli wcaJ , fucK and fucI genes and gene body insertion of persistent expression cassettes. Cassette for fucose permease (fucP) from E. coli with SEQ ID NO 13, bifunctional fucokinase/fucose-1-guanosine phosphate from Bacteroides fragilis with SEQ ID NO 14 Acyltransferase (fkp), LgtA from Neisseria meningitidis with SEQ ID NO 27 and WbgO from E. coli O55:H7 with SEQ ID NO 28. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LNB, LN3, sialylated lactose, LNB, fucosylated and sialylated LNB in all culture broth samples LN3, LNT, and an oligosaccharide mixture of fucosylated and sialylated LNT structures (Table 18) were evaluated for novel strains with cultures containing sucrose as carbon source and lactose as precursor. Table 18 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contained sucrose as Carbon source and lactose as precursors. strain plastid that exists * Oligosaccharide SF118 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LNB, 2'FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF119 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LNB, 4-FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LSTa SF120 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LNB, 2'FLNB, 6'SLNB, LN3, 6'S-LN3, LNT, LNFP-I SF121 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LNB, 4-FLNB, 6'SLNB, LN3, 6'S-LN3, LNT SF122 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LNB, 2'FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa SF123 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LNB, 4-FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S- LN3, LNT, LSTa SF124 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF125 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 6'SLNB, LN3, 6'S-LN3, LNT, LNFP-I SF126 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LNB, 2' FLNB, 4-FLNB, Di-FLNB, 3'SLNB, 6'SLNB, LN3, LNT, 3'S-LN3, 6'S-LN3, LNFP-I, LSTa *See Table 2 for plastid information . Example 71. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for sialic acid production as described in Example 1 were further modified with gene body deletion of E. coli wcaJ , fucK and fucI genes and gene body insertion of persistent expression cassettes. Cassettes were used for fucP from E. coli with SEQ ID NO 13, fkp from B. fragilis with SEQ ID NO 14, LgtA from N. meningitidis with SEQ ID NO 27 and from LgtA with SEQ ID NO 29 The LgtB of Neisseria meningitidis. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LacNAc, LN3, sialylated LN3, LNnT, and an oligosaccharide mixture of fucosylated and sialylated LNnT structures (Table 19) were evaluated for novel strains in which cultures contained sucrose as carbon source and lactose as precursor. Table 19 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF127 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LSTd SF128 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LacNAc, 3'FlacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF129 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LSTc SF130 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LacNAc, 3'FlacNAc, 6'SlacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF131 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LacNAc, 2'FLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LSTc, LSTd SF132 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LacNAc, 3'FlacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S- LN3, LNnT, LNFP-III, LSTc, LSTd SF133 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF134 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF135 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2' FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plastid information . Example 72. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Siallylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as described in Example 1 were further modified with gene body deletion of the E. coli nagA , nagB , ushA and galT genes and gene body insertion of a persistent expression cassette. Isocontinuous expression cassettes were used for galE from E. coli with SEQ ID NO 30, mutant glmS*54 from E. coli with SEQ ID NO 19, GNA1 from Saccharomyces cerevisiae with SEQ ID NO 16, from Saccharomyces cerevisiae with SEQ ID NO 16 LgtA of N. meningitidis of SEQ ID NO 27 and WbgO from E. coli O55:H7 of SEQ ID NO 28. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LNB, LN3, sialylated lactose, LNB, fucosylated and sialylated LNB in all culture broth samples LN3, LNT, and an oligosaccharide mixture of fucosylated and sialylated LNT structures (Table 20) were evaluated for novel strains with cultures containing sucrose as carbon source and sialic acid and lactose as precursors. Table 20 : Evaluation of production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the culture medium contains Sucrose as carbon source and sialic acid and lactose as precursors. strain plastid that exists * Oligosaccharide SF136 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LNB, 2'FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF137 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LNB, 4-FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LSTa SF138 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LNB, 2'FLNB, 6'SLNB, LN3, 6'S-LN3, LNT, LNFP-I SF139 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LNB, 4-FLNB, 6'SLNB, LN3, 6'S-LN3, LNT SF140 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LNB, 2'FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa SF141 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LNB, 4-FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S- LN3, LNT, LSTa SF142 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF143 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 6'SLNB, LN3, 6'S-LN3, LNT, LNFP-I SF144 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LNB, 2' FLNB, 4-FLNB, Di-FLNB, 3'SLNB, 6'SLNB, LN3, LNT, 3'S-LN3, 6'S-LN3, LNFP-I, LSTa *See Table 2 for plastid information . Example 73. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as described in Example 1 were further modified with gene body deletion of the E. coli nagA , nagB , ushA and galT genes and gene body insertion of a persistent expression cassette. Isocontinuous expression cassettes were used for galE from E. coli with SEQ ID NO 30, mutant glmS*54 from E. coli with SEQ ID NO 19, GNA1 from Saccharomyces cerevisiae with SEQ ID NO 16, from Saccharomyces cerevisiae with SEQ ID NO 16 LgtA from N. meningitidis of SEQ ID NO 27 and LgtB from N. meningitidis with SEQ ID NO 29. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LacNAc, LN3, sialylated LN3, LNnT, and an oligosaccharide mixture of fucosylated and sialylated LNnT structures (Table 21) evaluated novel strains with cultures containing sucrose as carbon source and sialic acid and lactose as precursors. Table 21 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and sialic acid and lactose as precursors. strain plastid that exists * Oligosaccharide SF145 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 3'SLacNAc, LN3, 3'SLN, LNnT, LSTd SF146 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LacNAc, 3'FlacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF147 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LSTc SF148 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LacNAc, 3'FlacNAc, 6'SlacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF149 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LacNAc, 2'FLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LSTc, LSTd SF150 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LacNAc, 3'FlacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S- LN3, LNnT, LNFP-III, LSTc, LSTd SF151 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF152 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF153 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2' FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plastid information . Example 74. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as described in Example 1 were further genetically knocked out of the E. coli nagA , nagB , nanA , nanE , nanK , ushA and galT genes and subjected to a persistent expression cassette Gene body insertion modification, these persistent expression cassettes are used for galE from E. coli with SEQ ID NO 30, mutant glmS*54 from E. coli with SEQ ID NO 19, from brew with SEQ ID NO 16 GNA1 of yeast, N-acetylglucosamine 2-epimerase (AGE) of Bacteroides ovale with SEQ ID NO 17, neuB of Neisseria meningitidis with SEQ ID NO 18, from Bacillus ovale with SEQ ID NO 18 LgtA from Neisseria meningitidis of ID NO 27 and WbgO from E. coli O55:H7 with SEQ ID NO 28. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LNB, LN3, sialylated lactose, LNB, fucosylated and sialylated LNB in all culture broth samples LN3, LNT, and an oligosaccharide mixture of fucosylated and sialylated LNT structures (Table 22) were evaluated for novel strains with cultures containing sucrose as carbon source and lactose as precursor. Table 22 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF154 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LNB, 2'FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF155 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LNB, 4-FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LSTa SF156 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LNB, 2'FLNB, 6'SLNB, LN3, 6'S-LN3, LNT, LNFP-I SF157 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LNB, 4-FLNB, 6'SLNB, LN3, 6'S-LN3, LNT SF158 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LNB, 2'FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa SF159 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LNB, 4-FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S- LN3, LNT, LSTa SF160 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF161 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 6'SLNB, LN3, 6'S-LN3, LNT, LNFP-I SF162 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LNB, 2' FLNB, 4-FLNB, Di-FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa *See Table 2 for plastid information . Example 75. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as described in Example 1 were further genetically knocked out of the E. coli nagA , nagB , nanA , nanE , nanK , ushA and galT genes and subjected to a persistent expression cassette Gene body insertion modification, these persistent expression cassettes are used for galE from E. coli with SEQ ID NO 30, mutant glmS*54 from E. coli with SEQ ID NO 19, from brew with SEQ ID NO 16 GNA1 from yeast, AGE from Bacteroides ovale with SEQ ID NO 17, neuB from Neisseria meningitidis with SEQ ID NO 18, LgtA from Neisseria meningitidis with SEQ ID NO 27 and from Neisseria meningitidis with SEQ ID NO 27 LgtB of Neisseria meningitidis of SEQ ID NO 29. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LacNAc, LN3, sialylated LN3, LNnT, and an oligosaccharide mixture of fucosylated and sialylated LNnT structures (Table 23) were evaluated for novel strains with cultures containing sucrose as carbon source and lactose as precursor. Table 23 : Evaluation of production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF163 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LSTd SF164 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LacNAc, 3'FlacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF165 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LSTc SF166 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LacNAc, 3'FlacNAc, 6'SlacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF167 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LacNAc, 2'FLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LSTc, LSTd SF168 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LacNAc, 3'FlacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S- LN3, LNnT, LNFP-III, LSTc, LSTd SF169 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF170 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF171 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2' FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plastid information . Example 76. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Siallylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as described in Example 1 were further genetically knocked out of the E. coli nagA , nagB , nanA , nanE , nanK , ushA and galT genes and subjected to a persistent expression cassette Gene body insertion modification, these persistent expression cassettes are used for galE from E. coli with SEQ ID NO 30, mutant glmS*54 from E. coli with SEQ ID NO 19, from jejunum with SEQ ID NO 21 neuC from Campylobacter, neuB from Neisseria meningitidis with SEQ ID NO 18, LgtA from Neisseria meningitidis with SEQ ID NO 27, and WbgO from E. coli O55:H7 with SEQ ID NO 28. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In the growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LN3, sialylated LN3, LNT and fucosylated and sialylated lactose, LN3, sialylated LN3, LNT and fucosylated and sialylated in all broth samples Oligosaccharide mixtures of LNT structures (Table 24) evaluated novel strains in which cultures contained sucrose as carbon source and lactose as precursor. Table 24 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF172 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF173 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LN3, 3'S-LN3, LNT, LSTa SF174 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LN3, 6'S-LN3, LNT, LNFP-I SF175 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LN3, 6'S-LN3, LNT SF176 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa SF177 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, LN3, 3'S-LN3, 6'S-LN3, LNT, LSTa SF178 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF179 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LN3, 6'S-LN3, LNT, LNFP-I SF180 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LN3, 3'S-LN3, 6'S- LN3, LNT, LNFP-I, LSTa *See Table 2 for plastid information . Example 77. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Siallylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for GDP-fucose production as described in Example 1 were further genetically knocked out of the E. coli nagA , nagB , nanA , nanE , nanK , ushA and galT genes and subjected to a persistent expression cassette Gene body insertion modification, these persistent expression cassettes are used for galE from E. coli with SEQ ID NO 30, mutant glmS*54 from E. coli with SEQ ID NO 19, from jejunum with SEQ ID NO 21 neuC from Campylobacter, neuB from Neisseria meningitidis with SEQ ID NO 18, LgtA from Neisseria meningitidis with SEQ ID NO 27 and LgtB from Neisseria meningitidis with SEQ ID NO 29 . In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LN3, sialylated LN3, LNnT and fucosylated and sialylated lactose, LN3, sialylated LN3, LNnT and fucosylated and sialylated in all broth samples Oligosaccharide mixtures of LNnT structures (Table 25) evaluated novel strains in which cultures contained sucrose as carbon source and lactose as precursor. Table 25 : Evaluation of production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide mixture comprising SF181 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LN3, 3'S-LN3, LNnT, LSTd SF182 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF183 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LN3, 6'S-LN3, LNnT, LSTc SF184 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF185 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, LN3, 3'S-LN3, 6'S-LN3, LNnT, LSTc, LSTd SF186 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, LN3, 3'S-LN3, 6'S-LN3, LNnT, LNFP-III, LSTc, LSTd SF187 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF188 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF189 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LN3, 3'S-LN3, 6'S- LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plastid information . Example 78. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Siallylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for sialic acid production as described in Example 1 were further modified by gene body deletion of E. coli wcaJ , ushA and galT genes and gene body insertion of persistent expression cassettes. The cassettes were used for galE from E. coli with SEQ ID NO 30, LgtA from N. meningitidis with SEQ ID NO 27, and WbgO from E. coli O55:H7 with SEQ ID NO 28. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LNB, LN3, sialylated lactose, LNB, fucosylated and sialylated LNB in all culture broth samples LN3, LNT, and an oligosaccharide mixture of fucosylated and sialylated LNT structures (Table 26) were evaluated for novel strains with cultures containing sucrose as carbon source and lactose as precursor. Table 26 : Evaluation of production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF190 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LNB, 2'FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF191 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LNB, 4-FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LSTa SF192 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LNB, 2'FLNB, 6'SLNB, LN3, 6'S-LN3, LNT, LNFP-I SF193 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LNB, 4-FLNB, 6'SLNB, LN3, 6'S-LN3, LNT SF194 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LNB, 2'FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa SF195 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LNB, 4-FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S- LN3, LNT, LSTa SF196 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF197 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 6'SLNB, LN3, 6'SLN, LNT, LNFP-I SF198 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LNB, 2' FLNB, 4-FLNB, Di-FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa *See Table 2 for plastid information . Example 79. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for sialic acid production as described in Example 1 were further modified by gene body deletion of E. coli wcaJ , ushA and galT genes and gene body insertion of persistent expression cassettes. The cassettes were used for galE from E. coli with SEQ ID NO 30, LgtA from N. meningitidis with SEQ ID NO 27, and LgtB from N. meningitidis with SEQ ID NO 29. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LacNAc, LN3, sialylated LN3, LNnT and an oligosaccharide mixture of fucosylated and sialylated LNnT structures (Table 27) were evaluated for novel strains in which cultures contained sucrose as carbon source and lactose as precursor. Table 27 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF199 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LSTd SF200 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LacNAc, 3'FlacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF201 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LSTc SF202 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LacNAc, 3'FlacNAc, 6'SlacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF203 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LacNAc, 2'FLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LSTc, LSTd SF204 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LacNAc, 3'FlacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S- LN3, LNnT, LNFP-III, LSTc, LSTd SF205 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF206 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF207 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2' FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plastid information . Example 80. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for sialic acid production as described in Example 1 were further modified with gene body deletion of E. coli wcaJ , fucK , fucI , ushA and galT genes and gene body insertion of a persistent expression cassette, which Persistent expression cassettes for galE from E. coli with SEQ ID NO 30, fucP from E. coli with SEQ ID NO 13, fkp from B. fragilis with SEQ ID NO 14, fkp from B. fragilis with SEQ ID NO 27 LgtA from N. meningitidis and WbgO from E. coli O55:H7 with SEQ ID NO 28. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LNB, LN3, sialylated lactose, LNB, fucosylated and sialylated LNB in all culture broth samples LN3, LNT, and an oligosaccharide mixture of fucosylated and sialylated LNT structures (Table 28) were evaluated for novel strains with cultures containing sucrose as carbon source and lactose as precursor. Table 28 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF208 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LNB, 2'FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF209 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LNB, 4-FLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LSTa SF210 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LNB, 2'FLNB, 6'SLNB, LN3, 6'S-LN3, LNT, LNFP-I SF211 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LNB, 4-FLNB, 6'SLNB, LN3, 6'S-LN3, LNT SF212 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LNB, 2'FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa SF213 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LNB, 4-FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S- LN3, LNT, LSTa SF214 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 3'SLNB, LN3, 3'S-LN3, LNT, LNFP-I, LSTa SF215 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LNB, 2'FLNB, 4-FLNB, DiFLNB, 6'SLNB, LN3, 6'SLN, LNT, LNFP-I SF216 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LNB, 2' FLNB, 4-FLNB, Di-FLNB, 3'SLNB, 6'SLNB, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa *See Table 2 for plastid information . Example 81. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using Modified E. coli Hosts

E. coli strains adapted for sialic acid production as described in Example 1 were further modified with gene body deletion of E. coli wcaJ , fucK , fucI , ushA and galT genes and gene body insertion of a persistent expression cassette, which Persistent expression cassettes for galE from E. coli with SEQ ID NO 30, fucP from E. coli with SEQ ID NO 13, fkp from B. fragilis with SEQ ID NO 14, fkp from B. fragilis with SEQ ID NO 27 LgtA from N. meningitidis and LgtB from N. meningitidis having SEQ ID NO 28. In the next step, the novel strain is transformed with two compatible expression plastids, wherein the first plastid contains a persistent expression unit for one or two selected fucosyltransferases, and wherein the second plastid contains Persistent expression units for one or two selected sialyltransferases and NeuA from Pasteurella septicemia having SEQ ID NO 22. Table 2 presents a summary of the six plastids used. In growth experiments according to the culture conditions provided in Example 1, for the production of fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LacNAc, LN3, sialylated LN3, LNnT, and an oligosaccharide mixture of fucosylated and sialylated LNnT structures (Table 29) were evaluated for novel strains with cultures containing sucrose as carbon source and lactose as precursor. Table 29 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all cultures of mutant E. coli strains in growth experiments according to culture conditions as described in Example 1, wherein the medium contains Sucrose as carbon source and lactose as precursor. strain plastid that exists * Oligosaccharide SF217 pMF_1A, pMS_1A 2'FL, 3'SL, 3'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LSTd SF218 pMF_1B, pMS_1A 3-FL, 3'SL, 3'S-3-FL, LacNAc, 3'FlacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF219 pMF_1A, pMS_1B 2'FL, 6'SL, 6'S-2'FL, DiFL, LacNAc, 2'FLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LSTc SF220 pMF_1B, pMS_1B 3-FL, 6'SL, 6'S-3-FL, LacNAc, 3'FlacNAc, 6'SlacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF221 pMF_1A, pMS_2 2'FL, 3'SL, DiFL, 3'S-2'FL, 6'SL, 6'S-2'FL, Di-SL, LacNAc, 2'FLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LSTc, LSTd SF222 pMF_1B, pMS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL, Di-SL, LacNAc, 3'FlacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S- LN3, LNnT, LNFP-III, LSTc, LSTd SF223 pMF_2, pMS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd SF224 pMF_2, pMS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2'FLacNAc, 3'FLacNAc, DiFLacNAc, 6'SLacNAc, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc SF225 pMF_2, pMS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, Di-SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LacNAc, 2' FLacNAc, 3'FLacNAc, DiFLacNAc, 3'SLacNAc, 6'SLacNAc, LN3, 3'S-LN3, 6'S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plastid information , Example 82. Production of a Mammalian Milk Oligosaccharide Mixture Containing 2'FL , 3 - FL and DiFL Using a Modified Saccharomyces cerevisiae Host

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 unit: lactose permease (LAC12) from Kluyveromyces lactis with SEQ ID NO 31, GDP-mannose 4,6-dehydratase (gmd) from Escherichia coli with SEQ ID NO 11, from GDP-L-fucose synthase (fcl) from Escherichia coli having SEQ ID NO 12, α-1,2-fucosyltransferase from Helicobacter pylori having SEQ ID NO 04 and from Helicobacter pylori having SEQ ID NO 04 α-1,3-fucosyltransferase of Helicobacter pylori of NO 05. Mutant yeast strains were evaluated for the production of oligosaccharide mixtures comprising 2'FL, 3-FL and DiFL in growth experiments according to the culture conditions in Example 2 using SD CSM-Ura omission medium containing lactose as a precursor. Example 83. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Lactose Structures Using a Modified Saccharomyces cerevisiae Host

S. cerevisiae strains were adapted for GDP-fucose and CMP-saliva as described in Example 2 with a first yeast-expressed plastid (variant of p2a_2µ_Fuc) and a second yeast-expressed plastid (pRS420 plastid variant) Acid production and expression of one or more fucosyltransferases and one or more sialyltransferases, the first yeast expression plastid comprising a continuous transcription unit for LAC12 of Kluyveromyces lactis, gmd from E. coli having SEQ ID NO 11, fcl from E. coli having SEQ ID NO 12, and one or two selected fucosyltransferases; and the second yeast expression The plastid contains persistent transcription units for: mutant glmS*54 from E. coli with SEQ ID NO 19, phosphatase yqaB from E. coli with SEQ ID NO 20, phosphatase yqaB from E. coli with SEQ ID NO 17 AGE of Bacteroides ovale, neuB from Neisseria meningitidis with SEQ ID NO 18, neuA from Pasteurella septicemia with SEQ ID NO 22, and one or two selected sialyltransferases. Table 30 shows the fucosyltransferases and sialyltransferases selected in the plastids colonized in this experiment. SD CSM-Ura-Trp omission medium containing lactose as a precursor was used in growth experiments according to the culture conditions in Example 2, for the production of fucosylated and sialylated lactose-containing structures (as shown in Table 31) The oligosaccharide mixture was used to evaluate mutant yeast strains. Table 30 : Summary of plastids cloned with persistent transcription units for one or two fucosyltransferase genes or for one or two sialyltransferase genes plastid nr Fucosyltransferases selected from p2a_2µ_Fuc plastid variants a1,2 -bonded a1,3 -bonded pYF_1A SEQ ID NO 04 none pYF_1B none SEQ ID NO 05 pYF_2 SEQ ID NO 04 SEQ ID NO 05 plastid nr Sialyltransferases cloned from pRS420 plastid variants a2,3 -bonded a2,6 -bonded pYS_1A SEQ ID NO 23 none pYS_1B none SEQ ID NO 25 pYS_2 SEQ ID NO 23 SEQ ID NO 25 Table 31 : Oligosaccharide production assessed by mutant S. cerevisiae strains expressing selected fucosyltransferase and sialyltransferase genes and grown in SD CSM-Ura-Trp omission medium containing lactose as precursor . strain plastid that exists * Oligosaccharide SY1 p YF_1A, p YS_1A 2'FL, 3'SL, 3'S-2'FL SY2 p YF_1B, p YS_1A 3-FL, 3'SL, 3'S-3-FL SY3 p YF_1A, p YS_1B 2'FL, 6'SL, 6'S-2'FL SY4 p YF_1B, p YS_1B 3-FL, 6'SL, 6'S-3-FL SY5 p YF_1A, p YS_2 2'FL, 3'SL, 3'S-2'FL, 6'SL, 6'S-2'FL SY6 p YF_1B, p YS_2 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL SY7 p YF_2, p YS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL SY8 p YF_2, p YS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL SY9 p YF_2, p YS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL *For plastid information see Table 30Example 84. Production of Mammalian Milk Oligosaccharide Mixtures Containing LN3 , 3'- sialylated LN3 , LNT , LNB , 3'SL and LSTa Using a Modified Saccharomyces cerevisiae Host

Saccharomyces cerevisiae strains were adapted with a first yeast-expressing plastid and a second yeast-expressing plastid for CMP-sialic acid and LNT production and β-galactoside α-2,3-sialic acid as described in Example 2 Expression of a basal transferase, the first yeast expression plastid comprising a persistent transcription unit for: LAC12 from Kluyveromyces lactis with SEQ ID NO 31, mutations from Escherichia coli with SEQ ID NO 19 Type glmS*54, Phosphatase yqaB from E. coli with SEQ ID NO 20, AGE from Bacteroides ovale with SEQ ID NO 17, NeuB from Neisseria meningitidis with SEQ ID NO 18, from NeuA of Pasteurella septicemia having SEQ ID NO 22 and β-galactoside alpha-2,3-sialyltransferase from Pasteurella septicemia having SEQ ID NO 23; and the second yeast expression The plastids contain persistent transcription units for: galE from E. coli with SEQ ID NO 30, LgtA from N. meningitidis with SEQ ID NO 27, and from E. coli with SEQ ID NO 28 WbgO of O55:H7. SD CSM-Trp-His omission medium containing lactose as a precursor was used in growth experiments according to the culture conditions in Example 2 for the production of LN3, 3'-sialylated LN3, LNT, LNB, 3'SL and LSTa Oligosaccharide mixtures to evaluate mutant yeast strains. Example 85. Production of a mammalian milk oligosaccharide mixture comprising LN3 , 6' -sialylated LN3 , LNnT , LacNAc , 6'SL and LSTc using a modified S. cerevisiae host

Saccharomyces cerevisiae strains were adapted with a first yeast-expressing plastid and a second yeast-expressing plastid for the production of CMP-sialic acid and LNnT and β-galactoside α-2,6-sialic acid as described in Example 2 Expression of a basal transferase, the first yeast expression plastid comprising a persistent transcription unit for: LAC12 from Kluyveromyces lactis with SEQ ID NO 31, mutations from Escherichia coli with SEQ ID NO 19 Type glmS*54, Phosphatase yqaB from E. coli with SEQ ID NO 20, AGE from Bacteroides ovale with SEQ ID NO 17, NeuB from Neisseria meningitidis with SEQ ID NO 18, from NeuA of Pasteurella septicemia having SEQ ID NO 22 and β-galactoside alpha-2,6-sialyltransferase from P. mermaidii having SEQ ID NO 25 and the second yeast expression plastid comprises Continuous transcription units for: galE from E. coli with SEQ ID NO 30, LgtA from Neisseria meningitidis with SEQ ID NO 27 and from Neisseria meningitidis with SEQ ID NO 29 Bacterial LgtB. SD CSM-Trp-His omission medium containing lactose as a precursor was used in growth experiments according to the culture conditions in Example 2 for the production of LN3, 6'-sialylated LN3, LNnT, LAcNAc, 6'SL and LSTc Oligosaccharide mixtures to evaluate mutant yeast strains. Example 86. Production of a mammalian milk oligosaccharide mixture comprising LN3 , 3'- sialylated LN3 , LNnT , LacNAc , 3'SL and LSTd using a modified S. cerevisiae host

Saccharomyces cerevisiae strains were adapted with a first yeast-expressing plastid and a second yeast-expressing plastid for the production of CMP-sialic acid and LNnT and β-galactoside α-2,3-sialic acid as described in Example 2 Expression of a basal transferase, the first yeast expression plastid comprising a persistent transcription unit for: LAC12 from Kluyveromyces lactis with SEQ ID NO 31, mutations from Escherichia coli with SEQ ID NO 19 Type glmS*54, Phosphatase yqaB from E. coli with SEQ ID NO 20, AGE from Bacteroides ovale with SEQ ID NO 17, NeuB from Neisseria meningitidis with SEQ ID NO 18, from NeuA of Pasteurella septicemia with SEQ ID NO 22 and β-galactoside alpha-2,3-sialyltransferase from Pasteurella septicemia with SEQ ID NO 23 and the second yeast expression The body contains persistent transcription units for: galE from E. coli with SEQ ID NO 30, LgtA from Neisseria meningitidis with SEQ ID NO 27 and from meningitidis with SEQ ID NO 29 LgtB of S. cerevisiae. SD CSM-Trp-His omission medium containing lactose as a precursor was used in growth experiments according to the culture conditions in Example 2 for the production of LN3, 3'-sialylated LN3, LNnT, LacNAc, 3'SL and LSTd Oligosaccharide mixtures to evaluate mutant yeast strains. Example 87. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2'FL , DiFL , LN3 , LNT and LNFP-I Using a Modified Saccharomyces cerevisiae Host

Saccharomyces cerevisiae strains were adapted with a first yeast-expressing plastid and a second yeast-expressing plastid for the production of GDP-fucose and LNT and a-1,2-fucosyltransferase as described in Example 2 expression, the first yeast expression plastid comprises a persistent transcription unit for: LAC12 from Kluyveromyces lactis with SEQ ID NO 31, gmd from E. coli with SEQ ID NO 11, gmd from Escherichia coli with SEQ ID NO 11 The fcl of E. coli of SEQ ID NO 12 and the alpha-1,2-fucosyltransferase from Helicobacter pylori of SEQ ID NO 04 and the second yeast expression plastid comprise persistent transcription for Units: galE from E. coli with SEQ ID NO 30, LgtA from N. meningitidis with SEQ ID NO 27 and WbgO from E. coli O55:H7 with SEQ ID NO 28. SD CSM-Ura-His omission medium containing lactose as a precursor was used in the growth experiments according to the culture conditions in Example 2, for production of 2'FL, DiFL, LN3, LNT and lacto-N-fucopentose I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) oligosaccharide mixture to evaluate mutant yeast strains. Example 88. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2'FL , DiFL , LN3 , LNT and LNFP-II Using a Modified Saccharomyces cerevisiae Host

Saccharomyces cerevisiae strains were adapted with a first yeast-expressing plastid and a second yeast-expressing plastid for the production of GDP-fucose and LNT and al,3/4-fucosidase and a1,3/4-fucosidase as described in Example 2 - Expression of 1,2-fucosyltransferase, the first yeast expression plastid comprising a continuous transcription unit for LAC12 from Kluyveromyces lactis with SEQ ID NO 31, from LAC12 with SEQ ID NO 31 gmd of E. coli with ID NO 11, fcl from E. coli with SEQ ID NO 12, mutant al,3/4 fucosidase from Bifidobacterium longum subsp. infantis ATCC 15697 with SEQ ID NO 39 and The alpha-1,2-fucosyltransferase from Helicobacter pylori with SEQ ID NO 04 and the second yeast expression plastid comprise a persistent transcription unit for: from large intestine with SEQ ID NO 30 Bacillus galE, LgtA from Neisseria meningitidis with SEQ ID NO 27, and WbgO from Escherichia coli O55:H7 with SEQ ID NO 28. SD CSM-Ura-His omission medium containing lactose as a precursor was used in the growth experiments according to the culture conditions in Example 2 for the production of 2'FL, DiFL, LN3, LNT and lacto-N-fucopentose II (LNFP-II, Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc) oligosaccharide mixture to evaluate mutant yeast strains. Example 89. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3-FL , LN3 , LNT and LNFP-V Using a Modified Saccharomyces cerevisiae Host

Saccharomyces cerevisiae strains were adapted with a first yeast-expressing plastid and a second yeast-expressing plastid for GDP-fucose and LNT production and a-1,3-fucosyltransferase as described in Example 2 expression, the first yeast expression plastid comprises a persistent transcription unit for: LAC12 from Kluyveromyces lactis with SEQ ID NO 31, gmd from E. coli with SEQ ID NO 11, gmd from Escherichia coli with SEQ ID NO 11 The fcl of E. coli of SEQ ID NO 12 and the truncated al,3-fucosyltransferase from Helicobacter pylori of SEQ ID NO 06 and the second yeast expression plastid comprise a persistent form for Transcription units: galE from E. coli with SEQ ID NO 30, LgtA from N. meningitidis with SEQ ID NO 27, and WbgO from E. coli O55:H7 with SEQ ID NO 28. SD CSM-Ura-His omission medium containing lactose as a precursor was used in the growth experiments according to the culture conditions in Example 2, for the production of -V,Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuca1,3)-Glc) oligosaccharide mixture to evaluate mutant yeast strains. Example 90. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3-FL , LN3 , LNnT and LNFP-III Using a Modified Saccharomyces cerevisiae Host

