PRODUCTION OF A SIALYLATED OLIGOSACCHARIDE MIXTURE BY A CELL

Abstract

This disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, this disclosure is in the technical field of cultivation or fermentation of metabolically engineered cells. This disclosure describes a cell metabolically engineered for production of a mixture of at least three different sialylated oligosaccharides. Furthermore, this disclosure provides a method for the production of a mixture of at least three different sialylated oligosaccharides by a cell as well as the purification of at least one of the sialylated oligosaccharides from the cultivation.

Claims

1.-100. (canceled)

101. A metabolically engineered cell that produces a mixture of at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide, wherein the cell is metabolically engineered for the production of the mixture, expresses a glycosyltransferase which is a sialyltransferase, is capable of synthesizing the nucleotide-sugar CMP-N-acetylneuraminic acid (CMP-Neu5Ac), expresses at least one glycosyltransferase in addition to sialyltransferase, and is capable of synthesizing one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for the additional glycosyltransferase.

102. The cell of claim 101, wherein the mixture comprises at least four different sialylated oligosaccharides.

103. The cell of claim 101, wherein an additional glycosyltransferase in addition to sialyltransferase is selected from the group consisting of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases, and fucosaminyltransferases.

104. The cell of claim 101, wherein the cell is modified in the expression or activity of at least one of the glycosyltransferases.

105. The cell of claim 101, wherein the cell uses at least one precursor for the production of any one or more of the sialylated oligosaccharides, the precursor(s) being fed to the cell from a cultivation medium.

106. The cell of claim 101, wherein the cell produces at least one precursor for the production of any one of the sialylated oligosaccharides.

107. The cell of claim 101, wherein the cell is further genetically modified for i) modified expression of an endogenous membrane protein, ii) modified activity of an endogenous membrane protein, iii) expression of a homologous membrane protein, or iv) expression of a heterologous membrane protein, wherein the membrane protein is involved in the secretion of any one of the sialylated oligosaccharides from the mixture outside the cell or (ii) uptake of a precursor or acceptor for the synthesis of any one of the sialylated oligosaccharide of the mixture.

108. The cell of claim 101, wherein the membrane protein provides improved production, enabled efflux, or enhanced efflux of any one of the sialylated oligosaccharides.

109. The cell of claim 101, wherein all of the sialylated oligosaccharides are mammalian milk oligosaccharides.

110. The cell of claim 101, wherein the cell is a bacterium, fungus, yeast, plant cell, animal cell, or protozoan cell.

111. The cell of claim 101, wherein the sialylated oligosaccharides are produced intracellularly.

112. The cell of claim 101, wherein the sialyltransferase is alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase or alpha-2,8-sialyltransferase.

113. The cell of claim 101, wherein the sialyltransferase is from an organism selected from the group consisting of Pasteurella species, Photobacterium species, Porphyromonas species, Streptococcus species, Neisseria meningitidis, Campylobacter jejuni, Haemophilus species, Vibrio species, Alistipes species, Actinobacillus species, Homo sapiens, and Mus musculus.

114. The cell of claim 101, wherein the relative abundance of the sialylated oligosaccharides in the mixture is at least 10%.

115. The cell of claim 101, wherein the mixture comprises at least three different sialylated mammalian milk oligosaccharides.

116. The cell of claim 101, wherein all oligosaccharides in the mixture are sialylated.

117. The cell of claim 101, wherein the cell expresses at least two additional glycosyltransferases.

118. The cell of claim 106, wherein the precursor is: lactose for the production of a lactose-based oligosaccharide, lacto-N-biose (LNB) for the production of a LNB-based oligosaccharide, and/or N-acetyllactosamine (LacNAc) for the production of a LacNAc-based oligosaccharide.

119. A method of producing a mixture of at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide, the method comprising: cultivating the cell of claim 101 so as to produce the mixture.

120. A method of producing a mixture of at least three different sialylated oligosaccharides by a cell, wherein the mixture comprises more than one mammalian milk oligosaccharide, the method comprising: i) providing a cell (a) expressing a glycosyltransferase which is a sialyltransferase and capable of synthesizing the nucleotide-sugar CMP-Neu5Ac, (b) expressing at least one additional glycosyltransferase, and (c) capable of synthesizing at least one nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for the additional glycosyltransferases, and ii) cultivating the cell under conditions permissive to express the glycosyltransferases and to synthesize the nucleotide-sugars, resulting in the cell producing the mixture of at least three different sialylated oligosaccharides.

121. The method according to claim 120, wherein the cell is a metabolically engineered cell that produces a mixture of at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide, wherein the cell is metabolically engineered for the production of the mixture, expresses a glycosyltransferase being a sialyltransferase, is capable to synthesize the nucleotide-sugar CMP-N-acetylneuraminic acid (CMP-Neu5Ac), expresses at least one additional glycosyltransferase, and is capable of synthesizing one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for the additional glycosyltransferase.

122. The method according to claim 120, wherein the mixture comprises at least four different sialylated oligosaccharides.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0163] The disclosure will be described in more detail in the examples and the attached figures, in which:

[0164] FIG. 1 shows the chromatogram plot obtained of a whole broth sample taken from the LSTc producing E. coli strain S2 (Table 8) expressing the ?-2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 with SEQ ID NO: 25 and analyzed via the Dionex method for presence of oligosaccharides, as described in Example 1. Peaks indicated represent following glycans (with 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, 6SL (31.984 min).

[0165] FIG. 2 shows the chromatogram plot obtained of a whole broth sample taken from an LSTd producing E. coli strain S5 (Table 9) expressing the ?-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22 and analyzed via the Dionex method for presence of oligosaccharides, as described in Example 1. Peaks indicated represent following glycans (with 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, 3SL (33.050 min).

DETAILED DESCRIPTION

[0166] According to a first aspect, this disclosure provides a metabolically engineered cell for the production of a mixture comprising at least three different sialylated oligosaccharides wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different mammalian milk oligosaccharides, i.e., a cell that is metabolically engineered for the production of a mixture comprising at least three different sialylated oligosaccharides wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different mammalian milk oligosaccharides. Herein, a single metabolically engineered cell is provided that is capable to express, preferably expresses, a glycosyltransferase being a sialyltransferase and is capable of synthesizing the nucleotide-sugar CMP-N-acetylneuraminic acid (CMP-Neu5Ac), and that expresses at least one additional glycosyltransferase and is capable of synthesizing one or more sugar-nucleotide(s) that is/are donor(s) for the additional glycosyltransferase. Throughout the disclosure, unless explicitly stated otherwise, a genetically modified cell or metabolically engineered cell preferably means a cell that is genetically modified or metabolically engineered, respectively, for the production of the mixture comprising at least three different sialylated oligosaccharides according to the disclosure. In the context of the disclosure, the at least three different oligosaccharides of the mixture as disclosed herein preferably do not occur in the wild type progenitor of the metabolically engineered cell.

[0167] According to second aspect, this disclosure provides a method for the production of a mixture comprising at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different mammalian milk oligosaccharides. The method comprises the steps of: [0168] providing a cell, preferably a single cell, that is capable to express, preferably expresses, a glycosyltransferase being a sialyltransferase and is capable of synthesizing the nucleotide-sugar CMP-N-acetylneuraminic acid (CMP-Neu5Ac), and that expresses at least one additional glycosyltransferase and is capable of synthesizing one or more sugar-nucleotide(s) that is/are donor(s) for the additional glycosyltransferase, and [0169] cultivating the cell under conditions permissive to express the glycosyltransferases and to synthesize the nucleotide-sugar(s), resulting in the cell producing the mixture of at least three different sialylated oligosaccharides, [0170] preferably, separating at least one of the oligosaccharides from the cultivation, more preferably separating all of the oligosaccharides from the cultivation.

[0171] The sialyltransferase used in this disclosure can be any of the sialyltransferases as defined herein.

[0172] In the scope of this disclosure, permissive conditions are understood to be conditions relating to physical or chemical parameters including but not limited to temperature, pH, pressure, osmotic pressure and product/precursor/acceptor concentration.

[0173] In a particular embodiment, the permissive conditions may include a temperature-range of 30+/?20 degrees centigrade, a pH-range of 7+/?3.

[0174] In the context of the disclosure, it should be understood that the cell produces the oligosaccharides intracellularly. The skilled person will further understand that a fraction or substantially all of the produced oligosaccharides remains intracellularly and/or is excreted outside the cell via passive or active transport.

[0175] According to the disclosure, the method for the production of a mixture comprising at least three different sialylated oligosaccharides wherein the mixture comprises more than one mammalian milk oligosaccharide can make use of a non-metabolically engineered cell or can make use of a metabolically engineered cell, i.e., a cell that is metabolically engineered for the production of the mixture comprising at least three different sialylated oligosaccharides.

[0176] According to a preferred embodiment of the method and cell according to the disclosure, the metabolically engineered cell is modified with gene expression modules wherein the expression from any one of the expression modules is constitutive or is created by a natural inducer.

[0177] The expression modules are also known as transcriptional units and comprise polynucleotides for expression of recombinant genes including coding gene sequences and appropriate transcriptional and/or translational control signals that are operably linked to the coding genes. The control signals comprise promoter sequences, untranslated regions, ribosome binding sites, terminator sequences. The expression modules can contain elements for expression of one single recombinant gene but can also contain elements for expression of more recombinant genes or can be organized in an operon structure for integrated expression of two or more recombinant genes. The polynucleotides may be produced by recombinant DNA technology using techniques well-known in the art. Methods that are well known to those skilled in the art to construct expression modules include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989 and yearly updates).

[0178] According to a preferred embodiment of this disclosure, the cell is modified with one or more expression modules. The expression modules can be integrated in the genome of the cell or can be presented to the cell on a vector. The vector can be present in the form of a plasmid, cosmid, phage, liposome, or virus, which is to be stably transformed/transfected into the metabolically engineered cell. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. These vectors may contain selection markers such as but not limited to antibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNA sense/antisense markers. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., see above. For recombinant production, cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the disclosure. Introduction of a polynucleotide into the cell can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986), and Sambrook et al., 1989, supra.

[0179] As used herein, an expression module comprises polynucleotides for expression of at least one recombinant gene. The recombinant gene is involved in the expression of a polypeptide acting in the synthesis of the oligosaccharide mixture; or the recombinant gene is linked to other pathways in the host cell that are not involved in the synthesis of the mixture of three or more oligosaccharides. The recombinant genes encode endogenous proteins with a modified expression or activity, preferably the endogenous proteins are overexpressed; or the recombinant genes encode heterologous proteins that are heterogeneously introduced and expressed in the modified cell, preferably overexpressed. The endogenous proteins can have a modified expression in the cell that also expresses a heterologous protein.

[0180] According to a preferred embodiment of this disclosure, the expression of each of the expression modules is constitutive or created by a natural inducer. As used herein, constitutive expression should be understood as expression of a gene that is transcribed continuously in an organism. Expression that is created by a natural inducer should be understood as a facultative or regulatory expression of a gene that is only expressed upon a certain natural condition of the host (e.g., organism being in labor, or during lactation), as a response to an environmental change (e.g., including but not limited to hormone, heat, cold, light, oxidative or osmotic stress/signaling), or dependent on the position of the developmental stage or the cell cycle of the host cell including but not limited to apoptosis and autophagy.

[0181] This disclosure provides different types of cells for the production of an oligosaccharide mixture comprising three or more sialylated oligosaccharides wherein the mixture comprises more than one mammalian milk oligosaccharide with a single metabolically engineered cell. For example, this disclosure provides a cell wherein the cell expresses two different glycosyltransferases and the cell synthesizes one single nucleotide-sugar that is donor for both the expressed glycosyltransferases. This disclosure also provides a cell wherein the cell expresses three different glycosyltransferases and the cell synthesizes one single nucleotide-sugar that is donor for all of the three expressed glycosyltransferases. This disclosure also provides a cell wherein the cell expresses two different glycosyltransferases and the cell synthesizes two different nucleotide-sugars whereby a first nucleotide-sugar is donor for the first glycosyltransferase and a second nucleotide-sugar is donor for the second glycosyltransferase. This disclosure also provides a cell wherein the cell expresses three or more glycosyltransferases and the cell synthesizes one or more different nucleotide-sugar(s) that is/are donor(s) for all of the expressed glycosyltransferases.

[0182] In the method and cell described herein, the cell preferably comprises multiple copies of the same coding DNA sequence encoding for one protein. In the context of this disclosure, the protein can be a glycosyltransferase, a membrane protein or any other protein as disclosed herein. Throughout the disclosure, the feature multiple means at least 2, preferably at least 3, more preferably at least 4, even more preferably at least 5.

[0183] In an embodiment of the method and/or cell according to the disclosure, 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 sialylated oligosaccharides.

[0184] In an additional embodiment of the method and/or cell according to the disclosure, the mixture comprises more than one mammalian milk oligosaccharide.

[0185] In another embodiment of the method and/or cell according to the disclosure, at least one of the sialylated oligosaccharides in the mixture is a mammalian milk oligosaccharide (MMO), preferably lactose-based mammalian milk oligosaccharide, more preferably human milk oligosaccharide (HMO). In a preferred embodiment, the cell produces more than two mammalian milk oligosaccharides in the produced mixture of at least three different sialylated oligosaccharides. In an even more preferred embodiment, all the oligosaccharides in the produced mixture of at least three different sialylated oligosaccharides are mammalian milk oligosaccharides.

[0186] In another embodiment of the method and/or cell of the disclosure, at least one of the oligosaccharides in the mixture is an antigen of the human ABO blood group system. In an embodiment, the cell produces one antigen of the human ABO blood group system in the produced mixture of at least three different sialylated oligosaccharides. In a preferred embodiment, the cell produces more than one antigen of the human ABO blood group system in the produced mixture of at least three different sialylated oligosaccharides. In a more preferred embodiment of the method and/or cell according to the disclosure, the mixture comprises at least three different antigens of the human ABO blood group system.

[0187] In the context of the disclosure, the mixture of at least three different sialylated oligosaccharides according to the disclosure can further comprise neutral oligosaccharides such as neutral fucosylated oligosaccharides and neutral non-fucosylated oligosaccharides as described herein. Neutral oligosaccharides are non-sialylated oligosaccharides, and thus do not contain an acidic monosaccharide subunit. Neutral oligosaccharides comprise non-charged fucosylated oligosaccharides that contain one or more fucose subunits in their glycan structure as well as non-charged non-fucosylated oligosaccharides that lack any fucose subunit. Such neutral oligosaccharides can be, for example, lactose-based oligosaccharides, LNB-based oligosaccharides, LacNAc-based oligosaccharides, GalNAc-Glc-based oligosaccharides and/or GalNAc-GlcNAc-based oligosaccharides as described herein.

[0188] In a preferred embodiment of the method and/or cell according to the disclosure, the mixture comprises at least three different sialylated oligosaccharides as disclosed herein and optionally at least one, preferably at least two, more preferably at least three antigens of the human ABO blood group system. In another preferred embodiment of the method and/or cell according to the disclosure, the mixture comprises at least three different charged oligosaccharides as disclosed herein and optionally at least one, preferably at least two, more preferably at least three, even more preferably at least four, different LNB-based oligosaccharides (the LNB-based oligosaccharides are neutral and/or charged, preferably charged, more preferably sialylated), and optionally at least one, preferably at least two, more preferably at least three, even more preferably at least four, different LacNAc-based oligosaccharides (the LacNAc-based oligosaccharides are neutral and/or charged, preferably charged, more preferably sialylated).

[0189] In another more preferred embodiment of the method and/or cell according to the disclosure, 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 sialylated mammalian milk oligosaccharides (MMOs), preferably lactose-based mammalian milk oligosaccharides, more preferably human milk oligosaccharides (HMOs). Throughout the disclosure, unless explicitly stated otherwise, the feature mixture comprising at least three different sialylated oligosaccharides is preferably replaced with mixture comprising at least three different sialylated MMOs, preferably lactose-based MMOs, more preferably HMOs, likewise it is preferred to replace mixture comprising at least four different sialylated oligosaccharides with mixture comprising at least four different sialylated MMOs, preferably lactose-based MMOs, more preferably HMOs etc. In the context of the disclosure, the mixture of at least three different sialylated mammalian milk oligosaccharides according to a preferred embodiment of the disclosure can comprise further oligosaccharides such as mammalian milk oligosaccharides and/or non-mammalian milk oligosaccharides. The further oligosaccharides can be neutral or charged (preferably sialylated) oligosaccharides. Charged oligosaccharides are oligosaccharide structures that contain one or more negatively charged monosaccharide subunits including N-acetylneuraminic acid (Neu5Ac), commonly known as sialic acid, N-glycolylneuraminic acid (Neu5Gc), glucuronate and galacturonate. Charged oligosaccharides are also referred to as acidic oligosaccharides. Throughout the disclosure, the charged oligosaccharides are preferably sialylated oligosaccharides. Throughout the disclosure, the charged oligosaccharides are more preferably sialylated oligosaccharides that are not sialylated ganglioside oligosaccharides except for GM3 (i.e., 3sialyllactose). Throughout the disclosure, the charged oligosaccharides are even more preferably sialylated oligosaccharides that are not sialylated ganglioside oligosaccharides. Sialic acid belongs to the family of derivatives of neuraminic acid (5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid). Neu5Gc is a derivative of sialic acid, which is formed by hydroxylation of the N-acetyl group at C5 of Neu5Ac. The further oligosaccharides can be, for example, lactose-based oligosaccharides, LNB-based oligosaccharides and/or LacNAc-based oligosaccharides as described herein. In a preferred embodiment of the method and/or cell according to the disclosure, the mixture comprises at least three different sialylated MMOs as disclosed herein and optionally at least one, preferably at least two, more preferably at least three antigens of the human ABO blood group system. In another preferred embodiment of the method and/or cell according to the disclosure, the mixture comprises at least three different charged MMOs as disclosed herein and optionally at least one, preferably at least two, more preferably at least three, even more preferably at least four, different LNB-based oligosaccharides (the LNB-based oligosaccharides are neutral and/or charged, preferably charged, more preferably sialylated), and optionally at least one, preferably at least two, more preferably at least three, even more preferably at least four, different LacNAc-based oligosaccharides (the LacNAc-based oligosaccharides are neutral and/or charged, preferably charged, more preferably sialylated). In an alternative and/or additional preferred embodiment of the method and/or cell according to the disclosure, mammalian milk oligosaccharides constitute at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, of the oligosaccharide mixture according to the disclosure. In a more preferred embodiment of the method and/or cell according to the disclosure, all the oligosaccharides in the mixture are MMOs, preferably lactose-based MMOs, more preferably HMOs. As already stated herein, it is preferred that the mixture as disclosed herein is the direct result of metabolically engineering a cell as described herein.

[0190] Throughout the disclosure, unless explicitly stated otherwise, the feature at least one is preferably replaced with one, likewise the feature at least two is preferably replaced with two, etc.

[0191] In an optional embodiment of the method and/or cell according to the disclosure, the mixture according to the disclosure further comprises LacdiNAc (i.e., GalNAc-b1,4-GlCNAc) and/or GalNAc-b1,4-glucose.

[0192] In an additional and/or alternative embodiment of the method and/or cell according to the disclosure, the oligosaccharide mixture comprises at least three different sialylated oligosaccharides differing 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, the degree of polymerization of an oligosaccharide is three (DP3) or more, the latter comprising any one of 4 (DP4), 5 (DP5), 6 (DP6) or longer. The oligosaccharide mixture as described herein preferably comprises at least three different sialylated oligosaccharides wherein all oligosaccharides present in the mixture have a different degree of polymerization from each other. For example, the oligosaccharide mixture comprises three sialylated oligosaccharides, wherein the first oligosaccharide is a trisaccharide with a degree of polymerization of 3 (DP3), the second oligosaccharide is a tetrasaccharide with a degree of polymerization of 4 (DP4) and the third oligosaccharide is a pentasaccharide with a degree of polymerization of 5 (DP5).

[0193] In an embodiment of the method and/or cell according to the disclosure, the oligosaccharide mixture is composed of at least one neutral oligosaccharide in addition to three or more sialylated oligosaccharides.

[0194] According to one aspect of the method and/or cell of the disclosure, the cell produces a mixture comprising four different sialylated oligosaccharides or more than four different sialylated oligosaccharides. In one embodiment, such mixture comprises at least four different oligosaccharides wherein three of the oligosaccharides have a different degree of polymerization. In one embodiment, all of the oligosaccharides in the mixture have a different degree of polymerization as described herein.

[0195] According to the method and/or cell of the disclosure, at least one of the oligosaccharides of the mixture is fucosylated, sialylated, galactosylated, glucosylated, xylosylated, mannosylated, contains an N-acetylglucosamine, contains an N-acetylneuraminate, contains an N-glycolylneuraminate, contains an N-acetylgalactosamine, contains a rhamnose, contains a glucuronate, contains a galacturonate, and/or contains an N-acetylmannosamine.

[0196] According to the method and/or cell of the disclosure, at least one of the sialylated oligosaccharides of the mixture is fucosylated, sialylated, galactosylated, glucosylated, xylosylated, mannosylated, contains an N-acetylglucosamine, contains an N-acetylneuraminate, contains an N-glycolylneuraminate, contains an N-acetylgalactosamine, contains a rhamnose, contains a glucuronate, contains a galacturonate, and/or contains an N-acetylmannosamine.

[0197] Preferably, the oligosaccharide mixture comprises at least one fucosylated oligosaccharide as defined herein.

[0198] Alternatively or additionally, the mixture of oligosaccharides comprises at least one oligosaccharide of 3 or more monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of the monosaccharide residues is an N-acetylglucosamine (GlcNAc) residue. The oligosaccharide can contain more than one GlcNAc residue, e.g., two, three or more. The oligosaccharide can be a neutral oligosaccharide or a charged oligosaccharide, e.g., 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 comprising galactose, fucose, Neu5Ac, Neu5Gc.

[0199] Alternatively or additionally, the oligosaccharide mixture comprises at least one galactosylated oligosaccharide and contains at least one galactose monosaccharide subunit. The galactosylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of the monosaccharide subunit is a galactose. The galactosylated oligosaccharide can contain more than one galactose residue, e.g., two, three or more. The galactosylated oligosaccharide can be a neutral oligosaccharide or a charged oligosaccharide, e.g., also comprising sialic acid structures. Galactose can be linked to other monosaccharide subunits comprising glucose, GlcNAc, fucose, sialic acid.

[0200] In an additional and/or alternative preferred embodiment of the method and/or cell according to the disclosure, the oligosaccharide mixture according to the disclosure comprises sialylated oligosaccharides with a relative abundance in the mixture of at least 10%, preferably at least 15%, more preferably at last 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, most preferably all oligosaccharides in the mixture of the disclosure are sialylated. The skilled person will understand that if the relative abundance of the sialylated oligosaccharides in the mixture is defined, inevitably the remainder fraction of oligosaccharides in the mixture are neutral oligosaccharides. Throughout the disclosure, unless otherwise stated, the features oligosaccharide and oligosaccharides are preferably replaced with MMO and MMOs, respectively, more preferably replaced with lactose-based MMO and lactose-based MMO's, respectively, even more preferably replaced with HMO and HMOs, respectively.

[0201] In an additional and/or alternative embodiment of the method and/or cell according to the disclosure, the oligosaccharide mixture as described herein further comprises neutral oligosaccharides, wherein the relative abundance of the neutral oligosaccharides in the mixture is preferably less than 90%, more preferably less than 80%, even more preferably less than 70%, even more preferably less than 60%, even more preferably less than 50%, even more preferably less than 40%, even more preferably less than 30%, even more preferably less than 20%, even more preferably less than 10%, most preferably all oligosaccharides in the mixture of the disclosure are charged (preferably sialylated) oligosaccharides.

[0202] As such, in an additional and/or alternative embodiment of the method and/or cell according to the disclosure, the oligosaccharide mixture as described herein is composed of charged (preferably sialylated) and neutral oligosaccharides, wherein the relative abundance of the charged (preferably sialylated) oligosaccharides in the mixture is preferably 5-20%, preferably 5-15%, more preferably 10-15%, even more preferably 12-14%, most preferably reflecting the relative abundance of charged oligosaccharides in human breast milk and/or colostrum.

[0203] In an additional and/or alternative embodiment of the method and/or cell according to the disclosure, the oligosaccharide mixture as described herein comprises fucosylated oligosaccharide(s) with a relative abundance in the mixture of at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, most preferably at least 55%. Preferably, the relative abundance of the fucosylated oligosaccharides in the mixture is less than 90%, preferably less than 80%, more preferably less than 70%, even more preferably less than 60%. As such, the relative abundance of the fucosylated oligosaccharides in the mixture is preferably 10-90%, preferably 20-80%, more preferably 30-60%, even more preferably 40-55%, most preferably reflecting the relative abundance of fucosylated oligosaccharides in human breast milk and/or colostrum.

[0204] In an additional and/or alternative embodiment of the method and/or cell according to the disclosure, the oligosaccharide mixture as described herein further comprises neutral oligosaccharides selected from neutral fucosylated oligosaccharides and/or neutral non-fucosylated oligosaccharides. In a preferred embodiment of the method and/or cell according to the disclosure, the neutral oligosaccharides do not comprise non-fucosylated oligosaccharides. In an alternative preferred embodiment, the neutral oligosaccharides do not comprise fucosylated oligosaccharides. In a more preferred embodiment, the neutral oligosaccharides comprise fucosylated oligosaccharide(s) and non-fucosylated oligosaccharide(s). In an even more preferred embodiment, the relative abundance of fucosylated oligosaccharides in the neutral oligosaccharides fraction 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 oligosaccharides in the neutral oligosaccharides fraction of the mixture is 10-60%, preferably 20-60%, more preferably 30-60%, even more preferably 30-50%, most preferably reflecting the relative abundance of fucosylated oligosaccharides in the neutral oligosaccharides fraction in human breast milk and/or colostrum.

[0205] In an additional and/or alternative embodiment of the method and/or cell according to the disclosure, the relative abundance of each oligosaccharide in the mixture as described herein is at least 5%, preferably at least 10%.

[0206] In the context of this disclosure, the oligosaccharide mixture as disclosed herein is preferably the direct result of metabolically engineering a cell as described herein. This means that preferably at least one, more preferably at least two, even more preferably at least three, most preferably all, of the oligosaccharides in the mixture according to the disclosure are not produced by the wild type progenitor of the metabolically engineered cell.

[0207] The names of the oligosaccharides as described herein are in accordance with the oligosaccharide names and formulae as published by Urashima et al. (Trends in Glycoscience and Glycotechnology, 2018, vol 30, no 72, pag SE51-SE65) and references therein and as published in Prebiotics and Probiotics in human milk. Origins and Functions of Milk-Borne Oligosaccharides and Bacteria, Chapters 2 & 3, Eds M. McGuire, M. McGuire, L. Bode, Elsevier, Academic Press, pag 506).

[0208] In a more preferred embodiment of the method and/or cell according to the disclosure, the mixture comprises, consists essentially of or consists of at least three, 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 sialylated oligosaccharides preferably selected from: [0209] lactose-based sialylated oligosaccharides, preferably any one of 3-sialyllactose, 6-sialyllactose, 3,6-disialyllactose, 6,6-disialyllactose, 8,3-disialyllactose, 3'S-2FL, 6'S-2FL, 6'S-3-FL, pentasaccharide LSTD (Neu5Ac?-2,3Gal?-1,4GlcNAc?-1,3Gal?-1,4Glc), sialylated lacto-N-triose, sialylated lacto-N-tetraose comprising LSTa and LSTb, sialyllacto-N-neotetraose comprising LSTc and LSTd, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a, disialyllacto-N-hexaose I, sialyllacto-N-tetraose b, 3-sialyl-3-fucosyllactose (3'S-3-FL), disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose, FS Gal-LNnH (Gal-a1,3-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), DFSGal-LNnH (Gal-a1,3-[Fuc?1,2]-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), FS-LNnH (Fuc?1,2-Gal-b1,4-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), MSDF-para-LNnH (Neu5Ac?2,3-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3-Gal-b1,4-Glc), GD3 (Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,4Glc), GT3 (Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,4Glc); GM2 GalNAc?-1,4(Neu5Ac?-2,3)Gal?-1,4Glc, GM1 (Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,3)Gal?-1,4Glc), GD1a (Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,3)Gal?-1,4Glc), GT1a (Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,3)Gal?-1,4Glc), GD2 (GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GT2 (GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GD1b, (Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GT1b (Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GQ1b (Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,3GalNAc ?-1,4(Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GT1c (Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GQ1c (Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GP1c (Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GD1a (Neu5Ac?-2,3Gal?-1,3(Neu5Ac?-2,6)GalNAc?-1,4Gal?-1,4Glc), Fucosyl-GM1 (Fuc?-1,2Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,3)Gal ?-1,4Glc), Neu5Ac?2,3-Galb1,3-Gal-b1,4-Glc, Galb1,3-[Neu5Ac?2,6]-Gal-b1,4-Glc, Neu5Gca2,8-Neu5 Ac?2,3-Gal-b1,4-Glc, Neu5Ac?2,8-Neu5Gca2,3-Gal-b1,4-Glc, Neu5Ac?2,8-Neu5Ac?2,3-Gal-b1,4-Glc, Neu5Gca2,8-Neu5Gca2,3-Gal-b1,4-Glc, Neu5Ac?2,3-Gal-b1,3-[Neu5Ac?2,6]-Gal-b1,4-Glc, Galb1,6-[Neu5Ac?2,3]-Gal-b1,4-Glc, Gal-b1,3-[Neu5Ac?2,6]-Gal-b1,4-Glc, Neu5Gca2,3-Gal-b1,3-Gal-b1,4-Glc, Neu5Gca2,3-Gal-b1,3-[Neu5Ac?2,6]-Gal-b1,4-Glc, Galb1,4-GlcNAc-b16-[Neu5Ac2,3-Gal-b1,3]-Gal-b1,4-Glc, Neu5Ac2,6-Gal-b1,4-GlcNAc-b1,6-[Galb1,3]-Gal-b1,4-Glc, Neu5Gca2,3-Gal-b1,4-Glc, Neu5Gca2,6-Gal-b1,4-Glc, GalMSLNnH (Gal?1,3-Gal-b1,4-GlcNAc-b1,6-[Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), F-LSTa, F-LSTb, F-LSTc, 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, DF-para-LNH sulfate I, DF-para-LNH sulfate II, TF-para-LNH sulfate, Neu5GcLNnT, GM2 tetrasaccharide, SLNOa, S-LNH I, S-LNH II, S-LNnH I, S-LNnH II, S-para-LNnH, DS-LNH II, 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, FDS-LNT I, FDS-LNT II, FDS-LNH I, FDS-LNH II, FDS-LNH III, FDS-LNnH, TS-LNH, SLNnD, FS-novo-LNP I, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, Gal-b1,3-[Neu5Gc-a2,6]-Gal-b1,4-Glc, more preferably any one of 3-sialyllactose, 6-sialyllactose, 3,6-disialyllactose, 6,6-disialyllactose, 8,3-disialyllactose, 3'S-2FL, 6'S-2FL, 6'S-3-FL, pentasaccharide LSTD (Neu5Ac?-2,3Gal?-1,4GlcNAc?-1,3Gal?-1,4Glc), sialylated lacto-N-triose, sialylated lacto-N-tetraose comprising LSTa and LSTb, sialyllacto-N-neotetraose comprising LSTc and LSTd, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a, disialyllacto-N-hexaose I, sialyllacto-N-tetraose b, 3-sialyl-3-fucosyllactose (3'S-3-FL), disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose, FS Gal-LNnH (Gal-a1,3-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), DFSGal-LNnH (Gal-a1,3-[Fuc?1,2]-Gal-b1,4-[Fuca 1,3]-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), FS-LNnH (Fuc?1,2-Gal-b1,4-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), MSDF-para-LNnH (Neu5Ac?2,3-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3-Gal-b1,4-Glc), most preferably any one of 3-sialyllactose, 6-sialyllactose, 3,6-disialyllactose, 6,6-disialyllactose, 8,3-disialyllactose, 3'S-2FL, 6'S-2FL, 6'S-3-FL, pentasaccharide LSTD (Neu5Ac?-2,3Gal?-1,4GlcNAc?-1,3Gal?-1,4Glc), sialylated lacto-N-triose, sialylated lacto-N-tetraose, sialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a, disialyllacto-N-hexaose I, sialyllacto-N-tetraose b, 3-sialyl-3-fucosyllactose (3'S-3-FL), disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose; and/or [0210] LNB-based sialylated oligosaccharides, preferably any one of 3-sialyllacto-N-biose (3SLNB), 6-sialyllacto-N-biose (6SLNB), monofucosylmonosialyllacto-N-octaose (sialyl Lea); and/or [0211] LacNAc-based sialylated oligosaccharides, preferably any one of 3-sialyllactosamine (3SLacNAc), 6-sialyllactosamine (6SLacNAc), sialyl Lex, Neu5Gc-a2,3-Gal-b1,4-GlcNAc.

[0212] Preferred mixtures in this context of the disclosure comprise mixtures comprising, consisting essentially of or consisting of at least three, 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 sialylated oligosaccharides chosen from the list comprising 3-sialyllactose, 6-sialyllactose, 3,6-disialyllactose, 6,6-disialyllactose, 8,3-disialyllactose, 3'S-2FL, 6'S-2FL, 6'S-3-FL, pentasaccharide LSTD (Neu5Ac?-2,3Gal?-1,4GlcNAc?-1,3Gal?-1,4Glc), sialylated lacto-N-triose, sialylated lacto-N-tetraose comprising LSTa and LSTb, sialyllacto-N-neotetraose comprising LSTc and LSTd, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a, disialyllacto-N-hexaose I, sialyllacto-N-tetraose b, 3-sialyl-3-fucosyllactose (3'S-3-FL), disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose, FS Gal-LNnH (Gal-a1,3-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), DFSGal-LNnH (Gal-a1,3-[Fuc?1,2]-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), FS-LNnH (Fuc?1,2-Gal-b1,4-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), MSDF-para-LNnH (Neu5Ac?2,3-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3-Gal-b1,4-Glc), GD3 (Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,4Glc), GT3 (Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,4Glc); GM2 GalNAc?-1,4(Neu5Ac?-2,3)Gal?-1,4Glc, GM1 (Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,3)Gal?-1,4Glc), GD1a (Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,3)Gal?-1,4Glc), GT1a (Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,3)Gal?-1,4Glc), GD2 (GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GT2 (GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GD1b, (Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GT1b (Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GQ1b (Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,3GalNAc ?-1,4(Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GT1c (Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GQ1c (Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GP1c (Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GD1a (Neu5Ac?-2,3Gal?-1,3(Neu5Ac?-2,6)GalNAc?-1,4Gal?-1,4Glc), Fucosyl-GM1 (Fuc?-1,2Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,3)Gal ?-1,4Glc), Neu5Ac?2,3-Galb 1,3-Gal-b1,4-Glc, Galb 1,3-[Neu5Ac?2,6]-Gal-b1,4-Glc, Neu5Gca2,8-Neu5Ac?2,3-Gal-b1,4-Glc, Neu5Ac?2,8-Neu5Gca2,3-Gal-b1,4-Glc, Neu5Ac?2,8-Neu5Ac?2,3-Gal-b1,4-Glc, Neu5Gca2,8-Neu5Gca2,3-Gal-b1,4-Glc, Neu5Ac?2,3-Gal-b1,3-[Neu5Ac?2,6]-Gal-b1,4-Glc, Galb 1,6-[Neu5Ac?2,3]-Gal-b1,4-Glc, Gal-b1,3-[Neu5 Ac?2,6]-Gal-b1,4-Glc, Neu5Gca2,3-Gal-b1,3-Gal-b1,4-Glc, Neu5Gca2,3-Gal-b1,3-[Neu5Ac?2,6]-Gal-b1,4-Glc, Galb 1,4-GlcNAc-b16-[Neu5Ac2,3-Gal-b1,3]-Gal-b1,4-Glc, Neu5Ac2,6-Gal-b1,4-GlcNAc-b1,6-[Galb 1,3]-Gal-b1,4-Glc, Neu5Gca2,3-Gal-b1,4-Glc, Neu5Gca2,6-Gal-b1,4-Glc, GalMSLNnH (Gal?1,3-Gal-b1,4-GlcNAc-b1,6-[Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), F-LSTa, F-LSTb, F-LSTc, 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, DF-para-LNH sulfate I, DF-para-LNH sulfate II, TF-para-LNH sulfate, Neu5GcLNnT, GM2 tetrasaccharide, SLNOa, S-LNH I, S-LNH II, S-LNnH I, S-LNnH II, S-para-LNnH, DS-LNH II, 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, FDS-LNT I, FDS-LNT II, FDS-LNH I, FDS-LNH II, FDS-LNH III, FDS-LNnH, TS-LNH, SLNnD, FS-novo-LNP I, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, Gal-b1,3-[Neu5Gc-a2,6]-Gal-b1,4-Glc, 3-sialyllacto-N-biose (3SLNB), 6-sialyllacto-N-biose (6SLNB), monofucosylmonosialyllacto-N-octaose (sialyl Lea), 3-sialyllactosamine (3SLacNAc), 6-sialyllactosamine (6SLacNAc), sialyl Lex and Neu5Gc-a2,3-Gal-b1,4-GlcNAc.

[0213] More preferred mixtures in this context of the disclosure comprise mixtures comprising, consisting essentially of or consisting of at least three, 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 sialylated oligosaccharides chosen from the list comprising 3-sialyllactose, 6-sialyllactose, 3,6-disialyllactose, 6,6-disialyllactose, 8,3-disialyllactose, 3'S-2FL, 6'S-2FL, 6'S-3-FL, pentasaccharide LSTD (Neu5Ac?-2,3Gal?-1,4GlcNAc?-1,3Gal?-1,4Glc), sialylated lacto-N-triose, sialylated lacto-N-tetraose comprising LSTa and LSTb, sialyllacto-N-neotetraose comprising LSTc and LSTd, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a, disialyllacto-N-hexaose I, sialyllacto-N-tetraose b, 3-sialyl-3-fucosyllactose (3'S-3-FL), disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose, FS Gal-LNnH (Gal-a1,3-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), DFSGal-LNnH (Gal-a1,3-[Fuc?1,2]-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), FS-LNnH (Fuc?1,2-Gal-b1,4-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), MSDF-para-LNnH (Neu5Ac?2,3-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3-Gal-b1,4-Glc), 3-sialyllacto-N-biose (3SLNB), 6-sialyllacto-N-biose (6SLNB), monofucosylmonosialyllacto-N-octaose (sialyl Lea), 3-sialyllactosamine (3SLacNAc), 6-sialyllactosamine (6SLacNAc), sialyl Lex and Neu5Gc-a2,3-Gal-b1,4-GlcNAc.

[0214] An example of the preferred mixtures is a mixture comprising at least three sialylated oligosaccharides chosen from the list comprising 3-sialyllactose, 6-sialyllactose, 3,6-disialyllactose, 6,6-disialyllactose, 8,3-disialyllactose, 3'S-2FL, 6'S-2FL, 6'S-3-FL, 3-sialyl-3-fucosyllactose (3'S-3-FL), sialylated lacto-N-triose, sialylated lacto-N-tetraose comprising LSTa and LSTb, sialyllacto-N-neotetraose comprising LSTc and LSTd, 3-sialyllacto-N-biose (3SLNB), 6-sialyllacto-N-biose (6SLNB), monofucosylmonosialyllacto-N-octaose (sialyl Lea), 3-sialyllactosamine (3SLacNAc), 6-sialyllactosamine (6SLacNAc) and sialyl Lex.

[0215] Exemplary mixtures in this context of the disclosure are described in the Examples section.

[0216] Optionally, 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 oligosaccharides are present in the mixture according to the disclosure. Preferably, the neutral fucoslated oligosaccharides are preferably selected from: [0217] lactose-based neutral fucosylated oligosaccharides, preferably any one of 2-fucosyllactose (2FL), 3-fucosyllactose (3-FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL or LDFT), Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[Fuc-a1,3-[Gal-b1,4]-GlcNAc-b1,6]-Gal-b1,4-Glc, Lacto-N-fucopentaose I (LNFP-I; Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), GalNAc-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), Lacto-N-fucopentaose II (LNFP-II; Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc), Lacto-N-fucopentaose III (LNFP III; Gal-b1,4-(Fuc-a1,3)-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-fucopentaose VI (LNFP-VI; Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc), lacto-N-neofucopentaose I (LNnFP I; Fuc-a1,2-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), lacto-N-difucohexaose I (LNDFH I; Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-Gal-b1,4-Glc), lacto-N-difucohexaose II (LNDFH II; Fuc-a1,4-(Gal-b1,3)-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc), Monofucosyllacto-N-hexaose III, Difucosyllacto-N-hexaose, difucosyl-lacto-N-neohexaose, LNnDFH (Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc), A-tetrasaccharide (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc), more preferably any one of 2-fucosyllactose (2FL), 3-fucosyllactose (3-FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL or LDFT), Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[Fuc-a1,3-[Gal-b1,4]-GlcNAc-b1,6]-Gal-b1,4-Glc, Lacto-N-fucopentaose I (LNFP-I; Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), GalNAc-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), Lacto-N-fucopentaose II (LNFP-II; Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc), Lacto-N-fucopentaose III (LNFP III; Gal-b1,4-(Fuc-a1,3)-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-fucopentaose VI (LNFP-VI; Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc), most preferably any one of 2-fucosyllactose (2FL), 3-fucosyllactose (3-FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL or LDFT), Gal-LNFP-III, 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-novo-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-novo-LND, 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-isoglobotriose, 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; and/or [0218] LNB-based neutral fucosylated oligosaccharides, preferably any one of 2FLNB, 4-FLNB, Leb (Fuc-a1,2-Gal-b1,3-(Fuc-a1,4)-GlcNAc); and/or [0219] LacNAc-based neutral fucosylated oligosaccharides, preferably any one of 2FLacNAc, 3-FLacNAc, Ley (Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-GlcNAc).