Saccharomyces cerevisiae strains were adapted with a first yeast-expressing plastid and a second yeast-expressing plastid for the production of GDP-fucose and LNnT and the expression of a-1,3-fucosyltransferase in Example 2 , the first yeast expression plastid comprises a persistent transcription unit for: LAC12 from Kluyveromyces lactis with SEQ ID NO 31, gmd from E. coli with SEQ ID NO 11, gmd from Escherichia coli with SEQ ID NO 11 The fcl of E. coli of NO 12 and the truncated al,3-fucosyltransferase from Helicobacter pylori with SEQ ID NO 06 and the second yeast expression plastid comprise a persistent transcription unit for : galE from E. coli with SEQ ID NO 30, LgtA from Neisseria meningitidis with SEQ ID NO 27 and LgtB from Neisseria meningitidis with SEQ ID NO 29. SD CSM-Ura-His omission medium containing lactose as a precursor was used in the growth experiments according to the culture conditions in Example 2, for the production of -III, Oligosaccharide mixture of Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1,3-Gal-b1,4-Glc) to evaluate mutant yeast strains. Example 91. Production of mammalian milk oligosaccharide mixtures comprising fucosylated and sialylated oligosaccharide structures using a modified S. cerevisiae host

Saccharomyces cerevisiae strains were adapted for GDP- Production of fucose, CMP-sialic acid and LNT and expression of selected fucosyltransferases and sialyltransferases, the first yeast expression plastid comprising a persistent transcription unit for LAC12 of Kluyveromyces lactis with ID NO 31, gmd from E. coli with SEQ ID NO 11, fcl from E. coli with SEQ ID NO 12 and one or two selected fucosyltransferases (see Table 30), and the second yeast expression plastid comprises a persistent transcription unit for: mutant glmS*54 from Escherichia coli with SEQ ID NO 19, phosphatase from Escherichia coli with SEQ ID NO 20 yqaB, AGE from Bacteroides ovale with SEQ ID NO 17, neuB from Neisseria meningitidis with SEQ ID NO 18, neuA from Pasteurella septicemia with SEQ ID NO 22, and one or both A selected sialyltransferase (see Table 30), and the third yeast-expressed plastid comprises a continuous transcription unit for: galE from E. coli with SEQ ID NO 30, from galE with SEQ ID NO 27 LgtA from N. meningitidis and WbgO from E. coli O55:H7 with SEQ ID NO 28. SD CSM-Ura-Trp-His omission medium containing lactose as a precursor was used in the growth experiments according to the culture conditions in Example 2, for the production of fucosylated and sialylated lactose, LNB, sialylated LNB, LN3 Mutant yeast strains were evaluated for oligosaccharide mixtures of , sialylated LN3, LNT, and fucosylated and sialylated LNT structures (Table 32). Table 32 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all broths of mutant S. cerevisiae strains expressing selected fucosyltransferase and sialyltransferase genes and Cultured in SD CSM-Ura-Trp-His sparing medium containing lactose as a precursor. strain plastid that exists * Oligosaccharide SY10 pYF_1C, pYS_1A 2'FL, DiFL, 3'SL, 3'S-2'FL, LN3, 3'S-LN3, LNT, LNFP-I, LSTa, 3'SLNB SY11 pYF_1D, pYS_1A 3-FL, 3'SL, 3'S-3-FL, LN3, 3'S-LN3, LNT, LSTa, 3'SLNB SY12 pYF_1C, pYS_1B 2'FL, DiFL, 6'SL, 6'S-2'FL, LN3, 6'S-LN3, LNT, LNFP-I, 6'SLNB SY13 pYF_1D, pYS_1B 3-FL, 6'SL, 6'S-3-FL, LN3, LNT, 6'S-LN3, 6'SLNB SY14 pYF_1C, pYS_2 2'FL, DiFL, 3'SL, 3'S-2'FL, 6'SL, 6'S-2'FL, LN3, 3'S-LN3, 6'S-LN3, LNT, LNFP-I, LSTa, 3'SLNB, 6' SLNB SY15 pYF_1D, pYS_2 2'FL, 3'SL, 3'S-2'FL, 6'SL, 6'S-2'FL, LN3, 3'S-LN3, 6'S-LN3, LNT, LSTa, 3'SLNB, 6'SLNB SY16 pYF_2CD, pYS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LN3, 3'S-LN3, LNT, LNFP-I, LSTa, 3'SLNB SY17 pYF_2CD, pYS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LN3, 6'S-LN3, LNT, LNFP-I, 6'SLNB SY18 pYF_2CD, pYS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LN3, 3'S-LN3, 6'S- LN3, LNT, LNFP-I, LSTa, 3'SLNB, 6'SLNB *See Table 30 for plastid information , Example 92. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using a Modified Saccharomyces cerevisiae Host

Saccharomyces cerevisiae strains were adapted for GDP- Production of fucose, CMP-sialic acid and LNnT and expression of selected fucosyltransferases and sialyltransferases, the first yeast expressing plastid comprising a persistent transcription unit for LAC12 of Kluyveromyces lactis with ID NO 31, gmd from E. coli with SEQ ID NO 11, fcl from E. coli with SEQ ID NO 12 and one or two selected fucosyltransferases (see Table 30), and the second yeast expression plastid comprises a persistent transcription unit for: mutant glmS*54 from Escherichia coli with SEQ ID NO 19, phosphatase from Escherichia coli with SEQ ID NO 20 yqaB, AGE from Bacteroides ovale with SEQ ID NO 17, neuB from Neisseria meningitidis with SEQ ID NO 18, neuA from Pasteurella septicemia with SEQ ID NO 22, and one or both A selected sialyltransferase (see Table 30), and the third yeast-expressed plastid comprises a continuous transcription unit for: galE from E. coli with SEQ ID NO 30, from galE with SEQ ID NO 27 LgtA from N. meningitidis and LgtB from N. meningitidis having SEQ ID NO 29. SD CSM-Ura-Trp-His omission medium containing lactose as a precursor was used in the growth experiments according to the culture conditions in Example 2 for production of fucosylated and sialylated lactose, LacNAc, sialylated LacNAc, LN3 Mutant yeast strains were evaluated for oligosaccharide mixtures of , sialylated LN3, LNnT, and fucosylated and sialylated LNnT structures (Table 33). Table 33 : Evaluation of the production of mixtures comprising tri-, tetra- and penta-oligosaccharides in all broths of mutant S. cerevisiae strains expressing selected fucosyltransferase and sialyltransferase genes and Cultured in SD CSM-Ura-Trp-His sparing medium containing lactose as a precursor. strain plastid that exists * Oligosaccharide SY19 pYF_1C, pYS_1A 2'FL, DiFL, 3'SL, 3'S-2'FL, LN3, 3'S-LN3, LNnT, LSTd, 3'SLacNAc SY20 pYF_1D, pYS_1A 3-FL, 3'SL, 3'S-3-FL, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd, 3'SLacNAc SY21 pYF_1C, pYS_1B 2'FL, DiFL, 6'SL, 6'S-2'FL, LN3, 6'S-LN3, LNnT, LSTc, 6'SLacNAc SY22 pYF_1D, pYS_1B 3-FL, 6'SL, 6'S-3-FL, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc, 6'SLacNAc SY23 pYF_1C, pYS_2 2'FL, DiFL, 3'SL, 3'S-2'FL, 6'SL, 6'S-2'FL, LN3, 3'S-LN3, 6'S-LN3, LNnT, LSTc, LSTd, 3'SLacNAc, 6'SLacNAc SY24 pYF_1D, pYS_2 2'FL, 3'SL, 3'S-2'FL, 6'SL, 6'S-2'FL, LN3, 3'S-LN3, 6'S-LN3, LNnT, LNFP-III, LSTc, LSTd, 3'SLacNAc, 6' SLacNAc SY25 pYF_2CD, pYS_1A 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL, LN3, 3'S-LN3, LNnT, LNFP-III, LSTd, 3'SLacNAc SY26 pYF_2CD, pYS_1B 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL, LN3, 6'S-LN3, LNnT, LNFP-III, LSTc, 6'SLacNAc SY27 pYF_2CD, pYS_2 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL, LN3, 3'S-LN3, 6'S- LN3, LNnT, LNFP-III, LSTc, LSTd, 3'SLacNAc, 6'SLacNAc *See Table 30 for plastid information Example 93. Production of Mammalian Milk Oligosaccharide Mixtures Containing Fucosylated and Sialylated Oligosaccharide Structures Using a Modified Saccharomyces cerevisiae Host

Adaptation of Saccharomyces cerevisiae strains with yeast artificial chromosome (YAC) for production of GDP-fucose and CMP-sialic acid and interaction of one or more fucosyltransferases and one or more sialyltransferases It was shown that the yeast artificial chromosome comprises a persistent transcription unit for: LAC12 from Kluyveromyces lactis with SEQ ID NO 31, gmd from E. coli with SEQ ID NO 11, gmd from Escherichia coli with SEQ ID NO 12 fcl from Escherichia coli, mutant glmS*54 from Escherichia coli with SEQ ID NO 19, phosphatase yqaB from Escherichia coli with SEQ ID NO 20, AGE from Bacteroides ovale with SEQ ID NO 17, neuB from Neisseria meningitidis with SEQ ID NO 18, neuA from Pasteurella septicemia with SEQ ID NO 22, one or two selected fucosyltransferases and one or two selected sialic acids base transferase. Table 34 shows selected fucosyltransferases and sialyltransferases among the YACs produced in this experiment. SD CSM medium containing lactose as a precursor was used in growth experiments according to the culture conditions in Example 2, evaluated for the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose structures (as shown in Table 35) mutant yeast strains. Table 34 : Summary of fucosyltransferase genes and sialyltransferase genes colonized in different yeast artificial chromosomes yeast artificial chromosome Fucosyltransferase sialyltransferase a1,2 -bonded a1,3 -bonded a2,3 -bonded a2,6 -bonded YAC1 SEQ ID NO 04 none SEQ ID NO 23 none YAC2 none SEQ ID NO 05 SEQ ID NO 23 none YAC3 SEQ ID NO 04 none none SEQ ID NO 25 YAC4 none SEQ ID NO 05 none SEQ ID NO 25 YAC5 SEQ ID NO 04 none SEQ ID NO 23 SEQ ID NO 25 YAC6 none SEQ ID NO 05 SEQ ID NO 23 SEQ ID NO 25 YAC7 SEQ ID NO 04 SEQ ID NO 05 SEQ ID NO 23 none YAC8 SEQ ID NO 04 SEQ ID NO 05 none SEQ ID NO 25 YAC9 SEQ ID NO 04 SEQ ID NO 05 SEQ ID NO 23 SEQ ID NO 25 Table 35 : Evaluation of oligosaccharide production in all cultures of mutant S. cerevisiae strains expressing selected fucosyltransferase and sialyltransferase genes from yeast artificial chromosomes and cultured in cultures containing lactose as precursor in SD CSM medium. strain YAC* that exists Oligosaccharide SY28 YAC1 2'FL, 3'SL, 3'S-2'FL SY29 YAC2 3-FL, 3'SL, 3'S-3-FL SY30 YAC3 2'FL, 6'SL, 6'S-2'FL SY31 YAC4 3-FL, 6'SL, 6'S-3-FL SY32 YAC5 2'FL, 3'SL, 3'S-2'FL, 6'SL, 6'S-2'FL SY33 YAC6 3-FL, 3'SL, 3'S-3-FL, 6'SL, 6'S-3-FL SY34 YAC7 2'FL, 3-FL, DiFL, 3'SL, 3'S-2'FL, 3'S-3-FL SY35 YAC8 2'FL, 3-FL, DiFL, 6'SL, 6'S-2'FL, 6'S-3-FL SY36 YAC9 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3-FL, 6'S-2'FL, 6'S-3-FL *See Table 34 for an overview of YAC , Example 94. 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 was composed of 0.735 g/L CaCl2.2H2O, 0.1 g/L MnCl2.2H2O, 0.033 g/L CuCl2.2H2O, 0.06 g/L CoCl2.6H2O, 0.17 g/L ZnCl2, 0.0311 g/L H3BO4, 0.4 g/L Na2EDTA.2H2O and 0.06 g/L Na2MoO4. The ferric citrate solution contained 0.135 g/L FeCl3.6H2O, 1 g/L sodium citrate (Hoch 1973 PMC1212887).

Luria Broth (LB) medium 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, LNB or LacNAc can be added.

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 is performed 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. Suitable promoters for expression can be derived from the Component Repository (iGem): 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 E. coli lacY having SEQ ID NO 15. In one embodiment of 2'FL, 3FL and/or diFL production, an alpha-1,2- and/or a-1,3-fucosyltransferase expression construct is additionally added to the strain. In one example of LN3 production, a galactoside beta-1,3-N-acetylglucosaminyltransferase (such as, for example, IgtA (SEQ ID NO 27) from Neisseria meningitidis) will be included A persistent transcription unit was additionally added to the strain. In one embodiment of LNT production, the LN3-producing strain is further treated with an N-acetylglucosamine beta-1,3-galactosyltransferase (such as, for example, WbgO (SEQ ID NO 28) from E. coli O55:H7 )) persistent transcriptional unit modification. In one example of LNnT production, the LN3-producing strain is further treated with an enzyme comprising N-acetylglucosamine beta-1,4-galactosyltransferase (such as, for example, lgtB (SEQ ID NO. 29)) of the persistent transcription unit modification.

In one example of sialic acid production, mutant B. subtilis strains enhance cellularity by overexpressing fructose-6-P-aminotransferase such as native fructose-6-P-aminotransferase (UniProt ID P0CI73). produced from the pool of glucosamine-6-phosphate. In addition, the enzymatic activities of the genes nagA, nagB and gamA are disrupted by gene knockout and such as eg glucosamine-6-P-aminotransferase (SEQ ID NO 16) of Saccharomyces cerevisiae, as eg N- Acetylglucosamine-2-epimerase (SEQ ID NO 17) and N-acetylneuraminic acid synthase (SEQ ID NO 18) as for example from Neisseria meningitidis are overexpressed in the gene body superior. To allow sialylated oligosaccharide production, the sialic acid-producing strain is further modified with a persistent transcription unit comprising an N-acyl neuraminic acid cytidine group such as, for example, the NeuA enzyme from Pasteurella septicemia Transferase (SEQ ID NO 22) and one or more copies of β-galactoside α-2,3-sialyltransferase (UniProt ID Q9CLP3) such as, for example, PmultST3 from Pasteurella septicaemia or by having A PmultST3-like polypeptide (SEQ ID NO 23) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 with β-galactoside α-2,3-sialyltransferase activity or from Neisseria meningitidis. NmeniST3 (SEQ ID NO 24) or PmultST2 (GenBank NO. AAK02592.1) from Pasteurella septicemia subsp. Pasteurella strain Pm70, such as, for example, β-galactoside alpha-2 from PdST6 of Luminus mermaidii, 6-Sialyltransferase (UniProt ID O66375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID O66375 with β-galactoside α-2,6-sialyltransferase activity (SEQ ID NO 25) or P-JT-ISH-224-ST6 (UniProt ID A8QYL1) from Luminus sp. A P-JT-ISH-224-ST6-like polypeptide (SEQ ID NO 26) consisting of amino acid residues 18 to 514 of the enzymatically active UniProt ID A8QYL1 and/or such as, for example, α-2,8-saliva of Mus musculus Acid transferase (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 95. Production of Mammalian Milk Oligosaccharide Mixtures Containing 2'FL , 3-FL and DiFL Using a Modified Bacillus subtilis Host

Bacillus subtilis strains were modified as described in Example 94 by gene body insertion of persistent transcription units for lactose permease (LacY) from E. coli with SEQ ID NO 15 and with SEQ ID NO 15 The alpha-1,2-fucosyltransferase HpFutC of ID NO 04 and the alpha-1,3-fucosyltransferase HpFucT of SEQ ID NO 05. Novel strains were evaluated for production of 2'FL, 3-FL and DiFL in growth experiments on MMsf medium containing lactose according to the culture conditions provided in Example 94. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 96. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2'FL , DiFL , LNFP-I , LNT and LN3 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 from Escherichia coli Lactose permease (LacY) (SEQ ID NO 15), native fructose-6-P-aminotransferase (UniProt ID P0CI73), galactoside β-1,3-N- from Neisseria meningitidis Acetylglucosaminyltransferase LgtA (SEQ ID NO 27), sucrose transporter (CscB) from Escherichia coli W (SEQ ID NO 01), fructokinase (Frk) from Zymomonas mobilis (SEQ ID NO 02) and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (SEQ ID NO 03). In the next step, the mutant strain was further transformed with a gene comprising a continuous transcription unit of N-acetylglucosamine β-1,3-galactosyltransferase WbgO (SEQ ID NO 28) from E. coli O55:H7 Body intercalation modification to generate LNT. In a subsequent step, the LNT-producing strain was transformed with an expression plastid comprising a persistent transcription unit for the alpha-1,2-fucosyltransferase HpFutC (SEQ ID NO 04) from H. pylori. Novel strains were evaluated for production of oligosaccharide mixtures comprising 2'FL, DiFL, LN3, LNT and LNFP-I in growth experiments on MMsf medium containing lactose as a precursor according to the culture conditions provided in Example 94. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 97. Production of a mammalian milk oligosaccharide mixture comprising 3'SL , LN3 , LNT , sialylated LN3 , LSTa using a modified Bacillus subtilis host

LNT-producing mutant B. subtilis strains as described in Example 96 were further genetically knocked out of the nagA gene and modified with a second compatible expression plastid comprising the TRP1 selectable marker and a persistent transcription unit For: two copies of mutant L-glutamic acid-D-fructose-6-phosphate aminotransferase (glmS*54) (SEQ ID NO 19) from Escherichia coli; phosphatase, such as for example selected from Contains 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 , phosphatases of E. coli genes of YrfG and YbiU or PsMupP from Pseudomonas sputum, ScDOG1 from Saccharomyces cerevisiae or BsAraL from Bacillus subtilis (as described in WO18122225); N- Acetylglucosamine 2-epimerase (AGE) (SEQ ID NO 17); N-acetylneuraminic acid synthase (NeuB) from Neisseria meningitidis (SEQ ID NO 18); from Pasteurella septicemia's N-acylneuraminic acid cytidine acyltransferase NeuA (SEQ ID NO 22); Three copies of a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 with transferase activity. Growth experiments were performed on MMsf medium containing lactose as a precursor according to the culture conditions provided in Example 94 for production of 3'SL, LN3, sialylated LN3, LNT and LSTa (Neu5Ac-a2,3-Gal-b1 , 3-GlcNAc-b1,3-Gal-b1,4-Glc) mixtures were evaluated for novel strains. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 98. Production of an oligosaccharide mixture comprising 2'FL , DiFL , LNFP-I , LNFP-II , LNT and LN3 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 from Escherichia coli Lactose permease (LacY) (SEQ ID NO 15), native fructose-6-P-aminotransferase (UniProt ID P0CI73), galactoside β-1,3-N- from Neisseria meningitidis Acetylglucosaminyltransferase LgtA (SEQ ID NO 27), sucrose transporter (CscB) from Escherichia coli W (SEQ ID NO 01), fructokinase (Frk) from Zymomonas mobilis (SEQ ID NO 02) and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (SEQ ID NO 03). In the next step, the mutant strain was further transformed with a gene comprising a continuous transcription unit of N-acetylglucosamine β-1,3-galactosyltransferase WbgO (SEQ ID NO 28) from E. coli O55:H7 Body intercalation modification to generate LNT. In a subsequent step, the LNT-producing strain was transformed with an expression plastid containing the α-1,2-fucosyltransferase HpFutC (SEQ ID NO 04) for the α-1,2-fucosyltransferase from H. Continuous transcription unit of mutant al,3/4 fucosidase of Bifidobacterium longum subsp. infantis ATCC 15697 of SEQ ID NO 39. Evaluation for the production of oligosaccharide mixtures comprising LN3, LNT, LNFP-I, LNFP-II, 2'FL and DiFL in growth experiments on MMsf medium containing lactose as a precursor according to the culture conditions provided in Example 94 Novel strains. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 99. Production of 2'FL , 3 - FL , DiFL , 3'SL , 6'SL , 3'S - 2'FL , 3'S - 3 - FL , 6 ' using a modified Bacillus subtilis host Oligosaccharide mixture of S-2 ' FL and 6 ' S-3-FL

Bacillus subtilis strains modified by gene body deletion of nagA , nagB , glmS and gamA genes and gene body insertion of persistent transcription units for lactose permease from Escherichia coli with SEQ ID NO 15 (LacY), sucrose transporter (CscB) from Escherichia coli W (SEQ ID NO 01), fructokinase (Frk) from Zymomonas mobilis (SEQ ID NO 02), sucrose phosphate from Bifidobacterium adolescentis Enzyme (BaSP) (SEQ ID NO 03), native fructose-6-P-aminotransferase (UniProt ID P0CI73), glucosamine 6-phosphate N-acetyltransferase GNA1 (SEQ ID NO) from Saccharomyces cerevisiae 16), mutant L-glutamic acid-D-fructose-6-phosphate aminotransferase (glmS*54) (SEQ ID NO 19) from Escherichia coli; phosphatase, such as 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 Phosphatase from the E. coli gene or PsMupP from Pseudomonas putida, ScDOG1 from Saccharomyces cerevisiae or BsAraL from Bacillus subtilis (as described in WO18122225); N-acetylglucose from Bacillus ovale Amine 2-epimerase (AGE) (SEQ ID NO 17); N-acetylneuraminic acid synthase (NeuB) (SEQ ID NO 18) from Neisseria meningitidis; and NeuA (SEQ ID NO 22). In the next step, the strain is transformed with an expression plastid comprising a persistent transcription unit for the Three copies of a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 with transferase activity; 25) Three copies of a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID O66375. In another step, the mutant strain is transformed with a second compatible expression plastid comprising HpFutC for the alpha-1,2-fucosyltransferase having SEQ ID NO 04 and a continuous transcription unit with the α-1,3-fucosyltransferase HpFucT of SEQ ID NO 05. In growth experiments on MMsf medium containing lactose according to the culture conditions provided in Example 94, for the production of 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S- 3-FL, 6'S-2'FL, 6'S-3-FL evaluated novel strains. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 100. Production of LN3 , LNnT , GalNAc-b1,3-LNnT , Gal-b1,3-GalNAc-b1,3-LNnT , GalNAc -b1,3- lactose, Gal- Oligosaccharide mixture of b1,3-GalNAc-b1,3- lactose and (GalNAc) -poly- LNnT

Bacillus subtilis strains were first modified by gene body deletion of the lacZ , nagB , gamA , glk , galE , galT , galK and galM genes and gene body insertion comprising the persistent transcription unit of the genes encoding lactose from E. coli Permease (LacY) (SEQ ID NO 15), native fructose-6-P-aminotransferase (UniProt ID POCI73), sucrose transporter protein (CscB) from Escherichia coli W (SEQ ID NO 01), from locomotor Zymomonas fructokinase (Frk) (SEQ ID NO 02) and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (SEQ ID NO 03). The mutant strain thus obtained is further modified by gene body insertion of persistent transcription units comprising lgtB (SEQ ID NO 29) from Neisseria meningitidis, galE (SEQ ID NO: 29) from Escherichia coli NO 30), IgtA (SEQ ID NO 27) from Neisseria meningitidis, mutant glmS*54 (SEQ ID NO 19) from Escherichia coli, WbpP (SEQ ID NO 34) from Pseudomonas aeruginosa ) and LgtD (SEQ ID NO 35) from Haemophilus influenzae. In growth experiments on MMsf medium containing lactose as a precursor according to the culture conditions provided in Example 94, for the production of LN3, LNnT, GalNAc-b1,3-LNnT, Gal-b1,3-GalNAc-b1,3 - Oligosaccharide mixtures of LNnT, GalNAc-b1,3-lactose and Gal-b1,3-GalNAc-b1,3-lactose and poly-LNnT structures and GalNAc-ylated poly-LNnT structures were evaluated for novel strains. Example 101. Production of LacNAc , GalNAc- b1,3- lactose, Gal-b1,3-GalNAc-b1,3- lactose, LN3 , LNnT , GalNAc-b1,3-LacNAc , Oligosaccharide mixture of Gal-b1,3-GalNAc-b1,3-LacNAc , poly -LacNAc (Gal-b1,4-GlcNAc)n and GalNAc - ylated polyLacNAc structures

Bacillus subtilis strains were first modified by gene body deletion of the lacZ , nagB , gamA , glk , galE , galT , galK and galM genes and gene body insertion comprising the persistent transcription unit of the genes encoding lactose from E. coli Permease (LacY) (SEQ ID NO 15), native fructose-6-P-aminotransferase (UniProt ID POCI73), mutant glmS*54 (SEQ ID NO 19) from E. coli, GNA1 from Saccharomyces cerevisiae (SEQ ID NO 16), phosphatase yqaB (SEQ ID NO 20) from Escherichia coli, galactoside β-1,3-N-acetylglucosaminyltransferase (LgtA) from Neisseria meningitidis ) (SEQ ID NO 27), LgtB from Neisseria meningitidis (SEQ ID NO 29), 4-epimerase (WbpP) from Pseudomonas aeruginosa (SEQ ID NO 34) and from influenza β1,3-N-Acetylgalactosaminyltransferase (LgtD) of Haemophilus (SEQ ID NO 35). Growth experiments were performed on MMsf medium containing lactose as a precursor according to the culture conditions provided in Example 94 for production of LacNAc, LN3, LNnT, GalNAc-b1,3-lactose, Gal-b1,3-GalNAc-b1 ,3-Lactose, GalNAc-b1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, GalNAc-b1,3-LacNAc, Gal-b1,3-GalNAc-b1,3 - Oligosaccharide mixture of LacNAc, poly-LacNAc structures ie (Gal-bl,4-GlcNAc)n and GalNAc-ylated poly-LacNAc structures to evaluate novel strains. Example 102. 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 was composed of 10 g/L CaCl2, 10 g/L FeSO4.7H2O, 10 g/L MnSO4.H2O, 1 g/L ZnSO4.7H2O, 0.2 g/L CuSO4, 0.02 g/L NiCl2.6H2O, 0.2 g/L biotin (pH 7.0) and 0.03 g/L protocatechuic acid.

Minimal medium 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 to the medium.

TY medium was composed 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., 2005 Apr, 67(2):225-33) and as described by Okibe et al. (Journal of Microbiological Methods 85, 2011, 155-163) described the thermosensitive shuttle vector was 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 having SEQ ID NO 15). In one embodiment of 2'FL, 3FL and/or diFL production, an alpha-1,2- and/or a-1,3-fucosyltransferase expression construct is additionally added to the strain.

In one example of LN3 production, a galactoside beta-1,3-N-acetylglucosaminyltransferase (such as, for example, IgtA (SEQ ID NO 27) from Neisseria meningitidis) will be included A persistent transcription unit was additionally added to the strain. In one embodiment of LNT production, the LN3-producing strain is further treated with an N-acetylglucosamine beta-1,3-galactosyltransferase (such as, for example, WbgO (SEQ ID NO 28) from E. coli O55:H7 )) persistent transcriptional unit modification. In one example of LNnT production, the LN3-producing strain is further treated with an enzyme comprising N-acetylglucosamine beta-1,4-galactosyltransferase (such as, for example, lgtB (SEQ ID NO. 29)) of the persistent transcription unit modification.