[0220] Preferred mixtures in this context of the disclosure comprise mixtures comprising, consisting essentially of or consisting 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 oligosaccharides chosen from the list comprising 2-fucosyllactose (2FL), 3-fucosyllactose (3-FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL or LDFT), Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[Fuc-a1,3-[Gal-b1,4]-GlcNAc-b1,6]-Gal-b1,4-Glc, Lacto-N-fucopentaose I (LNFP-I; Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), GalNAc-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), Lacto-N-fucopentaose II (LNFP-II; Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc), Lacto-N-fucopentaose III (LNFP III; Gal-b1,4-(Fuc-a1,3)-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-fucopentaose VI (LNFP-VI; Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc), lacto-N-neofucopentaose I (LNnFP I; Fuc-a1,2-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), lacto-N-difucohexaose I (LNDFH I; Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-Gal-b1,4-Glc), lacto-N-difucohexaose II (LNDFH II; Fuc-a1,4-(Gal-b1,3)-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc), Monofucosyllacto-N-hexaose III, Difucosyllacto-N-hexaose, difucosyl-lacto-N-neohexaose, LNnDFH (Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc), A-tetrasaccharide (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc), Gal-LNFP-III, 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-novo-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-novo-LND, 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-isoglobotriose, 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, 2FLNB, 4-FLNB, Leb (Fuc-a1,2-Gal-b1,3-(Fuc-a1,4)-GlcNAc), 2FLacNAc, 3-FLacNAc and Ley (Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-GlcNAc.

[0221] Exemplary mixtures in this context of the disclosure are described in the Examples section.

[0222] Optionally, 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 non-fucosylated oligosaccharides are present in the mixture according to the disclosure. Preferably, the neutral non-fucosylated oligosaccharides are preferably selected from: [0223] lactose-based neutral non-fucosylated oligosaccharides, preferably any one of Lacto-N-triose II (LN3), Lacto-N-neotetraose (LNnT), Lacto-N-tetraose (LNT), para-Lacto-N-neopentaose, para-Lacto-N-pentaose, para-Lacto-N-neohexaose, para-Lacto-N-hexaose, beta-(1,3)Galactosyl-para-Lacto-N-neopentaose, beta-(1,4)Galactosyl-para-Lacto-N-pentaose, Gal-a1,4-Gal-b1,4-Glc (Gal-a1,4-lactose), ?3-galactosyllactose, B6-galactosyllactose, Gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, Gal-a1,4-Gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, Gal-b1,3-Galb1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Galb1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Galb1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Galb1,3-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 (globo-N-tetraose), Gal-b1,3-GalNAc-b1,3-Gal-a1,4-Gal-b1,4-Glc, GalNAc-b1,3-LNT, Gal-b1,3-GalNAc-b1,3-LNT, novo-LNT (GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,4-Glc), Gal-novo-LNP I (Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3-Gal-b1,3]-Gal-b1,4-Glc), Gal-novo-LNP II (Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,3-Gal-b1,4-Glc), Gal-novo-LNP III (Gal-b1,3-Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,4-Glc), novo-LNO, GalNAc-b1,3-LNnT, Gal-b1,3-GalNAc-b1,3-LNnT, LNH, LNnH, iso-LNO, novo-LNO, novo-LNnO, LND, iso-LND, GalNAc-a1,3-Gal-b1,4-Glc, novo-LNP I, iso-LNT, DGalLNnH, galilipentasaccharide, more preferably any one of Lacto-N-triose II (LN3), Lacto-N-neotetraose (LNnT), Lacto-N-tetraose (LNT), para-Lacto-N-neopentaose, para-Lacto-N-pentaose, para-Lacto-N-neohexaose, para-Lacto-N-hexaose, beta-(1,3)Galactosyl-para-Lacto-N-neopentaose, beta-(1,4)Galactosyl-para-Lacto-N-pentaose, Gal-a1,4-Gal-b1,4-Glc (Gal-a1,4-lactose), ?3-galactosyllactose, ?6-galactosyllactose, GalNAc-b1,3-Lactose, globo-N-tetraose, most preferably any one of Lacto-N-triose II (LN3), Lacto-N-neotetraose (LNnT), Lacto-N-tetraose (LNT), para-Lacto-N-neopentaose, para-Lacto-N-pentaose, para-Lacto-N-neohexaose, para-Lacto-N-hexaose; and/or [0224] LNB-based neutral non-fucosylated oligosaccharides; and/or [0225] LacNAc-based neutral non-fucosylated oligosaccharides, like e.g., LacDiNAc and poly-LacNAc.

[0226] Preferred mixtures in this context of the disclosure comprise mixtures comprising, consisting essentially of or consisting 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 non-fucosylated oligosaccharides chosen from the list comprising Lacto-N-triose II (LN3), Lacto-N-neotetraose (LNnT), Lacto-N-tetraose (LNT), para-Lacto-N-neopentaose, para-Lacto-N-pentaose, para-Lacto-N-neohexaose, para-Lacto-N-hexaose, beta-(1,3)Galactosyl-para-Lacto-N-neopentaose, beta-(1,4)Galactosyl-para-Lacto-N-pentaose, Gal-a1,4-Gal-b1,4-Glc (Gal-a1,4-lactose), ?3-galactosyllactose, B6-galactosyllactose, Gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, Gal-a1,4-Gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, Gal-b1,3-Galb1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Galb1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Galb1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Galb1,3-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 (globo-N-tetraose), Gal-b1,3-GalNAc-b1,3-Gal-a1,4-Gal-b1,4-Glc, GalNAc-b1,3-LNT, Gal-b1,3-GalNAc-b1,3-LNT, novo-LNT (GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,4-Glc), Gal-novo-LNP I (Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3-Gal-b1,3]-Gal-b1,4-Glc), Gal-novo-LNP II (Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,3-Gal-b1,4-Glc), Gal-novo-LNP III (Gal-b1,3-Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,4-Glc), novo-LNO, GalNAc-b1,3-LNnT, Gal-b1,3-GalNAc-b1,3-LNnT, LNH, LNnH, iso-LNO, novo-LNO, novo-LNnO, LND, iso-LND, GalNAc-a1,3-Gal-b1,4-Glc, novo-LNP I, iso-LNT, DGalLNnH, galilipentasaccharide, LacDiNAc and poly-LacNAc.

[0227] Exemplary mixtures in this context of the disclosure are described in the Examples section.

[0228] In an even more preferred embodiment of the method and/or cell according to the disclosure, the mixture according to the disclosure comprises, consists essentially of or consists of at least three, 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 sialylated oligosaccharides chosen from the list comprising 3-sialyllactose, 6-sialyllactose, 3,6-disialyllactose, 6,6-disialyllactose, 8,3-disialyllactose, 3'S-2FL, 6'S-2FL, 6'S-3-FL, pentasaccharide LSTD (Neu5Ac?-2,3Gal?-1,4GlcNAc?-1,3Gal?-1,4Glc), sialylated lacto-N-triose, sialylated lacto-N-tetraose comprising LSTa and LSTb, sialyllacto-N-neotetraose comprising LSTc and LSTd, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a, disialyllacto-N-hexaose I, sialyllacto-N-tetraose b, 3-sialyl-3-fucosyllactose (3'S-3-FL), disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose, FS Gal-LNnH (Gal-a1,3-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), DFSGal-LNnH (Gal-a 1,3-[Fuc?1,2]-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), FS-LNnH (Fuc?1,2-Gal-b1,4-GlcNAc-b1,6-[Neu5Ac?2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), MSDF-para-LNnH (Neu5Ac?2,3-Gal-b1,4-[Fuc?1,3]-GlcNAc-b1,3-Gal-b1,4-[Fuc-a1,3]-GlcNAc-b1,3-Gal-b1,4-Glc), GD3 (Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,4Glc), GT3 (Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,4Glc); GM2 GalNAc?-1,4(Neu5Ac?-2,3)Gal?-1,4Glc, GM1 (Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,3)Gal?-1,4Glc), GD1a (Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,3)Gal?-1,4Glc), GT1a (Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,3)Gal?-1,4Glc), GD2 (GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GT2 (GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GD1b, (Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GT1b (Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GQ1b (Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,3GalNAc ?-1,4(Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GT1c (Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GQ1c (Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GP1c (Neu5Ac?-2,8Neu5Ac?-2,3Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,8Neu5Ac?-2,8Neu5Ac?2,3)Gal?-1,4Glc), GD1a (Neu5Ac?-2,3Gal?-1,3(Neu5Ac?-2,6)GalNAc?-1,4Gal?-1,4Glc), Fucosyl-GM1 (Fuc?-1,2Gal?-1,3GalNAc?-1,4(Neu5Ac?-2,3)Gal ?-1,4Glc), Neu5Ac?2,3-Galb1,3-Gal-b1,4-Glc, Galb1,3-[Neu5Ac?2,6]-Gal-b1,4-Glc, Neu5Gca2,8-Neu5Ac?2,3-Gal-b1,4-Glc, Neu5Ac?2,8-Neu5Gca2,3-Gal-b1,4-Glc, Neu5Ac?2,8-Neu5Ac?2,3-Gal-b1,4-Glc, Neu5Gca2,8-Neu5Gca2,3-Gal-b1,4-Glc, Neu5Ac?2,3-Gal-b1,3-[Neu5Ac?2,6]-Gal-b1,4-Glc, Galb 1,6-[Neu5Ac?2,3]-Gal-b1,4-Glc, Gal-b1,3-[Neu5Ac?2,6]-Gal-b1,4-Glc, Neu5Gca2,3-Gal-b1,3-Gal-b1,4-Glc, Neu5Gca2,3-Gal-b1,3-[Neu5Ac?2,6]-Gal-b1,4-Glc, Galb 1,4-GlcNAc-b16-[Neu5Ac2,3-Gal-b1,3]-Gal-b1,4-Glc, Neu5Ac2,6-Gal-b1,4-GlcNAc-b1,6-[Galb 1,3]-Gal-b1,4-Glc, Neu5Gca2,3-Gal-b1,4-Glc, Neu5Gca2,6-Gal-b1,4-Glc, GalMSLNnH (Gal?1,3-Gal-b1,4-GlcNAc-b1,6-[Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3]-Gal-b1,4-Glc), F-LSTa, F-LSTb, F-LSTc, 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, DF-para-LNH sulfate I, DF-para-LNH sulfate II, TF-para-LNH sulfate, Neu5GcLNnT, GM2 tetrasaccharide, SLNOa, S-LNH I, S-LNH II, S-LNnH I, S-LNnH II, S-para-LNnH, DS-LNH II, 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, FDS-LNT I, FDS-LNT II, FDS-LNH I, FDS-LNH II, FDS-LNH III, FDS-LNnH, TS-LNH, SLNnD, FS-novo-LNP I, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5 Ac-a2,6-[GlcNAc-b1,3]-Gal-b1,4-Glc, Gal-b1,3-[Neu5Gc-a2,6]-Gal-b1,4-Glc, 3-sialyllacto-N-biose (3SLNB), 6-sialyllacto-N-biose (6SLNB), monofucosylmonosialyllacto-N-octaose (sialyl Lea), 3-sialyllactosamine (3SLacNAc), 6-sialyllactosamine (6SLacNAc), sialyl Lex and Neu5Gc-a2,3-Gal-b1,4-GlcNAc, combined 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 neutral fucosylated oligosaccharides chosen from the list comprising 2-fucosyllactose (2FL), 3-fucosyllactose (3-FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL or LDFT), Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-[Fuc-a1,3-[Gal-b1,4]-GlcNAc-b1,6]-Gal-b1,4-Glc, Lacto-N-fucopentaose I (LNFP-I; Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), GalNAc-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), Lacto-N-fucopentaose II (LNFP-II; Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc), Lacto-N-fucopentaose III (LNFP III; Gal-b1,4-(Fuc-a1,3)-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-fucopentaose VI (LNFP-VI; Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc), lacto-N-neofucopentaose I (LNnFP I; Fuc-a1,2-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), lacto-N-difucohexaose I (LNDFH I; Fuc-a1,2-Gal-b1,3-[Fuc-a1,4]-GlcNAc-b1,3-Gal-b1,4-Glc), lacto-N-difucohexaose II (LNDFH II; Fuc-a1,4-(Gal-b1,3)-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc), Monofucosyllacto-N-hexaose III, Difucosyllacto-N-hexaose, difucosyl-lacto-N-neohexaose, LNnDFH (Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc), A-tetrasaccharide (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc), Gal-LNFP-III, 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-novo-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-novo-LND, 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-isoglobotriose, 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, 2FLNB, 4-FLNB, Leb (Fuc-a1,2-Gal-b1,3-(Fuc-a1,4)-GlcNAc), 2FLacNAc, 3-FLacNAc and Ley (Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-GlcNAc and/or combined 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 neutral non-fucosylated oligosaccharides chosen from the list comprising Lacto-N-triose II (LN3), Lacto-N-neotetraose (LNnT), Lacto-N-tetraose (LNT), para-Lacto-N-neopentaose, para-Lacto-N-pentaose, para-Lacto-N-neohexaose, para-Lacto-N-hexaose, beta-(1,3)Galactosyl-para-Lacto-N-neopentaose, beta-(1,4)Galactosyl-para-Lacto-N-pentaose, Gal-a1,4-Gal-b1,4-Glc (Gal-a1,4-lactose), ?3-galactosyllactose, ?6-galactosyllactose, Gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, Gal-a1,4-Gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, Gal-b1,3-Galb1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Galb1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Galb1,3-Gal-b1,4-Glc, Gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1,3-Galb1,3-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 (globo-N-tetraose), Gal-b1,3-GalNAc-b1,3-Gal-a1,4-Gal-b1,4-Glc, GalNAc-b1,3-LNT, Gal-b1,3-GalNAc-b1,3-LNT, novo-LNT (GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,4-Glc), Gal-novo-LNP I (Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3-Gal-b1,3]-Gal-b1,4-Glc), Gal-novo-LNP II (Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,3-Gal-b1,4-Glc), Gal-novo-LNP III (Gal-b1,3-Gal-b1,4-GlcNAc-b1,6-[Gal-b1,3]-Gal-b1,4-Glc), novo-LNO, GalNAc-b1,3-LNnT, Gal-b1,3-GalNAc-b1,3-LNnT, LacDiNAc, poly-LacNAc, LNH, LNnH, iso-LNO, novo-LNO, novo-LNnO, LND, iso-LND, GalNAc-a1,3-Gal-b1,4-Glc, novo-LNP I, iso-LNT, DGalLNnH, galilipentasaccharide.

[0229] An exemplary mixture in this context comprises, consists of or consists essentially of 3-sialyllactose, 6-sialyllactose, 3'S-2FL, 6'S-2FL, 6'S-3-FL, 3-sialyl-3-fucosyllactose (3'S-3-FL), 2FL, 3-FL and DiFL. Another exemplary mixture in this context comprises, consists of or consists essentially of LN3, LNT, LSTa, 3SL, 6SL, LSTb. Another exemplary mixture in this context comprises, consists of or consists essentially of LN3, LNnT, LSTc, LSTd, 3SL and 6SL. Another exemplary mixture in this context comprises, consists of or consists essentially of 2FL, 3-FL, DiFL, LN3, LNT, LNnT, 3SL, 6SL, LNFP-I and LSTc. Another exemplary mixture in this context comprises, consists of or consists essentially of 2FL, 3-FL, DiFL, 3SL, 6SL, LN3, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LSTa, LSTc and LSTd. Another exemplary mixture in this context comprises, consists of or consists essentially of 2FL, 3-FL, monofucosylmonosialyllacto-N-octaose (sialyl Lea), Fuc-a1,2-Gal-b1,3-(Fuc-a1,4)-GlcNAc (Leb), 3SL and 6SL. Another exemplary mixture in this context comprises, consists of or consists essentially of 2FL, 3-FL, 3SL, 6SL, sialyl Lex and Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-GlcNAc (Ley).

[0230] Exemplary mixtures in this context of the disclosure are described in the Examples section.

[0231] For the production of lactose-based oligosaccharides as described herein and in an embodiment of the method and/or cell according to the disclosure, lactose can be added to the cultivation so that the cell can take it up passively or through active transport; or lactose can be produced by the cell (for example, upon metabolically engineering the cell for this purpose as known to the skilled person), preferably intracellularly. Lactose can hence be used as an acceptor in the synthesis of a mammalian milk oligosaccharide or human milk oligosaccharide, preferably all of the lactose-based MMOs or HMOs, which is/are preferably comprised in the oligosaccharide mixture according to the disclosure as described herein. A cell producing lactose can be obtained by expression of an N-acetylglucosamine beta-1,4-galactosyltransferase and an UDP-glucose 4-epimerase. More preferably, the cell is modified for enhanced lactose production. The modification can be any one or more chosen from the group comprising over-expression of an N-acetylglucosamine beta-1,4-galactosyltransferase, over-expression of an UDP-glucose 4-epimerase. Alternatively, a cell using lactose as acceptor in glycosylation reactions preferably has a transporter for the uptake of lactose from the cultivation. More preferably, the cell is optimized for lactose uptake. The optimization can be over-expression of a lactose transporter like a lactose permease from e.g., E. coli, Kluyveromyces lactis or Lactobacillus casei BL23. It is preferred to constitutively express the lactose permease. The lactose can be added at the start of the cultivation or it can be added as soon as enough biomass has been formed during the growth phase of the cultivation, i.e., the MMO production phase (initiated by the addition of lactose to the cultivation) is decoupled form the growth phase. In a preferred embodiment, the lactose is added at the start and/or during the cultivation, i.e., the growth phase and production phase are not decoupled.

[0232] In a preferred embodiment of the method and/or cell according to the disclosure, the cell resists the phenomenon of lactose killing when grown in an environment in which lactose is combined with one or more other carbon source(s). With the term lactose killing is meant the hampered growth of the cell in medium in which lactose is present together with another carbon source. In a preferred embodiment, the cell is genetically modified such that it retains at least 50% of the lactose influx without undergoing lactose killing, even at high lactose concentrations, as is described in WO 2016/075243. The genetic modification comprises expression and/or over-expression of an exogenous and/or an endogenous lactose transporter gene by a heterologous promoter that does not lead to a lactose killing phenotype and/or modification of the codon usage of the lactose transporter to create an altered expression of the lactose transporter that does not lead to a lactose killing phenotype. The content of WO 2016/075243 in this regard is incorporated by reference.

[0233] For the production of LNB-based oligosaccharides as described herein and in an additional and/or alternative embodiment of the method and/or cell according to the disclosure, LNB (i.e., lacto-N-biose, Gal-b1,3-GlcNAc) can be added to the cultivation so that the cell can take it up passively or through active transport; or LNB can be produced by the cell (for example, upon metabolically engineering the cell for this purpose as known to the skilled person), preferably intracellularly. LNB can hence be used as an acceptor in the synthesis of a LNB-based oligosaccharide, preferably all of the LNB-based oligosaccharides, which is/are preferably comprised in the oligosaccharide mixture according to the disclosure as described herein. A cell producing LNB can be obtained by expression of an N-acetylglucosamine beta-1,3-galactosyltransferase that can modify GlcNAc (produced in the cell and/or taken up passively or through active transport) to form LNB. Preferably, a cell producing LNB is capable to express, preferably expresses, enzymes required for the synthesis of GlcNAc, such as glucosamine 6-phosphate N-acetyltransferase, phosphatase, N-acetylglucosamine beta-1,3-galactosyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase, preferably a glucosamine 6-phosphate N-acetyltransferase and a phosphatase, preferably a HAD-like phosphatase, such as any one of the E. coli genes 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 or PsMupP from Pseudomonas putida, ScDOG1 from S. cerevisiae and BsAraL from Bacillus subtilis as described in WO 2018122225). Preferably, the cell is metabolically engineered for production of LNB. More preferably, the cell is metabolically engineered for enhanced production of LNB. The cell is preferably modified to express and/or over-express any one or more of the polypeptides comprising glucosamine 6-phosphate N-acetyltransferase, phosphatase, N-acetylglucosamine beta-1,3-galactosyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase.

[0234] A cell using LNB as acceptor in glycosylation reactions preferably has a transporter for the uptake of LNB from the cultivation. More preferably, the cell is optimized for LNB uptake. The optimization can be over-expression of a LNB transporter like a lactose permease from E. coli, Kluyveromyces lactis or Lactobacillus casei BL23. It is preferred to constitutively express the lactose permease. The LNB can be added at the start of the cultivation or it can be added as soon as enough biomass has been formed during the growth phase of the cultivation, i.e., the oligosaccharide production phase (initiated by the addition of LNB to the cultivation) is decoupled form the growth phase. In a preferred embodiment, the LNB is added at the start and/or during the cultivation, i.e., the growth phase and production phase are not decoupled.

[0235] For the production of LacNAc-based oligosaccharides as described herein and in an additional and/or alternative embodiment of the method and/or cell according to the disclosure, LacNAc (i.e., N-acetyllactosamine, Gal-b1,4-GlcNAc) can be added to the cultivation so that the cell can take it up passively or through active transport; or LacNAc can be produced by the cell (for example, upon metabolically engineering the cell for this purpose as known to the skilled person), preferably intracellularly. LacNAc can hence be used as an acceptor in the synthesis of a LacNAc-based oligosaccharide, preferably all of the LacNAc-based oligosaccharides, which is/are preferably comprised in the oligosaccharide mixture according to the disclosure as described herein. A cell producing LacNAc can be obtained by expression of an N-acetylglucosamine beta-1,4-galactosyltransferase that can modify GlcNAc (produced in the cell and/or taken up passively or through active transport) to form LacNAc. Preferably, a cell producing LacNAc is capable to express, preferably expresses, enzymes required for the synthesis of GlcNAc, such as glucosamine 6-phosphate N-acetyltransferase, phosphatase, N-acetylglucosamine beta-1,4-galactosyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase, preferably a glucosamine 6-phosphate N-acetyltransferase and a phosphatase (preferably a HAD-like phosphatase). Preferably, the cell is metabolically engineered for production of LacNAc. More preferably, the cell is metabolically engineered for enhanced production of LacNAc. The cell is preferably modified to express and/or over-express any one or more of the polypeptides comprising glucosamine 6-phosphate N-acetyltransferase, phosphatase, N-acetylglucosamine beta-1,4-galactosyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase.

[0236] A cell using LacNAc as acceptor in glycosylation reactions preferably has a transporter for the uptake of LacNAc from the cultivation. More preferably, the cell is optimized for LacNAc uptake. The optimization can be over-expression of a LNB transporter like a lactose permease from E. coli, Kluyveromyces lactis or Lactobacillus casei BL23. It is preferred to constitutively express the lactose permease. The LacNAc can be added at the start of the cultivation or it can be added as soon as enough biomass has been formed during the growth phase of the cultivation, i.e., the oligosaccharide production phase (initiated by the addition of LacNAc to the cultivation) is decoupled form the growth phase. In a preferred embodiment, the LacNAc is added at the start and/or during the cultivation, i.e., the growth phase and production phase are not decoupled.

[0237] In an additional and/or alternative embodiment of the method and/or cell according to the disclosure, the cell is (i) capable to express, preferably expresses, a sialyltransferase, preferably chosen from alpha-2,3-sialyltransferases, alpha-2,6-sialyltransferases and alpha-2,8-sialyltransferases, and (ii) capable to express, preferably expresses, at least one, preferably at least two, preferably at least three, more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, additional glycosyltransferase(s) preferably chosen from the list comprising fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases as defined herein.

[0238] In a preferred embodiment, the fucosyltransferase is chosen from the list comprising alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase. In another preferred embodiment, the sialyltransferase is chosen from the list comprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase. In another preferred embodiment, the galactosyltransferase is chosen from the list comprising beta-1,3-galactosyltransferase, N-acetylglucosamine beta-1,3-galactosyltransferase, beta-1,4-galactosyltransferase, N-acetylglucosamine beta-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase and alpha-1,4-galactosyltransferase. In another preferred embodiment, the glucosyltransferase is chosen from the list comprising alpha-glucosyltransferase, beta-1,2-glucosyltransferase, beta-1,3-glucosyltransferase and beta-1,4-glucosyltransferase. In another preferred embodiment, the mannosyltransferase is chosen from the list comprising alpha-1,2-mannosyltransferase, alpha-1,3-mannosyltransferase and alpha-1,6-mannosyltransferase. In another preferred embodiment, the N-acetylglucosaminyltransferase is chosen from the list comprising galactoside beta-1,3-N-acetylglucosaminyltransferase and beta-1,6-N-acetylglucosaminyltransferase. In another preferred embodiment, the N-acetylgalactosaminyltransferase is chosen from the list comprising alpha-1,3-N-acetylgalactosaminyltransferase and beta-1,3-N-acetylgalactosaminyltransferase.

[0239] In a further embodiment of the method and/or cell of the disclosure, the cell is modified in the expression or activity of at least one, preferably at least two, more preferably all, of the glycosyltransferases. In a preferred embodiment, the glycosyltransferase is an endogenous protein of the cell with a modified expression or activity, preferably the endogenous glycosyltransferase is overexpressed; alternatively the glycosyltransferase is a heterologous protein that is heterogeneously introduced and expressed in the cell, preferably overexpressed. The endogenous glycosyltransferase can have a modified expression in the cell that also expresses a heterologous glycosyltransferase.

[0240] In another embodiment of the method and/or cell of this disclosure, at least one, preferably at least two, of the glycosyltransferases is a fucosyltransferase and the cell is capable of synthesizing GDP-Fuc. The GDP-fucose can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing GDP-fucose can express an enzyme converting, e.g., fucose, which is to be added to the cell, to GDP-fucose. This enzyme may be, e.g., a bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase, like Fkp from Bacteroides fragilis, or the combination of one separate fucose kinase together with one separate fucose-1-phosphate guanylyltransferase like they are known from several species including Homo sapiens, Sus scrofa and Rattus norvegicus. In a preferred embodiment of the method and/or cell of this disclosure, the cell is capable of expressing at least one, preferably at least two, fucosyltransferase(s) selected from alpha-1,2-fucosyltransferases, alpha-1,3/1,4-fucosyltransferases and alpha-1,6-fucosyltransferases. Preferably, the fucosyltransferases are selected from organisms like e.g., Helicobacter species like e.g., Helicobacter pylori, Helicobacter mustelae, Akkermansia species like e.g., Akkermansia muciniphila, Bacteroides species like e.g., Bacteroides fragilis, Bacteroides vulgatus, Bacteroides ovatus, E. coli species like e.g., E. coli O126, E. coli 055:H7, Lachnospiraceae species, Tannerella species, Clostridium species, Salmonella species like e.g., Salmonella enterica, Methanosphaerula palustries, Butyrivibrio species, Prevotella species, Porphyromonas species like e.g., Porphyromonas catoniae, Arabidopsis thaliana, Homo sapiens, Mus musculus. In a more preferred embodiment of the method and/or cell of this disclosure, the fucosyltransferases are selected from the list comprising alpha-1,2-fucosyltransferases and alpha-1,3/1,4-fucosyltransferases.

[0241] Preferably, the cell is modified to produce GDP-fucose. More preferably, the cell is modified for enhanced GDP-fucose production. The modification can be any one or more chosen from the group comprising knock-out of an UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase encoding gene, over-expression of a GDP-L-fucose synthase encoding gene, over-expression of a GDP-mannose 4,6-dehydratase encoding gene, over-expression of a mannose-1-phosphate guanylyltransferase encoding gene, over-expression of a phosphomannomutase encoding gene and over-expression of a mannose-6-phosphate isomerase encoding gene. Throughout the disclosure, unless explicitly stated otherwise, the feature enhanced and/or optimized production preferably means that the modification(s) and/or metabolic engineering introduced in a cell as described herein results in a higher production yield compared to the wild type progenitor of the modified cell or metabolically engineered cell. For example, an enhanced GDP-fucose production preferably means that the intracellular production of GDP-fucose is higher in the modified cell compared to the wild type progenitor that does not contain these specific modifications.

[0242] Preferably, the cell in this context comprises a fucosylation pathway as described herein.

[0243] In another embodiment of the method and/or cell of the disclosure, at least one, preferably at least two, of the glycosyltransferases is a sialyltransferase and the cell is capable of synthesizing CMP-Neu5Ac. The CMP-Neu5Ac can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing CMP-Neu5 Ac can express an enzyme converting, e.g., sialic acid, which is to be added to the cell, to CMP-Neu5Ac. This enzyme may be a CMP-sialic acid synthetase, like the N-acylneuraminate cytidylyltransferase from several species including Homo sapiens, Neisseria meningitidis, and Pasteurella multocida. In a preferred embodiment of the method and/or cell of this disclosure, the cell is capable of expressing at least one, preferably at least two, sialyltransferase(s) selected from alpha-2,3-sialyltransferases, alpha-2,6-sialyltransferases and alpha-2,8-sialyltransferases. Preferably, the sialyltransferases are selected from organisms like e.g., Pasteurella species like e.g., Pasteurella multocida, Pasteurella dagmatis, Photobacterium species like e.g., Photobacterium damselae, Photobacterium sp. JT-ISH-224, Photobacterium phosphoreum, Photobacterium leiognathi, Porphyromonas species like e.g., Porphyromonas catoniae, Streptococcus species like e.g., Streptococcus suis, Streptococcus agalactiae, Streptococcus entericus, Neisseria meningitidis, Campylobacter jejuni, Haemophilus species like e.g., Haemophilus somnus, Haemophilus ducreyi, Haemophilus parahaemolyticus, Haemophilus parasuis, Vibrio species, Alistipes species like e.g., Alistipes sp. CAG:268, Alistipes sp. AL-1, Alistipes shahii, Alistipes timonensis, Actinobacillus species like e.g., Actinobacillus suis, Actinobacillus capsulatus, Homo sapiens, Mus musculus. In a more preferred embodiment of the method and/or cell of this disclosure, the sialyltransferases are selected from the list comprising alpha-2,3-sialyltransferases and alpha-2,6-sialyltransferases.

[0244] Preferably, the cell is modified to produce CMP-Neu5Ac. More preferably, the cell is modified for enhanced CMP-Neu5Ac production. The modification can be any one or more chosen from the group comprising knock-out of an N-acetylglucosamine-6-phosphate deacetylase, knock-out of a glucosamine-6-phosphate deaminase, over-expression of a sialate synthase encoding gene, and over-expression of an N-acetyl-D-glucosamine-2-epimerase encoding gene. Optionally, the cell is modified to produce GlcNAc and/or UDP-GlcNAc.

[0245] Preferably, the cell in this context comprises a sialylation pathway as described herein.

[0246] In another embodiment of the method and/or cell of the disclosure, at least one, preferably at least two, of the additional glycosyltransferases is an N-acetylglucosaminyltransferase and the cell is capable of synthesizing UDP-GlcNAc. The UDP-GlcNAc can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing an UDP-GlcNAc can express enzymes converting, e.g., GlcNAc, which is to be added to the cell, to UDP-GlcNAc. These enzymes may be an N-acetyl-D-glucosamine kinase, an N-acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, and an N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli. Alternatively, a cell can (preferably metabolically engineered to) express enzymes required for the synthesis of GlcNAc, such as glucosamine 6-phosphate N-acetyltransferase, phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase, preferably a glucosamine 6-phosphate N-acetyltransferase and a phosphatase (preferably a HAD-like phosphatase). In a preferred embodiment of the method and/or cell of this disclosure, the cell is capable of expressing at least one, preferably at least two, N-acetylglucosaminyltransferase(s) selected from beta-1,3-N-acetylglucosaminyltransferases and beta-1,6-N-acetylglucosaminyltransferases. Preferably, the N-acetylglucosaminyltransferases are selected from organisms like e.g., Neisseria species, like e.g., Neisseria meningitidis, Neisseria lactamica, Neisseria polysaccharea, Neisseria elongata, Neisseria gonorrhoeae, Neisseria subflava, Pasteurella species like e.g., Pasteurella dagmatis, Neorhizobium species like e.g., Neorhizobium galegae, Haemophilus species like e.g., Haemophilus parainfluenzae, Haemophilus ducreyi, Homo sapiens, Mus musculus.

[0247] Preferably, the cell is modified to produce UDP-GlcNAc. More preferably, the cell is modified for enhanced UDP-GlcNAc production. The modification can be any one or more chosen from the group comprising knock-out of an N-acetylglucosamine-6-phosphate deacetylase, over-expression of an L-glutamine-D-fructose-6-phosphate aminotransferase, over-expression of a phosphoglucosamine mutase, and over-expression of an N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase. Optionally, the cell is modified to produce GlcNAc.

[0248] Preferably, the cell in this context comprises an N-acetylglucosamine carbohydrate pathway as described herein.

[0249] In another embodiment of the method and/or cell of the disclosure, at least one, preferably at least two, of the glycosyltransferases is a galactosyltransferase and the cell is capable of synthesizing UDP-Gal. The UDP-Gal can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing UDP-Gal can express an enzyme converting, e.g., UDP-glucose, to UDP-Gal. This enzyme may be, e.g., the UDP-glucose-4-epimerase GalE like as known from several species including Homo sapiens, Escherichia coli, and Rattus norvegicus. In a preferred embodiment of the method and/or cell of this disclosure, the cell is capable of expressing at least one, preferably at least two, galactosyltransferase(s) selected from beta-1,3-galactosyltransferases and beta-1,4-galactosyltransferases, and/or the cell is capable of expressing at least one, preferably at least two, galactosyltransferases selected from alpha-1,3-galactosyltransferases and alpha-1,4-galactosyltransferases. Preferably, the galactosyltransferases are chosen from organisms like e.g., E. coli species like e.g., E. coli 055:H7, E. coli DEC1B, E. coli DEC1D, Neisseria species like e.g., Neisseria meningitidis, Neisseria lactamica, Neisseria polysaccharea, Neisseria elongata, Neisseria gonorrhoeae, Neisseria subflava, Kingella species like e.g., Kingella denitrificans, Brucella species like e.g., Brucella canis, Brucella suis, Salmonella species, like e.g., Salmonella enterica, Pseudogulbenkiana ferrooxidans, Corynebacterium glutamicum, Streptococcus species, Arabidopsis thaliana, Homo sapiens, Mus musculus.

[0250] Preferably, the cell is modified to produce UDP-Gal. More preferably, the cell is modified for enhanced UDP-Gal production. The modification can be any one or more chosen from the group comprising knock-out of an bifunctional 5-nucleotidase/UDP-sugar hydrolase encoding gene, knock-out of a galactose-1-phosphate uridylyltransferase encoding gene and over-expression of an UDP-glucose-4-epimerase encoding gene.

[0251] Preferably, the cell in this context comprises a galactosylation pathway as described herein.

[0252] In another embodiment of the method and/or cell of the disclosure, at least one, preferably at least two, of the glycosyltransferases is an N-acetylgalactosaminyltransferase and the cell is capable of synthesizing UDP-GalNAc. The UDP-GalNAc can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing UDP-GalNAc can express an enzyme converting, e.g., UDP-glucose, to UDP-Gal. This enzyme may be, e.g., the UDP-glucose-4-epimerase GalE like as known from several species including Homo sapiens, Escherichia coli, and Rattus norvegicus. In a preferred embodiment of the method and/or cell of this disclosure, the cell is capable of expressing at least one, preferably at least two, N-acetylgalactosaminyltransferase(s) selected from alpha-1,3-N-acetylgalactosaminyltransferases and beta-1,3-N-acetylgalactosaminyltransferases. Preferably, the N-acetylgalactosaminyltransferases are chosen from organisms like e.g., Helicobacter species like e.g., Helicobacter mustelae, Haemophilus species like e.g., Haemophilus influenzae, Neisseria species like e.g., Neisseria meningitidis, Neisseria lactamica, Neisseria polysaccharea, Neisseria elongata, Neisseria gonorrhoeae, Neisseria subflava, Rickettsia species like e.g., Rickettsia bellii, Rickettsia prowazekii, Rickettsia japonica, Rickettsia conorii, Rickettsia felis, Rickettsia massiliae, Homo sapiens, Mus musculus.

[0253] Preferably, the cell is modified to produce UDP-GalNAc. More preferably, the cell is modified for enhanced UDP-GalNAc production. The modification can be any one or more chosen from the group comprising knock-out of a bifunctional 5-nucleotidase/UDP-sugar hydrolase encoding gene, knock-out of a galactose-1-phosphate uridylyltransferase encoding gene and over-expression of an UDP-glucose-4-epimerase encoding gene.

[0254] Preferably, the cell in this context comprises an N-acetylgalactosaminylation pathway as described herein.

[0255] Throughout the disclosure, whenever a protein is disclosed, e.g., by referring to a SEQ ID NO, an unique database number (e.g., UNIPROT number) or by referring to the specific organism of origin, the protein embodiment can be preferably replaced with any, preferably all, of the following embodiments (and hence the protein is considered to be disclosed according to all of the following embodiment): [0256] protein (e.g., by referring to a SEQ ID NO, an unique database number (e.g., UNIPROT number) or by referring to the specific organism of origin), [0257] a functional homologue, variant or derivative of the protein having at least 80% overall sequence identity to the full length of the protein, [0258] a functional fragment of the protein and having the same activity, or [0259] comprises a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of the protein and having the same activity.

[0260] For example, when H. pylori alpha-1,3-fucosyltransferase with SEQ ID NO: 05 is disclosed, the embodiment is preferably replaced with any, preferably all, of the following embodiments: [0261] H. pylori alpha-1,3-fucosyltransferase with SEQ ID NO: 05, [0262] alpha-1,3-fucosyltransferase comprising a polypeptide sequence according to SEQ ID NO: 05, [0263] a functional homologue, variant or derivative of SEQ ID NO: 05 having at least 80% overall sequence identity to the full length of SEQ ID NO: 05 and having alpha-1,3-fucosyltransferase activity, [0264] a functional fragment of SEQ ID NO: 05 and having alpha-1,3-fucosyltransferase activity, or [0265] comprises a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of the SEQ ID NO: 05 and having alpha-1,3-fucosyltransferase activity.

[0266] In another embodiment of the method and/or cell of the disclosure, the cell is capable of synthesizing any one of the nucleotide-sugars chosen from the list comprising GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy--L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy--L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose. In a preferred embodiment, the cell is capable of synthesizing two nucleotide-sugars. In a more preferred embodiment, the cell is capable of synthesizing at least three nucleotide-activated sugars. In an even more preferred embodiment, the cell is capable of synthesizing at least four nucleotide-activated sugars. In a most preferred embodiment, the cell is capable of synthesizing at least five nucleotide-activated sugars. In another preferred embodiment, the cell is metabolically engineered for the production of a nucleotide-sugar. In another preferred embodiment, the cell is modified and/or engineered for the optimized production of a nucleotide-sugar i.e., enhanced production of a nucleotide-sugar 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.

[0267] In another embodiment of the method and/or cell of this disclosure, the cell expresses one or more polypeptides chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, GDP-fucose pyrophosphorylase, fucose-1-phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 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 uridylyltransferase, glucosamine-1-phosphate acetyltransferase, N-acetylneuraminate synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphate phosphatase, N-acylneuraminate cytidylyltransferase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridylyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase.

[0268] In a preferred embodiment of the method and/or cell according to the disclosure, the mixture of at least three different sialylated oligosaccharides according to the disclosure can be produced by providing a cell that, for the production of lactose-based sialylated non-fucosylated oligosaccharides, is 1) capable takeoff taking up lactose from the cultivation as described herein or is able to produce lactose after uptake of glucose by the action of a b-1,4-galactosyltransferase as described herein; and 2) capable of expressing at least one, preferably at least two, sialyltransferase(s) as described herein, chosen from the list comprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase; 3) optionally capable of expressing an N-acetylglucosaminyltransferase as described herein, preferably a galactoside beta-1,3-N-acetylglucosaminyltransferase; 4) optionally capable of expressing at least one, preferably at least two, galactosyltransferase(s) as described herein, chosen from the list comprising N-acetylglucosamine beta-1,3-galactosyltransferase, N-acetylglucosamine beta-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase, alpha-1,4-galactosyltransferase; 5) optionally capable of expressing at least one, preferably at least two, N-acetylgalactosaminyltransferase(s) as described herein, chosen from the list comprising alpha-1,3-N-acetylgalactosaminyltransferase and beta-1,3-N-acetylgalactosaminyltransferase; 6) capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein, and 7) capable of synthesizing the nucleotide-sugar of each of the glycosyltransferase if present.

[0269] In another and/or additional preferred embodiment of the method and/or cell according to the disclosure, the mixture of at least three different sialylated oligosaccharides according to the disclosure can be produced by providing a cell that, for the production of LNB-based sialylated non-fucosylated oligosaccharides, is 1) capable of taking up LNB from the cultivation as described herein or is able to produce LNB as described herein; and 2) capable of expressing at least one, preferably at least two sialyltransferase(s) as described herein, chosen from the list comprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase; 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.

[0270] In another and/or additional preferred embodiment of the method and/or cell according to the disclosure, the mixture of at least three different sialylated oligosaccharides according to the disclosure can be produced by providing a cell that, for the production of LacNAc-based sialylated non-fucosylated oligosaccharides, is 1) capable of taking up LacNAc from the cultivation as described herein or is able to produce LacNAc as described herein; and 2) capable of expressing at least one, preferably at least two sialyltransferase(s) as described herein, chosen from the list comprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase; 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.