In one example of sialic acid production, mutant Corynebacterium glutamicum strains were developed by overexpressing fructose-6-P-aminotransferase such as native fructose-6-P-aminotransferase (UniProt ID Q8NND3) Produced by enhancing the intracellular glucosamine-6-phosphate pool. In addition, the enzymatic activities of the genes nagA, nagB and gamA are disrupted by gene knockout and such as eg glucosamine-6-P-aminotransferase (SEQ ID NO 16) of Saccharomyces cerevisiae, as eg N- Acetylglucosamine-2-epimerase (SEQ ID NO 17) and N-acetylneuraminic acid synthase (SEQ ID NO 18) as for example from Neisseria meningitidis are overexpressed in the gene body superior. To allow sialylated oligosaccharide production, the sialic acid-producing strain is further modified with a persistent transcription unit comprising an N-acyl neuraminic acid cytidine group such as, for example, the NeuA enzyme from Pasteurella septicemia Transferase (SEQ ID NO 22) and one or more copies of β-galactoside α-2,3-sialyltransferase (UniProt ID Q9CLP3) such as, for example, PmultST3 from Pasteurella septicaemia or by having A PmultST3-like polypeptide (SEQ ID NO 23) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 with β-galactoside α-2,3-sialyltransferase activity or from Neisseria meningitidis. NmeniST3 (SEQ ID NO 24) or PmultST2 (GenBank NO. AAK02592.1) from Pasteurella septicemia subsp. Pasteurella strain Pm70, such as, for example, β-galactoside alpha-2 from PdST6 of Luminus mermaidii, 6-Sialyltransferase (UniProt ID O66375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID O66375 with β-galactoside α-2,6-sialyltransferase activity (SEQ ID NO 25) or P-JT-ISH-224-ST6 (UniProt ID A8QYL1) from Luminus sp. A P-JT-ISH-224-ST6-like polypeptide (SEQ ID NO 26) consisting of amino acid residues 18 to 514 of the enzymatically active UniProt ID A8QYL1 and/or such as, for example, α-2,8-saliva of Mus musculus Acid transferase (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 was determined by dividing the measured oligosaccharide concentration in the complete broth 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 103. Production of a mammalian milk oligosaccharide mixture comprising 2'FL , 3-FL , DiFL , 3'SL and 6'SL using a modified Corynebacterium glutamicum host

The Corynebacterium glutamicum strain was modified as described in Example 102 by the gene body deletion of the Corynebacterium glutamicum genes ldh , cgl2645 , nagB , gamA and nagA and the gene body insertion of the persistent transcription unit, the persistent Type transcription units for lactose permease (LacY) from Escherichia coli with SEQ ID NO 15, sucrose permease CscB from Escherichia coli W with SEQ ID NO 01, from mobilis fermentase with SEQ ID NO 02 Fructokinase Frk from Bacillus sp., sucrose phosphorylase BaSP from Bifidobacterium adolescentis with SEQ ID NO 03, native fructose-6-P-aminotransferase (UniProt ID Q8NND3), glucosamine from Saccharomyces cerevisiae- 6-P-aminotransferase (SEQ ID NO 16), N-acetylglucosamine-2-epimerase (SEQ ID NO 17) from Bacteroides ovale and N-acetylglucosamine-2-epimerase from Neisseria meningitidis N-Acetylneuraminic acid synthase (SEQ ID NO 18). In the next step, the novel strain is transformed with expressive plastids comprising persistent transcription units for the NeuA enzyme (SEQ ID NO 22) from Pasteurella septicemia, β-galactoside α-2,3-sialyltransferase PmultST3 (UniProt ID Q9CLP3), β-galactoside α-2,6-sialyltransferase PdST6 (UniProt ID O66375) ), α-1,2-fucosyltransferase HpFutC (SEQ ID NO 04) from Helicobacter pylori and α-1,3-fucosyltransferase HpFucT (SEQ ID NO 05) from Helicobacter pylori ). Novel strains were evaluated for production of oligosaccharide mixtures comprising 2'FL, 3-FL, DiFL, 3'SL and 6'SL in growth experiments on MMsf medium containing lactose according to the culture conditions provided in Example 102. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 104. Production of a mammalian milk oligosaccharide mixture comprising 3-FL , LNFP-III , LNnT and LN3 using a modified Corynebacterium glutamicum host

Corynebacterium glutamicum strain modified as described in Example 102 by gene body deletion of nagB , glmS and gamA genes and gene body insertion comprising the gene's persistent transcription unit for LN3 production and growth on sucrose , these genes encode lactose permease (LacY) (SEQ ID NO 15) from Escherichia coli, native fructose-6-P-aminotransferase (UniProt ID Q8NND3), galactoside from Neisseria meningitidis β-1,3-N-acetylglucosaminyltransferase LgtA (SEQ ID NO 27), sucrose transport protein (CscB) (SEQ ID NO 01) from Escherichia coli W, from Zymomonas mobilis Fructokinase (Frk) (SEQ ID NO 02) and sucrose phosphorylase (BaSP) (SEQ ID NO 03) from Bifidobacterium adolescentis. In the next step, the mutant strain was further transformed with a continuous transcription unit comprising N-acetylglucosamine beta-1,4-galactosyltransferase LgtB (SEQ ID NO 29) from Neisseria meningitidis Gene body insertion modification to generate LNnT. In a subsequent step, the LNnT-producing strain was transformed with an expression plastid comprising a persistent transcription unit for the alpha-1,3-fucosyltransferase HpFucT (SEQ ID NO 05) from H. pylori. Novel strains were evaluated for the production of oligosaccharide mixtures comprising 3-FL, LN3, LNnT and LNFP-III in growth experiments on MMsf medium containing lactose as a precursor according to the culture conditions provided in Example 102. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 105. Production of a mammalian milk oligosaccharide mixture comprising 2'FL , 3-FL , DiFL , LN3 , LNnT , LNFP-III and lacto -N- neohexasaccharide using a modified Corynebacterium glutamicum host

Corynebacterium glutamicum strains are modified for LN3 production and growth on sucrose by gene body deletion of nagB , glmS and gamA genes and gene body insertion including persistent transcription units of the genes encoding from the large intestine Bacillus lactose permease (LacY) (SEQ ID NO 15), native fructose-6-P-aminotransferase (UniProt ID Q8NND3), galactoside β-1,3-N from Neisseria meningitidis -Acetylglucosaminyltransferase LgtA (SEQ ID NO 27), sucrose transporter (CscB) from Escherichia coli W (SEQ ID NO 01), fructokinase (Frk) from Zymomonas mobilis (SEQ ID NO 01) ID NO 02) and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (SEQ ID NO 03). In the next step, the mutant strain was further transformed with a continuous transcription unit comprising N-acetylglucosamine beta-1,4-galactosyltransferase LgtB (SEQ ID NO 29) from Neisseria meningitidis Gene body insertion modification to generate LNnT. In a subsequent step, the LNnT-producing strain was transformed with an expression plastid containing a persistent transcription unit for the alpha-1,2-fucosyltransferase HpFutC (SEQ ID) from Helicobacter pylori NO 04) and α-1,3-fucosyltransferase HpFucT (SEQ ID NO 05) from Helicobacter pylori. In growth experiments on MMsf medium containing lactose as a precursor according to the culture conditions provided in Example 102, for the production of 2'FL, 3-FL, DiFL, LN3, LNnT, LNFP-III and milk-N- Oligosaccharide mixtures of new hexasaccharides to evaluate novel strains. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 106. Production of an oligosaccharide mixture comprising LN3 , sialylated LN3 , 6'SL , LNnT and LSTc using a modified Corynebacterium glutamicum host

Corynebacterium glutamicum strains were modified as described in Example 102 for LN3 production and in Growth on sucrose, the genes encoding lactose permease (LacY) from Escherichia coli (SEQ ID NO 15), native fructose-6-P-aminotransferase (UniProt ID Q8NND3), from Neisseria meningitidis Galactoside β-1,3-N-acetylglucosaminyltransferase LgtA (SEQ ID NO 27), sucrose transport protein (CscB) (SEQ ID NO 01) from Escherichia coli W, Fructokinase (Frk) from Monascus (SEQ ID NO 02) and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (SEQ ID NO 03). In the next step, the mutant strain was further transformed with a continuous transcription unit comprising N-acetylglucosamine beta-1,4-galactosyltransferase LgtB (SEQ ID NO 29) from Neisseria meningitidis Gene body insertion modification to generate LNnT. In the next step, the mutant strain was further modified by gene body insertion of a persistent transcription unit comprising a native fructose-6-P-aminotransferase (UniProt ID Q8NND3) from Saccharomyces cerevisiae to produce sialic acid GNA1 (SEQ ID NO 16), AGE (SEQ ID NO 17) from Bacteroides ovale, and N-acetylneuraminic acid synthase (SEQ ID NO 18) from Neisseria meningitidis. In the next step, the novel strain was transformed with an expressive plastid containing persistent transcription units for NeuA enzyme (SEQ ID NO 22) from Pasteurella septicemia and β from P. mermaidii - Galactoside alpha-2,6-sialyltransferase PdST6 (UniProt ID O66375). In growth experiments on MMsf medium containing lactose according to the culture conditions provided in Example 102, for the production of LN3, 6'-sialylated LN3 (Neu5Ac-a2,6-(GlcNAc-b1,3)-Gal- The oligosaccharide mixture of b1,4-Glc), 6'SL, LNnT and LSTc was evaluated for novel strains. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 107. Production of an oligosaccharide mixture comprising 3'SL , 6'SL , LNB , 3'- sialylated LNB and 6' - sialylated LNB using a modified Corynebacterium glutamicum host

Corynebacterium glutamicum strain as described in Example 102 by gene body deletion of ldh , cgl2645 , nagB , gamA and nagA genes and gene body insertion modification comprising the persistent transcription unit of the genes encoding from the large intestine Lactose permease (LacY) from Bacillus (SEQ ID NO 15), WbgO from Escherichia coli O55:H7 with SEQ ID NO 28, galE from Escherichia coli with SEQ ID NO 30, native fructose-6-P-amine Syltransferase (UniProt ID Q8NND3), glmS*54 with SEQ ID NO 19, glucosamine-6-P-aminotransferase (SEQ ID NO 16) from Saccharomyces cerevisiae, N-ethyltransferase from Bacillus ovale Acetylglucosamine-2-epimerase (SEQ ID NO 17) and N-acetylneuraminic acid synthase (SEQ ID NO 18) from Neisseria meningitidis. In the next step, the novel strain is transformed with an expressive plastid containing persistent transcription units for the NeuA enzyme (SEQ ID NO 22) from Pasteurella septicemia, β-galactoside α-2,3-sialyltransferase PmultST3 (UniProt ID Q9CLP3) and β-galactoside α-2,6-sialyltransferase PdST6 (UniProt ID O66375) ). In growth experiments on MMsf medium containing lactose and glucose according to the culture conditions provided in Example 102, for the production of 3'SL, 6'SL, LNB, 3'-sialylated LNB (3'SLNB) and 6' Oligosaccharide mixture of '-sialylated LNB (6'SLNB) to evaluate novel strains. After 72 h of incubation, the broth was collected and analyzed for sugars on UPLC. Example 108. Production of LNB , LN3 , LNT , GalNAc-b1,3- lactose, Gal- b1,3-GalNAc-b1,3- lactose, GalNAc-b1,3 in mutant Corynebacterium glutamicum strains - Oligosaccharide mixture of LNB and Gal-b1,3-GalNAc-b1,3-LNB

The wild type C. glutamicum strain was first modified by gene body deletion of the C. glutamicum genes ldh , cgl2645 , nagB , gamA and nagA and gene body insertion comprising the persistent transcription unit of the gene to produce LNB, which The genes encode phosphatase yqaB (SEQ ID NO 20) from E. coli, GNA1 (SEQ ID NO 16) from Saccharomyces cerevisiae and WbgO (SEQ ID NO 28) from E. coli O55:H7. In the next step, LNB production was further treated with 4-epimerase (WbpP) for Pseudomonas aeruginosa (SEQ ID NO 34), galactoside β-1,3 from Neisseria meningitidis -N-acetylglucosaminyltransferase (LgtA) (SEQ ID NO 27) and β1,3-N-acetylgalactosaminyltransferase (LgtD) (SEQ ID NO 35) from Haemophilus influenzae ) embedded modification of persistent representation units. In growth experiments on MMsf medium containing lactose as a precursor according to the culture conditions provided in Example 102, for the production of LNB, LN3, LNT, GalNAc-b1,3-lactose, Gal-b1,3-GalNAc- b1,3-lactose, GalNAc-b1,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, GalNAc-b1,3-LNB and Gal-b1,3-GalNAc-b1, Oligosaccharide mixture of 3-LNB to evaluate novel strains. Example 109. Materials and Methods of Chlamydomonas reinhardtii Medium

Chlamydomonas reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7.0). TAP medium uses 1000x stock Hutner's trace element mix. Hutner's trace element mixture is composed 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(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, compounds such as galactose, glucose, fructose, fucose, lactose, LacNAc, LNB 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 min. Finally, the cell suspension was transferred to a 50 ml conical centrifuge tube containing 10 mL of freshly prepared liquid TAP medium with 60 mM sorbitol and allowed to recover overnight in the dark with slow shaking. After overnight recovery, cells were re-harvested and seeded by starch embedding onto selective 1.5% (w/v) agar-TAP dishes containing either ampicillin (100 mg/L) or chloramphenicol (100 mg/v). L). 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 embodiment of the production of UDP-galactose, the Chlamydomonas reinhardtii cells are modified with a transcription unit comprising genes encoding for example galactokinase from Arabidopsis (KIN, UniProt ID Q9SEE5) and as for example from Arabidopsis ( UDP-sugar pyrophosphorylase (UniProt ID Q9C5I1) of USP of A. thaliana ).

In one example of LN3 production, a galactoside beta-1,3-N-acetylglucosaminyltransferase (such as, for example, IgtA (SEQ ID NO 27) from Neisseria meningitidis) will be included A persistent transcription unit was additionally added to the strain. In one embodiment of LNT production, the LN3-producing strain is further treated with an N-acetylglucosamine beta-1,3-galactosyltransferase (such as, for example, WbgO (SEQ ID NO 28) from E. coli O55:H7 )) persistent transcriptional unit modification. In one example of LNnT production, the LN3-producing strain is further treated with an enzyme comprising N-acetylglucosamine beta-1,4-galactosyltransferase (such as, for example, lgtB (SEQ ID NO. 29)) of the persistent transcription unit modification.

In one embodiment of the production of GDP-fucose, C. reinhardtii cells are modified with a transcription unit such as for example, GDP-fucose synthase from Arabidopsis (GER1, UniProt ID 049213).

In one example of fucosylation, Chlamydomonas reinhardtii cells can be modified with an expression plastid comprising a persistent transcription unit for alpha-1,2 such as, for example, HpFutC from H. pylori - Fucosyltransferase (SEQ ID NO 04) and/or alpha-1,3-fucosyltransferase (SEQ ID NO 05) such as eg HpFucT from Helicobacter pylori.

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-acylneuraminocytidine 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 Homo sapiens, Golgi-localized sialyltransferase in Mus musculus and brown rat. 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 cultures, cells can be grown in a closed system as described by Chen et al. (Bioresour. Technol. 2011, 102: 71-81) and Johnson et al. (Biotechnol. Prog. 2018, 34: 811-827). , as for example in vertical or horizontal tube photobioreactors, stirred tank photobioreactors or flat plate photobioreactors. Example 110. Production of a mammalian milk oligosaccharide mixture comprising 2'FL , 3 - FL , DiFL , LacNAc , 2'FLacNAc and 3-FLacNAc in mutant Chlamydomonas reinhardtii cells

Chlamydomonas reinhardtii cells were engineered for the production of UDP-Gal as described in Example 109 with gene body insertion of a persistent transcription unit comprising encoding galactose kinase (KIN, UniProt ID Q9SEE5) and Arabidopsis gene for UDP-sugar pyrophosphorylase (USP) (UniProt ID Q9C5I1). In the next step, cells are modified with gene body insertion of persistent transcription units comprising b1,4-galactosyltransferase (SEQ ID NO 29) from Neisseria meningitidis, GDP-fucose synthase from mustard (GER1, UniProt ID 049213), α-1,2-fucosyltransferase HpFutC (SEQ ID NO 04) from Helicobacter pylori and α-1 from Helicobacter pylori , 3-fucosyltransferase HpFucT (SEQ ID NO 05). In culture experiments on TAP agar plates containing galactose and N-acetylglucosamine as precursors according to the culture conditions provided in Example 109, for the production of 2'FL, 3-FL, DiFL, LacNAc, Oligosaccharide mixtures of 2'FLacNAc and 3-FLacNAc were evaluated for novel strains. After 5 days of incubation, cells were harvested and analyzed for sugar production on UPLC. Example 111. Production of 2'FL , 3 - FL , DiFL , LacNAc , 2'FLacNAc , 3 -FLacNAc , LN3 , LNnT , LNFP - III , and difucosyl - lacNAc in mutant Chlamydomonas reinhardtii cells Mammalian Milk Oligosaccharide Mixture of N- Neohexasaccharide

Mutant C. reinhardtii cells as described in Example 110 were further subjected to beta-1,3-N-acetylglucosamine transfer of galactoside from Neisseria meningitidis having SEQ ID NO 27 Gene body-insertion adaptation of the continuous transcription unit of the enzyme (lgtA). Culture experiments were performed on TAP agar plates containing galactose and N-acetylglucosamine as precursors according to the culture conditions provided in Example 109 for the production of 2'FL, 3-FL, DiFL, LacNAc, 2' FL, 3-FL, DiFL, LacNAc, 2 'The oligosaccharide mixture of FLacNAc, 3-FLacNAc, LN3, LNnT, LNFP-III and difucosyl-lacto-N-neohexasaccharide evaluated novel strains. After 5 days of incubation, cells were harvested and analyzed for sugar production on UPLC. Example 112. Production of oligosaccharide mixtures comprising sialylated LNB and sialylated LacNAc structures in mutant Chlamydomonas reinhardtii cells

Chlamydomonas reinhardtii cells were engineered for the production of CMP-sialic acid as described in Example 109 with gene body inserts of persistent transcription units comprising a different source from the gene than the native polypeptide with the R263L mutation. Human UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase GNE mutant form (UniProt ID Q9Y223), N-acetylneuraminic acid from Homo sapiens -9-phosphate synthase NANS (UniProt ID Q9NR45) and N-acylneuraminic acid cytidine syltransferase CMAS (UniProt ID Q8NFW8) from Homo sapiens. In the next step, the cells were modified by gene body insertion of persistent transcription units comprising CMP-sialic acid transporter CST (UniProt ID Q61420) from Mus musculus, α-2, 3-Sialyltransferase (UniProt ID P61943 and E9PSJ1) and α-2,6-sialyltransferase from Brown rat (UniProt ID P13721). In the final step, cells were transformed with gene body inserts comprising the persistent transcription unit of Arabidopsis genes encoding galactose kinase (KIN, UniProt ID Q9SEE5) and UDP-sugar pyrophosphorylase (USP) (UniProt ID Q9C5I1), and N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from Escherichia coli O55:H7 with SEQ ID NO 28 and Neisseria meningitidis from SEQ ID NO 29 Bacterial N-acetylglucosamine β-1,4-galactosyltransferase LgtB. Culture experiments were performed on TAP agar plates containing galactose, glucose and N-acetylglucosamine as precursors according to the culture conditions provided in Example 109 for the production of Sugar (3'SLNB), 6'-Sialyllacto-N-disaccharide (6'SLNB), 3'-Sialyllactosamine (3'SLacNAc) and 6'-Sialyllactosamine (6' SLacNAc) oligosaccharide mixture to evaluate novel strains. After 5 days of incubation, cells were harvested and analyzed for sugar production on UPLC. Example 113. Production of GlcNAc-b1,3-Gal-b1,4-GlcNAc , β - Gal-(1,4) -β- GlcNAc-(1,3)-[β-Gal-(1,4)-β- GlcNAc-(1,3)-[ β ] in mutant Chlamydomonas reinhardtii cells -GlcNAc-(1,6)]- β- Gal-(1,4)-GlcNAc and poly- LacNAc structure oligosaccharide mixture

Chlamydomonas reinhardtii cells were engineered as described in Example 109 to contain gene body insertions comprising persistent transcriptional units of Arabidopsis genes encoding galactokinase (KIN, UniProt ID Q9SEE5) and UDP-sugar Phosphorylase (USP) (UniProt ID Q9C5I1), mutant glmS*54 from E. coli (different from wild-type glmS (UniProt ID P17169) by A39T, R250C and G472S mutations), phosphatase yqaB from E. coli (UniProt ID NP_417175.1), galE from Escherichia coli (UniProt ID P09147), LgtA from Neisseria meningitidis (UniProt ID Q9JXQ6), LgtB from Neisseria meningitidis (UniProt ID Q51116) and human N-Acetyllactosamine sugar β-1,6-N-Acetylglucosaminyltransferase GCNT2 (UniProt ID Q8N0V5). In culture experiments on TAP agar plates containing galactose and GlcNAc as precursors according to the culture conditions provided in Example 109, for the production of GlcNAc-bl,3-Gal-bl,4-GlcNAc, β-Gal-( 1,4)-β-GlcNAc-(1,3)-[β-GlcNAc-(1,6)]-β-Gal-(1,4)-GlcNAc and Poly-LacNAc Constructed Oligosaccharide Mixtures to Assess Novel Strains . Example 114. 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, pigs, sheep, chickens, ducks, catfish, snakes, frogs) or from 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 et al. 1999; Animal Cell Technology': Basic & Applied Aspects, Springer, Dordrecht. 141-145; Williams et al. 2009, Breast Cancer Res 11(3): 26-43; and Arevalo et al. 2015, Am J Physiol Cell Physiol. 310( 5): C348-C356; each of which is incorporated herein by reference in its entirety for all purposes.

For 2D cultures, isolated cells were initially seeded in culture dishes in growth medium supplemented with 10 ng/ml epithelial growth factor and 5 pg/ml insulin. At confluence, cells were fed for 48 h with growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U/ml penicillin, 100 ug/ml streptomycin) and 5 pg/ml insulin. To induce differentiation, cells were fed 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 culture, isolated cells were trypsinized and cultured in Matrigel, hyaluronic acid, or ultra-low attachment surface culture dishes for six days, and incubated by adding epithelial growth factor supplemented with 10 ng/ml and 5 pg/ml insulin. Growth medium to induce differentiation and lactation. At confluence, cells were fed for 48 h with growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U/ml penicillin, 100 ug/ml streptomycin) and 5 pg/ml insulin. To induce differentiation, cells were fed 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 (obtained from Stem Cell Technologies) or breast cell enrichment medium (DMEM, 3% FBS, estrogen, progesterone, heparin, corticosterone, insulin, EGF) to This is transformed into mammary gland-like cells from which the expression 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 included high glucose DMEM/F12, 10% FBS, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, and 5 pg/ml hydrocortisone. Complete lactation medium including high glucose DMEM/F12, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, 5 pg/ml hydrocortisone, and 1 pg/ml Prolactin (5ug/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 desired concentration of lactation medium 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 hormones and other growth factors used can be purified by resins, such as selective extraction using nickel resins to remove His-tagged growth factors, to further reduce the level of contaminants in the lactated product. Example 115. Production of Mammalian Milk Oligosaccharide Mixtures Containing 2'FL , 3-FL and DiFL in Non-Mammary Adult Stem Cells

Isolated mesenchymal cells and reprogrammed into mammary-like cells as described in Example 114 were modified by CRISPR-CAS to overexpress GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), from Helicobacter pylori α-1,2-fucosyltransferase HpFutC (SEQ ID NO 04) from Bacillus and α-1,3-fucosyltransferase HpFucT (SEQ ID NO 05) from Helicobacter pylori. 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 for about 7 days. After culturing as described in Example 114, cells were subjected to UPLC to analyze the production of oligosaccharide mixtures comprising 2'FL, 3-FL and DiFL. Example 116. Production of a mammalian milk oligosaccharide mixture comprising 2'FL , 3-FL , DiFL , LN3 , LNT and LNFP-I in non-mammary adult stem cells

Isolated mesenchymal cells and reprogrammed into mammary-like cells as described in Example 114 were modified by CRISPR-CAS to overexpress GlcN6P synthase from Homo sapiens (UniProt ID Q06210), glucosamine 6- Phosphate N-acetyltransferase (UniProt ID Q96EK6), phosphoacetylglucosamine mutase from Homo sapiens (UniProt ID 095394), UDP-N-acetylhexosamine pyrophosphorylase (UniProt ID Q16222), galactoside β-1,3-N-acetylglucosaminyltransferase LgtA from Neisseria meningitidis with SEQ ID NO 27, from Neisseria meningitidis with SEQ ID NO 29 Bacterial N-acetylglucosamine β-1,4-galactosyltransferase LgtB, GDP-fucose synthase GFUS (UniProt ID Q13630) from Homo sapiens, α-1 from Helicobacter pylori, 2-fucosyltransferase HpFutC (SEQ ID NO 04) and alpha-1,3-fucosyltransferase HpFucT (SEQ ID NO 05) from Helicobacter pylori. For host cells, all genes introduced are codon-optimized. 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 for about 7 days. After culturing as described in Example 114, cells were subjected to UPLC to analyze the production of oligosaccharide mixtures comprising 2'FL, 3-FL, DiFL, LN3, LNT and LNFP-I. Example 117. Evaluation of production in non-mammary adult stem cells comprising LNB , LN3 , LNT , GalNAc- b1,3- lactose, Gal- b1,3-GalNAc-b1,3- lactose, GalNAc-b1,3-LNB and Oligosaccharide mixture of Gal-b1,3-GalNAc-b1,3-LNB

Isolated mesenchymal cells and reprogrammed into mammary-like cells as described in Example 114 were modified by CRISPR-CAS to overexpress β-1,4-galactosyltransferase 1 B4GalT1 from Homo sapiens (UniProt ID P15291 ), phosphatase ScDOG1 (UniProt ID P38774) from Saccharomyces cerevisiae, GNA1 (SEQ ID NO 16) from Saccharomyces cerevisiae and WbgO (SEQ ID NO 28) from Escherichia coli O55:H7, 4 of Pseudomonas aeruginosa - Epimerase (WbpP) (SEQ ID NO 34), galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from Neisseria meningitidis (SEQ ID NO 27) ) and β1,3-N-acetylgalactosaminyltransferase (LgtD) (SEQ ID NO 35) from Haemophilus influenzae. For the host, all genes introduced into the cell are codon-optimized. 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 for about 7 days. After culturing as described in Example 114, cells were subjected to UPLC and for production comprising LNB, LN3, LNT, GalNAc-b1,3-lactose, Gal-b1,3-GalNAc-b1,3-lactose, GalNAc-b1,3 - Oligosaccharide mixtures of Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc, GalNAc-bl,3-LNB and Gal-bl,3-GalNAc-bl,3-LNB were evaluated.

none

The invention will be described in more detail in the examples and the accompanying drawings, in which the drawings.

[ Fig. 1] shows a chromatogram plot obtained with respect to a sample of a whole culture broth obtained from the LSTc-producing E. coli strain S2 (Table 8), which appears to be derived from the genus Luminobacterium having SEQ ID NO 26 ( Photobacterium sp.) a-2,6-sialyltransferase of JT-ISH-224 and analysis of the presence of oligosaccharides by the Dionex method, as described in Example 1. The indicated peaks represent the following glycans (and retention times): 1, lactose (5.325 min); 2, LN3 (6.825 min); 3, LNnT (9.625 min); 4, sialic acid (20.359 min); 5, LSTc ( 30.809 min); 6,6'SL (31.984 min).

[ Fig. 2 ] Shows a chromatographic chart plot obtained with respect to a whole culture fluid sample obtained from the LSTd-producing Escherichia coli strain S5 (Table 9), which appears to be derived from Pasteurella septicaemia ( P. multocida ) having SEQ ID NO 23 ) a-2,3-sialyltransferase and analyzed for the presence of oligosaccharides by the Dionex method, as described in Example 1. The indicated peaks represent the following glycans (and retention times): 1, lactose (5.334 min); 2, LN3 (6.825 min); 3, LNnT (9.567 min); 4, sialic acid (20.017 min); 5, LSTd ( 31.709 min); 6, 3' SL (33.050 min).

The following examples serve to further illustrate and illustrate the invention and are not intended to be limiting.

                                  
           <![CDATA[ <110> Inbiose N.V., Belgium]]>
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          Val Glu Ile Ala Ser Lys Val Asp Arg Val Tyr Asp Phe Ala Leu Pro
                          245 250 255
          Pro Leu Leu Leu His Ala Leu Ser Thr Gly His Val Glu Pro Val Ala
                      260 265 270
          His Trp Thr Asp Ile Arg Pro Asn Asn Ala Val Thr Val Leu Asp Thr
                  275 280 285
          His Asp Gly Ile Gly Val Ile Asp Ile Gly Ser Asp Gln Leu Asp Arg
              290 295 300
          Ser Leu Lys Gly Leu Val Pro Asp Glu Asp Val Asp Asn Leu Val Asn
          305 310 315 320
          Thr Ile His Ala Asn Thr His Gly Glu Ser Gln Ala Ala Thr Gly Ala
                          325 330 335
          Ala Ala Ser Asn Leu Asp Leu Tyr Gln Val Asn Ser Thr Tyr Tyr Ser
                      340 345 350
          Ala Leu Gly Cys Asn Asp Gln His Tyr Ile Ala Ala Arg Ala Val Gln
                  355 360 365
          Phe Phe Leu Pro Gly Val Pro Gln Val Tyr Tyr Val Gly Ala Leu Ala
              370 375 380
          Gly Lys Asn Asp Met Glu Leu Leu Arg Lys Thr Asn Asn Gly Arg Asp
          385 390 395 400
          Ile Asn Arg His Tyr Tyr Ser Thr Ala Glu Ile Asp Glu Asn Leu Lys
                          405 410 415
          Arg Pro Val Val Lys Ala Leu Asn Ala Leu Ala Lys Phe Arg Asn Glu
                      420 425 430
          Leu Asp Ala Phe Asp Gly Thr Phe Ser Tyr Thr Thr Asp Asp Asp Thr
                  435 440 445
          Ser Ile Ser Phe Thr Trp Arg Gly Glu Thr Ser Gln Ala Thr Leu Thr
              450 455 460
          Phe Glu Pro Lys Arg Gly Leu Gly Val Asp Asn Thr Thr Pro Val Ala
          465 470 475 480
          Met Leu Glu Trp Glu Asp Ser Ala Gly Asp His Arg Ser Asp Asp Leu
                          485 490 495
          Ile Ala Asn Pro Pro Val Val Ala
                      500
           <![CDATA[ <210> 4]]>
           <![CDATA[ <211> 299]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Helicobacter pylori UA1234]]>
           <![CDATA[ <400> 4]]>
          Met Ala Phe Lys Val Val Gln Ile Cys Gly Gly Leu Gly Asn Gln Met
          1 5 10 15
          Phe Gln Tyr Ala Phe Ala Lys Ser Leu Gln Lys His Ser Asn Thr Pro
                      20 25 30
          Val Leu Leu Asp Ile Thr Ser Phe Asp Trp Ser Asn Arg Lys Met Gln
                  35 40 45
          Leu Glu Leu Phe Pro Ile Asp Leu Pro Tyr Ala Ser Glu Lys Glu Ile
              50 55 60
          Ala Ile Ala Lys Met Gln His Leu Pro Lys Leu Val Arg Asn Val Leu
          65 70 75 80
          Lys Cys Met Gly Phe Asp Arg Val Ser Gln Glu Ile Val Phe Glu Tyr
                          85 90 95
          Glu Pro Lys Leu Leu Lys Thr Ser Arg Leu Thr Tyr Phe Tyr Gly Tyr
                      100 105 110
          Phe Gln Asp Pro Arg Tyr Phe Asp Ala Ile Ser Pro Leu Ile Lys Gln
                  115 120 125
          Thr Phe Thr Leu Pro Pro Pro Pro Glu Asn Gly Asn Asn Lys Lys Lys
              130 135 140
          Glu Glu Glu Tyr His Arg Lys Leu Ala Leu Ile Leu Ala Ala Lys Asn
          145 150 155 160
          Ser Val Phe Val His Ile Arg Arg Gly Asp Tyr Val Gly Ile Gly Cys
                          165 170 175
          Gln Leu Gly Ile Asp Tyr Gln Lys Lys Ala Leu Glu Tyr Met Ala Lys
                      180 185 190
          Arg Val Pro Asn Met Glu Leu Phe Val Phe Cys Glu Asp Leu Glu Phe
                  195 200 205
          Thr Gln Asn Leu Asp Leu Gly Tyr Pro Phe Met Asp Met Thr Thr Arg
              210 215 220
          Asp Lys Glu Glu Glu Ala Tyr Trp Asp Met Leu Leu Met Gln Ser Cys
          225 230 235 240
          Lys His Gly Ile Ile Ala Asn Ser Thr Tyr Ser Trp Trp Ala Ala Tyr
                          245 250 255
          Leu Ile Asn Asn Pro Glu Lys Ile Ile Ile Gly Pro Lys His Trp Leu
                      260 265 270
          Phe Gly His Glu Asn Ile Leu Cys Lys Glu Trp Val Lys Ile Glu Ser
                  275 280 285
          His Phe Glu Val Lys Ser Gln Lys Tyr Asn Ala
              290 295
           <![CDATA[ <210> 5]]>
           <![CDATA[ <211> 478]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Helicobacter pylori UA1234]]>
           <![CDATA[ <400> 5]]>
          Met Phe Gln Pro Leu Leu Asp Ala Tyr Val Glu Ser Ala Ser Ile Glu
          1 5 10 15
          Lys Met Ala Ser Lys Ser Pro Pro Pro Leu Lys Ile Ala Val Ala Asn
                      20 25 30
          Trp Trp Gly Asp Glu Glu Ile Lys Glu Phe Lys Asn Ser Val Leu Tyr
                  35 40 45
          Phe Ile Leu Ser Gln Arg Tyr Thr Ile Thr Leu His Gln Asn Pro Asn
              50 55 60
          Glu Phe Ser Asp Leu Val Phe Gly Asn Pro Leu Gly Ser Ala Arg Lys
          65 70 75 80
          Ile Leu Ser Tyr Gln Asn Ala Lys Arg Val Phe Tyr Thr Gly Glu Asn
                          85 90 95
          Glu Ser Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp Glu
                      100 105 110
          Leu Asp Phe Asn Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Asp Arg
                  115 120 125
          Leu His His Lys Ala Glu Ser Val Asn Asp Thr Thr Ala Pro Tyr Lys
              130 135 140
          Leu Lys Asp Asn Ser Leu Tyr Ala Leu Lys Lys Pro Ser His Cys Phe
          145 150 155 160
          Lys Glu Lys His Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser Asp
                          165 170 175
          Pro Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Pro Asn Ala
                      180 185 190
          Pro Ile Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro Val
                  195 200 205
          Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Asn Val Lys Asn
              210 215 220
          Lys Asn Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu Asn
          225 230 235 240
          Thr Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Ile Asp Ala Tyr Phe
                          245 250 255
          Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys Asp
                      260 265 270
          Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Lys Asn Phe Asp
                  275 280 285
          Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His Lys Asn Ala Tyr
              290 295 300
          Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys Ala
          305 310 315 320
          Tyr Phe Tyr Gln Asn Leu Ser Phe Lys Lys Ile Leu Ala Phe Phe Lys
                          325 330 335
          Thr Ile Leu Glu Asn Asp Thr Ile Tyr His Asp Asn Pro Phe Ile Phe
                      340 345 350
          Cys Arg Asp Leu Asn Glu Pro Leu Val Thr Ile Asp Asp Leu Arg Val
                  355 360 365
          Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Ile Asn Tyr
              370 375 380
          Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Ile Asn Tyr Asp Asp
          385 390 395 400
          Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg
                          405 410 415
          Ile Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn
                      420 425 430
          Tyr Glu Arg Leu Leu Ser Lys Ala Thr Pro Leu Leu Glu Leu Ser Gln
                  435 440 445
          Asn Thr Thr Ser Lys Ile Tyr Arg Lys Ala Tyr Gln Lys Ser Leu Pro
              450 455 460
          Leu Leu Arg Ala Ile Arg Arg Trp Val Lys Lys Leu Gly Leu
          465 470 475
           <![CDATA[ <210> 6]]>
           <![CDATA[ <211> 412]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Helicobacter pylori UA1234]]>
           <![CDATA[ <400> 6]]>
          Met Phe Gln Pro Leu Leu Asp Ala Tyr Val Glu Ser Ala Ser Ile Glu
          1 5 10 15
          Lys Met Ala Ser Lys Ser Pro Pro Pro Leu Lys Ile Ala Val Ala Asn
                      20 25 30
          Trp Trp Gly Asp Glu Glu Ile Lys Glu Phe Lys Asn Ser Val Leu Tyr
                  35 40 45
          Phe Ile Leu Ser Gln Arg Tyr Thr Ile Thr Leu His Gln Asn Pro Asn
              50 55 60
          Glu Phe Ser Asp Leu Val Phe Gly Asn Pro Leu Gly Ser Ala Arg Lys
          65 70 75 80
          Ile Leu Ser Tyr Gln Asn Ala Lys Arg Val Phe Tyr Thr Gly Glu Asn
                          85 90 95
          Glu Ser Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp Glu
                      100 105 110
          Leu Asp Phe Asn Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Asp Arg
                  115 120 125
          Leu His His Lys Ala Glu Ser Val Asn Asp Thr Thr Ala Pro Tyr Lys
              130 135 140
          Leu Lys Asp Asn Ser Leu Tyr Ala Leu Lys Lys Pro Ser His Cys Phe
          145 150 155 160
          Lys Glu Lys His Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser Asp
                          165 170 175
          Pro Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Pro Asn Ala
                      180 185 190
          Pro Ile Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro Val
                  195 200 205
          Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Asn Val Lys Asn
              210 215 220
          Lys Asn Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu Asn
          225 230 235 240
          Thr Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Ile Asp Ala Tyr Phe
                          245 250 255
          Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys Asp
                      260 265 270
          Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Lys Asn Phe Asp
                  275 280 285
          Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His Lys Asn Ala Tyr
              290 295 300
          Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys Ala
          305 310 315 320
          Tyr Phe Tyr Gln Asn Leu Ser Phe Lys Lys Ile Leu Ala Phe Phe Lys
                          325 330 335
          Thr Ile Leu Glu Asn Asp Thr Ile Tyr His Asp Asn Pro Phe Ile Phe
                      340 345 350
          Cys Arg Asp Leu Asn Glu Pro Leu Val Thr Ile Asp Asp Leu Arg Val
                  355 360 365
          Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Ile Asn Tyr
              370 375 380
          Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Ile Asn Tyr Asp Asp
          385 390 395 400
          Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr
                          405 410
           <![CDATA[ <210> 7]]>
           <![CDATA[ <211> 264]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 7]]>
          Met Lys Gln Tyr Leu Glu Leu Met Gln Lys Val Leu Asp Glu Gly Thr
          1 5 10 15
          Gln Lys Asn Asp Arg Thr Gly Thr Gly Thr Leu Ser Ile Phe Gly His
                      20 25 30
          Gln Met Arg Phe Asn Leu Gln Asp Gly Phe Pro Leu Val Thr Thr Lys
                  35 40 45
          Arg Cys His Leu Arg Ser Ile Ile His Glu Leu Leu Trp Phe Leu Gln
              50 55 60
          Gly Asp Thr Asn Ile Ala Tyr Leu His Glu Asn Asn Val Thr Ile Trp
          65 70 75 80
          Asp Glu Trp Ala Asp Glu Asn Gly Asp Leu Gly Pro Val Tyr Gly Lys
                          85 90 95
          Gln Trp Arg Ala Trp Pro Thr Pro Asp Gly Arg His Ile Asp Gln Ile
                      100 105 110
          Thr Thr Val Leu Asn Gln Leu Lys Asn Asp Pro Asp Ser Arg Arg Ile
                  115 120 125
          Ile Val Ser Ala Trp Asn Val Gly Glu Leu Asp Lys Met Ala Leu Ala
              130 135 140
          Pro Cys His Ala Phe Phe Gln Phe Tyr Val Ala Asp Gly Lys Leu Ser
          145 150 155 160
          Cys Gln Leu Tyr Gln Arg Ser Cys Asp Val Phe Leu Gly Leu Pro Phe
                          165 170 175
          Asn Ile Ala Ser Tyr Ala Leu Leu Val His Met Met Ala Gln Gln Cys
                      180 185 190
          Asp Leu Glu Val Gly Asp Phe Val Trp Thr Gly Gly Asp Thr His Leu
                  195 200 205
          Tyr Ser Asn His Met Asp Gln Thr His Leu Gln Leu Ser Arg Glu Pro
              210 215 220
          Arg Pro Leu Pro Lys Leu Ile Ile Lys Arg Lys Pro Glu Ser Ile Phe
          225 230 235 240
          Asp Tyr Arg Phe Glu Asp Phe Glu Ile Glu Gly Tyr Asp Pro His Pro
                          245 250 255
          Gly Ile Lys Ala Pro Val Ala Ile
                      260
           <![CDATA[ <210> 8]]>
           <![CDATA[ <211> 391]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 8]]>
          Met Gln Lys Leu Ile Asn Ser Val Gln Asn Tyr Ala Trp Gly Ser Lys
          1 5 10 15
          Thr Ala Leu Thr Glu Leu Tyr Gly Met Glu Asn Pro Ser Ser Gln Pro
                      20 25 30
          Met Ala Glu Leu Trp Met Gly Ala His Pro Lys Ser Ser Ser Arg Val
                  35 40 45
          Gln Asn Ala Ala Gly Asp Ile Val Ser Leu Arg Asp Val Ile Glu Ser
              50 55 60
          Asp Lys Ser Thr Leu Leu Gly Glu Ala Val Ala Lys Arg Phe Gly Glu
          65 70 75 80
          Leu Pro Phe Leu Phe Lys Val Leu Cys Ala Ala Gln Pro Leu Ser Ile
                          85 90 95
          Gln Val His Pro Asn Lys His Asn Ser Glu Ile Gly Phe Ala Lys Glu
                      100 105 110
          Asn Ala Ala Gly Ile Pro Met Asp Ala Ala Glu Arg Asn Tyr Lys Asp
                  115 120 125
          Pro Asn His Lys Pro Glu Leu Val Phe Ala Leu Thr Pro Phe Leu Ala
              130 135 140
          Met Asn Ala Phe Arg Glu Phe Ser Glu Ile Val Ser Leu Leu Gln Pro
          145 150 155 160
          Val Ala Gly Ala His Pro Ala Ile Ala His Phe Leu Gln Gln Pro Asp
                          165 170 175
          Ala Glu Arg Leu Ser Glu Leu Phe Ala Ser Leu Leu Asn Met Gln Gly
                      180 185 190
          Glu Glu Lys Ser Arg Ala Leu Ala Ile Leu Lys Ser Ala Leu Asp Ser
                  195 200 205
          Gln Gln Gly Glu Pro Trp Gln Thr Ile Arg Leu Ile Ser Glu Phe Tyr
              210 215 220
          Pro Glu Asp Ser Gly Leu Phe Ser Pro Leu Leu Leu Asn Val Val Lys
          225 230 235 240
          Leu Asn Pro Gly Glu Ala Met Phe Leu Phe Ala Glu Thr Pro His Ala
                          245 250 255
          Tyr Leu Gln Gly Val Ala Leu Glu Val Met Ala Asn Ser Asp Asn Val
                      260 265 270
          Leu Arg Ala Gly Leu Thr Pro Lys Tyr Ile Asp Ile Pro Glu Leu Val
                  275 280 285
          Ala Asn Val Lys Phe Glu Ala Lys Pro Ala Asn Gln Leu Leu Thr Gln
              290 295 300
          Pro Val Lys Gln Gly Ala Glu Leu Asp Phe Pro Ile Pro Val Asp Asp
          305 310 315 320
          Phe Ala Phe Ser Leu His Asp Leu Ser Asp Lys Glu Thr Thr Ile Ser
                          325 330 335
          Gln Gln Ser Ala Ala Ile Leu Phe Cys Val Glu Gly Asp Ala Thr Leu
                      340 345 350
          Trp Lys Gly Ser Gln Gln Leu Gln Leu Lys Pro Gly Glu Ser Ala Phe
                  355 360 365
          Ile Ala Ala Asn Glu Ser Pro Val Thr Val Lys Gly His Gly Arg Leu
              370 375 380
          Ala Arg Val Tyr Asn Lys Leu
          385 390
           <![CDATA[ <210> 9]]>
           <![CDATA[ <211> 456]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 9]]>
          Met Lys Lys Leu Thr Cys Phe Lys Ala Tyr Asp Ile Arg Gly Lys Leu
          1 5 10 15
          Gly Glu Glu Leu Asn Glu Asp Ile Ala Trp Arg Ile Gly Arg Ala Tyr
                      20 25 30
          Gly Glu Phe Leu Lys Pro Lys Thr Ile Val Leu Gly Gly Asp Val Arg
                  35 40 45
          Leu Thr Ser Glu Thr Leu Lys Leu Ala Leu Ala Lys Gly Leu Gln Asp
              50 55 60
          Ala Gly Val Asp Val Leu Asp Ile Gly Met Ser Gly Thr Glu Glu Ile
          65 70 75 80
          Tyr Phe Ala Thr Phe His Leu Gly Val Asp Gly Gly Ile Glu Val Thr
                          85 90 95
          Ala Ser His Asn Pro Met Asp Tyr Asn Gly Met Lys Leu Val Arg Glu
                      100 105 110
          Gly Ala Arg Pro Ile Ser Gly Asp Thr Gly Leu Arg Asp Val Gln Arg
                  115 120 125
          Leu Ala Glu Ala Asn Asp Phe Pro Pro Val Asp Glu Thr Lys Arg Gly
              130 135 140
          Arg Tyr Gln Gln Ile Asn Leu Arg Asp Ala Tyr Val Asp His Leu Phe
          145 150 155 160
          Gly Tyr Ile Asn Val Lys Asn Leu Thr Pro Leu Lys Leu Val Ile Asn
                          165 170 175
          Ser Gly Asn Gly Ala Ala Gly Pro Val Val Asp Ala Ile Glu Ala Arg
                      180 185 190
          Phe Lys Ala Leu Gly Ala Pro Val Glu Leu Ile Lys Val His Asn Thr
                  195 200 205
          Pro Asp Gly Asn Phe Pro Asn Gly Ile Pro Asn Pro Leu Leu Pro Glu
              210 215 220
          Cys Arg Asp Asp Thr Arg Asn Ala Val Ile Lys His Gly Ala Asp Met
          225 230 235 240
          Gly Ile Ala Phe Asp Gly Asp Phe Asp Arg Cys Phe Leu Phe Asp Glu
                          245 250 255
          Lys Gly Gln Phe Ile Glu Gly Tyr Tyr Ile Val Gly Leu Leu Ala Glu
                      260 265 270
          Ala Phe Leu Glu Lys Asn Pro Gly Ala Lys Ile Ile His Asp Pro Arg
                  275 280 285
          Leu Ser Trp Asn Thr Val Asp Val Val Thr Ala Ala Gly Gly Thr Pro
              290 295 300
          Val Met Ser Lys Thr Gly His Ala Phe Ile Lys Glu Arg Met Arg Lys
          305 310 315 320
          Glu Asp Ala Ile Tyr Gly Gly Glu Met Ser Ala His His Tyr Phe Arg
                          325 330 335
          Asp Phe Ala Tyr Cys Asp Ser Gly Met Ile Pro Trp Leu Leu Val Ala
                      340 345 350
          Glu Leu Val Cys Leu Lys Asp Lys Thr Leu Gly Glu Leu Val Arg Asp
                  355 360 365
          Arg Met Ala Ala Phe Pro Ala Ser Gly Glu Ile Asn Ser Lys Leu Ala
              370 375 380
          Gln Pro Val Glu Ala Ile Asn Arg Val Glu Gln His Phe Ser Arg Glu
          385 390 395 400
          Ala Leu Ala Val Asp Arg Thr Asp Gly Ile Ser Met Thr Phe Ala Asp
                          405 410 415
          Trp Arg Phe Asn Leu Arg Thr Ser Asn Thr Glu Pro Val Val Arg Leu
                      420 425 430
          Asn Val Glu Ser Arg Gly Asp Val Pro Leu Met Glu Ala Arg Thr Arg
                  435 440 445
          Thr Leu Leu Thr Leu Leu Asn Glu
              450 455
           <![CDATA[ <210> 10]]>
           <![CDATA[ <211> 478]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 10]]>
          Met Ala Gln Ser Lys Leu Tyr Pro Val Val Met Ala Gly Gly Ser Gly
          1 5 10 15
          Ser Arg Leu Trp Pro Leu Ser Arg Val Leu Tyr Pro Lys Gln Phe Leu
                      20 25 30
          Cys Leu Lys Gly Asp Leu Thr Met Leu Gln Thr Thr Ile Cys Arg Leu
                  35 40 45
          Asn Gly Val Glu Cys Glu Ser Pro Val Val Ile Cys Asn Glu Gln His
              50 55 60
          Arg Phe Ile Val Ala Glu Gln Leu Arg Gln Leu Asn Lys Leu Thr Glu
          65 70 75 80
          Asn Ile Ile Leu Glu Pro Ala Gly Arg Asn Thr Ala Pro Ala Ile Ala
                          85 90 95
          Leu Ala Ala Leu Ala Ala Lys Arg His Ser Pro Glu Ser Asp Pro Leu
                      100 105 110
          Met Leu Val Leu Ala Ala Asp His Val Ile Ala Asp Glu Asp Ala Phe
                  115 120 125
          Arg Ala Ala Val Arg Asn Ala Met Pro Tyr Ala Glu Ala Gly Lys Leu
              130 135 140
          Val Thr Phe Gly Ile Val Pro Asp Leu Pro Glu Thr Gly Tyr Gly Tyr
          145 150 155 160
          Ile Arg Arg Gly Glu Val Ser Ala Gly Glu Gln Asp Met Val Ala Phe
                          165 170 175
          Glu Val Ala Gln Phe Val Glu Lys Pro Asn Leu Glu Thr Ala Gln Ala
                      180 185 190
          Tyr Val Ala Ser Gly Glu Tyr Tyr Trp Asn Ser Gly Met Phe Leu Phe
                  195 200 205
          Arg Ala Gly Arg Tyr Leu Glu Glu Leu Lys Lys Tyr Arg Pro Asp Ile
              210 215 220
          Leu Asp Ala Cys Glu Lys Ala Met Ser Ala Val Asp Pro Asp Leu Asn
          225 230 235 240
          Phe Ile Arg Val Asp Glu Glu Ala Phe Leu Ala Cys Pro Glu Glu Ser
                          245 250 255
          Val Asp Tyr Ala Val Met Glu Arg Thr Ala Asp Ala Val Val Val Pro
                      260 265 270
          Met Asp Ala Gly Trp Ser Asp Val Gly Ser Trp Ser Ser Leu Trp Glu
                  275 280 285
          Ile Ser Ala His Thr Ala Glu Gly Asn Val Cys His Gly Asp Val Ile
              290 295 300
          Asn His Lys Thr Glu Asn Ser Tyr Val Tyr Ala Glu Ser Gly Leu Val
          305 310 315 320
          Thr Thr Val Gly Val Lys Asp Leu Val Val Val Gln Thr Lys Asp Ala
                          325 330 335
          Val Leu Ile Ala Asp Arg Asn Ala Val Gln Asp Val Lys Lys Val Val
                      340 345 350
          Glu Gln Ile Lys Ala Asp Gly Arg His Glu His Arg Val His Arg Glu
                  355 360 365
          Val Tyr Arg Pro Trp Gly Lys Tyr Asp Ser Ile Asp Ala Gly Asp Arg
              370 375 380
          Tyr Gln Val Lys Arg Ile Thr Val Lys Pro Gly Glu Gly Leu Ser Val
          385 390 395 400
          Gln Met His His His Arg Ala Glu His Trp Val Val Val Ala Gly Thr
                          405 410 415
          Ala Lys Val Thr Ile Asp Gly Asp Ile Lys Leu Leu Gly Glu Asn Glu
                      420 425 430
          Ser Ile Tyr Ile Pro Leu Gly Ala Thr His Cys Leu Glu Asn Pro Gly
                  435 440 445
          Lys Ile Pro Leu Asp Leu Ile Glu Val Arg Ser Gly Ser Tyr Leu Glu
              450 455 460
          Glu Asp Asp Val Val Arg Phe Ala Asp Arg Tyr Gly Arg Val
          465 470 475
           <![CDATA[ <210> 11]]>
           <![CDATA[ <211> 373]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 11]]>
          Met Ser Lys Val Ala Leu Ile Thr Gly Val Thr Gly Gln Asp Gly Ser
          1 5 10 15
          Tyr Leu Ala Glu Phe Leu Leu Glu Lys Gly Tyr Glu Val His Gly Ile
                      20 25 30
          Lys Arg Arg Ala Ser Ser Phe Asn Thr Glu Arg Val Asp His Ile Tyr
                  35 40 45
          Gln Asp Pro His Thr Cys Asn Pro Lys Phe His Leu His Tyr Gly Asp
              50 55 60
          Leu Ser Asp Thr Ser Asn Leu Thr Arg Ile Leu Arg Glu Val Gln Pro
          65 70 75 80
          Asp Glu Val Tyr Asn Leu Gly Ala Met Ser His Val Ala Val Ser Phe
                          85 90 95
          Glu Ser Pro Glu Tyr Thr Ala Asp Val Asp Ala Met Gly Thr Leu Arg
                      100 105 110
          Leu Leu Glu Ala Ile Arg Phe Leu Gly Leu Glu Lys Lys Thr Arg Phe
                  115 120 125
          Tyr Gln Ala Ser Thr Ser Glu Leu Tyr Gly Leu Val Gln Glu Ile Pro
              130 135 140
          Gln Lys Glu Thr Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Ala Val Ala
          145 150 155 160
          Lys Leu Tyr Ala Tyr Trp Ile Thr Val Asn Tyr Arg Glu Ser Tyr Gly
                          165 170 175
          Met Tyr Ala Cys Asn Gly Ile Leu Phe Asn His Glu Ser Pro Arg Arg
                      180 185 190
          Gly Glu Thr Phe Val Thr Arg Lys Ile Thr Arg Ala Ile Ala Asn Ile
                  195 200 205
          Ala Gln Gly Leu Glu Ser Cys Leu Tyr Leu Gly Asn Met Asp Ser Leu
              210 215 220
          Arg Asp Trp Gly His Ala Lys Asp Tyr Val Lys Met Gln Trp Met Met
          225 230 235 240
          Leu Gln Gln Glu Gln Pro Glu Asp Phe Val Ile Ala Thr Gly Val Gln
                          245 250 255
          Tyr Ser Val Arg Gln Phe Val Glu Met Ala Ala Ala Gln Leu Gly Ile
                      260 265 270
          Lys Leu Arg Phe Glu Gly Thr Gly Val Glu Glu Lys Gly Ile Val Val
                  275 280 285
          Ser Val Thr Gly His Asp Ala Pro Gly Val Lys Pro Gly Asp Val Ile
              290 295 300
          Ile Ala Val Asp Pro Arg Tyr Phe Arg Pro Ala Glu Val Glu Thr Leu
          305 310 315 320
          Leu Gly Asp Pro Thr Lys Ala His Glu Lys Leu Gly Trp Lys Pro Glu
                          325 330 335
          Ile Thr Leu Arg Glu Met Val Ser Glu Met Val Ala Asn Asp Leu Glu
                      340 345 350
          Ala Ala Lys Lys His Ser Leu Leu Lys Ser His Gly Tyr Asp Val Ala
                  355 360 365
          Ile Ala Leu Glu Ser
              370
           <![CDATA[ <210> 12]]>
           <![CDATA[ <211> 321]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 12]]>
          Met Ser Lys Gln Arg Val Phe Ile Ala Gly His Arg Gly Met Val Gly
          1 5 10 15
          Ser Ala Ile Arg Arg Gln Leu Glu Gln Arg Gly Asp Val Glu Leu Val
                      20 25 30
          Leu Arg Thr Arg Asp Glu Leu Asn Leu Leu Asp Ser Arg Ala Val His
                  35 40 45
          Asp Phe Phe Ala Ser Glu Arg Ile Asp Gln Val Tyr Leu Ala Ala Ala
              50 55 60
          Lys Val Gly Gly Ile Val Ala Asn Asn Thr Tyr Pro Ala Asp Phe Ile
          65 70 75 80
          Tyr Gln Asn Met Met Ile Glu Ser Asn Ile Ile His Ala Ala His Gln
                          85 90 95
          Asn Asp Val Asn Lys Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pro
                      100 105 110
          Lys Leu Ala Lys Gln Pro Met Ala Glu Ser Glu Leu Leu Gln Gly Thr
                  115 120 125
          Leu Glu Pro Thr Asn Glu Pro Tyr Ala Ile Ala Lys Ile Ala Gly Ile
              130 135 140
          Lys Leu Cys Glu Ser Tyr Asn Arg Gln Tyr Gly Arg Asp Tyr Arg Ser
          145 150 155 160
          Val Met Pro Thr Asn Leu Tyr Gly Pro His Asp Asn Phe His Pro Ser
                          165 170 175
          Asn Ser His Val Ile Pro Ala Leu Leu Arg Arg Phe His Glu Ala Thr
                      180 185 190
          Ala Gln Asn Ala Pro Asp Val Val Val Trp Gly Ser Gly Thr Pro Met
                  195 200 205
          Arg Glu Phe Leu His Val Asp Asp Met Ala Ala Ala Ser Ile His Val
              210 215 220
          Met Glu Leu Ala His Glu Val Trp Leu Glu Asn Thr Gln Pro Met Leu
          225 230 235 240
          Ser His Ile Asn Val Gly Thr Gly Val Asp Cys Thr Ile Arg Glu Leu
                          245 250 255
          Ala Gln Thr Ile Ala Lys Val Val Gly Tyr Lys Gly Arg Val Val Phe
                      260 265 270
          Asp Ala Ser Lys Pro Asp Gly Thr Pro Arg Lys Leu Leu Asp Val Thr