[0271] In another and/or additional preferred embodiment of the method and/or cell according to the disclosure, the mixture of at least three different sialylated oligosaccharides according to the disclosure can be produced by providing a cell that, for the production of lactose-based sialylated fucosylated oligosaccharides, is 1) capable of taking up lactose from the cultivation as described herein or is able to produce lactose after uptake of glucose by the action of a b-1,4-galactosyltransferase as described herein; and 2) capable of expressing at least one, preferably at least two, fucosyltransferase(s) as described herein, chosen from the list comprising alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase; 3) capable of expressing at least one, preferably at least two, sialyltransferase(s) as described herein, chosen from the list comprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase; 4) optionally capable of expressing a N-acetylglucosaminyltransferase as described herein, preferably a galactoside beta-1,3-N-acetylglucosaminyltransferase; 5) optionally capable of expressing at least one, preferably at least two, galactosyltransferase(s) as described herein, chosen from the list comprising N-acetylglucosamine beta-1,3-galactosyltransferase, N-acetylglucosamine beta-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase, alpha-1,4-galactosyltransferase; 6) optionally capable of expressing at least one, preferably at least two, N-acetylgalactosaminyltransferase(s) as described herein, chosen from the list comprising alpha-1,3-N-acetylgalactosaminyltransferase and beta-1,3-N-acetylgalactosaminyltransferase; 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) capable of synthesizing the nucleotide-sugar of each of the glycosyltransferase if present.

[0272] In another and/or additional preferred embodiment of the method and/or cell according to the disclosure, the mixture of at least three different sialylated oligosaccharides according to the disclosure can be produced by providing a cell that, for the production of LNB-based sialylated fucosylated oligosaccharides, is 1) capable to of taking up LNB from the cultivation as described herein or is able to produce LNB as described herein; and 2) capable of expressing at least one, preferably at least two, fucosyltransferase(s) as described herein, chosen from the list comprising alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase; 3) capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein; 4) capable of expressing at least one, preferably at least two sialyltransferase(s) as described herein, chosen from the list comprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase; 5) capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein; and 6) optionally capable of producing UDP-galactose.

[0273] In another and/or additional preferred embodiment of the method and/or cell according to the disclosure, the mixture of at least three different sialylated oligosaccharides according to the disclosure can be produced by providing a cell that, for the production of LacNAc-based sialylated fucosylated oligosaccharides, is 1) capable of taking up LacNAc from the cultivation as described herein or is able to produce LacNAc as described herein; and 2) capable of expressing at least one, preferably at least two, fucosyltransferase(s) as described herein, chosen from the list comprising alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase; 3) capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein; 4) capable of expressing at least one, preferably at least two sialyltransferase(s) as described herein, chosen from the list comprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase; 5) capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein; and 6) optionally capable of producing UDP-galactose.

[0274] In another and/or additional preferred embodiment of the method and/or cell according to the disclosure, a cell is provided that is additionally adapted for the production of lactose-based neutral non-fucosylated oligosaccharides, lactose-based neutral fucosylated oligosaccharides, LNB-based neutral non-fucosylated oligosaccharides, LNB-based neutral fucosylated oligosaccharides, LacNAc-based neutral non-fucosylated oligosaccharides and/or LacNAc-based neutral fucosylated oligosaccharides.

[0275] A cell adapted for the production of lactose-based neutral non-fucosylated oligosaccharides is 1) capable of taking up lactose from the cultivation as described herein or is able to produce lactose after uptake of glucose by the action of a b-1,4-galactosyltransferase as described herein; and 2) capable of expressing an N-acetylglucosaminyltransferase as described herein, preferably a galactoside beta-1,3-N-acetylglucosaminyltransferase; 3) optionally capable of expressing at least one, preferably at least two, galactosyltransferase(s) as described herein, chosen from the list comprising an N-acetylglucosamine beta-1,3-galactosyltransferase, an N-acetylglucosamine beta-1,4-galactosyltransferase, an alpha-1,3-galactosyltransferase, an alpha-1,4-galactosyltransferase; and 4) optionally capable of expressing at least one, preferably at least two, N-acetylgalactosaminyltransferase(s) as described herein, chosen from the list comprising an alpha-1,3-N-acetylgalactosaminyltransferase and a beta-1,3-N-acetylgalactosaminyltransferase and 5) capable of synthesizing the nucleotide-sugar of each of the glycosyltransferases if present.

[0276] A cell adapted for the production of lactose-based neutral fucosylated oligosaccharides is 1) capable to take up lactose from the cultivation as described herein or is able to produce lactose after uptake of glucose by the action of a b-1,4-galactosyltransferase as described herein; and 2) capable of expressing at least one, preferably at least two, fucosyltransferase(s) as described herein, chosen from the list comprising alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase; 3) optionally capable of expressing an N-acetylglucosaminyltransferase as described herein, preferably a galactoside beta-1,3-N-acetylglucosaminyltransferase; 4) optionally capable of expressing at least one, preferably at least two, galactosyltransferase(s) as described herein, chosen from the list comprising N-acetylglucosamine beta-1,3-galactosyltransferase, N-acetylglucosamine beta-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase, alpha-1,4-galactosyltransferase; 5) optionally capable of expressing at least one, preferably at least two, N-acetylgalactosaminyltransferase(s) as described herein, chosen from the list comprising alpha-1,3-N-acetylgalactosaminyltransferase and beta-1,3-N-acetylgalactosaminyltransferase; 6) capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein, and 7) capable of synthesizing the nucleotide-sugar of each of the glycosyltransferase if present.

[0277] A cell adapted for the production of LNB-based neutral non-fucosylated oligosaccharides is able to produce LNB as described herein or is capable to take up LNB from the cultivation as described herein; and capable of synthesizing UDP-Gal.

[0278] A cell adapted for the production of LNB-based neutral fucosylated oligosaccharides is 1) capable to take up LNB from the cultivation as described herein or is able to produce LNB as described herein; and 2) capable of expressing at least one, preferably at least two, fucosyltransferase(s) as described herein, chosen from the list comprising alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase; 3) capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein, and 4) optionally capable to produce UDP-galactose.

[0279] A cell adapted for the production of LacNAc-based neutral non-fucosylated oligosaccharides is able to produce LacNAc as described herein; and capable of synthesizing UDP-Gal.

[0280] A cell adapted for the production of LacNAc-based neutral fucosylated oligosaccharides is 1) capable to take up LacNAc from the cultivation as described herein or is able to produce LacNAc as described herein; and 2) capable of expressing at least one, preferably at least two, fucosyltransferase(s) as described herein, chosen from the list comprising alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase; 3) capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein, and 4) optionally capable to produce UDP-galactose.

[0281] In another more preferred embodiment of the method and/or cell according to the disclosure, a mixture of at least three different sialylated oligosaccharides comprising sialylated lactose-based oligosaccharides like e.g., sialyllactose(s) and sialylated lacto-N-triose and sialylated Lacto-N-tetraose(s) and/or sialylated lacto-N-neotetraose(s) and no fucosylated oligosaccharides can be produced by providing a cell that is 1) capable to take up lactose from the cultivation as described herein or is able to produce lactose after uptake of glucose by the action of a b-1,4-galactosyltransferase as described herein; and 2) capable of expressing at least one, preferably at least two, sialyltransferase(s) as described herein, chosen from the list comprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase; 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 a galactoside beta-1,3-N-acetylglucosaminyltransferase; 5) capable of expressing at least one, preferably at least two, galactosyltransferase(s) as described herein, chosen from the list comprising N-acetylglucosamine beta-1,3-galactosyltransferase, N-acetylglucosamine beta-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase, alpha-1,4-galactosyltransferase; 6) capable of synthesizing UDP-GlcNAc, preferably the cell has an N-acetylglucosaminylation pathway as defined herein; 7) capable of synthesizing UDP-Gal, preferably the cell has a galactosylation pathway as defined herein.

[0282] In another more preferred embodiment of the method and/or cell according to the disclosure, a mixture of at least three different sialylated oligosaccharides comprising sialylated and neutral lactose-based oligosaccharides like e.g., fucosyllactose(s), sialyllactose(s), LN3, fucosylated LNT and/or LNnT, sialylated lacto-N-triose and sialylated Lacto-N-tetraose(s) and/or sialylated lacto-N-neotetraose(s) can be produced by providing a cell that is 1) capable to take up lactose from the cultivation as described herein or is able to produce lactose after uptake of glucose by the action of a b-1,4-galactosyltransferase as described herein; and 2) capable of expressing at least one, preferably at least two, sialyltransferase(s) as described herein, chosen from the list comprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase; 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, fucosyltransferase(s) as described herein, chosen from the list comprising alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase; 5) capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein; 6) capable of expressing an N-acetylglucosaminyltransferase as described herein, preferably a galactoside beta-1,3-N-acetylglucosaminyltransferase; 7) capable of expressing at least one, preferably at least two, galactosyltransferase(s) as described herein, chosen from the list comprising N-acetylglucosamine beta-1,3-galactosyltransferase, N-acetylglucosamine beta-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase, alpha-1,4-galactosyltransferase; 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 galactosylation pathway as defined herein.

[0283] Exemplary methods and cells according to the disclosure are described in the Examples section. It is emphasized that these examples show at least one way to produce the specific mixtures. The person skilled in the art will understand that any of the expressed enzymes can be replaced by another enzyme if it has the same catalytic activity, preferably to a similar extent, which can be readily assessed through routine experimentation wherein the activity of an enzyme is compared with the activity of a reference enzyme as disclosed herein (e.g., in vitro conversion of a substrate).

[0284] In a preferred embodiment of the method and/or cell of the disclosure, any one of the oligosaccharides, more preferably all of the oligosaccharides, is/are translocated to the outside of the cell by a passive transport i.e., without means of an active transport system consuming energy from the cell.

[0285] In a preferred embodiment of the method and/or cell of the disclosure, the cell uses at least one precursor for the production of any one or more of the oligosaccharides. The term precursor should be understood as explained in the definitions as disclosed herein. In a more preferred embodiment, the cell uses two or more precursors for the production of any one or more of the oligosaccharides.

[0286] In a preferred embodiment of the method of the disclosure, the cultivation is fed with a precursor and/or acceptor for the synthesis of any one of the oligosaccharides in the mixture. The term acceptor should be understood as explained in the definitions as disclosed herein. In a further preferred embodiment of the method, the cultivation is fed with at least two precursors and/or acceptors for the synthesis of any one or more, preferably all, of the oligosaccharides in the mixture. This can be useful if two or more glycosyltransferases of the same classification (e.g., a2,3-sialyltransferases) are used that have a different affinity (e.g., one sialyltransferase having affinity to lactose and the other sialyltransferase having affinity to LNB) for the production of a mixture of oligosaccharides according to this disclosure.

[0287] In another embodiment of the method and/or cell as described herein, the cell is producing a precursor for the production of any one of the oligosaccharides. In a preferred embodiment, the cell is producing one or more precursors for the synthesis of the oligosaccharide mixture. In a more preferred embodiment, the cell is modified for optimized production of any one of the precursors for the synthesis of any one of the oligosaccharides.

[0288] In a preferred embodiment of the method and/or cell of the disclosure, at least one precursor for the production of any one of the oligosaccharides is completely converted into any one of the oligosaccharides. In a more preferred embodiment, the cell completely converts any one of the precursors into any one of the oligosaccharides.

[0289] In another preferred embodiment of the method and/or cell of the disclosure, the cell is further metabolically engineered for: [0290] i) modified expression of an endogenous membrane protein, and/or [0291] ii) modified activity of an endogenous membrane protein, and/or [0292] iii) expression of a homologous membrane protein, and/or [0293] iv) expression of a heterologous membrane protein, [0294] wherein the membrane protein is involved in the secretion of any one of the oligosaccharides outside the cell. The cell can express one of the membrane proteins that is involved in the secretion of any one of the oligosaccharides from the cell to the outside of the cell. The cell can also express more than one of the membrane proteins. Any one of the membrane proteins can translocate one or more of the oligosaccharides to the outside of the cell. The cell producing a mixture of at least three oligosaccharides can translocate any one of the oligosaccharides comprising passive diffusion, channel membrane proteins, membrane transporter proteins, membrane carrier proteins. [0295] i) In another preferred embodiment of the method and/or cell of the disclosure, the cell is further metabolically engineered for: [0296] ii) modified expression of an endogenous membrane protein, and/or [0297] iii) modified activity of an endogenous membrane protein, and/or [0298] iv) expression of a homologous membrane protein, and/or expression of a heterologous membrane protein, [0299] wherein the membrane protein is involved in the uptake of a precursor and/or an acceptor for the synthesis of any one of the oligosaccharides. The cell can express one of the membrane proteins that is involved in the uptake of any type of precursor and/or acceptor used in the synthesis of any one of the oligosaccharides. The cell can also express more than one of the membrane proteins, involved in the uptake of at least one of the precursors and/or acceptors. The cell can be modified for the uptake of more than one precursor and/or acceptor for the synthesis of any one of the oligosaccharides. In a preferred embodiment, the cell is modified for the uptake of all the required precursors. In another preferred embodiment, the cell is modified for the uptake of all the acceptors.

[0300] In a more preferred embodiment of the method and/or cell of the disclosure, the membrane protein is chosen from the list comprising porters, P-P-bond-hydrolysis-driven transporters, ?-barrel porins, auxiliary transport proteins, putative transport proteins and phosphotransfer-driven group translocators. In an even more preferred embodiment of the method and/or cell of the disclosure, the porters comprise MFS transporters, sugar efflux transporters and siderophore exporters. In another more preferred embodiment of the method and/or cell of the disclosure, the P-P-bond-hydrolysis-driven transporters comprise ABC transporters and siderophore exporters.

[0301] In another preferred embodiment of the method and/or cell of the disclosure, the membrane protein provides improved production of any one of the oligosaccharides, preferably all of the oligosaccharides. In an alternative and/or additional preferred embodiment of the method and/or cell of the disclosure, the membrane protein provides enabled efflux of any one of the oligosaccharides, preferably all of the oligosaccharides. In an alternative and/or additional preferred embodiment of the method and/or cell of the disclosure, the membrane protein provides enhanced efflux of any one of the oligosaccharides, preferably all of the oligosaccharides.

[0302] In a more preferred embodiment of the method and/or cell of this disclosure, the cell expresses a membrane protein belonging to the family of MFS transporters like e.g., an MdfA polypeptide of the multidrug transporter MdfA family from species comprising E. coli (UniProt ID POAEY8), Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), Citrobacter youngae (UniProt ID D4BC23) and Yokenella regensburgei (UniProt ID G9Z5F4). In another more preferred embodiment of the method and/or cell of this disclosure, the cell expresses a membrane protein belonging to the family of sugar efflux transporters like e.g., a SetA polypeptide of the SetA family from species comprising E. coli (UniProt ID P31675), Citrobacter koseri (UniProt ID A0A078LM16) and Klebsiella pneumoniae (UniProt ID A0A0C4MGS7). In another more preferred embodiment of the method and/or cell of this disclosure, the cell expresses a membrane protein belonging to the family of siderophore exporters like e.g., the E. coli entS (UniProt ID P24077) and the E. coli iceT (UniProt ID A0A024L207). In another more preferred embodiment of the method and/or cell of this disclosure, the cell expresses a membrane protein belonging to the family of ABC transporters like e.g., oppF from E. coli (UniProt ID P77737), ImrA from Lactococcus lactis subsp. lactis bv. diacetylactis (UniProt ID A0A1VONEL4) and Blon 2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4).

[0303] In a preferred embodiment of the method and/or cell of the disclosure, the cell confers enhanced bacteriophage resistance. The enhancement of bacteriophage resistance can be derived from reduced expression of an endogenous membrane protein and/or mutation of an endogenous membrane protein encoding gene. The term phage insensitive or phage resistant or phage resistance or phage resistant profile is understood to mean a bacterial strain that is less sensitive, and preferably insensitive to infection and/or killing by phage and/or growth inhibition. As used herein, the terms anti-phage activity or resistant to infection by at least one phage refers to an increase in resistance of a bacterial cell expressing a functional phage resistance system to infection by at least one phage family in comparison to a bacterial cell of the same species under the same developmental stage (e.g., culture state) that does not express a functional phage resistance system, as may be determined by e.g., bacterial viability, phage lysogeny, phage genomic replication and phage genomic degradation. The phage can be a lytic phage or a temperate (lysogenic) phage. Membrane proteins involved in bacteriophage resistance of a cell comprise OmpA, OmpC, OmpF, OmpT, BtuB, TolC, LamB, FhuA, TonB, FadL, Tsx, FepA, YncD, PhoE, and NfrA and homologs thereof.

[0304] In a preferred embodiment of the method and/or cell of the disclosure, the cell confers reduced viscosity. Reduced viscosity of a cell can be obtained by a modified cell wall biosynthesis. Cell wall biosynthesis can be modified comprising reduced or abolished synthesis of, for example, poly-N-acetyl-glucosamine, the enterobacterial common antigen, cellulose, colanic acid, core oligosaccharides, osmoregulated periplasmic glucans and glucosylglycerol, glycan, and trehalose.

[0305] According to another embodiment of the method and/or cell of this disclosure, the cell is capable to produce phosphoenolpyruvate (PEP). In a preferred embodiment of the method and/or cell of this disclosure, the cell is modified for enhanced production and/or supply of PEP compared to a non-modified progenitor.

[0306] In a preferred embodiment and as a means for enhanced production and/or supply of PEP, one or more PEP-dependent, sugar-transporting phosphotransferase system(s) is/are disrupted such as but not limited to: 1) the N-acetyl-D-glucosamine Npi-phosphotransferase (EC 2.7.1.193), which is encoded, for instance, by the nagE gene (or the cluster nagABCD) in E. coli or Bacillus species, 2) ManXYZ that encodes the Enzyme Il Man complex (mannose PTS permease, protein-Npi-phosphohistidine-D-mannose phosphotransferase) that imports exogenous hexoses (mannose, glucose, glucosamine, fructose, 2-deoxyglucose, mannosamine, N-acetylglucosamine, etc.) and releases the phosphate esters into the cell cytoplasm, 3) the glucose-specific PTS transporter (for instance, encoded by PtsG/Crr) that takes up glucose and forms glucose-6-phosphate in the cytoplasm, 4) the sucrose-specific PTS transporter that takes up sucrose and forms sucrose-6-phosphate in the cytoplasm, 5) the fructose-specific PTS transporter (for instance, encoded by the genes fruA and fruB and the kinase fruk that takes up fructose and forms in a first step fructose-1-phosphate and in a second step fructose1,6 bisphosphate, 6) the lactose PTS transporter (for instance, encoded by lacE in Lactococcus casei) that takes up lactose and forms lactose-6-phosphate, 7) the galactitol-specific PTS enzyme that takes up galactitol and/or sorbitol and forms galactitol-1-phosphate or sorbitol-6-phosphate, respectively, 8) the mannitol-specific PTS enzyme that takes up mannitol and/or sorbitol and forms mannitol-1-phosphate or sorbitol-6-phosphate, respectively, and 9) the trehalose-specific PTS enzyme that takes up trehalose and forms trehalose-6-phosphate.

[0307] In another and/or additional preferred embodiment and as a means for enhanced production and/or supply of PEP, the full PTS system is disrupted by disrupting the PtsIH/Crr gene cluster. PtsI (Enzyme I) is a cytoplasmic protein that serves as the gateway for the phosphoenolpyruvate:sugar phosphotransferase system (PTSsugar) of E. coli K-12. PtsI is one of two (PtsI and PtsH) sugar non-specific protein constituents of the PTSsugar that along with a sugar-specific inner membrane permease effects a phosphotransfer cascade that results in the coupled phosphorylation and transport of a variety of carbohydrate substrates. HPr (histidine containing protein) is one of two sugar-non-specific protein constituents of the PTSsugar. It accepts a phosphoryl group from phosphorylated Enzyme I (PtsI-P) and then transfers it to the EIIA domain of any one of the many sugar-specific enzymes (collectively known as Enzymes II) of the PTSsugar. Crr or EIIAGlc is phosphorylated by PEP in a reaction requiring PtsH and PtsI.