                  275 280 285
          Arg Leu His Gln Leu Gly Trp Tyr His Glu Ile Ser Leu Glu Ala Gly
              290 295 300
          Leu Ala Ser Thr Tyr Gln Trp Phe Leu Glu Asn Gln Asp Arg Phe Arg
          305 310 315 320
          Gly
           <![CDATA[ <210> 13]]>
           <![CDATA[ <211> 438]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 13]]>
          Met Gly Asn Thr Ser Ile Gln Thr Gln Ser Tyr Arg Ala Val Asp Lys
          1 5 10 15
          Asp Ala Gly Gln Ser Arg Ser Tyr Ile Ile Pro Phe Ala Leu Leu Cys
                      20 25 30
          Ser Leu Phe Phe Leu Trp Ala Val Ala Asn Asn Leu Asn Asp Ile Leu
                  35 40 45
          Leu Pro Gln Phe Gln Gln Ala Phe Thr Leu Thr Asn Phe Gln Ala Gly
              50 55 60
          Leu Ile Gln Ser Ala Phe Tyr Phe Gly Tyr Phe Ile Ile Pro Ile Pro
          65 70 75 80
          Ala Gly Ile Leu Met Lys Lys Leu Ser Tyr Lys Ala Gly Ile Ile Thr
                          85 90 95
          Gly Leu Phe Leu Tyr Ala Leu Gly Ala Ala Leu Phe Trp Pro Ala Ala
                      100 105 110
          Glu Ile Met Asn Tyr Thr Leu Phe Leu Val Gly Leu Phe Ile Ile Ala
                  115 120 125
          Ala Gly Leu Gly Cys Leu Glu Thr Ala Ala Asn Pro Phe Val Thr Val
              130 135 140
          Leu Gly Pro Glu Ser Ser Gly His Phe Arg Leu Asn Leu Ala Gln Thr
          145 150 155 160
          Phe Asn Ser Phe Gly Ala Ile Ile Ala Val Val Phe Gly Gln Ser Leu
                          165 170 175
          Ile Leu Ser Asn Val Pro His Gln Ser Gln Asp Val Leu Asp Lys Met
                      180 185 190
          Ser Pro Glu Gln Leu Ser Ala Tyr Lys His Ser Leu Val Leu Ser Val
                  195 200 205
          Gln Thr Pro Tyr Met Ile Ile Val Ala Ile Val Leu Leu Val Ala Leu
              210 215 220
          Leu Ile Met Leu Thr Lys Phe Pro Ala Leu Gln Ser Asp Asn His Ser
          225 230 235 240
          Asp Ala Lys Gln Gly Ser Phe Ser Ala Ser Leu Ser Arg Leu Ala Arg
                          245 250 255
          Ile Arg His Trp Arg Trp Ala Val Leu Ala Gln Phe Cys Tyr Val Gly
                      260 265 270
          Ala Gln Thr Ala Cys Trp Ser Tyr Leu Ile Arg Tyr Ala Val Glu Glu
                  275 280 285
          Ile Pro Gly Met Thr Ala Gly Phe Ala Ala Asn Tyr Leu Thr Gly Thr
              290 295 300
          Met Val Cys Phe Phe Ile Gly Arg Phe Thr Gly Thr Trp Leu Ile Ser
          305 310 315 320
          Arg Phe Ala Pro His Lys Val Leu Ala Ala Tyr Ala Leu Ile Ala Met
                          325 330 335
          Ala Leu Cys Leu Ile Ser Ala Phe Ala Gly Gly His Val Gly Leu Ile
                      340 345 350
          Ala Leu Thr Leu Cys Ser Ala Phe Met Ser Ile Gln Tyr Pro Thr Ile
                  355 360 365
          Phe Ser Leu Gly Ile Lys Asn Leu Gly Gln Asp Thr Lys Tyr Gly Ser
              370 375 380
          Ser Phe Ile Val Met Thr Ile Ile Gly Gly Gly Ile Val Thr Pro Val
          385 390 395 400
          Met Gly Phe Val Ser Asp Ala Ala Gly Asn Ile Pro Thr Ala Glu Leu
                          405 410 415
          Ile Pro Ala Leu Cys Phe Ala Val Ile Phe Ile Phe Ala Arg Phe Arg
                      420 425 430
          Ser Gln Thr Ala Thr Asn
                  435
           <![CDATA[ <210> 14]]>
           <![CDATA[ <211> 949]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bacteroides fragilis NCTC 9343]]>
           <![CDATA[ <400> 14]]>
          Met Gln Lys Leu Leu Ser Leu Pro Ser Asn Leu Val Gln Ser Phe His
          1 5 10 15
          Glu Leu Glu Arg Val Asn Arg Thr Asp Trp Phe Cys Thr Ser Asp Pro
                      20 25 30
          Val Gly Lys Lys Leu Gly Ser Gly Gly Gly Thr Ser Trp Leu Leu Glu
                  35 40 45
          Glu Cys Tyr Asn Glu Tyr Ser Asp Gly Ala Thr Phe Gly Glu Trp Leu
              50 55 60
          Glu Lys Glu Lys Arg Ile Leu Leu His Ala Gly Gly Gln Ser Arg Arg
          65 70 75 80
          Leu Pro Gly Tyr Ala Pro Ser Gly Lys Ile Leu Thr Pro Val Pro Val
                          85 90 95
          Phe Arg Trp Glu Arg Gly Gln His Leu Gly Gln Asn Leu Leu Ser Leu
                      100 105 110
          Gln Leu Pro Leu Tyr Glu Lys Ile Met Ser Leu Ala Pro Asp Lys Leu
                  115 120 125
          His Thr Leu Ile Ala Ser Gly Asp Val Tyr Ile Arg Ser Glu Lys Pro
              130 135 140
          Leu Gln Ser Ile Pro Glu Ala Asp Val Val Cys Tyr Gly Leu Trp Val
          145 150 155 160
          Asp Pro Ser Leu Ala Thr His His Gly Val Phe Ala Ser Asp Arg Lys
                          165 170 175
          His Pro Glu Gln Leu Asp Phe Met Leu Gln Lys Pro Ser Leu Ala Glu
                      180 185 190
          Leu Glu Ser Leu Ser Lys Thr His Leu Phe Leu Met Asp Ile Gly Ile
                  195 200 205
          Trp Leu Leu Ser Asp Arg Ala Val Glu Ile Leu Met Lys Arg Ser His
              210 215 220
          Lys Glu Ser Ser Glu Glu Leu Lys Tyr Tyr Asp Leu Tyr Ser Asp Phe
          225 230 235 240
          Gly Leu Ala Leu Gly Thr His Pro Arg Ile Glu Asp Glu Glu Val Asn
                          245 250 255
          Thr Leu Ser Val Ala Ile Leu Pro Leu Pro Gly Gly Glu Phe Tyr His
                      260 265 270
          Tyr Gly Thr Ser Lys Glu Leu Ile Ser Ser Thr Leu Ser Val Gln Asn
                  275 280 285
          Lys Val Tyr Asp Gln Arg Arg Ile Met His Arg Lys Val Lys Pro Asn
              290 295 300
          Pro Ala Met Phe Val Gln Asn Ala Val Val Arg Ile Pro Leu Cys Ala
          305 310 315 320
          Glu Asn Ala Asp Leu Trp Ile Glu Asn Ser His Ile Gly Pro Lys Trp
                          325 330 335
          Lys Ile Ala Ser Arg His Ile Ile Thr Gly Val Pro Glu Asn Asp Trp
                      340 345 350
          Ser Leu Ala Val Pro Ala Gly Val Cys Val Asp Val Val Pro Met Gly
                  355 360 365
          Asp Lys Gly Phe Val Ala Arg Pro Tyr Gly Leu Asp Asp Val Phe Lys
              370 375 380
          Gly Asp Leu Arg Asp Ser Lys Thr Thr Leu Thr Gly Ile Pro Phe Gly
          385 390 395 400
          Glu Trp Met Ser Lys Arg Gly Leu Ser Tyr Thr Asp Leu Lys Gly Arg
                          405 410 415
          Thr Asp Asp Leu Gln Ala Val Ser Val Phe Pro Met Val Asn Ser Val
                      420 425 430
          Glu Glu Leu Gly Leu Val Leu Arg Trp Met Leu Ser Glu Pro Glu Leu
                  435 440 445
          Glu Glu Gly Lys Asn Ile Trp Leu Arg Ser Glu His Phe Ser Ala Asp
              450 455 460
          Glu Ile Ser Ala Gly Ala Asn Leu Lys Arg Leu Tyr Ala Gln Arg Glu
          465 470 475 480
          Glu Phe Arg Lys Gly Asn Trp Lys Ala Leu Ala Val Asn His Glu Lys
                          485 490 495
          Ser Val Phe Tyr Gln Leu Asp Leu Ala Asp Ala Ala Glu Asp Phe Val
                      500 505 510
          Arg Leu Gly Leu Asp Met Pro Glu Leu Leu Pro Glu Asp Ala Leu Gln
                  515 520 525
          Met Ser Arg Ile His Asn Arg Met Leu Arg Ala Arg Ile Leu Lys Leu
              530 535 540
          Asp Gly Lys Asp Tyr Arg Pro Glu Glu Gln Ala Ala Phe Asp Leu Leu
          545 550 555 560
          Arg Asp Gly Leu Leu Asp Gly Ile Ser Asn Arg Lys Ser Thr Pro Lys
                          565 570 575
          Leu Asp Val Tyr Ser Asp Gln Ile Val Trp Gly Arg Ser Pro Val Arg
                      580 585 590
          Ile Asp Met Ala Gly Gly Trp Thr Asp Thr Pro Pro Tyr Ser Leu Tyr
                  595 600 605
          Ser Gly Gly Asn Val Val Asn Leu Ala Ile Glu Leu Asn Gly Gln Pro
              610 615 620
          Pro Leu Gln Val Tyr Val Lys Pro Cys Lys Asp Phe His Ile Val Leu
          625 630 635 640
          Arg Ser Ile Asp Met Gly Ala Met Glu Ile Val Ser Thr Phe Asp Glu
                          645 650 655
          Leu Gln Asp Tyr Lys Lys Lys Ile Gly Ser Pro Phe Ser Ile Pro Lys Ala
                      660 665 670
          Ala Leu Ser Leu Ala Gly Phe Ala Pro Ala Phe Ser Ala Val Ser Tyr
                  675 680 685
          Ala Ser Leu Glu Glu Gln Leu Lys Asp Phe Gly Ala Gly Ile Glu Val
              690 695 700
          Thr Leu Leu Ala Ala Ile Pro Ala Gly Ser Gly Leu Gly Thr Ser Ser
          705 710 715 720
          Ile Leu Ala Ser Thr Val Leu Gly Ala Ile Asn Asp Phe Cys Gly Leu
                          725 730 735
          Ala Trp Asp Lys Asn Glu Ile Cys Gln Arg Thr Leu Val Leu Glu Gln
                      740 745 750
          Leu Leu Thr Thr Gly Gly Gly Trp Gln Asp Gln Tyr Gly Gly Val Leu
                  755 760 765
          Gln Gly Val Lys Leu Leu Gln Thr Glu Ala Gly Phe Ala Gln Ser Pro
              770 775 780
          Leu Val Arg Trp Leu Pro Asp His Leu Phe Thr His Pro Glu Tyr Lys
          785 790 795 800
          Asp Cys His Leu Leu Tyr Tyr Thr Gly Ile Thr Arg Thr Ala Lys Gly
                          805 810 815
          Ile Leu Ala Glu Ile Val Ser Ser Met Phe Leu Asn Ser Ser Leu His
                      820 825 830
          Leu Asn Leu Leu Ser Glu Met Lys Ala His Ala Leu Asp Met Asn Glu
                  835 840 845
          Ala Ile Gln Arg Gly Ser Phe Val Glu Phe Gly Arg Leu Val Gly Lys
              850 855 860
          Thr Trp Glu Gln Asn Lys Ala Leu Asp Ser Gly Thr Asn Pro Pro Ala
          865 870 875 880
          Val Glu Ala Ile Ile Asp Leu Ile Lys Asp Tyr Thr Leu Gly Tyr Lys
                          885 890 895
          Leu Pro Gly Ala Gly Gly Gly Gly Tyr Leu Tyr Met Val Ala Lys Asp
                      900 905 910
          Pro Gln Ala Ala Val Arg Ile Arg Lys Ile Leu Thr Glu Asn Ala Pro
                  915 920 925
          Asn Pro Arg Ala Arg Phe Val Glu Met Thr Leu Ser Asp Lys Gly Phe
              930 935 940
          Gln Val Ser Arg Ser
          945
           <![CDATA[ <210> 15]]>
           <![CDATA[ <211> 417]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 15]]>
          Met Tyr Tyr Leu Lys Asn Thr Asn Phe Trp Met Phe Gly Leu Phe Phe
          1 5 10 15
          Phe Phe Tyr Phe Phe Ile Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile
                      20 25 30
          Trp Leu His Asp Ile Asn His Ile Ser Lys Ser Asp Thr Gly Ile Ile
                  35 40 45
          Phe Ala Ala Ile Ser Leu Phe Ser Leu Leu Phe Gln Pro Leu Phe Gly
              50 55 60
          Leu Leu Ser Asp Lys Leu Gly Leu Arg Lys Tyr Leu Leu Trp Ile Ile
          65 70 75 80
          Thr Gly Met Leu Val Met Phe Ala Pro Phe Phe Ile Phe Ile Phe Gly
                          85 90 95
          Pro Leu Leu Gln Tyr Asn Ile Leu Val Gly Ser Ile Val Gly Gly Ile
                      100 105 110
          Tyr Leu Gly Phe Cys Phe Asn Ala Gly Ala Pro Ala Val Glu Ala Phe
                  115 120 125
          Ile Glu Lys Val Ser Arg Arg Ser Asn Phe Glu Phe Gly Arg Ala Arg
              130 135 140
          Met Phe Gly Cys Val Gly Trp Ala Leu Cys Ala Ser Ile Val Gly Ile
          145 150 155 160
          Met Phe Thr Ile Asn Asn Gln Phe Val Phe Trp Leu Gly Ser Gly Cys
                          165 170 175
          Ala Leu Ile Leu Ala Val Leu Leu Phe Phe Ala Lys Thr Asp Ala Pro
                      180 185 190
          Ser Ser Ala Thr Val Ala Asn Ala Val Gly Ala Asn His Ser Ala Phe
                  195 200 205
          Ser Leu Lys Leu Ala Leu Glu Leu Phe Arg Gln Pro Lys Leu Trp Phe
              210 215 220
          Leu Ser Leu Tyr Val Ile Gly Val Ser Cys Thr Tyr Asp Val Phe Asp
          225 230 235 240
          Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ala Thr Gly Glu Gln
                          245 250 255
          Gly Thr Arg Val Phe Gly Tyr Val Thr Thr Met Gly Glu Leu Leu Asn
                      260 265 270
          Ala Ser Ile Met Phe Phe Ala Pro Leu Ile Ile Asn Arg Ile Gly Gly
                  275 280 285
          Lys Asn Ala Leu Leu Leu Ala Gly Thr Ile Met Ser Val Arg Ile Ile
              290 295 300
          Gly Ser Ser Phe Ala Thr Ser Ala Leu Glu Val Val Ile Leu Lys Thr
          305 310 315 320
          Leu His Met Phe Glu Val Pro Phe Leu Leu Val Gly Cys Phe Lys Tyr
                          325 330 335
          Ile Thr Ser Gln Phe Glu Val Arg Phe Ser Ala Thr Ile Tyr Leu Val
                      340 345 350
          Cys Phe Cys Phe Phe Lys Gln Leu Ala Met Ile Phe Met Ser Val Leu
                  355 360 365
          Ala Gly Asn Met Tyr Glu Ser Ile Gly Phe Gln Gly Ala Tyr Leu Val
              370 375 380
          Leu Gly Leu Val Ala Leu Gly Phe Thr Leu Ile Ser Val Phe Thr Leu
          385 390 395 400
          Ser Gly Pro Gly Pro Leu Ser Leu Leu Arg Arg Gln Val Asn Glu Val
                          405 410 415
          Ala
           <![CDATA[ <210> 16]]>
           <![CDATA[ <211> 159]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Saccharomyces cerevisiae]]>
           <![CDATA[ <400> 16]]>
          Met Ser Leu Pro Asp Gly Phe Tyr Ile Arg Arg Met Glu Glu Gly Asp
          1 5 10 15
          Leu Glu Gln Val Thr Glu Thr Leu Lys Val Leu Thr Thr Val Gly Thr
                      20 25 30
          Ile Thr Pro Glu Ser Phe Ser Lys Leu Ile Lys Tyr Trp Asn Glu Ala
                  35 40 45
          Thr Val Trp Asn Asp Asn Glu Asp Lys Lys Ile Met Gln Tyr Asn Pro
              50 55 60
          Met Val Ile Val Asp Lys Arg Thr Glu Thr Val Ala Ala Thr Gly Asn
          65 70 75 80
          Ile Ile Ile Glu Arg Lys Ile Ile His Glu Leu Gly Leu Cys Gly His
                          85 90 95
          Ile Glu Asp Ile Ala Val Asn Ser Lys Tyr Gln Gly Gln Gly Leu Gly
                      100 105 110
          Lys Leu Leu Ile Asp Gln Leu Val Thr Ile Gly Phe Asp Tyr Gly Cys
                  115 120 125
          Tyr Lys Ile Ile Leu Asp Cys Asp Glu Lys Asn Val Lys Phe Tyr Glu
              130 135 140
          Lys Cys Gly Phe Ser Asn Ala Gly Val Glu Met Gln Ile Arg Lys
          145 150 155
           <![CDATA[ <210> 17]]>
           <![CDATA[ <211> 421]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bacteroides ovale]]>
           <![CDATA[ <400> 17]]>
          Met Asp Ser Lys Asn Asn Ile Gly His Ser Ala Asp Ile Ser Leu Thr
          1 5 10 15
          Ala Glu Leu Pro Ile Pro Ile Tyr Asn Gly Asn Thr Ile Met Asp Phe
                      20 25 30
          Lys Lys Leu Ala Ser Leu Tyr Lys Asp Glu Leu Leu Asp Asn Val Leu
                  35 40 45
          Pro Phe Trp Leu Glu His Ser Gln Asp His Glu Tyr Gly Gly Tyr Phe
              50 55 60
          Thr Cys Leu Asp Arg Glu Gly Lys Val Phe Asp Thr Asp Lys Phe Ile
          65 70 75 80
          Trp Leu Gln Ser Arg Glu Val Trp Met Phe Ser Met Leu Tyr Asn Lys
                          85 90 95
          Val Glu Lys Arg Gln Glu Trp Leu Asp Cys Ala Ile Gln Gly Gly Glu
                      100 105 110
          Phe Leu Lys Lys Tyr Gly His Asp Gly Asn Tyr Asn Trp Tyr Phe Ser
                  115 120 125
          Leu Asp Arg Ser Gly Arg Pro Leu Val Glu Pro Tyr Asn Ile Phe Ser
              130 135 140
          Tyr Thr Phe Ala Thr Met Ala Phe Gly Gln Leu Ser Leu Thr Thr Gly
          145 150 155 160
          Asn Gln Glu Tyr Ala Asp Ile Ala Lys Lys Thr Phe Asp Ile Ile Leu
                          165 170 175
          Ser Lys Val Asp Asn Pro Lys Gly Arg Trp Asn Lys Leu His Pro Gly
                      180 185 190
          Thr Arg Asn Leu Lys Asn Phe Ala Leu Pro Met Ile Leu Cys Asn Leu
                  195 200 205
          Ala Leu Glu Ile Glu His Leu Leu Asp Glu Thr Tyr Leu Arg Glu Thr
              210 215 220
          Met Asp Thr Cys Ile His Glu Val Met Glu Val Phe Tyr Arg Pro Glu
          225 230 235 240
          Leu Gly Gly Ile Ile Val Glu Asn Val Asp Ile Asp Gly Asn Leu Val
                          245 250 255
          Asp Cys Phe Glu Gly Arg Gln Val Thr Pro Gly His Ala Ile Glu Ala
                      260 265 270
          Met Trp Phe Ile Met Asp Leu Gly Lys Arg Leu Asn Arg Pro Glu Leu
                  275 280 285
          Ile Glu Lys Ala Lys Glu Thr Thr Leu Thr Met Leu Asn Tyr Gly Trp
              290 295 300
          Asp Lys Gln Tyr Gly Gly Ile Tyr Tyr Phe Met Asp Arg Asn Gly Cys
          305 310 315 320
          Pro Pro Gln Gln Leu Glu Trp Asp Gln Lys Leu Trp Trp Val His Ile
                          325 330 335
          Glu Thr Leu Ile Ser Leu Leu Lys Gly Tyr Gln Leu Thr Gly Asp Lys
                      340 345 350
          Lys Cys Leu Glu Trp Phe Glu Lys Val His Asp Tyr Thr Trp Glu His
                  355 360 365
          Phe Lys Asp Lys Glu Tyr Pro Glu Trp Tyr Gly Tyr Leu Asn Arg Arg
              370 375 380
          Gly Glu Val Leu Leu Pro Leu Lys Gly Gly Lys Trp Lys Gly Cys Phe
          385 390 395 400
          His Val Pro Arg Gly Leu Tyr Gln Cys Trp Lys Thr Leu Glu Glu Ile
                          405 410 415
          Lys Asn Ile Val Ser
                      420
           <![CDATA[ <210> 18]]>
           <![CDATA[ <211> 349]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Neisseria meningitidis]]>
           <![CDATA[ <400> 18]]>
          Met Gln Asn Asn Asn Glu Phe Lys Ile Gly Asn Arg Ser Val Gly Tyr
          1 5 10 15
          Asn His Glu Pro Leu Ile Ile Cys Glu Ile Gly Ile Asn His Glu Gly
                      20 25 30
          Ser Leu Lys Thr Ala Phe Glu Met Val Asp Ala Ala Tyr Asn Ala Gly
                  35 40 45
          Ala Glu Val Val Lys His Gln Thr His Ile Val Glu Asp Glu Met Ser
              50 55 60
          Asp Glu Ala Lys Gln Val Ile Pro Gly Asn Ala Asp Val Ser Ile Tyr
          65 70 75 80
          Glu Ile Met Glu Arg Cys Ala Leu Asn Glu Glu Asp Glu Ile Lys Leu
                          85 90 95
          Lys Glu Tyr Val Glu Ser Lys Gly Met Ile Phe Ile Ser Thr Pro Phe
                      100 105 110
          Ser Arg Ala Ala Ala Leu Arg Leu Gln Arg Met Asp Ile Pro Ala Tyr
                  115 120 125
          Lys Ile Gly Ser Gly Glu Cys Asn Asn Tyr Pro Leu Ile Lys Leu Val
              130 135 140
          Ala Ser Phe Gly Lys Pro Ile Ile Leu Ser Thr Gly Met Asn Ser Ile
          145 150 155 160
          Glu Ser Ile Lys Lys Ser Val Glu Ile Ile Arg Glu Ala Gly Val Pro
                          165 170 175
          Tyr Ala Leu Leu His Cys Thr Asn Ile Tyr Pro Thr Pro Tyr Glu Asp
                      180 185 190
          Val Arg Leu Gly Gly Met Asn Asp Leu Ser Glu Ala Phe Pro Asp Ala
                  195 200 205
          Ile Ile Gly Leu Ser Asp His Thr Leu Asp Asn Tyr Ala Cys Leu Gly
              210 215 220
          Ala Val Ala Leu Gly Gly Ser Ile Leu Glu Arg His Phe Thr Asp Arg
          225 230 235 240
          Met Asp Arg Pro Gly Pro Asp Ile Val Cys Ser Met Asn Pro Asp Thr
                          245 250 255
          Phe Lys Glu Leu Lys Gln Gly Ala His Ala Leu Lys Leu Ala Arg Gly
                      260 265 270
          Gly Lys Lys Asp Thr Ile Ile Ala Gly Glu Lys Pro Thr Lys Asp Phe
                  275 280 285
          Ala Phe Ala Ser Val Val Ala Asp Lys Asp Ile Lys Lys Gly Glu Leu
              290 295 300
          Leu Ser Gly Asp Asn Leu Trp Val Lys Arg Pro Gly Asn Gly Asp Phe
          305 310 315 320
          Ser Val Asn Glu Tyr Glu Thr Leu Phe Gly Lys Val Ala Ala Cys Asn
                          325 330 335
          Ile Arg Lys Gly Ala Gln Ile Lys Lys Thr Asp Ile Glu
                      340 345
           <![CDATA[ <210> 19]]>
           <![CDATA[ <211> 609]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 19]]>
          Met Cys Gly Ile Val Gly Ala Ile Ala Gln Arg Asp Val Ala Glu Ile
          1 5 10 15
          Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala
                      20 25 30
          Gly Leu Ala Val Val Asp Thr Glu Gly His Met Thr Arg Leu Arg Arg
                  35 40 45
          Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu
              50 55 60
          His Gly Gly Thr Gly Ile Ala His Thr Arg Trp Ala Thr His Gly Glu
          65 70 75 80
          Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His Ile Val Val
                          85 90 95
          Val His Asn Gly Ile Ile Glu Asn His Glu Pro Leu Arg Glu Glu Leu
                      100 105 110
          Lys Ala Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val Ile
                  115 120 125
          Ala His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu
              130 135 140
          Ala Val Leu Arg Ala Ile Pro Gln Leu Arg Gly Ala Tyr Gly Thr Val
          145 150 155 160
          Ile Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Ala Arg Ser Gly
                          165 170 175
          Ser Pro Leu Val Ile Gly Leu Gly Met Gly Glu Asn Phe Ile Ala Ser
                      180 185 190
          Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe Ile Phe Leu Glu
                  195 200 205
          Glu Gly Asp Ile Ala Glu Ile Thr Arg Arg Ser Val Asn Ile Phe Asp
              210 215 220
          Lys Thr Gly Ala Glu Val Lys Arg Gln Asp Ile Glu Ser Asn Leu Gln
          225 230 235 240
          Tyr Asp Ala Gly Asp Lys Gly Ile Tyr Cys His Tyr Met Gln Lys Glu
                          245 250 255
          Ile Tyr Glu Gln Pro Asn Ala Ile Lys Asn Thr Leu Thr Gly Arg Ile
                      260 265 270
          Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Ala Asp Glu
                  275 280 285
          Leu Leu Ser Lys Val Glu His Ile Gln Ile Leu Ala Cys Gly Thr Ser
              290 295 300
          Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Ala Gly
          305 310 315 320
          Ile Pro Cys Asp Val Glu Ile Ala Ser Glu Phe Arg Tyr Arg Lys Ser
                          325 330 335
          Ala Val Arg Arg Asn Ser Leu Met Ile Thr Leu Ser Gln Ser Gly Glu
                      340 345 350
          Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr
                  355 360 365
          Leu Gly Ser Leu Ala Ile Cys Asn Val Pro Gly Ser Ser Leu Val Arg
              370 375 380
          Glu Ser Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu Ile Gly Val
          385 390 395 400
          Ala Ser Thr Lys Ala Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu
                          405 410 415
          Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser Ile Glu His
                      420 425 430
          Asp Ile Val His Gly Leu Gln Ala Leu Pro Ser Arg Ile Glu Gln Met
                  435 440 445
          Leu Ser Gln Asp Lys Arg Ile Glu Ala Leu Ala Glu Asp Phe Ser Asp
              450 455 460
          Lys His His Ala Leu Phe Leu Ser Arg Gly Asp Gln Tyr Pro Ile Ala
          465 470 475 480
          Leu Glu Gly Ala Leu Lys Leu Lys Glu Ile Ser Tyr Ile His Ala Glu
                          485 490 495
          Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu Ile Asp
                      500 505 510
          Ala Asp Met Pro Val Ile Val Val Ala Pro Asn Asn Glu Leu Leu Glu
                  515 520 525
          Lys Leu Lys Ser Asn Ile Glu Glu Val Arg Ala Arg Gly Gly Gln Leu
              530 535 540
          Tyr Val Phe Ala Asp Gln Asp Ala Gly Phe Val Ser Ser Asp Asn Met
          545 550 555 560
          His Ile Ile Glu Met Pro His Val Glu Glu Val Ile Ala Pro Ile Phe
                          565 570 575
          Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu Ile Lys
                      580 585 590
          Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val
                  595 600 605
          Glu
           <![CDATA[ <210> 20]]>
           <![CDATA[ <211> 188]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 20]]>
          Met Tyr Glu Arg Tyr Ala Gly Leu Ile Phe Asp Met Asp Gly Thr Ile
          1 5 10 15
          Leu Asp Thr Glu Pro Thr His Arg Lys Ala Trp Arg Glu Val Leu Gly
                      20 25 30
          His Tyr Gly Leu Gln Tyr Asp Ile Gln Ala Met Ile Ala Leu Asn Gly
                  35 40 45
          Ser Pro Thr Trp Arg Ile Ala Gln Ala Ile Ile Glu Leu Asn Gln Ala
              50 55 60
          Asp Leu Asp Pro His Ala Leu Ala Arg Glu Lys Thr Glu Ala Val Arg
          65 70 75 80
          Ser Met Leu Leu Asp Ser Val Glu Pro Leu Pro Leu Val Asp Val Val
                          85 90 95
          Lys Ser Trp His Gly Arg Arg Pro Met Ala Val Gly Thr Gly Ser Glu
                      100 105 110
          Ser Ala Ile Ala Glu Ala Leu Leu Ala His Leu Gly Leu Arg His Tyr
                  115 120 125
          Phe Asp Ala Val Val Ala Ala Asp His Val Lys His His Lys Pro Ala
              130 135 140
          Pro Asp Thr Phe Leu Leu Cys Ala Gln Arg Met Gly Val Gln Pro Thr
          145 150 155 160
          Gln Cys Val Val Phe Glu Asp Ala Asp Phe Gly Ile Gln Ala Ala Arg
                          165 170 175
          Ala Ala Gly Met Asp Ala Val Asp Val Arg Leu Leu
                      180 185
           <![CDATA[ <210> 21]]>
           <![CDATA[ <211> 373]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Campylobacter jejuni]]>
           <![CDATA[ <400> 21]]>
          Met Val Lys Lys Ile Leu Phe Ile Thr Gly Ser Arg Ala Asp Tyr Ser
          1 5 10 15
          Lys Ile Lys Ser Leu Met Tyr Arg Val Gln Asn Ser Ser Glu Phe Glu
                      20 25 30
          Leu Tyr Ile Phe Ala Thr Gly Met His Leu Ser Lys Asn Phe Gly Tyr
                  35 40 45
          Thr Val Lys Glu Leu Tyr Lys Asn Gly Phe Lys Asn Ile Tyr Glu Phe
              50 55 60
          Ile Asn Tyr Asp Lys Tyr Tyr Gln Thr Asp Lys Ala Leu Ala Thr Thr
          65 70 75 80
          Ile Asp Gly Phe Ser Arg Tyr Ala Asn Glu Leu Lys Pro Asp Leu Ile
                          85 90 95
          Val Val His Gly Asp Arg Ile Glu Pro Leu Ala Ala Ala Ile Val Gly
                      100 105 110
          Ala Leu Asn Asn Ile Leu Val Ala His Ile Glu Gly Gly Glu Ile Ser
                  115 120 125
          Gly Thr Ile Asp Asp Ser Leu Arg His Ala Ile Ser Lys Leu Ala His
              130 135 140
          Ile His Leu Val Asn Asp Glu Phe Ala Lys Arg Arg Leu Met Gln Leu
          145 150 155 160
          Gly Glu Asp Glu Lys Ser Ile Phe Ile Ile Gly Ser Pro Asp Leu Glu
                          165 170 175
          Leu Leu Asn Asp Asn Lys Ile Ser Leu Ser Glu Ala Lys Lys Tyr Tyr
                      180 185 190
          Asp Ile Asn Tyr Glu Asn Tyr Ala Leu Leu Met Phe His Pro Val Thr
                  195 200 205
          Thr Glu Ile Thr Ser Ile Lys Asn Gln Ala Asp Asn Leu Val Lys Ala
              210 215 220
          Leu Ile Gln Ser Asn Lys Asn Tyr Ile Val Ile Tyr Pro Asn Asn Asp
          225 230 235 240
          Leu Gly Phe Glu Leu Ile Leu Gln Ser Tyr Glu Glu Phe Lys Asn Asn
                          245 250 255
          Pro Arg Phe Lys Leu Phe Pro Ser Leu Arg Phe Glu Tyr Phe Ile Thr
                      260 265 270
          Leu Leu Lys Asn Ala Asp Phe Ile Ile Gly Asn Ser Ser Cys Ile Leu
                  275 280 285
          Lys Glu Ala Leu Tyr Leu Lys Thr Ala Gly Ile Leu Val Gly Ser Arg
              290 295 300
          Gln Asn Gly Arg Leu Gly Asn Glu Asn Thr Leu Lys Val Asn Ala Asn
          305 310 315 320
          Ser Asp Glu Ile Leu Lys Ala Ile Asn Thr Ile His Lys Lys Gln Asp
                          325 330 335
          Leu Phe Ser Ala Lys Leu Glu Ile Leu Asp Ser Ser Lys Leu Phe Phe
                      340 345 350
          Glu Tyr Leu Gln Ser Gly Asp Phe Phe Lys Leu Ser Thr Gln Lys Val
                  355 360 365
          Phe Lys Asp Ile Lys
              370
           <![CDATA[ <210> 22]]>
           <![CDATA[ <211> 223]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Pasteurella septicemia]]>
           <![CDATA[ <400> 22]]>
          Met Thr Asn Ile Ala Ile Ile Pro Ala Arg Ala Gly Ser Lys Gly Ile
          1 5 10 15
          Pro Asp Lys Asn Leu Gln Pro Val Gly Gly His Ser Leu Ile Gly Arg
                      20 25 30
          Ala Ile Leu Ala Ala Lys Asn Ala Asp Val Phe Asp Met Ile Val Val
                  35 40 45
          Thr Ser Asp Gly Asp Asn Ile Leu Arg Glu Ala Glu Lys Tyr Gly Ala
              50 55 60
          Leu Ala Leu Lys Arg Pro Ala Glu Leu Ala Gln Asp Asn Ser Arg Thr
          65 70 75 80
          Ile Asp Ala Ile Leu His Ala Leu Glu Ser Leu Asn Ile Arg Glu Gly
                          85 90 95
          Thr Cys Thr Leu Leu Gln Pro Thr Ser Pro Leu Arg Asp His Leu Asp
                      100 105 110
          Ile Lys Asn Ala Met Asp Met Tyr Val Asn Gly Gly Val His Ser Val
                  115 120 125
          Val Ser Ala Cys Glu Cys Glu His His Pro Tyr Lys Ala Phe Ala Leu
              130 135 140
          Ser Lys Asp His Glu Val Leu Pro Val Arg Glu Ile Ala Asp Phe Glu
          145 150 155 160
          Ala Ala Arg Gln Thr Leu Pro Lys Met Tyr Arg Ala Asn Gly Ala Ile
                          165 170 175
          Tyr Ile Asn Asp Ile Ala Gln Leu Leu Lys Glu Lys Tyr Phe Phe Ile
                      180 185 190
          Pro Pro Leu Lys Phe Tyr Leu Met Pro Thr Tyr Arg Ser Val Asp Ile
                  195 200 205
          Asp Val Lys Gln Asp Leu Glu Leu Ala Glu Ile Leu Ser Asn Lys
              210 215 220
           <![CDATA[ <210> 23]]>
           <![CDATA[ <211> 268]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Pasteurella septicemia]]>
           <![CDATA[ <400> 23]]>
          Met Asp Lys Phe Ala Glu His Glu Ile Pro Lys Ala Val Ile Val Ala
          1 5 10 15
          Gly Asn Gly Glu Ser Leu Ser Gln Ile Asp Tyr Arg Leu Leu Pro Lys
                      20 25 30
          Asn Tyr Asp Val Phe Arg Cys Asn Gln Phe Tyr Phe Glu Glu Arg Tyr
                  35 40 45
          Phe Leu Gly Asn Lys Ile Lys Ala Val Phe Phe Thr Pro Gly Val Phe
              50 55 60
          Leu Glu Gln Tyr Tyr Thr Leu Tyr His Leu Lys Arg Asn Asn Glu Tyr
          65 70 75 80
          Phe Val Asp Asn Val Ile Leu Ser Ser Phe Asn His Pro Thr Val Asp
                          85 90 95
          Leu Glu Lys Ser Gln Lys Ile Gln Ala Leu Phe Ile Asp Val Ile Asn
                      100 105 110
          Gly Tyr Glu Lys Tyr Leu Ser Lys Leu Thr Ala Phe Asp Val Tyr Leu
                  115 120 125
          Arg Tyr Lys Glu Leu Tyr Glu Asn Gln Arg Ile Thr Ser Gly Val Tyr
              130 135 140
          Met Cys Ala Val Ala Ile Ala Met Gly Tyr Thr Asp Ile Tyr Leu Thr
          145 150 155 160
          Gly Ile Asp Phe Tyr Gln Ala Ser Glu Glu Asn Tyr Ala Phe Asp Asn
                          165 170 175
          Lys Lys Pro Asn Ile Ile Arg Leu Leu Pro Asp Phe Arg Lys Glu Lys
                      180 185 190
          Thr Leu Phe Ser Tyr His Ser Lys Asp Ile Asp Leu Glu Ala Leu Ser
                  195 200 205
          Phe Leu Gln Gln His Tyr His Val Asn Phe Tyr Ser Ile Ser Pro Met
              210 215 220
          Ser Pro Leu Ser Lys His Phe Pro Ile Pro Thr Val Glu Asp Asp Cys
          225 230 235 240
          Glu Thr Thr Phe Val Ala Pro Leu Lys Glu Asn Tyr Ile Asn Asp Ile
                          245 250 255
          Leu Leu Pro Pro His Phe Val Tyr Glu Lys Leu Gly
                      260 265
           <![CDATA[ <210> 24]]>
           <![CDATA[ <211> 371]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Neisseria meningitidis]]>
           <![CDATA[ <400> 24]]>
          Met Gly Leu Lys Lys Ala Cys Leu Thr Val Leu Cys Leu Ile Val Phe
          1 5 10 15
          Cys Phe Gly Ile Phe Tyr Thr Phe Asp Arg Val Asn Gln Gly Glu Arg
                      20 25 30
          Asn Ala Val Ser Leu Leu Lys Glu Lys Leu Phe Asn Glu Glu Gly Glu
                  35 40 45
          Pro Val Asn Leu Ile Phe Cys Tyr Thr Ile Leu Gln Met Lys Val Ala
              50 55 60
          Glu Arg Ile Met Ala Gln His Pro Gly Glu Arg Phe Tyr Val Val Leu
          65 70 75 80
          Met Ser Glu Asn Arg Asn Glu Lys Tyr Asp Tyr Tyr Phe Asn Gln Ile
                          85 90 95
          Lys Asp Lys Ala Glu Arg Ala Tyr Phe Phe His Leu Pro Tyr Gly Leu
                      100 105 110
          Asn Lys Ser Phe Asn Phe Ile Pro Thr Met Ala Glu Leu Lys Val Lys
                  115 120 125
          Ser Met Leu Leu Pro Lys Val Lys Arg Ile Tyr Leu Ala Ser Leu Glu
              130 135 140
          Lys Val Ser Ile Ala Ala Phe Leu Ser Thr Tyr Pro Asp Ala Glu Ile
          145 150 155 160
          Lys Thr Phe Asp Asp Gly Thr Gly Asn Leu Ile Gln Ser Ser Ser Tyr
                          165 170 175
          Leu Gly Asp Glu Phe Ser Val Asn Gly Thr Ile Lys Arg Asn Phe Ala
                      180 185 190
          Arg Met Met Ile Gly Asp Trp Ser Ile Ala Lys Thr Arg Asn Ala Ser
                  195 200 205
          Asp Glu His Tyr Thr Ile Phe Lys Gly Leu Lys Asn Ile Met Asp Asp
              210 215 220
          Gly Arg Arg Lys Met Thr Tyr Leu Pro Leu Phe Asp Ala Ser Glu Leu
          225 230 235 240
          Lys Thr Gly Asp Glu Thr Gly Gly Thr Val Arg Ile Leu Leu Gly Ser
                          245 250 255
          Pro Asp Lys Glu Met Lys Glu Ile Ser Glu Lys Ala Ala Lys Asn Phe
                      260 265 270
          Lys Ile Gln Tyr Val Ala Pro His Pro Arg Gln Thr Tyr Gly Leu Ser
                  275 280 285
          Gly Val Thr Thr Leu Asn Ser Pro Tyr Val Ile Glu Asp Tyr Ile Leu
              290 295 300
          Arg Glu Ile Lys Lys Asn Pro His Thr Arg Tyr Glu Ile Tyr Thr Phe
          305 310 315 320
          Phe Ser Gly Ala Ala Leu Thr Met Lys Asp Phe Pro Asn Val His Val
                          325 330 335
          Tyr Ala Leu Lys Pro Ala Ser Leu Pro Glu Asp Tyr Trp Leu Lys Pro
                      340 345 350
          Val Tyr Ala Leu Phe Thr Gln Ser Gly Ile Pro Ile Leu Thr Phe Asp
                  355 360 365
          Asp Lys Asn
              370
           <![CDATA[ <210> 25]]>
           <![CDATA[ <211> 391]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Mermaid Light Fungus]]>
           <![CDATA[ <400> 25]]>
          Met Thr Val Val Ala Pro Thr Leu Glu Val Tyr Ile Asp His Ala Ser
          1 5 10 15
          Leu Pro Ser Leu Gln Gln Leu Ile His Ile Ile Gln Ala Lys Asp Glu
                      20 25 30
          Tyr Pro Ser Asn Gln Arg Phe Val Ser Trp Lys Arg Val Thr Val Asp
                  35 40 45
          Ala Asp Asn Ala Asn Lys Leu Asn Ile His Thr Tyr Pro Leu Lys Gly
              50 55 60
          Asn Asn Thr Ser Pro Glu Met Val Ala Ala Ile Asp Glu Tyr Ala Gln
          65 70 75 80
          Ser Lys Asn Arg Leu Asn Ile Glu Phe Tyr Thr Asn Thr Ala His Val
                          85 90 95
          Phe Asn Asn Leu Pro Pro Ile Ile Gln Pro Leu Tyr Asn Asn Glu Lys
                      100 105 110
          Val Lys Ile Ser His Ile Ser Leu Tyr Asp Asp Gly Ser Ser Glu Tyr
                  115 120 125
          Val Ser Leu Tyr Gln Trp Lys Asp Thr Pro Asn Lys Ile Glu Thr Leu
              130 135 140
          Glu Gly Glu Val Ser Leu Leu Ala Asn Tyr Leu Ala Gly Thr Ser Pro
          145 150 155 160
          Asp Ala Pro Lys Gly Met Gly Asn Arg Tyr Asn Trp His Lys Leu Tyr
                          165 170 175
          Asp Thr Asp Tyr Tyr Phe Leu Arg Glu Asp Tyr Leu Asp Val Glu Ala
                      180 185 190
          Asn Leu His Asp Leu Arg Asp Tyr Leu Gly Ser Ser Ala Lys Gln Met
                  195 200 205
          Pro Trp Asp Glu Phe Ala Lys Leu Ser Asp Ser Gln Gln Thr Leu Phe
              210 215 220
          Leu Asp Ile Val Gly Phe Asp Lys Glu Gln Leu Gln Gln Gln Tyr Ser
          225 230 235 240
          Gln Ser Pro Leu Pro Asn Phe Ile Phe Thr Gly Thr Thr Thr Trp Ala
                          245 250 255
          Gly Gly Glu Thr Lys Glu Tyr Tyr Ala Gln Gln Gln Val Asn Val Ile
                      260 265 270
          Asn Asn Ala Ile Asn Glu Thr Ser Pro Tyr Tyr Leu Gly Lys Asp Tyr
                  275 280 285
          Asp Leu Phe Phe Lys Gly His Pro Ala Gly Gly Val Ile Asn Asp Ile
              290 295 300
          Ile Leu Gly Ser Phe Pro Asp Met Ile Asn Ile Pro Ala Lys Ile Ser
          305 310 315 320
          Phe Glu Val Leu Met Met Thr Asp Met Leu Pro Asp Thr Val Ala Gly
                          325 330 335
          Ile Ala Ser Ser Leu Tyr Phe Thr Ile Pro Ala Asp Lys Val Asn Phe
                      340 345 350
          Ile Val Phe Thr Ser Ser Asp Thr Ile Thr Asp Arg Glu Glu Ala Leu
                  355 360 365
          Lys Ser Pro Leu Val Gln Val Met Leu Thr Leu Gly Ile Val Lys Glu
              370 375 380
          Lys Asp Val Leu Phe Trp Ala
          385 390
           <![CDATA[ <210> 26]]>
           <![CDATA[ <211> 498]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Photobacterium sp. JT-ISH-224]]>
           <![CDATA[ <400> 26]]>
          Met Ser Glu Glu Asn Thr Gln Ser Ile Ile Lys Asn Asp Ile Asn Lys
          1 5 10 15
          Thr Ile Ile Asp Glu Glu Tyr Val Asn Leu Glu Pro Ile Asn Gln Ser
                      20 25 30
          Asn Ile Ser Phe Thr Lys His Ser Trp Val Gln Thr Cys Gly Thr Gln
                  35 40 45
          Gln Leu Leu Thr Glu Gln Asn Lys Glu Ser Ile Ser Leu Ser Val Val
              50 55 60
          Ala Pro Arg Leu Asp Asp Asp Glu Lys Tyr Cys Phe Asp Phe Asn Gly
          65 70 75 80
          Val Ser Asn Lys Gly Glu Lys Tyr Ile Thr Lys Val Thr Leu Asn Val
                          85 90 95
          Val Ala Pro Ser Leu Glu Val Tyr Val Asp His Ala Ser Leu Pro Thr
                      100 105 110
          Leu Gln Gln Leu Met Asp Ile Ile Lys Ser Glu Glu Glu Asn Pro Thr
                  115 120 125
          Ala Gln Arg Tyr Ile Ala Trp Gly Arg Ile Val Pro Thr Asp Glu Gln
              130 135 140
          Met Lys Glu Leu Asn Ile Thr Ser Phe Ala Leu Ile Asn Asn His Thr
          145 150 155 160
          Pro Ala Asp Leu Val Gln Glu Ile Val Lys Gln Ala Gln Thr Lys His
                          165 170 175
          Arg Leu Asn Val Lys Leu Ser Ser Asn Thr Ala His Ser Phe Asp Asn
                      180 185 190
          Leu Val Pro Ile Leu Lys Glu Leu Asn Ser Phe Asn Asn Val Thr Val
                  195 200 205
          Thr Asn Ile Asp Leu Tyr Asp Asp Gly Ser Ala Glu Tyr Val Asn Leu
              210 215 220
          Tyr Asn Trp Arg Asp Thr Leu Asn Lys Thr Asp Asn Leu Lys Ile Gly
          225 230 235 240
          Lys Asp Tyr Leu Glu Asp Val Ile Asn Gly Ile Asn Glu Asp Thr Ser
                          245 250 255
          Asn Thr Gly Thr Ser Ser Val Tyr Asn Trp Gln Lys Leu Tyr Pro Ala
                      260 265 270
          Asn Tyr His Phe Leu Arg Lys Asp Tyr Leu Thr Leu Glu Pro Ser Leu
                  275 280 285
          His Glu Leu Arg Asp Tyr Ile Gly Asp Ser Leu Lys Gln Met Gln Trp
              290 295 300
          Asp Gly Phe Lys Lys Phe Asn Ser Lys Gln Gln Glu Leu Phe Leu Ser
          305 310 315 320
          Ile Val Asn Phe Asp Lys Gln Lys Leu Gln Asn Glu Tyr Asn Ser Ser
                          325 330 335
          Asn Leu Pro Asn Phe Val Phe Thr Gly Thr Thr Val Trp Ala Gly Asn
                      340 345 350
          His Glu Arg Glu Tyr Tyr Ala Lys Gln Gln Ile Asn Val Ile Asn Asn
                  355 360 365
          Ala Ile Asn Glu Ser Ser Pro His Tyr Leu Gly Asn Ser Tyr Asp Leu
              370 375 380
          Phe Phe Lys Gly His Pro Gly Gly Gly Ile Ile Asn Thr Leu Ile Met
          385 390 395 400
          Gln Asn Tyr Pro Ser Met Val Asp Ile Pro Ser Lys Ile Ser Phe Glu
                          405 410 415
          Val Leu Met Met Thr Asp Met Leu Pro Asp Ala Val Ala Gly Ile Ala
                      420 425 430
          Ser Ser Leu Tyr Phe Thr Ile Pro Ala Glu Lys Ile Lys Phe Ile Val
                  435 440 445
          Phe Thr Ser Thr Glu Thr Ile Thr Asp Arg Glu Thr Ala Leu Arg Ser
              450 455 460
          Pro Leu Val Gln Val Met Ile Lys Leu Gly Ile Val Lys Glu Glu Asn
          465 470 475 480
          Val Leu Phe Trp Ala Asp Leu Pro Asn Cys Glu Thr Gly Val Cys Ile
                          485 490 495
          Ala Val
           <![CDATA[ <210> 27]]>
           <![CDATA[ <211> 348]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Neisseria meningitidis]]>
           <![CDATA[ <400> 27]]>
          Met Pro Ser Glu Ala Phe Arg Arg His Arg Ala Tyr Arg Glu Asn Lys
          1 5 10 15
          Leu Gln Pro Leu Val Ser Val Leu Ile Cys Ala Tyr Asn Val Glu Lys
                      20 25 30
          Tyr Phe Ala Gln Ser Leu Ala Ala Val Val Asn Gln Thr Trp Cys Asn
                  35 40 45
          Leu Asp Ile Leu Ile Val Asp Asp Gly Ser Thr Asp Gly Thr Leu Ala
              50 55 60
          Ile Ala Gln Arg Phe Gln Glu Gln Asp Gly Arg Ile Lys Ile Leu Ala
          65 70 75 80
          Gln Ala Gln Asn Ser Gly Leu Ile Pro Ser Leu Asn Ile Gly Leu Asp
                          85 90 95
          Glu Leu Ala Lys Ser Gly Met Gly Glu Tyr Ile Ala Arg Thr Asp Ala
                      100 105 110
          Asp Asp Ile Ala Ala Pro Asp Trp Ile Glu Lys Ile Val Gly Glu Met
                  115 120 125
          Glu Lys Asp Arg Ser Ile Ile Ala Met Gly Ala Trp Leu Glu Val Leu
              130 135 140
          Ser Glu Glu Lys Asp Gly Asn Arg Leu Ala Arg His His Glu His Gly
          145 150 155 160
          Lys Ile Trp Lys Lys Pro Thr Arg His Glu Asp Ile Ala Asp Phe Phe
                          165 170 175
          Pro Phe Gly Asn Pro Ile His Asn Asn Thr Met Ile Met Arg Arg Ser
                      180 185 190
          Val Ile Asp Gly Gly Leu Arg Tyr Asn Thr Glu Arg Asp Trp Ala Glu
                  195 200 205
          Asp Tyr Gln Phe Trp Tyr Asp Val Ser Lys Leu Gly Arg Leu Ala Tyr
              210 215 220
          Tyr Pro Glu Ala Leu Val Lys Tyr Arg Leu His Ala Asn Gln Val Ser
          225 230 235 240
          Ser Lys Tyr Ser Ile Arg Gln His Glu Ile Ala Gln Gly Ile Gln Lys
                          245 250 255
          Thr Ala Arg Asn Asp Phe Leu Gln Ser Met Gly Phe Lys Thr Arg Phe
                      260 265 270
          Asp Ser Leu Glu Tyr Arg Gln Ile Lys Ala Val Ala Tyr Glu Leu Leu
                  275 280 285
          Glu Lys His Leu Pro Glu Glu Asp Phe Glu Arg Ala Arg Arg Phe Leu
              290 295 300
          Tyr Gln Cys Phe Lys Arg Thr Asp Thr Leu Pro Ala Gly Val Trp Leu
          305 310 315 320
          Asp Phe Ala Ala Asn Gly Arg Met Arg Arg Leu Phe Thr Leu Arg Gln
                          325 330 335
          Tyr Phe Gly Ile Leu His Arg Leu Leu Lys Asn Arg
                      340 345
           <![CDATA[ <210> 28]]>
           <![CDATA[ <211> 265]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli O55:H7]]>
           <![CDATA[ <400> 28]]>
          Met Ile Ile Asp Glu Ala Glu Ser Ala Glu Ser Thr His Pro Val Val
          1 5 10 15
          Ser Val Ile Leu Pro Val Asn Lys Lys Asn Pro Phe Leu Asp Glu Ala
                      20 25 30
          Ile Asn Ser Ile Leu Ser Gln Thr Phe Ser Ser Phe Glu Ile Ile Ile
                  35 40 45
          Val Ala Asn Cys Cys Thr Asp Asp Phe Tyr Asn Glu Leu Lys His Lys
              50 55 60
          Val Asn Asp Lys Ile Lys Leu Ile Arg Thr Asn Ile Ala Tyr Leu Pro
          65 70 75 80
          Tyr Ser Leu Asn Lys Ala Ile Asp Leu Ser Asn Gly Glu Phe Ile Ala
                          85 90 95
          Arg Met Asp Ser Asp Asp Ile Ser His Pro Asp Arg Phe Thr Lys Gln
                      100 105 110
          Val Asp Phe Leu Lys Asn Asn Pro Tyr Val Asp Val Val Gly Thr Asn
                  115 120 125
          Ala Ile Phe Ile Asp Asp Lys Gly Arg Glu Ile Asn Lys Thr Lys Leu
              130 135 140
          Pro Glu Glu Asn Leu Asp Ile Val Lys Asn Leu Pro Tyr Lys Cys Cys
          145 150 155 160
          Ile Val His Pro Ser Val Met Phe Arg Lys Lys Val Ile Ala Ser Ile
                          165 170 175
          Gly Gly Tyr Met Phe Ser Asn Tyr Ser Glu Asp Tyr Glu Leu Trp Asn
                      180 185 190
          Arg Leu Ser Leu Ala Lys Ile Lys Phe Gln Asn Leu Pro Glu Tyr Leu
                  195 200 205
          Phe Tyr Tyr Arg Leu His Glu Gly Gln Ser Thr Ala Lys Lys Asn Leu
              210 215 220
          Tyr Met Val Met Val Asn Asp Leu Val Ile Lys Met Lys Cys Phe Phe
          225 230 235 240
          Leu Thr Gly Asn Ile Asn Tyr Leu Phe Gly Gly Ile Arg Thr Ile Ala
                          245 250 255
          Ser Phe Ile Tyr Cys Lys Tyr Ile Lys
                      260 265
           <![CDATA[ <210> 29]]>
           <![CDATA[ <211> 275]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Neisseria meningitidis MC58]]>
           <![CDATA[ <400> 29]]>
          Met Gln Asn His Val Ile Ser Leu Ala Ser Ala Ala Glu Arg Arg Ala
          1 5 10 15
          His Ile Ala Asp Thr Phe Gly Arg His Gly Ile Pro Phe Gln Phe Phe
                      20 25 30
          Asp Ala Leu Met Pro Ser Glu Arg Leu Glu Gln Ala Met Ala Glu Leu
                  35 40 45
          Val Pro Gly Leu Ser Ala His Pro Tyr Leu Ser Gly Val Glu Lys Ala
              50 55 60
          Cys Phe Met Ser His Ala Val Leu Trp Lys Gln Ala Leu Asp Glu Gly
          65 70 75 80
          Leu Pro Tyr Ile Thr Val Phe Glu Asp Asp Val Leu Leu Gly Glu Gly
                          85 90 95
          Ala Glu Lys Phe Leu Ala Glu Asp Ala Trp Leu Gln Glu Arg Phe Asp
                      100 105 110
          Pro Asp Thr Ala Phe Ile Val Arg Leu Glu Thr Met Phe Met His Val
                  115 120 125
          Leu Thr Ser Pro Ser Gly Val Ala Asp Tyr Cys Gly Arg Ala Phe Pro
              130 135 140
          Leu Leu Glu Ser Glu His Trp Gly Thr Ala Gly Tyr Ile Ile Ser Arg
          145 150 155 160
          Lys Ala Met Arg Phe Phe Leu Asp Arg Phe Ala Ala Leu Pro Pro Glu
                          165 170 175
          Gly Leu His Pro Val Asp Leu Met Met Phe Ser Asp Phe Phe Asp Arg
                      180 185 190
          Glu Gly Met Pro Val Cys Gln Leu Asn Pro Ala Leu Cys Ala Gln Glu
                  195 200 205
          Leu His Tyr Ala Lys Phe His Asp Gln Asn Ser Ala Leu Gly Ser Leu
              210 215 220
          Ile Glu His Asp Arg Leu Leu Asn Arg Lys Gln Gln Arg Arg Asp Ser
          225 230 235 240
          Pro Ala Asn Thr Phe Lys His Arg Leu Ile Arg Ala Leu Thr Lys Ile
                          245 250 255
          Ser Arg Glu Arg Glu Lys Arg Arg Gln Arg Arg Glu Gln Phe Ile Val
                      260 265 270
          Pro Phe Gln
                  275
           <![CDATA[ <210> 30]]>
           <![CDATA[ <211> 338]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 30]]>
          Met Arg Val Leu Val Thr Gly Gly Ser Gly Tyr Ile Gly Ser His Thr
          1 5 10 15
          Cys Val Gln Leu Leu Gln Asn Gly His Asp Val Ile Ile Leu Asp Asn
                      20 25 30
          Leu Cys Asn Ser Lys Arg Ser Val Leu Pro Val Ile Glu Arg Leu Gly
                  35 40 45
          Gly Lys His Pro Thr Phe Val Glu Gly Asp Ile Arg Asn Glu Ala Leu
              50 55 60
          Met Thr Glu Ile Leu His Asp His Ala Ile Asp Thr Val Ile His Phe
          65 70 75 80
          Ala Gly Leu Lys Ala Val Gly Glu Ser Val Gln Lys Pro Leu Glu Tyr
                          85 90 95
          Tyr Asp Asn Asn Val Asn Gly Thr Leu Arg Leu Ile Ser Ala Met Arg
                      100 105 110
          Ala Ala Asn Val Lys Asn Phe Ile Phe Ser Ser Ser Ala Thr Val Tyr
                  115 120 125
          Gly Asp Gln Pro Lys Ile Pro Tyr Val Glu Ser Phe Pro Thr Gly Thr
              130 135 140
          Pro Gln Ser Pro Tyr Gly Lys Ser Lys Leu Met Val Glu Gln Ile Leu
          145 150 155 160
          Thr Asp Leu Gln Lys Ala Gln Pro Asp Trp Ser Ile Ala Leu Leu Arg
                          165 170 175
          Tyr Phe Asn Pro Val Gly Ala His Pro Ser Gly Asp Met Gly Glu Asp
                      180 185 190
          Pro Gln Gly Ile Pro Asn Asn Leu Met Pro Tyr Ile Ala Gln Val Ala
                  195 200 205
          Val Gly Arg Arg Asp Ser Leu Ala Ile Phe Gly Asn Asp Tyr Pro Thr
              210 215 220
          Glu Asp Gly Thr Gly Val Arg Asp Tyr Ile His Val Met Asp Leu Ala
          225 230 235 240
          Asp Gly His Val Val Ala Met Glu Lys Leu Ala Asn Lys Pro Gly Val
                          245 250 255
          His Ile Tyr Asn Leu Gly Ala Gly Val Gly Asn Ser Val Leu Asp Val
                      260 265 270
          Val Asn Ala Phe Ser Lys Ala Cys Gly Lys Pro Val Asn Tyr His Phe
                  275 280 285
          Ala Pro Arg Arg Glu Gly Asp Leu Pro Ala Tyr Trp Ala Asp Ala Ser
              290 295 300
          Lys Ala Asp Arg Glu Leu Asn Trp Arg Val Thr Arg Thr Leu Asp Glu
          305 310 315 320
          Met Ala Gln Asp Thr Trp His Trp Gln Ser Arg His Pro Gln Gly Tyr
                          325 330 335
          Pro Asp
           <![CDATA[ <210> 31]]>
           <![CDATA[ <211> 587]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Kluyveromyces lactis]]>
           <![CDATA[ <400> 31]]>
          Met Ala Asp His Ser Ser Ser Ser Ser Ser Ser Leu Gln Lys Lys Pro Ile
          1 5 10 15
          Asn Thr Ile Glu His Lys Asp Thr Leu Gly Asn Asp Arg Asp His Lys
                      20 25 30
          Glu Ala Leu Asn Ser Asp Asn Asp Asn Thr Ser Gly Leu Lys Ile Asn
                  35 40 45
          Gly Val Pro Ile Glu Asp Ala Arg Glu Glu Val Leu Leu Pro Gly Tyr
              50 55 60
          Leu Ser Lys Gln Tyr Tyr Lys Leu Tyr Gly Leu Cys Phe Ile Thr Tyr
          65 70 75 80
          Leu Cys Ala Thr Met Gln Gly Tyr Asp Gly Ala Leu Met Gly Ser Ile
                          85 90 95
          Tyr Thr Glu Asp Ala Tyr Leu Lys Tyr Tyr His Leu Asp Ile Asn Ser
                      100 105 110
          Ser Ser Gly Thr Gly Leu Val Phe Ser Ile Phe Asn Val Gly Gln Ile
                  115 120 125
          Cys Gly Ala Phe Phe Val Pro Leu Met Asp Trp Lys Gly Arg Lys Pro
              130 135 140
          Ala Ile Leu Ile Gly Cys Leu Gly Val Val Ile Gly Ala Ile Ile Ser
          145 150 155 160
          Ser Leu Thr Thr Thr Lys Ser Ala Leu Ile Gly Gly Arg Trp Phe Val
                          165 170 175
          Ala Phe Phe Ala Thr Ile Ala Asn Ala Ala Ala Pro Thr Tyr Cys Ala
                      180 185 190
          Glu Val Ala Pro Ala His Leu Arg Gly Lys Val Ala Gly Leu Tyr Asn
                  195 200 205
          Thr Leu Trp Ser Val Gly Ser Ile Val Ala Ala Phe Ser Thr Tyr Gly
              210 215 220
          Thr Asn Lys Asn Phe Pro Asn Ser Ser Lys Ala Phe Lys Ile Pro Leu
          225 230 235 240
          Tyr Leu Gln Met Met Phe Pro Gly Leu Val Cys Ile Phe Gly Trp Leu
                          245 250 255
          Ile Pro Glu Ser Pro Arg Trp Leu Val Gly Val Gly Arg Glu Glu Glu
                      260 265 270
          Ala Arg Glu Phe Ile Ile Lys Tyr His Leu Asn Gly Asp Arg Thr His
                  275 280 285
          Pro Leu Leu Asp Met Glu Met Ala Glu Ile Ile Glu Ser Phe His Gly
              290 295 300
          Thr Asp Leu Ser Asn Pro Leu Glu Met Leu Asp Val Arg Ser Leu Phe
          305 310 315 320
          Arg Thr Arg Ser Asp Arg Tyr Arg Ala Met Leu Val Ile Leu Met Ala
                          325 330 335
          Trp Phe Gly Gln Phe Ser Gly Asn Asn Val Cys Ser Tyr Tyr Leu Pro
                      340 345 350
          Thr Met Leu Arg Asn Val Gly Met Lys Ser Val Ser Leu Asn Val Leu
                  355 360 365
          Met Asn Gly Val Tyr Ser Ile Val Thr Trp Ile Ser Ser Ile Cys Gly
              370 375 380
          Ala Phe Phe Ile Asp Lys Ile Gly Arg Arg Glu Gly Phe Leu Gly Ser
          385 390 395 400
          Ile Ser Gly Ala Ala Leu Ala Leu Thr Gly Leu Ser Ile Cys Thr Ala
                          405 410 415
          Arg Tyr Glu Lys Thr Lys Lys Lys Lys Ser Ala Ser Asn Gly Ala Leu Val
                      420 425 430
          Phe Ile Tyr Leu Phe Gly Gly Ile Phe Ser Phe Ala Phe Thr Pro Met
                  435 440 445
          Gln Ser Met Tyr Ser Thr Glu Val Ser Thr Asn Leu Thr Arg Ser Lys
              450 455 460