[0308] In another and/or additional preferred embodiment, the cell is further modified to compensate for the deletion of a PTS system of a carbon source by the introduction and/or overexpression of the corresponding permease. These are e.g., permeases or ABC transporters that comprise but are not limited to transporters that specifically import lactose such as e.g., the transporter encoded by the LacY gene from E. coli, sucrose such as e.g., the transporter encoded by the cscB gene from E. coli, glucose such as e.g., the transporter encoded by the galP gene from E. coli, fructose such as e.g., the transporter encoded by the fruI gene from Streptococcus mutans, or the Sorbitol/mannitol ABC transporter such as the transporter encoded by the cluster SmoEFGK of Rhodobacter sphaeroides, the trehalose/sucrose/maltose transporter such as the transporter encoded by the gene cluster ThuEFGK of Sinorhizobium meliloti and the N-acetylglucosamine/galactose/glucose transporter such as the transporter encoded by NagP of Shewanella oneidensis. Examples of combinations of PTS deletions with overexpression of alternative transporters are: 1) the deletion of the glucose PTS system, e.g., ptsG gene, combined with the introduction and/or overexpression of a glucose permease (e.g., galP of glcP), 2) the deletion of the fructose PTS system, e.g., one or more of the fruB, fruA, fruk genes, combined with the introduction and/or overexpression of fructose permease, e.g., fruI, 3) the deletion of the lactose PTS system, combined with the introduction and/or overexpression of lactose permease, e.g., LacY, and/or 4) the deletion of the sucrose PTS system, combined with the introduction and/or overexpression of a sucrose permease, e.g., cscB. [0309] In a further preferred embodiment, the cell is modified to compensate for the deletion of a PTS system of a carbon source by the introduction of carbohydrate kinases, such as glucokinase (EC 2.7.1.1, EC 2.7.1.2, EC 2.7.1.63), galactokinase (EC 2.7.1.6), and/or fructokinase (EC 2.7.1.3, EC 2.7.1.4). Examples of combinations of PTS deletions with overexpression of alternative transporters and a kinase are: 1) the deletion of the glucose PTS system, e.g., ptsG gene, combined with the introduction and/or overexpression of a glucose permease (e.g., galP of glcP), combined with the introduction and/or overexpression of a glucokinase (e.g., glk), and/or 2) the deletion of the fructose PTS system, e.g., one or more of the fruB, fruA, fruk genes, combined with the introduction and/or overexpression of fructose permease, e.g., fruI, combined with the introduction and/or overexpression of a fructokinase (e.g., frk or mak).

[0310] In another and/or additional preferred embodiment and as a means for enhanced production and/or supply of PEP, the cell is modified by the introduction of or modification in any one or more of the list comprising phosphoenolpyruvate synthase activity (EC: 2.7.9.2 encoded in, for instance, E. coli by ppsA), phosphoenolpyruvate carboxykinase activity (EC 4.1.1.32 or EC 4.1.1.49 encoded in, for instance, Corynebacterium glutamicum by PCK or in E. coli by pckA, resp.), phosphoenolpyruvate carboxylase activity (EC 4.1.1.31 encoded in, for instance, E. coli by ppc), oxaloacetate decarboxylase activity (EC 4.1.1.112 encoded in, for instance, E. coli by eda), pyruvate kinase activity (EC 2.7.1.40 encoded in, for instance, E. coli by pykA and pykF), pyruvate carboxylase activity (EC 6.4.1.1 encoded in, for instance, B. subtilis by pyc) and malate dehydrogenase activity (EC 1.1.1.38 or EC 1.1.1.40 encoded in, for instance, E. coli by maeA or maeB, resp.).

[0311] In a more preferred embodiment, the cell is modified to overexpress any one or more of the polypeptides comprising ppsA from E. coli (UniProt ID P23538), PCK from C. glutamicum (UniProt ID Q6F5A5), pcka from E. coli (UniProt ID P22259), eda from E. coli (UniProt ID POA955), maeA from E. coli (UniProt ID P26616) and maeB from E. coli (UniProt ID P76558).

[0312] In another and/or additional preferred embodiment, the cell is modified to express any one or more polypeptide having phosphoenolpyruvate synthase activity, phosphoenolpyruvate carboxykinase activity, oxaloacetate decarboxylase activity, or malate dehydrogenase activity.

[0313] In another and/or additional preferred embodiment and as a means for enhanced production and/or supply of PEP, the cell is modified by a reduced activity of phosphoenolpyruvate carboxylase activity, and/or pyruvate kinase activity, preferably a deletion of the genes encoding for phosphoenolpyruvate carboxylase, the pyruvate carboxylase activity and/or pyruvate kinase.

[0314] In an exemplary embodiment, the cell is genetically modified by different adaptations such as the overexpression of phosphoenolpyruvate synthase combined with the deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate synthase combined with the deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the deletion of a pyruvate carboxylase gene, the overexpression of phosphoenolpyruvate carboxykinase combined with the deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate carboxykinase combined with the deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate carboxykinase combined with the deletion of a pyruvate carboxylase gene, the overexpression of oxaloacetate decarboxylase combined with the deletion of a pyruvate kinase gene, the overexpression of oxaloacetate decarboxylase combined with the deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of oxaloacetate decarboxylase combined with the deletion of a pyruvate carboxylase gene, the overexpression of malate dehydrogenase combined with the deletion of a pyruvate kinase gene, the overexpression of malate dehydrogenase combined with the deletion of a phosphoenolpyruvate carboxylase gene and/or the overexpression of malate dehydrogenase combined with the deletion of a pyruvate carboxylase gene.

[0315] In another exemplary embodiment, the cell is genetically modified by different adaptations such as the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of an oxaloacetate decarboxylase, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a malate dehydrogenase, the overexpression of phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase, the overexpression of phosphoenolpyruvate carboxykinase combined with the overexpression of a malate dehydrogenase, the overexpression of oxaloacetate decarboxylase combined with the overexpression of a malate dehydrogenase, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of a malate dehydrogenase, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase, the overexpression of phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase and/or the overexpression of phosphoenolpyruvate synthase combined with the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase.

[0316] In another exemplary embodiment, the cell is genetically modified by different adaptations such as the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase combined with the deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of an oxaloacetate decarboxylase combined with the deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase combined with the deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate carboxykinase combined with the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene, the overexpression of an oxaloacetate decarboxylase combined with the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase combined with the deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene, the overexpression of a phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene, the overexpression of phosphoenolpyruvate synthase combined the overexpression of oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene.

[0317] In another exemplary embodiment, the cell is genetically modified by different adaptations such as the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase combined with the deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of an oxaloacetate decarboxylase combined with the deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a malate dehydrogenase combined with the deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase combined with the deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate carboxykinase combined with the overexpression of a malate dehydrogenase combined with the deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of an oxaloacetate decarboxylase combined with the overexpression of a malate dehydrogenase combined with the deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase combined with the deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of a malate dehydrogenase combined with the deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a phosphoenolpyruvate carboxylase gene.

[0318] In another exemplary embodiment, the cell is genetically modified by different adaptations such as the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase combined with the deletion of a pyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of an oxaloacetate decarboxylase combined with the deletion of a pyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase combined with the deletion of a pyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate carboxykinase combined with the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate carboxylase gene, the overexpression of an oxaloacetate decarboxylase combined with the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase combined with the deletion of a pyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate carboxylase gene.

[0319] In another exemplary embodiment, the cell is genetically modified by different adaptations such as the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase combined with the deletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of an oxaloacetate decarboxylase combined with the deletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase combined with the deletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate carboxykinase combined with the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of an oxaloacetate decarboxylase combined with the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase combined with the deletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate synthase combined with the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene.

[0320] In another exemplary embodiment, the cell is genetically modified by different adaptations such as the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase combined with the deletion of a pyruvate kinase gene and a pyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of an oxaloacetate decarboxylase combined with the deletion of a pyruvate kinase gene and a pyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and a pyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase combined with the deletion of a pyruvate kinase gene and a pyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate carboxykinase combined with the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and a pyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of an oxaloacetate decarboxylase combined with the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and a pyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase combined with the deletion of a pyruvate kinase gene and a pyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and a pyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of a phosphoenolpyruvate carboxykinase and the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and pyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of a phosphoenolpyruvate carboxykinase combined with the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and a pyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase combined with the overexpression of an oxaloacetate decarboxylase and the overexpression of a malate dehydrogenase combined with the deletion of a pyruvate kinase gene and a pyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene.

[0321] According to another preferred embodiment of the method and/or cell of the disclosure, the cell comprises a modification for reduced production of acetate compared to a non-modified progenitor. The modification can be any one or more chosen from the group comprising overexpression of an acetyl-coenzyme A synthetase, a full or partial knock-out or rendered less functional pyruvate dehydrogenase and a full or partial knock-out or rendered less functional lactate dehydrogenase.

[0322] In a further embodiment of the method and/or cell of the disclosure, the cell is modified in the expression or activity of at least one acetyl-coenzyme A synthetase like e.g., acs from E. coli, S. cerevisiae, H. sapiens, M. musculus. In a preferred embodiment, the acetyl-coenzyme A synthetase is an endogenous protein of the cell with a modified expression or activity, preferably the endogenous acetyl-coenzyme A synthetase is overexpressed; alternatively, the acetyl-coenzyme A synthetase is a heterologous protein that is heterogeneously introduced and expressed in the cell, preferably overexpressed. The endogenous acetyl-coenzyme A synthetase can have a modified expression in the cell that also expresses a heterologous acetyl-coenzyme A synthetase. In a more preferred embodiment, the cell is modified in the expression or activity of the acetyl-coenzyme A synthetase acs from E. coli (UniProt ID P27550). In another and/or additional preferred embodiment, the cell is modified in the expression or activity of a functional homolog, variant or derivative of acs from E. coli (UniProt ID P27550) having at least 80% overall sequence identity to the full-length of the polypeptide from E. coli (UniProt ID P27550) and having acetyl-coenzyme A synthetase activity.

[0323] In an alternative and/or additional further embodiment of the method and/or cell of the disclosure, the cell is modified in the expression or activity of at least one pyruvate dehydrogenase like e.g., from E. coli, S. cerevisiae, H. sapiens and R. norvegicus. In a preferred embodiment, the cell has been modified to have at least one partially or fully knocked out or mutated pyruvate dehydrogenase encoding gene by means generally known by the person skilled in the art resulting in at least one protein with less functional or being disabled for pyruvate dehydrogenase activity. In a more preferred embodiment, the cell has a full knock-out in the poxB encoding gene resulting in a cell lacking pyruvate dehydrogenase activity.

[0324] In an alternative and/or additional further embodiment of the method and/or cell of the disclosure, the cell is modified in the expression or activity of at least one lactate dehydrogenase like e.g., from E. coli, S. cerevisiae, H. sapiens and R. norvegicus. In a preferred embodiment, the cell has been modified to have at least one partially or fully knocked out or mutated lactate dehydrogenase encoding gene by means generally known by the person skilled in the art resulting in at least one protein with less functional or being disabled for lactate dehydrogenase activity. In a more preferred embodiment, the cell has a full knock-out in the ldhA encoding gene resulting in a cell lacking lactate dehydrogenase activity.

[0325] According to another preferred embodiment of the method and/or cell of the disclosure, the cell comprises a lower or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the proteins comprising beta-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridylyltransferase, glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IcIR, lon protease, glucose-specific translocating phosphotransferase enzyme IIBC component ptsG, glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIAGlc, beta-glucoside specific PTS enzyme II, fructose-specific PTS multiphosphoryl transfer protein FruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, pyruvate decarboxylase compared to a non-modified progenitor.

[0326] According to another preferred embodiment of the method and/or cell of the disclosure, the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides that is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the production of any one of the oligosaccharides from the mixture.

[0327] Another embodiment of the disclosure provides for a method and a cell wherein a mixture comprising at least three different sialylated oligosaccharides is produced in and/or by a fungal, yeast, bacterial, insect, animal, plant and protozoan cell as described herein. The cell is chosen from the list comprising a bacterium, a yeast, a protozoan or a fungus, or, refers to a plant or animal cell. The latter bacterium preferably belongs to the phylum of the Proteobacteria or the phylum of the Firmicutes or the phylum of the Cyanobacteria or the phylum Deinococcus-Thermus. The latter bacterium belonging to the phylum Proteobacteria belongs preferably to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacterium preferably relates to any strain belonging to the species Escherichia coli such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to cultivated Escherichia coli strainsdesignated as E. coli K12 strainswhich are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. Well-known examples of the E. coli K12 strains are K12 Wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Hence, this disclosure specifically relates to a mutated and/or transformed Escherichia coli cell or strain as indicated above wherein the E. coli strain is a K12 strain. More preferably, the Escherichia coli K12 strain is E. coli MG1655. The latter bacterium belonging to the phylum Firmicutes belongs preferably to the Bacilli, preferably Lactobacilliales, with members such as Lactobacillus lactis, Leuconostoc mesenteroides, or Bacillales with members such as from the genus Bacillus, such as Bacillus subtilis or, B. amyloliquefaciens. The latter Bacterium belonging to the phylum Actinobacteria, preferably belonging to the family of the Corynebacteriaceae, with members Corynebacterium glutamicum or C. afermentans, or belonging to the family of the Streptomycetaceae with members Streptomyces griseus or S. fradiae. The latter yeast preferably belongs to the phylum of the Ascomycota or the phylum of the Basidiomycota or the phylum of the Deuteromycota or the phylum of the Zygomycetes. The latter yeast belongs preferably to the genus Saccharomyces (with members like e.g., Saccharomyces cerevisiae, S. bayanus, S. boulardii), Pichia (with members like e.g., Pichia pastoris, P. anomala, P. kluyveri), Komagataella, Hansenula, Kluyveromyces (with members like e.g., Kluyveromyces lactis, K. marxianus, K. thermotolerans), Debaryomyces, Yarrowia (like e.g., Yarrowia lipolytica) or Starmerella (like e.g., Starmerella bombicola). The latter yeast is preferably selected from Pichia pastoris, Yarrowia lipolitica, Saccharomyces cerevisiae and Kluyveromyces lactis. The latter fungus belongs preferably to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus. Plant cells include cells of flowering and non-flowering plants, as well as algal cells, for example, Chlamydomonas, Chlorella, etc. Preferably, the plant is a tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy, maize or corn plant. The latter animal cell is preferably derived from non-human mammals (e.g., cattle, buffalo, pig, sheep, mouse, rat), birds (e.g., chicken, duck, ostrich, turkey, pheasant), fish (e.g., swordfish, salmon, tuna, sea bass, trout, catfish), invertebrates (e.g., lobster, crab, shrimp, clams, oyster, mussel, sea urchin), reptiles (e.g., snake, alligator, turtle), amphibians (e.g., frogs) or insects (e.g., fly, nematode) or is a genetically modified cell line derived from human cells excluding embryonic stem cells. Both human and non-human mammalian cells are preferably chosen from the list comprising an epithelial cell like e.g., a mammary epithelial cell, an embryonic kidney cell (e.g., HEK293 or HEK 293T cell), a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell like e.g., an N20, SP2/0 or YB2/0 cell, an NIH-3T3 cell, a non-mammary adult stem cell or derivatives thereof such as described in WO 2021067641. The latter insect cell is preferably derived from Spodoptera frugiperda like e.g., Sf9 or Sf21 cells, Bombyx mori, Mamestra brassicae, Trichoplusia ni like e.g., BTI-TN-5B1-4 cells or Drosophila melanogaster like e.g., Drosophila S2 cells. The latter protozoan cell preferably is a Leishmania tarentolae cell.

[0328] In a preferred embodiment of the method and/or cell of the disclosure, the cell is a viable Gram-negative bacterium that comprises a reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose compared to a non-modified progenitor.

[0329] In a more preferred embodiment of the method and/or cell, the reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose is provided by a mutation in any one or more glycosyltransferases involved in the synthesis of any one of the poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose, wherein the mutation provides for a deletion or lower expression of any one of the glycosyltransferases. The glycosyltransferases comprise glycosyltransferase genes encoding poly-N-acetyl-D-glucosamine synthase subunits, UDP-N-acetylglucosamine-undecaprenyl-phosphate N-acetylglucosaminephosphotransferase, Fuc4NAc (4-acetamido-4,6-dideoxy-D-galactose) transferase, UDP-N-acetyl-D-mannosaminuronic acid transferase, the glycosyltransferase genes encoding the cellulose synthase catalytic subunits, the cellulose biosynthesis protein, colanic acid biosynthesis glucuronosyltransferase, colanic acid biosynthesis galactosyltransferase, colanic acid biosynthesis fucosyltransferase, UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase, putative colanic biosynthesis glycosyl transferase, UDP-glucuronate:LPS(HepIII) glycosyltransferase, ADP-heptose-LPS heptosyltransferase 2, ADP-heptose:LPS heptosyltransferase 1, putative ADP-heptose:LPS heptosyltransferase 4, lipopolysaccharide core biosynthesis protein, UDP-glucose:(glucosyl)LPS ?-1,2-glucosyltransferase, UDP-D-glucose:(glucosyl)LPS ?-1,3-glucosyltransferase, UDP-D-galactose:(glucosyl)lipopolysaccharide-1,6-D-galactosyltransferase, lipopolysaccharide glucosyltransferase I, lipopolysaccharide core heptosyltransferase 3, ?-1,6-galactofuranosyltransferase, undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase, lipid IVA 4-amino-4-deoxy-L-arabinosyltransferase, bactoprenol glucosyl transferase, putative family 2 glycosyltransferase, the osmoregulated periplasmic glucans (OPG) biosynthesis protein G, OPG biosynthesis protein H, glucosylglycerate phosphorylase, glycogen synthase, 1,4-?-glucan branching enzyme, 4-?-glucanotransferase and trehalose-6-phosphate synthase. In an exemplary embodiment, the cell is mutated in any one or more of the glycosyltransferases comprising pgaC, pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcal, wcaJ, wcaL, waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbl, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA and yaiP, wherein the mutation provides for a deletion or lower expression of any one of the glycosyltransferases.

[0330] In an alternative and/or additional preferred embodiment of the method and/or cell, the reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG) is provided by over-expression of a carbon storage regulator encoding gene, deletion of a Na+/H+antiporter regulator encoding gene and/or deletion of the sensor histidine kinase encoding gene.

[0331] The microorganism or cell as used herein is capable to grow on a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone, yeast extract or a mixture thereof like e.g., a mixed feedstock, preferably a mixed monosaccharide feedstock like e.g., hydrolyzed sucrose, as the main carbon source. With the term complex medium is meant a medium for which the exact constitution is not determined. With the term main is meant the most important carbon source for the bioproducts of interest, biomass formation, carbon dioxide and/or by-products formation (such as acids and/or alcohols, such as acetate, lactate, and/or ethanol), i.e., 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99% of all the required carbon is derived from the above-indicated carbon source. In one embodiment of the disclosure, the carbon source is the sole carbon source for the organism, i.e., 100% of all the required carbon is derived from the above-indicated carbon source. Common main carbon sources comprise but are not limited to glucose, glycerol, fructose, maltose, lactose, arabinose, malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, sucrose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvate. With the term complex medium is meant a medium for which the exact constitution is not determined. Examples are molasses, corn steep liquor, peptone, tryptone or yeast extract.

[0332] In a further preferred embodiment, the microorganism or cell described herein is using a split metabolism having a production pathway and a biomass pathway as described in WO 2012/007481, which is herein incorporated by reference. The organism can, for example, be genetically modified to accumulate fructose-6-phosphate by altering the genes selected from the phosphoglucoisomerase gene, phosphofructokinase gene, fructose-6-phosphate aldolase gene, fructose isomerase gene, and/or fructose:PEP phosphotransferase gene.

[0333] According to another embodiment of the method of the disclosure, the conditions permissive to produce the oligosaccharides in the mixture comprise the use of a culture medium to cultivate a cell of this disclosure for the production of the oligosaccharide mixture wherein the culture medium lacks any precursor and/or acceptor for the production of any one of the oligosaccharides and is combined with a further addition to the culture medium of at least one precursor and/or acceptor feed for the production of any one of the oligosaccharides, preferably for the production of all of the oligosaccharides in the mixture.

[0334] In a preferred embodiment, the method for the production of an oligosaccharide mixture as described herein comprises at least one of the following steps: [0335] i) Use of a culture medium comprising at least one precursor and/or acceptor; [0336] ii) Adding to the culture medium in a reactor at least one precursor and/or acceptor feed wherein the total reactor volume ranges from 250 mL (milliliter) to 10,000 m.sup.3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the culture medium before the addition of the precursor and/or acceptor feed; [0337] iii) Adding to the culture medium in a reactor at least one precursor and/or acceptor feed wherein the total reactor volume ranges from 250 mL (milliliter) to 10,000 m.sup.3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the culture medium before the addition of the precursor and/or acceptor feed and wherein preferably, the pH of the precursor and/or acceptor feed is set between 3 and 7 and wherein preferably, the temperature of the precursor and/or acceptor feed is kept between 20? C. and 80? C.; [0338] iv) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; [0339] v) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein preferably, the pH of the feeding solution is set between 3 and 7 and wherein preferably, the temperature of the feeding solution is kept between 20? C. and 80? ? C.; [0340] the method resulting in any one of the oligosaccharides with a concentration of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L in the final cultivation.

[0341] In another and/or additional preferred embodiment, the method for the production of an oligosaccharide mixture as described herein comprises at least one of the following steps: [0342] i) Use of a culture medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per liter of initial reactor volume wherein the reactor volume ranges from 250 mL to 10,000 m.sup.3 (cubic meter); [0343] ii) Adding to the culture medium at least one precursor and/or acceptor in one pulse or in a discontinuous (pulsed) manner wherein the total reactor volume ranges from 250 mL (milliliter) to 10,000 m.sup.3 (cubic meter), preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the culture medium before the addition of the precursor and/or acceptor feed pulse(s); [0344] iii) Adding to the culture medium in a reactor at least one precursor and/or acceptor feed in one pulse or in a discontinuous (pulsed) manner wherein the total reactor volume ranges from 250 mL (milliliter) to 10,000 m.sup.3 (cubic meter), preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture medium before the addition of the precursor and/or acceptor feed pulse(s) and wherein preferably, the pH of the precursor and/or acceptor feed pulse(s) is set between 3 and 7 and wherein preferably, the temperature of the precursor and/or acceptor feed pulse(s) is kept between 20? C. and 80? C.; [0345] iv) Adding at least one precursor and/or acceptor feed in a discontinuous (pulsed) manner to the culture medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; [0346] v) Adding at least one precursor and/or acceptor feed in a discontinuous (pulsed) manner to the culture medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein preferably, the pH of the feeding solution is set between 3 and 7 and wherein preferably, the temperature of the feeding solution is kept between 20? C. and 80? ? C.; [0347] the method resulting in any one of the oligosaccharides with a concentration of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L in the final cultivation.

[0348] In a further, more preferred embodiment, the method for the production of an oligosaccharide mixture as described herein comprises at least one of the following steps: [0349] i) Use of a culture medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per liter of initial reactor volume wherein the reactor volume ranges from 250 mL to 10,000 m.sup.3 (cubic meter); [0350] ii) Adding to the culture medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per liter of initial reactor volume wherein the total reactor volume ranges from 250 mL (milliliter) to 10,000 m.sup.3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture medium before the addition of the lactose feed; [0351] iii) Adding to the culture medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per liter of initial reactor volume wherein the reactor volume ranges from 250 mL to 10,000 m.sup.3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture medium before the addition of the lactose feed and wherein preferably the pH of the lactose feed is set between 3 and 7 and wherein preferably the temperature of the lactose feed is kept between 20? C. and 80? C.; [0352] iv) Adding a lactose feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; [0353] v) Adding a lactose feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein the concentration of the lactose feeding solution is 50 g/L, preferably 75 g/L, more preferably 100 g/L, more preferably 125 g/L, more preferably 150 g/L, more preferably 175 g/L, more preferably 200 g/L, more preferably 225 g/L, more preferably 250 g/L, more preferably 275 g/L, more preferably 300 g/L, more preferably 325 g/L, more preferably 350 g/L, more preferably 375 g/L, more preferably, 400 g/L, more preferably 450 g/L, more preferably 500 g/L, even more preferably, 550 g/L, most preferably 600 g/L; and wherein preferably the pH of the feeding solution is set between 3 and 7 and wherein preferably the temperature of the feeding solution is kept between 20? C. and 80? C.; [0354] the method resulting in any one of the oligosaccharides with a concentration of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L in the final cultivation.

[0355] Preferably the lactose feed is accomplished by adding lactose from the beginning of the cultivation at a concentration of at least 5 mM, preferably in a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably at a concentration>300 mM.

[0356] In another embodiment the lactose feed is accomplished by adding lactose to the culture medium in a concentration, such that throughout the production phase of the cultivation a lactose concentration of at least 5 mM, preferably 10 mM or 30 mM is obtained.

[0357] In a further embodiment of the methods described herein the host cells are cultivated for at least about 60, 80, 100, or about 120 hours or in a continuous manner.

[0358] In a preferred embodiment, a carbon source is provided, preferably sucrose, in the culture medium for 3 or more days, preferably up to 7 days; and/or provided, in the culture medium, at least 100, advantageously at least 105, more advantageously at least 110, even more advantageously at least 120 grams of sucrose per liter of initial culture volume in a continuous manner, so that the final volume of the culture medium is not more than three-fold, advantageously not more than two-fold, more advantageously less than two-fold of the volume of the culturing medium before the culturing.

[0359] Preferably, when performing the method as described herein, a first phase of exponential cell growth is provided by adding a carbon source, preferably glucose or sucrose, to the culture medium before the precursor, preferably lactose, is added to the cultivation in a second phase.

[0360] In another preferred embodiment of the method of this disclosure, a first phase of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the culture medium comprising a precursor, preferably lactose, followed by a second phase wherein only a carbon-based substrate, preferably glucose or sucrose, is added to the cultivation.

[0361] In another preferred embodiment of the method of this disclosure, a first phase of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the culture medium comprising a precursor, preferably lactose, followed by a second phase wherein a carbon-based substrate, preferably glucose or sucrose, and a precursor, preferably lactose, are added to the cultivation.

[0362] In an alternative preferable embodiment, in the method as described herein, the precursor is added already in the first phase of exponential growth together with the carbon-based substrate.

[0363] In another preferred embodiment of the method, the culture medium contains at least one precursor selected from the group comprising lactose, galactose, fucose, sialic acid, GlcNAc, GalNAc, lacto-N-biose (LNB) and N-acetyllactosamine (LacNAc).

[0364] According to this disclosure, the method as described herein preferably comprises a step of separating of any one or more of the oligosaccharides, preferably all of the oligosaccharides, from the cultivation.

[0365] The terms separating from the cultivation means harvesting, collecting, or retrieving any one of the oligosaccharides, preferably all of the oligosaccharides, from the cell and/or the medium of its growth.

[0366] Any one of the oligosaccharides can be separated in a conventional manner from the aqueous culture medium, in which the cell was grown. In case the oligosaccharide is still present in the cells producing the oligosaccharide mixture, conventional manners to free or to extract the oligosaccharide out of the cells can be used, such as cell destruction using high pH, heat shock, sonication, French press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergent, hydrolysis. The culture medium and/or cell extract together and separately can then be further used for separating the oligosaccharide. This preferably involves clarifying the oligosaccharide containing mixture to remove suspended particulates and contaminants, particularly cells, cell components, insoluble metabolites and debris produced by culturing the genetically modified cell. In this step, the 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 of separating the oligosaccharide from the oligosaccharide containing mixture preferably involves removing substantially all the proteins, as well as peptides, amino acids, RNA and DNA and any endotoxins and glycolipids that could interfere with the subsequent separation step, from the oligosaccharide containing mixture, 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, color, endotoxins and other related impurities are removed from the oligosaccharide containing mixture by ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, electrophoresis (e.g., using slab-polyacrylamide or sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE)), affinity chromatography (using affinity ligands including e.g., DEAE-Sepharose, poly-L-lysine and polymyxin-B, endotoxin-selective adsorber matrices), ion exchange chromatography (such as but not limited to cation exchange, anion exchange, mixed bed ion exchange, inside-out ligand attachment), hydrophobic interaction chromatography and/or gel filtration (i.e., size exclusion chromatography), particularly by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography. With the exception of size exclusion chromatography, proteins and related impurities are retained by a chromatography medium or a selected membrane, the oligosaccharide remains in the oligosaccharide containing mixture.

[0367] In a further preferred embodiment, the methods as described herein also provide for a further purification of any one or more of the oligosaccharide(s) from the oligosaccharide mixture. A further purification of the oligosaccharide(s) may be accomplished, for example, by use of (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange to remove any remaining DNA, protein, LPS, endotoxins, or other impurity. Alcohols, such as ethanol, and aqueous alcohol mixtures can also be used. Another purification step is accomplished by crystallization, evaporation or precipitation of the product. Another purification step is to dry, e.g., spray dry, lyophilize, spray freeze dry, freeze spray dry, band dry, belt dry, vacuum band dry, vacuum belt dry, drum dry, roller dry, vacuum drum dry or vacuum roller dry the produced oligosaccharide(s).

[0368] In an exemplary embodiment, the separation and purification of at least one, preferably all, of the produced oligosaccharides is made in a process, comprising the following steps in any order: [0369] a) contacting the cultivation or a clarified version thereof with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da ensuring the retention of produced oligosaccharide(s) and allowing at least a part of the proteins, salts, by-products, color and other related impurities to pass, [0370] b) conducting a diafiltration process on the retentate from step a), using the membrane, with an aqueous solution of an inorganic electrolyte, followed by optional diafiltration with pure water to remove excess of the electrolyte, [0371] c) and collecting the retentate enriched in the oligosaccharide(s) in the form of a salt from the cation of the electrolyte.

[0372] In an alternative exemplary embodiment, the separation and purification of at least one, preferably all, of the produced oligosaccharides is made in a process, comprising the following steps in any order: subjecting the cultivation or a clarified version thereof to two membrane filtration steps using different membranes, wherein: [0373] one membrane has a molecular weight cut-off of between about 300 to about 500 Dalton, and [0374] the other membrane as a molecular weight cut-off of between about 600 to about 800 Dalton.

[0375] In an alternative exemplary embodiment, the separation and purification of at least one, preferably all, of the produced oligosaccharides is made in a process, comprising the following steps in any order comprising the step of treating the cultivation or a clarified version thereof with a strong cation exchange resin in H+-form and a weak anion exchange resin in free base form.

[0376] In an alternative exemplary embodiment, the separation and purification of at least one of the produced oligosaccharides is made in the following way. The cultivation comprising the produced oligosaccharide, biomass, medium components and contaminants is applied to the following separation and purification steps: [0377] i) separation of biomass from the cultivation, [0378] ii) cationic ion exchanger treatment for the removal of positively charged material, [0379] iii) anionic ion exchanger treatment for the removal of negatively charged material, [0380] iv) nanofiltration step and/or electrodialysis step, [0381] wherein a purified solution comprising the produced oligosaccharide(s) at a purity of greater than or equal to 80 percent is provided. Optionally the purified solution is dried by any one or more drying steps chosen from the list comprising spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying and vacuum roller drying.

[0382] In an alternative exemplary embodiment, the separation and purification of at least one, preferably all, of the produced oligosaccharides is made in a process, comprising the following steps in any order: enzymatic treatment of the cultivation; removal of the biomass from the cultivation; ultrafiltration; nanofiltration; and a column chromatography step. Preferably such column chromatography is a single column or a multiple column. Further preferably the column chromatography step is simulated moving bed chromatography. Such simulated moving bed chromatography preferably comprises i) at least 4 columns, wherein at least one column comprises a weak or strong cation exchange resin; and/or ii) four zones I, II, III and IV with different flow rates; and/or iii) an eluent comprising water; and/or iv) an operating temperature of 15 degrees to 60 degrees centigrade.

[0383] In a specific embodiment, this disclosure provides the produced oligosaccharide or oligosaccharide mixture that is dried to powder by any one or more drying steps chosen from the list comprising spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying and vacuum roller drying, wherein the dried powder contains<15 percent-wt. of water, preferably <10 percent-wt. of water, more preferably <7 percent-wt. of water, most preferably <5 percent-wt. of water.

[0384] In a third aspect, this disclosure provides for the use of a metabolically engineered cell as described herein for the production of a mixture comprising at least three different sialylated oligosaccharides.

[0385] For identification of the oligosaccharides in the mixture comprising at least three different sialylated oligosaccharides produced in the cell as described herein, the monomeric building blocks (e.g., the monosaccharide or glycan unit composition), the anomeric configuration of side chains, the presence and location of substituent groups, degree of polymerization/molecular weight and the linkage pattern can be identified by standard methods known in the art, such as, e.g., methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas chromatography-mass spectrometry), MALDI-MS (Matrix-assisted laser desorption/ionization-mass spectrometry), ESI-MS (Electrospray ionization-mass spectrometry), HPLC (High-Performance Liquid chromatography with ultraviolet or refractive index detection), HPAEC-PAD (High-Performance Anion-Exchange chromatography with Pulsed Amperometric Detection), CE (capillary electrophoresis), IR (infrared)/Raman spectroscopy, and NMR (Nuclear magnetic resonance) spectroscopy techniques. The crystal structure can be solved using, e.g., solid-state NMR, FT-IR (Fourier transform infrared spectroscopy), and WAXS (wide-angle X-ray scattering). The degree of polymerization (DP), the DP distribution, and polydispersity can be determined by, e.g., viscosimetry and SEC (SEC-HPLC, high performance size-exclusion chromatography). To identify the monomeric components of the saccharide methods such as, e.g., acid-catalyzed hydrolysis, HPLC (high performance liquid chromatography) or GLC (gas-liquid chromatography) (after conversion to alditol acetates) may be used. To determine the glycosidic linkages, the saccharide is methylated with methyl iodide and strong base in DMSO, hydrolysis is performed, a reduction to partially methylated alditols is achieved, an acetylation to methylated alditol acetates is performed, and the analysis is carried out by GLC/MS (gas-liquid chromatography coupled with mass spectrometry). To determine the oligosaccharide sequence, a partial depolymerization is carried out using an acid or enzymes to determine the structures. To identify the anomeric configuration, the oligosaccharide is subjected to enzymatic analysis, e.g., it is contacted with an enzyme that is specific for a particular type of linkage, e.g., beta-galactosidase, or alpha-glucosidase, etc., and NMR may be used to analyze the products.

Products Comprising the Oligosaccharide Mixture

[0386] In some embodiments, an oligosaccharide mixture produced as described herein is incorporated into a food (e.g, human food or feed), dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine. In some embodiments, the oligosaccharide mixture is mixed with one or more ingredients suitable for food, feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine.

[0387] In some embodiments, the dietary supplement comprises at least one prebiotic ingredient and/or at least one probiotic ingredient.

[0388] A prebiotic is a substance that promotes growth of microorganisms beneficial to the host, particularly microorganisms in the gastrointestinal tract. In some embodiments, a dietary supplement provides multiple prebiotics, including the oligosaccharide mixture produced and/or purified by a process disclosed in this specification, to promote growth of one or more beneficial microorganisms. Examples of prebiotic ingredients for dietary supplements include other prebiotic molecules (such as HMOs) and plant polysaccharides (such as inulin, pectin, b-glucan and xylooligosaccharide). A probiotic product typically contains live microorganisms that replace or add to gastrointestinal microflora, to the benefit of the recipient. Examples of such microorganisms include Lactobacillus species (for example, L. acidophilus and L. bulgaricus), Bifidobacterium species (for example, B. animalis, B. longum and B. infantis (e.g., Bi-26)), and Saccharomyces boulardii. In some embodiments, an oligosaccharide mixture produced and/or purified by a process of this specification is orally administered in combination with such microorganism.

[0389] Examples of further ingredients for dietary supplements include disaccharides (such as lactose), monosaccharides (such as glucose and galactose), thickeners (such as gum arabic), acidity regulators (such as trisodium citrate), water, skimmed milk, and flavorings.

[0390] In some embodiments, the oligosaccharide mixture is incorporated into a human baby food (e.g., infant formula). Infant formula is generally a manufactured food for feeding to infants as a complete or partial substitute for human breast milk. In some embodiments, infant formula is sold as a powder and prepared for bottle- or cup-feeding to an infant by mixing with water. The composition of infant formula is typically designed to be roughly mimic human breast milk. In some embodiments, an oligosaccharide mixture produced and/or purified by a process in this specification is included in infant formula to provide nutritional benefits similar to those provided by the oligosaccharides in human breast milk. In some embodiments, the oligosaccharide mixture is mixed with one or more ingredients of the infant formula. Examples of infant formula ingredients include nonfat milk, carbohydrate sources (e.g., lactose), protein sources (e.g., whey protein concentrate and casein), fat sources (e.g., vegetable oilssuch as palm, high oleic safflower oil, rapeseed, coconut and/or sunflower oil; and fish oils), vitamins (such as vitamins A, Bb, Bi2, C and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate) and possibly human milk oligosaccharides (HMOs). Such HMOs may include, for example, DiFL, lacto-N-triose II, LNT, LNnT, lacto-N-fucopentaose I, lacto-N-neofucopentaose, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6-galactosyllactose, 3-galactosyllactose, lacto-N-hexaose and lacto-N-neohexaose.

[0391] In some embodiments, the one or more infant formula ingredients comprise non-fat milk, a carbohydrate source, a protein source, a fat source, and/or a vitamin and mineral.

[0392] In some embodiments, the one or more infant formula ingredients comprise lactose, whey protein concentrate and/or high oleic safflower oil.

[0393] In some embodiments, the oligosaccharide mixture's concentration in the infant formula is approximately the same concentration as the oligosaccharide's concentration generally present in human breast milk. In some embodiments, the concentration of each of the single oligosaccharides in the mixture of oligosaccharides in the infant formula is approximately the same concentration as the concentration of that oligosaccharide generally present in human breast milk.

[0394] In some embodiments, the oligosaccharide mixture is incorporated into a feed preparation, wherein the feed is chosen from the list comprising petfood, animal milk replacer, veterinary product, post weaning feed, or creep feed.

[0395] Each embodiment disclosed in the context of one aspect of the disclosure, is also disclosed in the context of all other aspects of the disclosure, unless explicitly stated otherwise.

[0396] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described above and below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, purification steps are performed according to the manufacturer's specifications.

[0397] Further advantages follow from the specific embodiments, the examples and the attached drawings. It goes without saying that the abovementioned features and the features that are still to be explained below can be used not only in the respectively specified combinations, but also in other combinations or on their own, without departing from the scope of this disclosure.

[0398] This disclosure relates to following specific embodiments:

[0399] 1. A metabolically engineered cell producing a mixture of at least three different sialylated oligosaccharides, wherein the cell: [0400] expresses a glycosyltransferase being a sialyltransferase, and [0401] is capable of synthesizing the nucleotide-sugar CMP-N-acetylneuraminic acid (CMP-Neu5Ac), and [0402] expresses at least one additional glycosyltransferase, and [0403] is capable of synthesizing one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for the additional glycosyltransferase.

[0404] 2. Cell according to embodiment 1, wherein the cell is modified with gene expression modules, wherein the expression from any of the expression modules is either constitutive or is created by a natural inducer.

[0405] 3. Cell according to any one of embodiments 1 and 2, wherein the cell produces a mixture of charged and neutral oligosaccharides.

[0406] 4. Cell according to any one of embodiments 1 to 3, wherein the oligosaccharide mixture comprises at least three different oligosaccharides differing in degree of polymerization.

[0407] 5. Cell according to any one of embodiments 1 to 4, wherein the cell produces four or more different sialylated oligosaccharides.

[0408] 6. Cell according to any one of embodiments 1 to 5, wherein any one of the additional glycosyltransferases is chosen from the list comprising fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases.

[0409] 7. Cell according to any one of embodiments 1 to 6 wherein the cell is modified in the expression or activity of at least one of the glycosyltransferases.

[0410] 8. Cell according to any one of embodiments 1 to 7 wherein any one of the additional glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-Neu5Ac.

[0411] 9. Cell according to any one of embodiments 1 to 8 wherein any one of the additional glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-Fucose (GDP-Fuc).

[0412] 10. Cell according to any one of embodiments 1 to 9 wherein any one of the additional glycosyltransferases is an N-acetylglucosaminyltransferase and one of the donor nucleotide-sugars is UDP-N-acetylglucosamine (UDP-GlcNAc).

[0413] 11. Cell according to any one of embodiments 1 to 10 wherein any one of the additional glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is UDP-galactose (UDP-Gal).

[0414] 12. Cell according to any one of embodiments 1 to 11, wherein any one of the nucleotide-sugars is chosen from the list comprising GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose.

[0415] 13. Cell according to any one of embodiments 1 to 12, wherein the oligosaccharide mixture comprises at least one neutral oligosaccharide in addition to three or more sialylated oligosaccharides.

[0416] 14. Cell according to any one of embodiments 1 to 13, wherein at least one of the sialylated oligosaccharides is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains an N-acetylglucosamine, contains an N-acetylneuraminate, contains an N-glycolylneuraminate, contains an N-acetylgalactosamine, contains a rhamnose, contains a glucuronate, contains a galacturonate, and/or contains an N-acetylmannosamine.

[0417] 15. Cell according to any one of embodiments 1 to 14, wherein the oligosaccharide mixture comprises at least one fucosylated oligosaccharide.

[0418] 16. Cell according to any one of embodiments 1 to 15, wherein the oligosaccharide mixture comprises at least one oligosaccharide that comprises an N-acetylglucosamine monosaccharide unit.

[0419] 17. Cell according to any one of embodiments 1 to 16, wherein the oligosaccharide mixture comprises at least one galactosylated oligosaccharide.

[0420] 18. Cell according to any one of embodiments 1 to 17, wherein the oligosaccharide mixture comprises at least one oligosaccharide that is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains an N-acetylglucosamine, contains an N-acetylneuraminate, contains an N-glycolylneuraminate, contains an N-acetylgalactosamine, contains a rhamnose, contains a glucuronate, contains a galacturonate, and/or contains an N-acetylmannosamine.

[0421] 19. Cell according to any one of embodiments 1 to 18, wherein the cell is further genetically modified for: [0422] i) modified expression of an endogenous membrane protein, and/or [0423] ii) modified activity of an endogenous membrane protein, and/or [0424] iii) expression of a homologous membrane protein, and/or [0425] iv) expression of a heterologous membrane protein, [0426] wherein the membrane protein is involved in the secretion of any one of the oligosaccharides from the mixture outside the cell.