          Ala Gln Leu Leu Asn Phe Val Val Ser Gly Val Ala Gln Phe Val Asn
          465 470 475 480
          Gln Phe Ala Thr Pro Lys Ala Met Lys Asn Ile Lys Tyr Trp Phe Tyr
                          485 490 495
          Val Phe Tyr Val Phe Phe Asp Ile Phe Glu Phe Ile Val Ile Tyr Phe
                      500 505 510
          Phe Phe Val Glu Thr Lys Gly Arg Ser Leu Glu Glu Leu Glu Val Val
                  515 520 525
          Phe Glu Ala Pro Asn Pro Arg Lys Ala Ser Val Asp Gln Ala Phe Leu
              530 535 540
          Ala Gln Val Arg Ala Thr Leu Val Gln Arg Asn Asp Val Arg Val Ala
          545 550 555 560
          Asn Ala Gln Asn Leu Lys Glu Gln Glu Pro Leu Lys Ser Asp Ala Asp
                          565 570 575
          His Val Glu Lys Leu Ser Glu Ala Glu Ser Val
                      580 585
           <![CDATA[ <210> 32]]>
           <![CDATA[ <211> 402]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 32]]>
          Met Met Gly Ser Trp Lys His Cys Leu Phe Ser Ala Ser Leu Ile Ser
          1 5 10 15
          Ala Leu Ile Phe Val Phe Val Tyr Asn Thr Glu Leu Trp Glu Asn Lys
                      20 25 30
          Arg Phe Leu Arg Ala Ala Leu Ser Asn Ala Ser Leu Leu Ala Glu Ala
                  35 40 45
          Cys His Gln Ile Phe Glu Gly Lys Val Phe Tyr Pro Thr Glu Asn Ala
              50 55 60
          Leu Lys Thr Thr Leu Asp Glu Ala Thr Cys Tyr Glu Tyr Met Val Arg
          65 70 75 80
          Ser His Tyr Val Thr Glu Thr Leu Ser Glu Glu Glu Ala Gly Phe Pro
                          85 90 95
          Leu Ala Tyr Thr Val Thr Ile His Lys Asp Phe Gly Thr Phe Glu Arg
                      100 105 110
          Leu Phe Arg Ala Ile Tyr Met Pro Gln Asn Val Tyr Cys Val His Leu
                  115 120 125
          Asp Gln Lys Ala Thr Asp Ala Phe Lys Gly Ala Val Lys Gln Leu Leu
              130 135 140
          Ser Cys Phe Pro Asn Ala Phe Leu Ala Ser Lys Lys Glu Ser Val Val
          145 150 155 160
          Tyr Gly Gly Ile Ser Arg Leu Gln Ala Asp Leu Asn Cys Leu Glu Asp
                          165 170 175
          Leu Val Ala Ser Glu Val Pro Trp Lys Tyr Val Ile Asn Thr Cys Gly
                      180 185 190
          Gln Asp Phe Pro Leu Lys Thr Asn Arg Glu Ile Val Gln Tyr Leu Lys
                  195 200 205
          Gly Phe Lys Gly Lys Asn Ile Thr Pro Gly Val Leu Pro Pro Asp His
              210 215 220
          Ala Val Gly Arg Thr Lys Tyr Val His Gln Glu Leu Leu Asn His Lys
          225 230 235 240
          Asn Ser Tyr Val Ile Lys Thr Thr Lys Leu Lys Thr Pro Pro Pro His
                          245 250 255
          Asp Met Val Ile Tyr Phe Gly Thr Ala Tyr Val Ala Leu Thr Arg Asp
                      260 265 270
          Phe Ala Asn Phe Val Leu Gln Asp Gln Leu Ala Leu Asp Leu Leu Ser
                  275 280 285
          Trp Ser Lys Asp Thr Tyr Ser Pro Asp Glu His Phe Trp Val Thr Leu
              290 295 300
          Asn Arg Ile Pro Gly Val Pro Gly Ser Met Pro Asn Ala Ser Trp Thr
          305 310 315 320
          Gly Asn Leu Arg Ala Ile Lys Trp Ser Asp Met Glu Asp Arg His Gly
                          325 330 335
          Gly Cys His Gly His Tyr Val His Gly Ile Cys Ile Tyr Gly Asn Gly
                      340 345 350
          Asp Leu Lys Trp Leu Val Asn Ser Pro Ser Leu Phe Ala Asn Lys Phe
                  355 360 365
          Glu Leu Asn Thr Tyr Pro Leu Thr Val Glu Cys Leu Glu Leu Arg His
              370 375 380
          Arg Glu Arg Thr Leu Asn Gln Ser Glu Thr Ala Ile Gln Pro Ser Trp
          385 390 395 400
          Tyr Phe
           <![CDATA[ <210> 33]]>
           <![CDATA[ <211> 290]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> European cattle]]>
           <![CDATA[ <400> 33]]>
          Met Glu Ser Lys Leu Lys Leu Ser Asp Trp Phe Asn Pro Phe Lys Arg
          1 5 10 15
          Pro Glu Val Val Thr Met Thr Lys Trp Lys Ala Pro Val Val Trp Glu
                      20 25 30
          Gly Thr Tyr Asn Arg Ala Val Leu Asp Asn Tyr Tyr Ala Lys Gln Lys
                  35 40 45
          Ile Thr Val Gly Leu Thr Val Phe Ala Val Gly Arg Tyr Ile Glu His
              50 55 60
          Tyr Leu Glu Glu Phe Leu Thr Ser Ala Asn Lys His Phe Met Val Gly
          65 70 75 80
          His Pro Val Ile Phe Tyr Ile Met Val Asp Asp Val Ser Arg Met Pro
                          85 90 95
          Leu Ile Glu Leu Gly Pro Leu Arg Ser Phe Lys Val Phe Lys Ile Lys
                      100 105 110
          Pro Glu Lys Arg Trp Gln Asp Ile Ser Met Met Arg Met Lys Thr Ile
                  115 120 125
          Gly Glu His Ile Val Ala His Ile Gln His Glu Val Asp Phe Leu Phe
              130 135 140
          Cys Met Asp Val Asp Gln Val Phe Gln Asp Lys Phe Gly Val Glu Thr
          145 150 155 160
          Leu Gly Glu Ser Val Ala Gln Leu Gln Ala Trp Trp Tyr Lys Ala Asp
                          165 170 175
          Pro Asn Asp Phe Thr Tyr Glu Arg Arg Lys Glu Ser Ala Ala Tyr Ile
                      180 185 190
          Pro Phe Gly Glu Gly Asp Phe Tyr Tyr His Ala Ala Ile Phe Gly Gly
                  195 200 205
          Thr Pro Thr Gln Val Leu Asn Ile Thr Gln Glu Cys Phe Lys Gly Ile
              210 215 220
          Leu Lys Asp Lys Lys Asn Asp Ile Glu Ala Gln Trp His Asp Glu Ser
          225 230 235 240
          His Leu Asn Lys Tyr Phe Leu Leu Asn Lys Pro Thr Lys Ile Leu Ser
                          245 250 255
          Pro Glu Tyr Cys Trp Asp Tyr His Ile Gly Leu Pro Ala Asp Ile Lys
                      260 265 270
          Leu Val Lys Met Ser Trp Gln Thr Lys Glu Tyr Asn Val Val Arg Asn
                  275 280 285
          Asn Val
              290
           <![CDATA[ <210> 34]]>
           <![CDATA[ <211> 341]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Pseudomonas aeruginosa]]>
           <![CDATA[ <400> 34]]>
          Met Met Ser Arg Tyr Glu Glu Leu Arg Lys Glu Leu Pro Ala Gln Pro
          1 5 10 15
          Lys Val Trp Leu Ile Thr Gly Val Ala Gly Phe Ile Gly Ser Asn Leu
                      20 25 30
          Leu Glu Thr Leu Leu Lys Leu Asp Gln Lys Val Val Gly Leu Asp Asn
                  35 40 45
          Phe Ala Thr Gly His Gln Arg Asn Leu Asp Glu Val Arg Ser Leu Val
              50 55 60
          Ser Glu Lys Gln Trp Ser Asn Phe Lys Phe Ile Gln Gly Asp Ile Arg
          65 70 75 80
          Asn Leu Asp Asp Cys Asn Asn Ala Cys Ala Gly Val Asp Tyr Val Leu
                          85 90 95
          His Gln Ala Ala Leu Gly Ser Val Pro Arg Ser Ile Asn Asp Pro Ile
                      100 105 110
          Thr Ser Asn Ala Thr Asn Ile Asp Gly Phe Leu Asn Met Leu Ile Ala
                  115 120 125
          Ala Arg Asp Ala Lys Val Gln Ser Phe Thr Tyr Ala Ala Ser Ser Ser
              130 135 140
          Thr Tyr Gly Asp His Pro Gly Leu Pro Lys Val Glu Asp Thr Ile Gly
          145 150 155 160
          Lys Pro Leu Ser Pro Tyr Ala Val Thr Lys Tyr Val Asn Glu Leu Tyr
                          165 170 175
          Ala Asp Val Phe Ser Arg Cys Tyr Gly Phe Ser Thr Ile Gly Leu Arg
                      180 185 190
          Tyr Phe Asn Val Phe Gly Arg Arg Gln Asp Pro Asn Gly Ala Tyr Ala
                  195 200 205
          Ala Val Ile Pro Lys Trp Thr Ser Ser Met Ile Gln Gly Asp Asp Val
              210 215 220
          Tyr Ile Asn Gly Asp Gly Glu Thr Ser Arg Asp Phe Cys Tyr Ile Glu
          225 230 235 240
          Asn Thr Val Gln Ala Asn Leu Leu Ala Ala Thr Ala Gly Leu Asp Ala
                          245 250 255
          Arg Asn Gln Val Tyr Asn Ile Ala Val Gly Gly Arg Thr Ser Leu Asn
                      260 265 270
          Gln Leu Phe Phe Ala Leu Arg Asp Gly Leu Ala Glu Asn Gly Val Ser
                  275 280 285
          Tyr His Arg Glu Pro Val Tyr Arg Asp Phe Arg Glu Gly Asp Val Arg
              290 295 300
          His Ser Leu Ala Asp Ile Ser Lys Ala Ala Lys Leu Leu Gly Tyr Ala
          305 310 315 320
          Pro Lys Tyr Asp Val Ser Ala Gly Val Ala Leu Ala Met Pro Trp Tyr
                          325 330 335
          Ile Met Phe Leu Lys
                      340
           <![CDATA[ <210> 35]]>
           <![CDATA[ <211> 352]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Haemophilus influenzae]]>
           <![CDATA[ <400> 35]]>
          Met Glu Asn Pro Pro Leu Val Ser Val Ile Val Cys Ala Tyr Asn Ala
          1 5 10 15
          Glu Gln Tyr Ile Asp Glu Ser Ile Ser Ser Ile Ile Asn Gln Ser Tyr
                      20 25 30
          Glu Asn Leu Glu Ile Ile Val Ile Asn Asp Gly Ser Thr Asp Leu Thr
                  35 40 45
          Leu Ser His Leu Glu Lys Ile Ser Lys Leu Asp Lys Arg Ile Lys Ile
              50 55 60
          Ile Ser Asn Lys Tyr Asn Leu Gly Phe Ile Asn Ser Leu Asn Ile Gly
          65 70 75 80
          Leu Gly Cys Phe Ser Gly Lys Tyr Ile Ala Arg Met Asp Ala Asp Asp
                          85 90 95
          Ile Ala Lys Pro Ser Trp Ile Glu Lys Ile Val Thr Tyr Leu Glu Lys
                      100 105 110
          Asn Asp His Ile Thr Thr Met Gly Ser Tyr Leu Glu Ile Ile Val Glu
                  115 120 125
          Lys Glu Cys Gly Ile Ile Gly Ser Gln Tyr Lys Thr Gly Asp Ile Trp
              130 135 140
          Lys Asn Pro Leu Leu His Asn Glu Ile Cys Glu Ala Met Leu Phe Tyr
          145 150 155 160
          Asn Pro Ile His Asn Asn Thr Met Ile Met Arg Ala Asn Val Tyr Arg
                          165 170 175
          Glu His Lys Leu Ile Phe Asn Lys Asp Tyr Pro Tyr Ala Glu Asp Tyr
                      180 185 190
          Lys Phe Trp Ser Glu Val Ser Arg Leu Gly Cys Leu Ala Asn Tyr Pro
                  195 200 205
          Glu Ala Leu Val Lys Tyr Arg Leu His Gly Asn Gln Ala Ser Ser Val
              210 215 220
          His Asn His Lys Gln Asn Glu Val Ala Lys Lys Ile Lys Arg Glu Asn
          225 230 235 240
          Ile Thr Tyr Tyr Leu Asn Lys Ile Gly Ile Asp Ile Asn Val Ile Asn
                          245 250 255
          Ser Val Ser Leu Leu Glu Ile Tyr His Val Asp Lys Ser Asn Lys Val
                      260 265 270
          Leu Lys Ser Ile Leu Tyr Asp Met Tyr Met Ser Leu Asp Lys Tyr Thr
                  275 280 285
          Ile Thr Ser Leu Leu His Phe Ile Lys Tyr His Leu Lys Leu Phe Asp
              290 295 300
          Leu Lys Gln Asn Phe Lys Ile Ile Lys Lys Phe Ile Arg Lys Ile Met
          305 310 315 320
          Leu Cys Tyr Val Met Leu Cys Tyr Val Met Leu Cys Phe Arg Trp Ser
                          325 330 335
          Val Gln Lys Gly Glu Glu Gln Ile Pro Ser Leu Ser Arg Thr Lys Phe
                      340 345 350
           <![CDATA[ <210> 36]]>
           <![CDATA[ <211> 306]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Neisseria gonorrhoeae]]>
           <![CDATA[ <400> 36]]>
          Met Asp Ile Val Phe Ala Ala Asp Asp Asn Tyr Ala Ala Tyr Leu Cys
          1 5 10 15
          Val Ala Ala Lys Ser Val Glu Ala Ala His Pro Asp Thr Glu Ile Arg
                      20 25 30
          Phe His Val Leu Asp Ala Gly Ile Ser Glu Glu Asn Arg Ala Ala Val
                  35 40 45
          Ala Ala Asn Leu Arg Gly Gly Gly Asn Ile Arg Phe Ile Asp Val Asn
              50 55 60
          Pro Glu Asp Phe Ala Gly Phe Pro Leu Asn Ile Arg His Ile Ser Ile
          65 70 75 80
          Thr Thr Tyr Ala Arg Leu Lys Leu Gly Glu Tyr Ile Ala Asp Cys Asp
                          85 90 95
          Lys Val Leu Tyr Leu Asp Thr Asp Val Leu Val Arg Asp Gly Leu Lys
                      100 105 110
          Pro Leu Trp Asp Thr Asp Leu Gly Gly Asn Trp Val Gly Ala Cys Ile
                  115 120 125
          Asp Leu Phe Val Glu Arg Gln Glu Gly Tyr Lys Gln Lys Ile Gly Met
              130 135 140
          Ala Asp Gly Glu Tyr Tyr Phe Asn Ala Gly Val Leu Leu Ile Asn Leu
          145 150 155 160
          Lys Lys Trp Arg Arg His Asp Ile Phe Lys Met Ser Cys Glu Trp Val
                          165 170 175
          Glu Gln Tyr Lys Asp Val Met Gln Tyr Gln Asp Gln Asp Ile Leu Asn
                      180 185 190
          Gly Leu Phe Lys Gly Gly Val Cys Tyr Ala Asn Ser Arg Phe Asn Phe
                  195 200 205
          Met Pro Thr Asn Tyr Ala Phe Met Ala Asn Gly Phe Ala Ser Arg His
              210 215 220
          Thr Asp Pro Leu Tyr Leu Asp Arg Thr Asn Thr Ala Met Pro Val Ala
          225 230 235 240
          Val Ser His Tyr Cys Gly Ser Ala Lys Pro Trp His Arg Asp Cys Thr
                          245 250 255
          Val Trp Gly Ala Glu Arg Phe Thr Glu Leu Ala Gly Ser Leu Thr Thr
                      260 265 270
          Val Pro Glu Glu Trp Arg Gly Lys Leu Ala Val Pro Pro Thr Lys Cys
                  275 280 285
          Met Leu Gln Arg Trp Arg Lys Lys Leu Ser Ala Arg Phe Leu Arg Lys
              290 295 300
          Ile Tyr
          305
           <![CDATA[ <210> 37]]>
           <![CDATA[ <211> 234]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 37]]>
          Met Val Ile Asn Ile Phe Tyr Ile Cys Thr Gly Glu Tyr Lys Arg Phe
          1 5 10 15
          Phe Asp Lys Phe Tyr Leu Ser Cys Glu Asp Lys Phe Ile Pro Glu Phe
                      20 25 30
          Gly Lys Lys Tyr Tyr Val Phe Thr Asp Ser Asp Arg Ile Tyr Phe Ser
                  35 40 45
          Lys Tyr Leu Asn Val Glu Val Ile Asn Val Glu Lys Asn Cys Trp Pro
              50 55 60
          Leu Asn Thr Leu Leu Arg Phe Ser Tyr Phe Leu Lys Val Ile Asp Lys
          65 70 75 80
          Leu Gln Thr Asn Ser Tyr Thr Phe Phe Phe Asn Ala Asn Ala Val Ile
                          85 90 95
          Val Lys Glu Ile Pro Phe Ser Thr Phe Met Glu Ser Asp Leu Ile Gly
                      100 105 110
          Val Ile His Pro Gly Tyr Lys Asn Arg Ile Ser Ile Leu Tyr Pro Trp
                  115 120 125
          Glu Arg Arg Lys Asn Ala Thr Cys Tyr Leu Gly Tyr Leu Lys Lys Gly
              130 135 140
          Ile Tyr Tyr Gln Gly Cys Phe Asn Gly Gly Lys Thr Ala Ser Phe Lys
          145 150 155 160
          Arg Leu Ile Gln Ile Cys Asn Met Met Thr Met Ala Asp Leu Lys Lys
                          165 170 175
          Asn Leu Ile Ala Lys Val His Asp Glu Ser Tyr Leu Asn Tyr Tyr Tyr
                      180 185 190
          Tyr Tyr Asn Lys Pro Leu Leu Leu Ser Glu Leu Tyr Ser Trp Pro Glu
                  195 200 205
          Lys Tyr Gly Glu Asn Lys Asp Ala Lys Ile Ile Met Arg Asp Lys Glu
              210 215 220
          Arg Glu Ser Trp Tyr Gly Asn Ile Lys Lys
          225 230
           <![CDATA[ <210> 38]]>
           <![CDATA[ <211> 306]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Helicobacter weasel (strain ATCC 43772)]]>
           <![CDATA[ <400> 38]]>
          Met Gln Ser Thr Ala Gln Asn Thr Gln Gln Asn Thr His Phe Ala Gly
          1 5 10 15
          Ser Ser Gln Thr Thr Pro Gln Ala Ala Gln Ser Val Gln Gln Ala Ser
                      20 25 30
          Leu Ala Leu Pro Lys Ser Ser Pro Thr Cys Tyr Lys Ile Ala Ile Leu
                  35 40 45
          Tyr Ile Cys Thr Gly Ala Tyr Ser Ile Phe Trp Gln Asp Phe Tyr Asp
              50 55 60
          Ser Ala Lys Val His Leu Leu Pro Ala His Arg Leu Thr Tyr Phe Val
          65 70 75 80
          Phe Thr Asp Ala Asp Ser Leu Tyr Ala Glu Glu Ala Ser Asp Val Arg
                          85 90 95
          Lys Ile Tyr Gln Glu Asn Leu Gly Trp Pro Phe Asn Thr Leu Lys Arg
                      100 105 110
          Phe Glu Met Phe Leu Gly Gln Glu Glu Ala Leu Arg Glu Phe Asp Phe
                  115 120 125
          Val Phe Phe Phe Asn Ala Asn Cys Leu Phe Phe Gln His Ile Gly Asp
              130 135 140
          Glu Phe Leu Pro Ile Glu Glu Asp Ile Leu Val Thr Gln His Tyr Gly
          145 150 155 160
          Phe Arg Asp Ala Ser Pro Glu Cys Phe Thr Tyr Glu Arg Asn Pro Lys
                          165 170 175
          Ser Leu Ala Tyr Val Pro Phe Gly Lys Gly Lys Ala Tyr Val Tyr Gly
                      180 185 190
          Ser Thr Asn Gly Gly Lys Ala Gly Ala Phe Leu Ala Leu Ala Arg Thr
                  195 200 205
          Leu Gln Glu Arg Ile Gln Glu Asp Leu Ser Arg Gly Ile Ile Ala Ile
              210 215 220
          Trp His Asp Glu Ser His Leu Asn Ala Tyr Ile Ile Asp His Pro Asn
          225 230 235 240
          Tyr Lys Met Leu Asp Tyr Gly Tyr Gly Phe Pro Glu Gly Tyr Gly Arg
                          245 250 255
          Val Pro Gly Gly Gly Val Tyr Ile Phe Leu Arg Asp Lys Ser Arg Val
                      260 265 270
          Ile Asp Val Asn Ala Ile Lys Gly Met Gly Ser Pro Ala Asn Arg Arg
                  275 280 285
          Leu Lys Asn Ala Leu Arg Lys Leu Lys His Phe Ser Lys Arg Leu Leu
              290 295 300
          Gly Arg
          305
           <![CDATA[ <210> 39]]>
           <![CDATA[ <211> 291]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bifidobacterium longum infantum subsp. ATCC 15697]]>
           <![CDATA[ <400> 39]]>
          Met Leu Phe Asn Pro Arg Asn Val Asp Val Asp Gln Trp Met Asp Ala
          1 5 10 15
          Leu Val Ala Gly Gly Met Ala Gly Val Ile Leu Thr Cys Lys His His
                      20 25 30
          Asp Gly Phe Cys Leu Trp Pro Ser Arg Leu Thr Arg His Thr Val Ala
                  35 40 45
          Ser Ser Pro Trp Arg Glu Gly Lys Gly Asp Leu Val Arg Glu Val Ser
              50 55 60
          Glu Ser Ala Arg Arg His Gly Leu Lys Phe Gly Val Tyr Leu Ser Pro
          65 70 75 80
          Trp Asp Arg Thr Glu Glu Ser Tyr Gly Lys Gly Lys Ala Tyr Asp Asp
                          85 90 95
          Phe Tyr Val Gly Gln Leu Thr Glu Leu Leu Thr Gln Tyr Gly Pro Ile
                      100 105 110
          Phe Ser Val Trp Leu Asp Gly Ala Asn Gly Glu Gly Lys Asn Gly Lys
                  115 120 125
          Thr Gln Tyr Tyr Asp Trp Asp Arg Tyr Tyr Asn Val Ile Arg Ser Leu
              130 135 140
          Gln Pro Asp Ala Val Ile Ser Val Cys Gly Pro Asp Val Arg Trp Ala
          145 150 155 160
          Gly Asn Glu Ala Gly His Val Arg Asp Asn Glu Trp Ser Val Val Pro
                          165 170 175
          Arg Arg Leu Arg Ser Ala Glu Leu Thr Met Glu Lys Ser Gln Gln Glu
                      180 185 190
          Asp Asp Ala Ser Phe Ala Thr Thr Val Ser Ser Gln Asp Asp Asp Leu
                  195 200 205
          Gly Ser Arg Glu Ala Val Ala Gly Tyr Gly Asp Asn Val Cys Trp Tyr
              210 215 220
          Pro Ala Glu Val Asp Thr Ser Ile Arg Pro Gly Trp Phe Tyr His Gln
          225 230 235 240
          Ser Glu Asp Asp Lys Val Met Ser Ala Asp Gln Leu Phe Asp Leu Trp
                          245 250 255
          Leu Ser Ala Val Gly Gly Asn Ser Ser Leu Leu Leu Asn Ile Pro Pro
                      260 265 270
          Ser Pro Glu Gly Leu Leu Ala Glu Pro Asp Val Gln Ser Leu Lys Gly
                  275 280 285
          Leu Gly Arg
              290
           <![CDATA[ <210> 40]]>
           <![CDATA[ <211> 292]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Sideroxydans lithotrophicus ES-1]]>
           <![CDATA[ <400> 40]]>
          Met Val Ile Ser Asn Ile Ile Gly Gly Leu Gly Asn Gln Met Phe Gln
          1 5 10 15
          Tyr Ala Ala Ala Arg Ala Leu Ser Leu Lys Leu Glu Val Pro Leu Lys
                      20 25 30
          Leu Asp Ile Ser Gly Phe Thr Asn Tyr Ala Leu His Gln Gly Phe Glu
                  35 40 45
          Leu Asp Arg Ile Phe Gly Cys Lys Ile Glu Ile Ala Ser Glu Ala Asp
              50 55 60
          Val His Glu Ile Leu Gly Trp Gln Ser Ala Ser Gly Ile Arg Arg Val
          65 70 75 80
          Val Ser Arg Pro Gly Met Ser Ile Phe Arg Arg Lys Gly Phe Val Val
                          85 90 95
          Glu Pro His Phe Ser Tyr Trp Asn Gly Ile Arg Lys Ile Thr Gly Asp
                      100 105 110
          Cys Tyr Leu Ala Gly Tyr Trp Gln Ser Glu Lys Tyr Phe Leu Asp Ala
                  115 120 125
          Ala Val Glu Ile Arg Lys Asp Phe Ser Phe Lys Leu Pro Leu Asp Ser
              130 135 140
          His Asn Ala Glu Leu Ala Glu Lys Ile Asp Gln Glu Asn Ala Val Ser
          145 150 155 160
          Leu His Ile Arg Arg Gly Asp Tyr Ala Asn Asn Pro Leu Thr Ala Ala
                          165 170 175
          Thr His Gly Leu Cys Ser Leu Asp Tyr Tyr Arg Lys Ser Ile Lys His
                      180 185 190
          Ile Ala Gly Gln Val Arg Asn Pro Tyr Phe Phe Val Phe Ser Asp Asp
                  195 200 205
          Ile Ala Trp Val Lys Asp Asn Leu Glu Ile Glu Phe Pro Ser Gln Tyr
              210 215 220
          Val Asp Tyr Asn His Gly Ser Met Ser Phe Asn Asp Met Arg Leu Met
          225 230 235 240
          Ser Leu Cys Lys His His His Ile Ile Ala Asn Ser Ser Phe Ser Trp Trp
                          245 250 255
          Gly Ala Trp Leu Asn Pro Asn Pro Glu Lys Val Val Ile Ala Pro Glu
                      260 265 270
          Arg Trp Phe Ala Asn Arg Thr Asp Val Gln Asp Leu Leu Pro Pro Gly
                  275 280 285
          Trp Val Lys Leu
              290
           <![CDATA[ <210> 41]]>
           <![CDATA[ <211> 264]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Salmonella enterica subsp. salamae serovar Greenside]]>
           <![CDATA[ <400> 41]]>
          Met Leu Thr Glu Val Arg Pro Val Ser Thr Thr Lys Pro Leu Val Ser
          1 5 10 15
          Val Ile Leu Pro Val Asn Lys Phe Asn Pro Tyr Leu Asp Arg Ala Ile
                      20 25 30
          His Ser Ile Leu Ser Gln Ser Tyr Pro Ser Ile Glu Leu Ile Ile Ile
                  35 40 45
          Ala Asn Asn Cys Thr Asn Asn Asp Phe Phe Asp Ala Leu Lys Lys Arg Glu
              50 55 60
          Cys Glu Thr Ile Lys Val Leu Arg Thr Asn Ile Ala Tyr Leu Pro Tyr
          65 70 75 80
          Cys Leu Asn Lys Gly Leu Asp Leu Cys Asn Gly Asp Phe Val Ala Arg
                          85 90 95
          Met Asp Ser Asp Asp Ile Ser His Pro Glu Arg Ile Asp Arg Gln Val
                      100 105 110
          Asp Phe Leu Ile Asn Asn Pro Asp Ile Asp Val Val Gly Thr Asn Ala
                  115 120 125
          Val Tyr Ile Asp Glu Asp Asp Ile Glu Leu Glu Lys Ser Asn Leu Pro
              130 135 140
          Val Asn Asn Asn Ala Ile Arg Lys Met Leu Pro Tyr Lys Cys Cys Leu
          145 150 155 160
          Val His Pro Ser Val Met Phe Arg Lys Asn Val Val Ile Thr Ser Gly
                          165 170 175
          Gly Tyr Met Phe Ala Asn Tyr Ser Glu Asp Tyr Glu Leu Trp Asn Arg
                      180 185 190
          Leu Ala Val Glu Gly Arg Asn Phe Tyr Asn Leu Ser Glu Tyr Leu Leu
                  195 200 205
          Tyr Tyr Arg Leu His Asn Asn Gln Ser Thr Ser Lys Asn Asn Leu Phe
              210 215 220
          Met Val Met Ala Asn Asp Val Ala Ile Lys Val Lys Tyr Phe Leu Leu
          225 230 235 240
          Thr Lys Lys Ile Ser Tyr Leu Leu Gly Ile Ile Arg Thr Val Phe Ser
                          245 250 255
          Val Phe Tyr Cys Lys Tyr Ile Lys
                      260
           <![CDATA[ <210> 42]]>
           <![CDATA[ <211> 445]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 42]]>
          Met Ser Asn Arg Lys Tyr Phe Gly Thr Asp Gly Ile Arg Gly Arg Val
          1 5 10 15
          Gly Asp Ala Pro Ile Thr Pro Asp Phe Val Leu Lys Leu Gly Trp Ala
                      20 25 30
          Ala Gly Lys Val Leu Ala Arg His Gly Ser Arg Lys Ile Ile Ile Gly
                  35 40 45
          Lys Asp Thr Arg Ile Ser Gly Tyr Met Leu Glu Ser Ala Leu Glu Ala
              50 55 60
          Gly Leu Ala Ala Ala Gly Leu Ser Ala Leu Phe Thr Gly Pro Met Pro
          65 70 75 80
          Thr Pro Ala Val Ala Tyr Leu Thr Arg Thr Phe Arg Ala Glu Ala Gly
                          85 90 95
          Ile Val Ile Ser Ala Ser His Asn Pro Phe Tyr Asp Asn Gly Ile Lys
                      100 105 110
          Phe Phe Ser Ile Asp Gly Thr Lys Leu Pro Asp Ala Val Glu Glu Ala
                  115 120 125
          Ile Glu Ala Glu Met Glu Lys Glu Ile Ser Cys Val Asp Ser Ala Glu
              130 135 140
          Leu Gly Lys Ala Ser Arg Ile Val Asp Ala Ala Gly Arg Tyr Ile Glu
          145 150 155 160
          Phe Cys Lys Ala Thr Phe Pro Asn Glu Leu Ser Leu Ser Glu Leu Lys
                          165 170 175
          Ile Val Val Asp Cys Ala Asn Gly Ala Thr Tyr His Ile Ala Pro Asn
                      180 185 190
          Val Leu Arg Glu Leu Gly Ala Asn Val Ile Ala Ile Gly Cys Glu Pro
                  195 200 205
          Asn Gly Val Asn Ile Asn Ala Glu Val Gly Ala Thr Asp Val Arg Ala
              210 215 220
          Leu Gln Ala Arg Val Leu Ala Glu Lys Ala Asp Leu Gly Ile Ala Phe
          225 230 235 240
          Asp Gly Asp Gly Asp Arg Val Ile Met Val Asp His Glu Gly Asn Lys
                          245 250 255
          Val Asp Gly Asp Gln Ile Met Tyr Ile Ile Ala Arg Glu Gly Leu Arg
                      260 265 270
          Gln Gly Gln Leu Arg Gly Gly Ala Val Gly Thr Leu Met Ser Asn Met
                  275 280 285
          Gly Leu Glu Leu Ala Leu Lys Gln Leu Gly Ile Pro Phe Ala Arg Ala
              290 295 300
          Lys Val Gly Asp Arg Tyr Val Leu Glu Lys Met Gln Glu Lys Gly Trp
          305 310 315 320
          Arg Ile Gly Ala Glu Asn Ser Gly His Val Ile Leu Leu Asp Lys Thr
                          325 330 335
          Thr Thr Gly Asp Gly Ile Val Ala Gly Leu Gln Val Leu Ala Ala Met
                      340 345 350
          Ala Arg Asn His Met Ser Leu His Asp Leu Cys Ser Gly Met Lys Met
                  355 360 365
          Phe Pro Gln Ile Leu Val Asn Val Arg Tyr Thr Ala Gly Ser Gly Asp
              370 375 380
          Pro Leu Glu His Glu Ser Val Lys Ala Val Thr Ala Glu Val Glu Ala
          385 390 395 400
          Ala Leu Gly Asn Arg Gly Arg Val Leu Leu Arg Lys Ser Gly Thr Glu
                          405 410 415
          Pro Leu Ile Arg Val Met Val Glu Gly Glu Asp Glu Ala Gln Val Thr
                      420 425 430
          Glu Phe Ala His Arg Ile Ala Asp Ala Val Lys Ala Val
                  435 440 445
          
      

Claims (99)

A metabolically engineered cell that produces a mixture of at least three different mammalian milk oligosaccharides, wherein the cell - metabolically engineered for the production of the mixture, and - Expresses at least two glycosyltransferases, and One or more nucleotide-sugars can be synthesized, wherein the nucleotide-sugar is the donor of the glycosyltransferase. The cell of claim 1, wherein the cell is capable of expressing, preferably at least five glycosyltransferases, preferably at least six, more preferably at least seven glycosyltransferases. The cell of any one of claims 1 or 2, wherein the mixture comprises neutral fucosylated and afucosylated oligosaccharides, neutral fucosylated and afucosylated oligosaccharides The saccharides consist of or consist essentially of neutral fucosylated and afucosylated oligosaccharides and uncharged oligosaccharides. The cell of any one of claims 1 or 2, wherein the mixture comprises, consists of, or consists essentially of neutral fucosylated oligosaccharides Sugar composition and no neutral afucosylated or charged oligosaccharides. The cell of any one of claims 1 or 2, wherein the mixture comprises, consists of, or consists essentially of neutral afucosylated oligosaccharides Sylated oligosaccharide composition and no neutral fucosylated or charged oligosaccharides. The cell of any one of claims 1 or 2, wherein the mixture comprises charged and neutral fucosylated and afucosylated oligosaccharides, charged and neutral fucosylated and non-fucosylated oligosaccharides Fucosylated oligosaccharides consist of or consist essentially of charged and neutral fucosylated and non-fucosylated oligosaccharides, preferably at least one of these charged oligosaccharides is sialylated Oligosaccharides. The cell of any one of claims 1 or 2, wherein the mixture comprises, consists or consists essentially of charged oligosaccharides and is free of neutral oligosaccharides, preferably the charged oligosaccharides At least one of them is a sialylated oligosaccharide. The cell of any one of claims 1 to 7, wherein the cell is modified with a gene expression module, characterized in that the expression from any of the expression modules is persistent or produced by a natural inducer . The cell of any one of claims 1 to 8, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein. The cell of any one of claims 1 to 9, wherein the oligosaccharide mixture comprises at least three different oligosaccharides that differ in degree of polymerization. The cell of any one of claims 1 to 10, wherein the cell produces four or more different mammalian milk oligosaccharides. The cell of any one of claims 1 to 11, wherein any one of the glycosyltransferases is selected from the list comprising: fucosyltransferase, sialyltransferase, transgalactosylase Enzymes, Glucosyltransferase, Mannosyltransferase, N-Acetylglucosaminyltransferase, N-Acetylgalactosaminyltransferase, N-Acetylmannosyltransferase, Xylose Syltransferase, glucuronyltransferase, galacturonyltransferase, glucosaminyltransferase, N-hydroxyacetylneuraminosyltransferase, rhamnosyltransferase, N-acetylmurine Lisosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP-N-acetylglucose Amine 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 selected from the list comprising: α-1,3-N-acetylgalactosaminyltransferase and β-1,3- N-Acetylgalactosaminyltransferase. The cell of any one of claims 1 to 12, wherein the cell is modified in the expression or activity of at least one of the glycosyltransferases. The cell of any one of claims 1 to 13, wherein one of the glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-fucoid Sugar (GDP-Fuc). The cell of any one of claims 1 to 14, wherein one of the glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-N-ethyl Acyl neuraminic acid (CMP-Neu5Ac). The cell of any one of claims 1 to 15, wherein one of the glycosyltransferases is N-acetylglucosaminyltransferase and one of the donor nucleotide-sugars is UDP-N-acetylglucosamine (UDP-GlcNAc). The cell of any one of claims 1 to 16, wherein one of the glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is UDP-galactose ( UDP-Gal). The cell of any one of claims 1 to 17, wherein the glycosyltransferase is N-acetylgalactosaminyltransferase and the donor nucleotide-sugar is UDP-N-acetylgalactose Amine (UDP-GalNAc). The cell of any one of claims 1 to 18, wherein the glycosyltransferase is N-acetylmannosylaminotransferase and the donor nucleotide-sugar is UDP-N-acetylmannose Amine (UDP-ManNAc). The cell of any one of claims 1 to 19, wherein any of the nucleotide-sugars 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-hydroxyacetyl neuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N ₃ , CMP-Neu4,5Ac ₂ , CMP-Neu5,7Ac ₂ , CMP-Neu5,9Ac ₂ , CMP -Neu5,7(8,9)Ac ₂ , UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose, UDP-2-acetamido-2,6 -Dideoxy--L-arabino (arabino)-4-hexylose, UDP-2-acetamido-2,6-dideoxy--L-lyxo (lyxo)-4-hexyl Ketose, 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-talose), UDP- N-Acetylmuramic acid, UDP-N-Acetyl-L-isorhamnosamine (quinovosamine) (UDP-L-QuiNAc or UDP-2-Acetylamino-2,6-dideoxy -L-glucose), GDP-L-isorhamnose. The cell of any one of claims 1 to 20, wherein the cell expresses one or more polypeptides selected from the list comprising: mannose-6-phosphate isomerase, phosphomannose metathesis Enzymes, mannose-1-phosphate guanosyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, GDP-rock Fucose pyrophosphorylase, fucose-1-phosphate guanosyltransferase, L-glutamic acid-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, Phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acetylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epiisomerase Constructing enzyme, N-acetylmannosamine-6-phosphate 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 transfer Enzymes, Glucosamine-1-Phosphate Acetyltransferase, N-Acetylneuraminic Acid Synthase, N-Acetylneuraminic Acid Lysase, N-Acetylneuraminic Acid-9-Phosphate Synthase, N-Acyl neuraminic acid-9-phosphate phosphatase, N-acyl neuraminic acid cytidine syltransferase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1- Uridine Phosphate Transferase, UDP-Glucose 4-Epimerase, Glucose-1-Phosphate Urinyl Transferase, Phosphoglucomutase, UDP-N-Acetyl Glucosamine 4-Epimerase Enzymes, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein the cell is modified in the expression or activity of any of these polypeptides . The cell of any one of claims 1 to 21, wherein the cell is capable of synthesizing at least two nucleotide-sugars, preferably at least three nucleotide-sugars, more preferably at least four nucleotide-sugars, even more Preferably at least five nucleotide-sugars. The cell of any one of claims 1 to 22, wherein at least one of the mammalian milk oligosaccharides is fucosylated, sialylated, galactosylated, glycosylated, xylosylated, Mannosylation; contains N-acetylglucosamine, contains N-acetylneuraminic acid, contains N-hydroxyacetylneuraminic acid, contains N-acetylgalactosamine, contains rhamnose, Contains glucuronide, galacturonate and/or N-acetylmannosamine. The cell of any one of claims 1 to 23, wherein the mammalian milk oligosaccharide mixture comprises at least one fucosylated mammalian milk oligosaccharide. The cell of any one of claims 1 to 24, wherein the mammalian milk oligosaccharide mixture comprises at least one sialylated mammalian milk oligosaccharide. The cell of any one of claims 1 to 25, wherein the mammalian milk oligosaccharide mixture comprises at least one mammalian milk oligosaccharide comprising N-acetylglucosamine monosaccharide units. The cell of any one of claims 1 to 26, wherein the mammalian milk oligosaccharide mixture comprises at least one galactosylated mammalian milk oligosaccharide. The cell of any one of claims 1 to 27, wherein the cell uses at least one precursor for the production of any one or more of the oligosaccharides, preferably the cell uses two or more A precursor for the production of any one or more of the oligosaccharides, the precursor(s) being fed into the cell from the culture medium. The cell of any one of claims 1 to 28, wherein the cell produces at least one precursor for the production of any of the oligosaccharides. The cell of any one of claims 1 to 29, wherein the at least one precursor for producing any of the oligosaccharides is fully converted to any of the oligosaccharides. The cell of any one of claims 1 to 30, wherein the cell produces the oligosaccharides intracellularly, and wherein some or substantially all of the produced oligosaccharides remain intracellularly and/or passively or actively Transport is excreted outside the cell. The cell of any one of claims 1 to 31, wherein the cell is further genetically modified for 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) expression of heterologous membrane proteins, wherein the membrane protein is involved in the secretion of any of the mammalian milk oligosaccharides from the mixture to the outside of the cell, preferably, wherein the membrane protein is involved in the secretion of any of the oligosaccharides from the mixture from the cell all. The cell of any one of claims 1 to 32, wherein the cell is further genetically modified for 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) expression of heterologous membrane proteins, wherein the membrane protein is involved in the absorption of precursors and/or receptors for any of the mammalian milk oligosaccharides used in the synthesis of 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 uptake of all these receptors. The cell of any one of claims 32 or 33, wherein the membrane protein is selected from the list comprising: transporter, P-P-bond hydrolysis driven transporter, β-barrel pore proteins, transport proteins, putative transport proteins and phosphotransfer driven group translocation proteins, Preferably, these transport proteins comprise MFS transport proteins, carbohydrate efflux transport proteins and chelating ferritin export proteins, Preferably, the P-P-bond hydrolysis-driven transport proteins comprise ABC transport proteins and chelatin export proteins. The cell of any one of claims 32 to 34, wherein the membrane protein provides improved production of and/or enables and/or enhances any of the oligosaccharides outflow. The cell of any one of claims 1 to 35, wherein the cell is resistant to lactose killing when grown in an environment where lactose is combined with one or more other carbon sources. The cell of any one of claims 1 to 36, wherein the cell comprises a modification for reducing acetate production compared to an unmodified precursor cell. The cell of claim 37, wherein the cell comprises a reduced or reduced expression and/or abrogated, attenuated, reduced or delayed activity of any one or more of the proteins comprising the following as compared to an unmodified precursor cell : β-Galactosidase, Galactoside O-Acetyltransferase, N-Acetylglucosamine-6-Phosphate Deacetylase, Glucosamine-6-Phosphate Deaminase, N-Acetyl Glucosamine inhibitor, 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-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 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 polyphosphoryl transfer Proteins FruA and FruB, alcohol dehydrogenase, aldehyde dehydrogenase, pyruvate-formate dissociation enzyme, acetate kinase, phosphoacetyltransferase, phosphoacetyltransferase, pyruvate decarboxylase. The cell of any one of claims 1 to 38, wherein the cell is capable of producing phosphoenolpyruvate (phosphoenolpyruvate; PEP). The cell of any one of claims 1 to 39, wherein the cell is modified to enhance the production and/or supply of phosphoenolpyruvate (PEP) compared to an unmodified precursor cell. The cell of any one of claims 1 to 40, wherein any of the mammalian milk oligosaccharides is a human milk oligosaccharide, preferably all of the oligosaccharides are human milk oligosaccharides. A method of producing a mixture of at least three different mammalian milk oligosaccharides by cells, the method comprising the steps of: a. providing a cell, preferably a single cell, expressing at least two glycosyltransferases and capable of synthesizing one or more nucleotide-sugars, wherein the nucleotide-sugar is a donor for the glycosyltransferases, and b. culturing the cell under conditions that allow expression of the glycosyltransferases and synthesis of the nucleotide-sugar such that the cell produces at least three different mammalian milk oligosaccharides, c. Preferably, at least one of the mammalian milk oligosaccharides is isolated from the culture, more preferably, all of the mammalian milk oligosaccharides are isolated from the culture. The method of claim 42, wherein the cell is capable of expressing, preferably at least five glycosyltransferases, preferably at least six, more preferably at least seven glycosyltransferases. The method of any one of claims 42 or 43, wherein the mixture comprises neutral fucosylated and afucosylated oligosaccharides, neutral fucosylated and afucosylated oligosaccharides The saccharides consist of or consist essentially of neutral fucosylated and afucosylated oligosaccharides and uncharged oligosaccharides. The method of any one of claims 42 or 43, wherein the mixture comprises, consists of, or consists essentially of neutral fucosylated oligosaccharides Sugar composition and no neutral afucosylated or charged oligosaccharides. The method of any one of claims 42 or 43, wherein the mixture comprises, consists of, or consists essentially of neutral afucosylated oligosaccharides Sylated oligosaccharide composition and no neutral fucosylated or charged oligosaccharides. The method of any one of claims 42 or 43, wherein the mixture comprises charged and neutral fucosylated and afucosylated oligosaccharides, charged and neutral fucosylated and non-fucosylated oligosaccharides Fucosylated oligosaccharides consist of or consist essentially of charged and neutral fucosylated and non-fucosylated oligosaccharides, preferably at least one of these charged oligosaccharides is sialylated Oligosaccharides. The method of any one of claims 42 or 43, wherein the mixture comprises, consists or consists essentially of charged oligosaccharides and is free of neutral oligosaccharides, preferably the charged oligosaccharides At least one of them is a sialylated oligosaccharide. The method of any one of claims 42 to 48, wherein the cell is a metabolically engineered cell of any one of embodiments 1 to 41. The method of claim 49, wherein the cell is modified with a gene expression module, characterized in that the expression from any of the expression modules is persistent or produced by a natural inducer. The method of any one of claims 49 or 50, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein. The method of any one of claims 42 to 51, wherein the oligosaccharide mixture comprises at least three different oligosaccharides that differ in degree of polymerization. The method of any one of claims 42 to 52, wherein the cell produces four or more different mammalian milk oligosaccharides. The method of any one of claims 42 to 53, wherein any of the glycosyltransferases is selected from the list comprising: fucosyltransferases, sialyltransferases, galactosyltransferases Transferase, Glucosyltransferase, Mannosyltransferase, N-Acetylglucosaminyltransferase, N-Acetylgalactosaminyltransferase, N-Acetylmannosyltransferase, Wood Glycosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosaminyltransferase, N-hydroxyacetylneuramidotransferase, rhamnosyltransferase, N-acetyltransferase Rhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP-N-acetylglucosaminene Alcohol acetonyltransferase and fucosyltransferase, - preferably, the fucosyltransferase is selected from the list comprising the following: 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 selected from the list comprising: α-1,3-N-acetylgalactosaminyltransferase and β-1,3- N-Acetylgalactosaminyltransferase, - Preferably, the cell is modified in the expression or activity of at least one of the glycosyltransferases. The method of any one of claims 42 to 54, wherein one of the glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-fucoid Sugar (GDP-Fuc). The method of any one of claims 42 to 55, wherein one of the glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-N-ethyl Acyl neuraminic acid (CMP-Neu5Ac). The method of any one of claims 42 to 56, wherein one of the glycosyltransferases is N-acetylglucosaminyltransferase and one of the donor nucleotide-sugars is UDP-N-acetylglucosamine (UDP-GlcNAc). The method of any one of claims 42 to 57, wherein one of the glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is UDP-galactose ( UDP-Gal). The method of any one of claims 42 to 58, 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 42 to 59, 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 42 to 60, wherein any of the nucleotide-sugars 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 ), 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-acetamido-2,6-diodes oxy-L-galactose), UDP-N-acetyl-L-neumosamine (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 42 to 61, wherein the cell expresses one or more polypeptides selected from the list comprising: mannose-6-phosphate isomerase, phosphomannose metathesis Enzymes, mannose-1-phosphate guanosyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, GDP-rock Fucose pyrophosphorylase, fucose-1-phosphate guanosyltransferase, L-glutamic acid-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, Phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acetylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epiisomerase Constructing enzyme, N-acetylmannosamine-6-phosphate 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 transfer Enzymes, Glucosamine-1-Phosphate Acetyltransferase, N-Acetylneuraminic Acid Synthase, N-Acetylneuraminic Acid Lysase, N-Acetylneuraminic Acid-9-Phosphate Synthase, N-Acyl neuraminic acid-9-phosphate phosphatase, N-acyl neuraminic acid cytidine syltransferase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1- Uridine Phosphate Transferase, UDP-Glucose 4-Epimerase, Glucose-1-Phosphate Urinyl Transferase, Phosphoglucomutase, UDP-N-Acetyl Glucosamine 4-Epimerase Enzymes, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein the cell is modified in the expression or activity of any of these polypeptides . The method of any one of claims 42 to 62, wherein the cell is capable of synthesizing at least two nucleotide-sugars, preferably at least three nucleotide-sugars, more preferably at least four nucleotide-sugars, even more Preferably at least five nucleotide-sugars. The method of any one of claims 42 to 63, wherein the mammalian milk oligosaccharide mixture comprises at least one fucosylated mammalian milk oligosaccharide. The method of any one of claims 42 to 64, wherein the mammalian milk oligosaccharide mixture comprises at least one sialylated mammalian milk oligosaccharide. The method of any one of claims 42 to 65, wherein the mammalian milk oligosaccharide mixture comprises at least one mammalian milk oligosaccharide comprising N-acetylglucosamine monosaccharide units. The method of any one of claims 42 to 66, wherein the mammalian milk oligosaccharide mixture comprises at least one galactosylated mammalian milk oligosaccharide. The method of any one of claims 42 to 67, wherein the mammalian milk oligosaccharide mixture comprises at least one mammalian milk oligosaccharide that is fucosylated, sialylated, galactose Glycosylation, Glycosylation, Xylosylation, Mannosylation, with N-acetylglucosamine, with N-acetylneuraminic acid, with N-hydroxyacetylneuraminic acid, with N- Acetylgalactosamine, with rhamnose, with glucuronide, with galacturonate, and/or with N-acetylmannosamine. The method of any one of claims 42 to 68, wherein the cell uses at least one precursor for the production of any one or more of the oligosaccharides, preferably the cell uses two or more A precursor for the production of any one or more of the oligosaccharides, which is fed into the cell from the culture medium. The method of any one of claims 42 to 69, wherein the cell produces at least one precursor for producing any of the mammalian milk oligosaccharides. The method of any one of claims 42 to 70, wherein the at least one precursor for producing any of the mammalian milk oligosaccharides is fully converted into any of the mammalian milk oligosaccharides By. The method of any one of claims 42 to 71, wherein the cell produces the oligosaccharides intracellularly, and wherein some or substantially all of the produced oligosaccharides remain intracellular and/or passively or actively Transport is excreted outside the cell. The method of any one of claims 42 to 72, wherein the cell is further genetically modified for 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) expression of heterologous membrane proteins, wherein the membrane protein is involved in the secretion of any of the mammalian milk oligosaccharides from the mixture to the outside of the cell, preferably, wherein the membrane protein is involved in the secretion of any of the oligosaccharides from the mixture from the cell all. The method of any one of claims 42 to 73, wherein the cell is further genetically modified for 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) expression of heterologous membrane proteins, wherein the membrane protein is involved in the absorption of precursors and/or receptors for any of the mammalian milk oligosaccharides used in the synthesis of 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 uptake of all these receptors. The method of any one of claims 73 or 74, wherein the membrane protein is selected from the list comprising transport proteins, P-P-bond hydrolysis-driven transport proteins, β-barrel porins, accessory transport proteins, Putative transport proteins and phosphate transfer driven group translocation proteins, preferably, these transport proteins comprise MFS transport proteins, sugar 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 73 to 75, wherein the membrane protein provides improved production of and/or enables and/or enhances any of the oligosaccharides outflow. The method of any one of claims 42 to 76, 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 42 to 77, wherein the cell comprises a modification for reducing acetate production compared to an unmodified precursor cell. The method of claim 78, wherein the cell comprises a reduced or reduced expression and/or abrogated, attenuated, reduced or delayed activity of any one or more of a protein comprising the following compared to an unmodified precursor cell : β-Galactosidase, Galactoside O-Acetyltransferase, N-Acetylglucosamine-6-Phosphate Deacetylase, Glucosamine-6-Phosphate Deaminase, N-Acetyl Glucosamine inhibitor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecyl isopentenyl-phosphoglucose-1-phosphotransferase, L-fucosokinase, L-fucoid Carbohydrate 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 transferase, glucose-1-phosphate adenosyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase with the same Dnzyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor 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 polyphosphoryl transfer proteins FruA and FruB, ethanol Dehydrogenase, aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacetyltransferase, phosphoacetyltransferase, pyruvate decarboxylase. The method of any one of claims 42 to 79, wherein the cell is capable of producing phosphoenolpyruvate (PEP). The method of any one of claims 42 to 80, wherein the cell is modified to enhance the production and/or supply of phosphoenolpyruvate (PEP) compared to an unmodified precursor cell. The method of any one of claims 42 to 81, wherein any of the mammalian milk oligosaccharides is a human milk oligosaccharide, preferably all of the mammalian milk oligosaccharides are human milk oligosaccharides. The method of any one of claims 42 to 82, wherein the conditions comprise: - using a medium comprising at least one precursor and/or acceptor for the production of any of these oligosaccharides, and/or - adding at least one precursor and/or acceptor feed to the medium for the production of any of the oligosaccharides. The method of any one of claims 42 to 83, comprising at least one of the following steps: i) using a medium comprising at least one precursor and/or acceptor; ii) adding at least one to the medium in the reactor A precursor and/or acceptor feed, wherein the total reactor volume is in the range of 250 mL (milliliters) to 10.000 ^m3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium does not vary. more than three times, preferably no more than two times, more preferably less than two times the volume of the medium before adding the precursor and/or acceptor feed; iii) adding at least one precursor and/or acceptor to the medium in the reactor body feed, wherein the total reactor volume is in the range of 250 mL (milliliters) to 10.000 ^m3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium does not exceed the addition of the precursor and/or or three times, preferably no more than twice, more preferably less than twice the volume of the medium before the acceptor feed, and wherein preferably the pH of the precursor and/or acceptor feed is set between 3 and 7 and wherein preferably, the temperature of the precursor and/or acceptor feed is maintained between 20°C and 80°C; iv) over a time course of 1 day, 2 days, 3 days, 4 days, 5 days Add at least one precursor and/or acceptor feed to the culture medium in a continuous manner by means of the feed solution; v) continuously by means of the feed solution over a time course of 1 day, 2 days, 3 days, 4 days, 5 days adding at least one precursor and/or acceptor feed to the culture medium, and wherein preferably the pH of the feed solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is maintained at between 20°C and 80°C; the method produces a concentration in the final culture of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 100 g/L 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L of any of these oligosaccharides. The method of any one of claims 42 to 83, comprising at least one of the following steps: i) using a method comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 1 per liter of initial reactor volume 120. More preferably at least 150 grams of lactose culture medium, wherein the reactor volume is in the range of 250 mL to 10.000 m ³ (cubic meters); ii) Add to the culture medium a medium containing at least 50, more preferably at least 50 per liter of initial reactor volume 75. Lactose feed of more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose, wherein the reactor volume is in the range of 250 mL to 10.000 ^m3 (cubic meters), preferably added in a continuous manner , and preferably such that the final volume of the medium is no more than three times, preferably no more than twice, more preferably less than two times the volume of the medium before the addition of the lactose feed; iii) the addition of the medium to the medium contains at least one per liter of the initial reactor volume 50. Lactose feed of more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose, wherein the reactor volume is in the range of 250 mL to 10.000 ^m3 (cubic meters), more preferably It is preferably added in a continuous manner, and preferably such that the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium before the addition of the lactose feed, and wherein preferably the lactose The pH of the feed is set between 3 and 7, and wherein preferably the temperature of the lactose feed is maintained between 20°C and 80°C; iv) over 1 day, 2 days, 3 days, 4 days, 5 Lactose feed was added to the medium by the feed solution in a continuous manner over the course of days; v) The medium was added in a continuous manner by the feed solution over the course of 1 day, 2 days, 3 days, 4 days, 5 days Lactose feed, and wherein the lactose feed solution has a concentration of 50 g/L, preferably 75 g/L, more preferably 100 g/L, more preferably 125 g/L, more preferably 150 g/L, more preferably 175 g/L g/L, better 200 g/L, better 225 g/L, better 250 g/L, better 275 g/L, better 300 g/L, better 325 g/L, better 350 g /L, better 375 g/L, better 400 g/L, better 450 g/L, better 500 g/L, even better 550 g/L, best 600 g/L, and better Preferably, the pH of the feed solution is set between 3 and 7, and wherein preferably, the temperature of the feed solution is maintained between 20°C and 80°C; the method yields a concentration of at least 50 g in the final culture /L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L , more preferably at least 200 g/L of any of these oligosaccharides. The method of claim 85, wherein the lactose feed is obtained by starting the culture at a concentration of at least 5 mM, preferably at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, More preferably this is achieved by adding lactose at a concentration of >300 mM. The method of any one of claims 85 or 86, wherein the lactose feed is carried out by feeding into the culture such that a lactose concentration of at least 5 mM, preferably 10 mM or 30 mM is obtained throughout the production phase of the culture The concentration of lactose was added to achieve. The method of any one of claims 42 to 87, wherein the host cells are cultured for at least about 60, 80, 100 or about 120 hours or in a continuous manner. The method of any one of claims 42 to 88, wherein the cells are cultured in a medium comprising a carbon source, the carbon source comprising monosaccharides, disaccharides, oligosaccharides, polysaccharides, polyols, glycerol; In a complex medium of corn infusion, protein gluten, pancreas gluten or yeast extract; preferably, wherein the carbon source is selected from the list comprising the following: glucose, glycerol, fructose, sucrose, maltose, lactose, arabinose, wheat Malto-oligosaccharide, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn infusion, high fruit Syrup, acetate, citrate, lactate and pyruvate. The method of any one of claims 42 to 89, wherein the culture medium contains at least one precursor selected from the group consisting of lactose, galactose, fucose, sialic acid, GlcNAc, GalNAc, milk-N-di Sugar (lacto-N-biose; LNB), N-acetyllactosamine (LacNAc). The method of any one of claims 42 to 90, wherein the first stage of exponential cell growth is carried out by adding a carbon-based substrate, preferably lactose, to the medium prior to adding the precursor, preferably lactose, to the culture medium in the second stage Preferably glucose or sucrose is added to the medium to provide. The method of any one of claims 42 to 91, wherein the first stage of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the medium comprising the precursor, preferably lactose , followed by a second phase in which only carbon-based substrates, preferably glucose or sucrose, are added to the medium. The method of any one of claims 42 to 92, wherein the first stage of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the medium comprising the precursor, preferably lactose , followed by a second stage in which a carbon-based substrate, preferably glucose or sucrose, and a precursor, preferably lactose, are added to the medium. The method of any one of claims 42 to 93, 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 42 to 94, further comprising purifying any of the mammalian milk oligosaccharides from the cell. The method of any one of claim 95, 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 Hydroalcoholic mixture, crystallization, evaporation, precipitation, drying, drying, freeze drying, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum belt drying, vacuum belt Type drying, drum drying, roller drying, vacuum roller drying or vacuum roller drying. The cell of any one of claims 1 to 41 or the method of any one of claims 42 to 96, wherein the cell is a bacterium, fungus, yeast, plant cell, animal cell or protozoal cell, - preferably Ground, the bacterium is Escherichia coli ( Escherichia coli ) strain, more preferably the Escherichia coli strain of K-12 strain, even more preferably the Escherichia coli K-12 strain is Escherichia coli MG1655, - preferably, the fungus belongs to the group selected from a genus comprising the group of Rhizopus , Dictyostelium , Penicillium , Mucor or Aspergillus , - preferably, the The yeast belongs to a genus selected from the group comprising: Saccharomyces , Zygosaccharomyces , Pichia , Komagataella , Hansenula , Yarrowia , Starmerella , Kluyveromyces or Debaromyces , - preferably, the plant cell is an algal cell or is derived from Tobacco, alfalfa, rice, tomato, cotton, rapeseed, soybean, maize or corn plants, - preferably, the animal cell is derived from a non-human mammal, bird, fish, invertebrate, reptile, amphibian or insect , or a genetically modified cell line derived from human cells excluding embryonic stem cells, more preferably the human and non-human mammalian cells are epithelial cells, embryonic kidney cells, fibroblasts, COS cells, Chinese hamster ovary (CHO ) cells, murine myeloma cells, NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, more preferably the insect cells are derived from Spodoptera frugiperda , Bombyx mori , Spodoptera cabbage ( Mamestra brassicae ), Trichoplusia ni or Drosophila melanogaster , - preferably, the protozoal cell is a Leishmania tarentolae cell. The cell of claim 97 or the method of claim 97, wherein the cell is a live Gram-negative bacterium comprising a reduced or eliminated synthesis of poly-N as compared to an unmodified precursor cell -Acetyl-glucosamine (PNAG), Enterobacterial common antigen (ECA), cellulose, colanic acid, core oligosaccharide, osmoregulated periplasmic Glucan (OPG), Glycerol glucosides, polysaccharides and/or trehalose. Use of a cell according to any one of claims 1 to 41, 97, 98 or a method according to any one of claims 42 to 98 for producing a mixture of at least three different mammalian milk oligosaccharides.

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