[0427] 20. Cell according to any one of embodiments 1 to 19, wherein the cell is further genetically modified for: [0428] i) modified expression of an endogenous membrane protein, and/or [0429] ii) modified activity of an endogenous membrane protein, and/or [0430] iii) expression of a homologous membrane protein, and/or [0431] iv) expression of a heterologous membrane protein, [0432] wherein the membrane protein is involved in the uptake of a precursor for the synthesis of any one of the oligosaccharides.

[0433] 21. Cell according to any one of embodiments 1 to 20, wherein the cell is producing a precursor for the synthesis of any one of the oligosaccharides.

[0434] 22. Cell according to any one of embodiments 1 to 21, wherein any one of the oligosaccharides is a mammalian milk oligosaccharide.

[0435] 23. Cell according to any one of embodiments 1 to 22, wherein all the oligosaccharides are mammalian milk oligosaccharides.

[0436] 24. Cell according to any one of embodiments 1 to 21, wherein any one of the oligosaccharides is an antigen of the human ABO blood group system.

[0437] 25. A method to produce a mixture of at least three different sialylated oligosaccharides by a cell, the method comprising the steps of: [0438] i) providing a cell (a) expressing a glycosyltransferase being a sialyltransferase and capable of synthesizing the nucleotide-sugar CMP-Neu5Ac, and (b) expressing at least one additional glycosyltransferase, and (c) capable of synthesizing at least one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for the additional glycosyltransferases, and [0439] ii) cultivating the cell under conditions permissive to express the glycosyltransferases and to synthesize the nucleotide-sugars, [0440] iii) preferably, separating at least one of the oligosaccharides from the cultivation.

[0441] 26. Method according to embodiment 25, wherein the cell is a metabolically engineered cell according to any one of embodiments 1 to 24.

[0442] 27. Method according to any one of embodiments 25 and 26, wherein the cell produces a mixture of charged and neutral oligosaccharides.

[0443] 28. Method according to any one of embodiments 25 to 27, wherein the oligosaccharide mixture comprises at least three different oligosaccharides differing in degree of polymerization.

[0444] 29. Method according to any one of embodiments 25 to 28, wherein the cell produces four or more different sialylated oligosaccharides.

[0445] 30. Method according to any one of embodiments 25 to 29, wherein any one of the additional glycosyltransferases is chosen from the list comprising fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases.

[0446] 31. Method according to any one of embodiments 25 to 30 wherein any one of the additional glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-N-acetylneuraminic acid (CMP-Neu5Ac).

[0447] 32. Method according to any one of embodiments 25 to 31 wherein any one of the additional glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-Fucose (GDP-Fuc).

[0448] 33. Method according to any one of embodiments 25 to 32 wherein any one of the additional glycosyltransferases is an N-acetylglucosaminyltransferase and one of the donor nucleotide-sugars is UDP-N-acetylglucosamine (UDP-GlcNAc).

[0449] 34. Method according to any one of embodiments 25 to 33 wherein any one of the additional glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is UDP-galactose (UDP-Gal).

[0450] 35. Method according to any one of embodiments 25 to 34, wherein any one of the nucleotide-sugars is chosen from the list comprising GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose.

[0451] 36. Method according to any one of embodiments 25 to 35, wherein the oligosaccharide mixture comprises at least one neutral oligosaccharide in addition to three or more sialylated oligosaccharides.

[0452] 37. Method according to any one of embodiments 25 to 36, wherein at least one of the sialylated oligosaccharides is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains an N-acetylglucosamine, contains an N-acetylneuraminate, contains an N-glycolylneuraminate, contains an N-acetylgalactosamine, contains a rhamnose, contains a glucuronate, contains a galacturonate, and/or contains an N-acetylmannosamine.

[0453] 38. Method according to any one of embodiments 25 to 37, wherein the oligosaccharide mixture comprises at least one fucosylated oligosaccharide.

[0454] 39. Method according to any one of embodiments 25 to 38, wherein the oligosaccharide mixture comprises at least one oligosaccharide that comprises an N-acetylglucosamine monosaccharide unit.

[0455] 40. Method according to any one of embodiments 25 to 39, wherein the oligosaccharide mixture comprises at least one galactosylated oligosaccharide.

[0456] 41. Method according to any one of embodiments 25 to 40, wherein the oligosaccharide mixture comprises at least one oligosaccharide that is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains an N-acetylglucosamine, contains an N-acetylneuraminate, contains an N-glycolylneuraminate, contains an N-acetylgalactosamine, contains a rhamnose, contains a glucuronate, contains a galacturonate, and/or contains an N-acetylmannosamine.

[0457] 42. Method according to any one of embodiments 25 to 41, wherein the cell uses at least one precursor for the synthesis of any one or more of the oligosaccharides, preferably the cell uses two or more precursors for the synthesis of any one or more of the oligosaccharides.

[0458] 43. Method according to any one of embodiments 25 to 42, wherein the cell is producing a precursor for the synthesis of any one of the oligosaccharides.

[0459] 44. Method according to any one of embodiments 25 to 43, wherein any one of the oligosaccharides is a mammalian milk oligosaccharide.

[0460] 45. Method according to any one of embodiments 25 to 44, wherein all the oligosaccharides are mammalian milk oligosaccharides.

[0461] 46. Method according to any one of embodiments 25 to 43, wherein any one of the oligosaccharide is an antigen of the human ABO blood group system.

[0462] 47. The method according to any one of embodiments 25 to 46, wherein the precursor for the synthesis of any one of the oligosaccharides is completely converted into any one of the oligosaccharides.

[0463] 48. The method according to any one of embodiments 25 to 47, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography.

[0464] 49. The method according to any one of embodiments 25 to 48, further comprising purification of any one of the oligosaccharides from the cell.

[0465] 50. The method according to any one of embodiments 25 to 49, wherein the purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying or lyophilization.

[0466] 51. The cell according to any one of embodiments 1 to 24 or method according to any one of embodiments 25 to 50, wherein the cell is selected from the group comprising microorganism, plant, or animal cells, preferably the microorganism is a bacterium, fungus or a yeast, preferably the plant is a rice, cotton, rapeseed, soy, maize or corn plant, preferably the animal is an insect, fish, bird or non-human mammal, preferably the animal cell is a mammalian cell line.

[0467] 52. The cell according to any one of embodiments 1 to 24 and 51, or method according to any one of embodiments 25 to 51, wherein the cell is a cell of a bacterium, preferably of an Escherichia coli strain, more preferably of an Escherichia coli strain that is a K-12 strain, even more preferably the Escherichia coli K-12 strain is E. coli MG1655.

[0468] 53. The cell according to any one of embodiments 1 to 24 and 51, or method according to any one of embodiments 25 to 51, wherein the cell is a yeast cell.

[0469] 54. Use of a cell according to any one of embodiments 1 to 24, 51 to 53, or method according to any one of embodiment 25 to 53 for the production of a mixture of at least three different sialylated oligosaccharides.

[0470] Moreover, this disclosure relates to the following preferred specific embodiments:

[0471] 1. A metabolically engineered cell producing a mixture of at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different sialylated mammalian milk oligosaccharides, [0472] wherein the cell [0473] is metabolically engineered for the production of the mixture, and [0474] expresses a glycosyltransferase being a sialyltransferase, and [0475] is capable of synthesizing the nucleotide-sugar CMP-N-acetylneuraminic acid (CMP-Neu5Ac), and [0476] expresses at least one additional glycosyltransferase, and [0477] is capable of synthesizing one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for the additional glycosyltransferase.

[0478] 2. Cell according to preferred embodiment 1, wherein the cell is modified with gene expression modules, wherein the expression from any of the expression modules is either constitutive or is created by a natural inducer.

[0479] 3. Cell according to any one of preferred embodiment 1 or 2, wherein the cell comprises multiple copies of the same coding DNA sequence encoding for one protein.

[0480] 4. Cell according to any one of preferred embodiments 1 to 3, wherein the cell produces a mixture of charged and neutral oligosaccharides.

[0481] 5. Cell according to any one of preferred embodiments 1 to 4, wherein the mixture comprises, consists or consists essentially of charged and neutral fucosylated and/or non-fucosylated oligosaccharides.

[0482] 6. Cell according to any one of preferred embodiments 1 to 5, wherein the oligosaccharide mixture comprises at least three different sialylated oligosaccharides differing in degree of polymerization.

[0483] 7. Cell according to any one of preferred embodiments 1 to 6, wherein the cell produces at least four, preferably at least five, more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different sialylated oligosaccharides.

[0484] 8. Cell according to any one of preferred embodiments 1 to 7, wherein any one of the additional glycosyltransferases is chosen from the list comprising fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases, [0485] preferably, the fucosyltransferase is chosen from the list comprising alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase, [0486] preferably, the sialyltransferase is chosen from the list comprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase [0487] preferably, the galactosyltransferase is chosen from the list comprising beta-1,3-galactosyltransferase, N-acetylglucosamine beta-1,3-galactosyltransferase, beta-1,4-galactosyltransferase, N-acetylglucosamine beta-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase and alpha-1,4-galactosyltransferase, [0488] preferably, the glucosyltransferase is chosen from the list comprising alpha-glucosyltransferase, beta-1,2-glucosyltransferase, beta-1,3-glucosyltransferase and beta-1,4-glucosyltransferase, [0489] preferably, the mannosyltransferase is chosen from the list comprising alpha-1,2-mannosyltransferase, alpha-1,3-mannosyltransferase and alpha-1,6-mannosyltransferase, [0490] preferably, the N-acetylglucosaminyltransferase is chosen from the list comprising galactoside beta-1,3-N-acetylglucosaminyltransferase and beta-1,6-N-acetylglucosaminyltransferase, [0491] preferably, the N-acetylgalactosaminyltransferase is chosen from the list comprising alpha-1,3-N-acetylgalactosaminyltransferase and beta-1,3-N-acetylgalactosaminyltransferase.

[0492] 9. Cell according to any one of preferred embodiments 1 to 8 wherein the cell is modified in the expression or activity of at least one of the glycosyltransferases.

[0493] 10. Cell according to any one of preferred embodiments 1 to 9 wherein any one of the additional glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-Neu5Ac.

[0494] 11. Cell according to any one of preferred embodiments 1 to 10 wherein any one of the additional glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-Fucose (GDP-Fuc).

[0495] 12. Cell according to any one of preferred embodiments 1 to 11 wherein any one of the additional glycosyltransferases is an N-acetylglucosaminyltransferase and one of the donor nucleotide-sugars is UDP-N-acetylglucosamine (UDP-GlcNAc).

[0496] 13. Cell according to any one of preferred embodiments 1 to 12 wherein any one of the additional glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is UDP-galactose (UDP-Gal).

[0497] 14. Cell according to any one of preferred embodiments 1 to 13, wherein any one of the additional glycosyltransferases is an N-acetylgalactosaminyltransferase and one of the donor nucleotide-sugars is UDP-N-acetylgalactosamine (UDP-GalNAc).

[0498] 15. Cell according to any one of preferred embodiments 1 to 14, wherein any one of the additional glycosyltransferases is an N-acetylmannosaminyltransferase and one of the donor nucleotide-sugars is UDP-N-acetylmannosamine (UDP-ManNAc).

[0499] 16. Cell according to any one of preferred embodiments 1 to 15, wherein any one of the nucleotide-sugars is chosen from the list comprising GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose, UDP-2-acetamido-2,6-dideoxy--L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy--L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose.

[0500] 17. Cell according to any one of preferred embodiments 1 to 16, wherein the cell expresses one or more polypeptides chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, GDP-fucose pyrophosphorylase, fucose-1-phosphate L-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 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 uridylyltransferase, glucosamine-1-phosphate acetyltransferase, N-acetylneuraminate synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphate phosphatase, N-acylneuraminate cytidylyltransferase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridylyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein the cell is modified in the expression or activity of any one of the polypeptides.

[0501] 18. Cell according to any one of preferred embodiments 1 to 17, 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.

[0502] 19. Cell according to any one of preferred embodiments 1 to 18, wherein the oligosaccharide mixture comprises at least one neutral oligosaccharide in addition to three or more sialylated oligosaccharides.

[0503] 20. Cell according to preferred embodiment 19, wherein the neutral oligosaccharide is chosen from the list comprising neutral fucosylated oligosaccharides and neutral non-fucosylated oligosaccharides.

[0504] 21. Cell according to any one of preferred embodiments 1 to 20, wherein at least one of the sialylated oligosaccharides is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains an N-acetylglucosamine, contains an N-acetylneuraminate, contains an N-glycolylneuraminate, contains an N-acetylgalactosamine, contains a rhamnose, contains a glucuronate, contains a galacturonate, and/or contains an N-acetylmannosamine.

[0505] 22. Cell according to any one of preferred embodiments 1 to 21, wherein the oligosaccharide mixture comprises at least one fucosylated oligosaccharide.

[0506] 23. Cell according to any one of preferred embodiments 1 to 22, wherein the oligosaccharide mixture comprises at least one oligosaccharide that comprises an N-acetylglucosamine monosaccharide unit.

[0507] 24. Cell according to any one of preferred embodiments 1 to 23, wherein the oligosaccharide mixture comprises at least one galactosylated oligosaccharide.

[0508] 25. Cell according to any one of preferred embodiments 1 to 24, wherein the oligosaccharide mixture comprises at least one oligosaccharide that is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains an N-acetylglucosamine, contains an N-acetylneuraminate, contains an N-glycolylneuraminate, contains an N-acetylgalactosamine, contains a rhamnose, contains a glucuronate, contains a galacturonate, and/or contains an N-acetylmannosamine.

[0509] 26. Cell according to any one of preferred embodiments 1 to 25, 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 precursors for the production of any one or more of the oligosaccharides, the precursor(s) being fed to the cell from the cultivation medium.

[0510] 27. Cell according to any one of preferred embodiments 1 to 26, wherein the cell is producing at least one precursor for the production of any one of the oligosaccharides.

[0511] 28. Cell according to any one of preferred embodiments 1 to 27, wherein the at least one precursor for the production of any one of the oligosaccharides is completely converted into any one of the oligosaccharides.

[0512] 29. Cell according to any one of preferred embodiments 1 to 28, wherein the cell produces the oligosaccharides intracellularly and wherein a fraction or substantially all of the produced oligosaccharides remains intracellularly and/or is excreted outside the cell via passive or active transport.

[0513] 30. Cell according to any one of preferred embodiments 1 to 29, wherein the cell is further genetically modified for [0514] i) modified expression of an endogenous membrane protein, and/or [0515] ii) modified activity of an endogenous membrane protein, and/or [0516] iii) expression of a homologous membrane protein, and/or [0517] iv) expression of a heterologous membrane protein, [0518] wherein the membrane protein is involved in the secretion of any one of the oligosaccharides from the mixture outside the cell, preferably wherein the membrane protein is involved in the secretion of all of the oligosaccharides from the mixture from the cell.

[0519] 31. Cell according to any one of preferred embodiments 1 to 30, wherein the cell is further genetically modified for [0520] i) modified expression of an endogenous membrane protein, and/or [0521] ii) modified activity of an endogenous membrane protein, and/or [0522] iii) expression of a homologous membrane protein, and/or [0523] iv) expression of a heterologous membrane protein, [0524] wherein the membrane protein is involved in the uptake of a precursor and/or acceptor for the synthesis of any one of the oligosaccharides of the mixture, preferably wherein the membrane protein is involved in the uptake of all of the required precursors, more preferably wherein the membrane protein is involved in the uptake of all of the acceptors.

[0525] 32. Cell according to any one of preferred embodiment 30 or 31, wherein the membrane protein is chosen from the list comprising porters, P-P-bond-hydrolysis-driven transporters, ?-barrel porins, auxiliary transport proteins, putative transport proteins and phosphotransfer-driven group translocators, [0526] preferably, the porters comprise MFS transporters, sugar efflux transporters and siderophore exporters, [0527] preferably, the P-P-bond-hydrolysis-driven transporters comprise ABC transporters and siderophore exporters.

[0528] 33. Cell according to any one of preferred embodiment 30 to 32, wherein the membrane protein provides improved production and/or enabled and/or enhanced efflux of any one of the oligosaccharides.

[0529] 34. Cell according to any one of preferred embodiment 1 to 33, wherein the cell resists the phenomenon of lactose killing when grown in an environment in which lactose is combined with one or more other carbon source(s).

[0530] 35. Cell according to any one of preferred embodiment 1 to 34, wherein the cell comprises a modification for reduced production of acetate compared to a non-modified progenitor.

[0531] 36. Cell according to preferred embodiment 35, wherein the cell comprises a lower or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the proteins comprising beta-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridylyltransferase, glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IcIR, lon protease, glucose-specific translocating phosphotransferase enzyme IIBC component ptsG, glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIAGIe, beta-glucoside specific PTS enzyme II, fructose-specific PTS multiphosphoryl transfer protein FruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, pyruvate decarboxylase compared to a non-modified progenitor.

[0532] 37. Cell according to any one of preferred embodiment 1 to 36, wherein the cell is capable to produce phosphoenolpyruvate (PEP).

[0533] 38. Cell according to any one of preferred embodiment 1 to 37, wherein the cell is modified for enhanced production and/or supply of phosphoenolpyruvate (PEP) compared to a non-modified progenitor.

[0534] 39. Cell according to any one of preferred embodiments 1 to 38, wherein any one of the oligosaccharides is a mammalian milk oligosaccharide.

[0535] 40. Cell according to any one of preferred embodiments 1 to 39, wherein all the oligosaccharides are mammalian milk oligosaccharides.

[0536] 41. Cell according to any one of preferred embodiments 1 to 38, wherein any one of the oligosaccharides is an antigen of the human ABO blood group system.

[0537] 42. A method to produce a mixture of at least three different sialylated oligosaccharides wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different sialylated mammalian milk oligosaccharides, by a cell, preferably a single cell, the method comprising the steps of: [0538] i) providing a cell (a) expressing a glycosyltransferase being a sialyltransferase and capable of synthesizing the nucleotide-sugar CMP-Neu5Ac, and (b) expressing at least one additional glycosyltransferase, and (c) capable of synthesizing at least one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for the additional glycosyltransferases, and [0539] ii) cultivating the cell under conditions permissive to express the glycosyltransferases and of synthesizing the nucleotide-sugars, resulting in the cell producing the mixture of at least three different sialylated oligosaccharides, [0540] iii) preferably, separating at least one of the oligosaccharides from the cultivation, more preferably separating all of the oligosaccharides from the cultivation.

[0541] 43. Method according to preferred embodiment 42, wherein the cell is a metabolically engineered cell according to any one of embodiments 1 to 41.

[0542] 44. Method according to preferred embodiment 43, wherein the cell is modified with gene expression modules, wherein the expression from any of the expression modules is either constitutive or is created by a natural inducer.

[0543] 45. Method according to any one of preferred embodiment 43 or 44, wherein the cell comprises multiple copies of the same coding DNA sequence encoding for one protein.

[0544] 46. Method according to any one of preferred embodiments 42 to 45, wherein the cell produces a mixture of charged and neutral oligosaccharides.

[0545] 47. Method according to any one of preferred embodiment 42 to 46, wherein the mixture comprises, consists or consists essentially of charged and neutral fucosylated and/or non-fucosylated oligosaccharides.

[0546] 48. Method according to any one of preferred embodiments 42 to 47, wherein the oligosaccharide mixture comprises at least three different sialylated oligosaccharides differing in degree of polymerization.

[0547] 49. Method according to any one of preferred embodiments 42 to 48, wherein the cell produces at least four, preferably at least five, more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different sialylated oligosaccharides.

[0548] 50. Method according to any one of preferred embodiments 42 to 49, wherein any one of the additional glycosyltransferases is chosen from the list comprising fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases, [0549] preferably, the fucosyltransferase is chosen from the list comprising alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase, [0550] preferably, the sialyltransferase is chosen from the list comprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase [0551] preferably, the galactosyltransferase is chosen from the list comprising beta-1,3-galactosyltransferase, N-acetylglucosamine beta-1,3-galactosyltransferase, beta-1,4-galactosyltransferase, N-acetylglucosamine beta-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase and alpha-1,4-galactosyltransferase, [0552] preferably, the glucosyltransferase is chosen from the list comprising alpha-glucosyltransferase, beta-1,2-glucosyltransferase, beta-1,3-glucosyltransferase and beta-1,4-glucosyltransferase, [0553] preferably, the mannosyltransferase is chosen from the list comprising alpha-1,2-mannosyltransferase, alpha-1,3-mannosyltransferase and alpha-1,6-mannosyltransferase, [0554] preferably, the N-acetylglucosaminyltransferase is chosen from the list comprising galactoside beta-1,3-N-acetylglucosaminyltransferase and beta-1,6-N-acetylglucosaminyltransferase, [0555] preferably, the N-acetylgalactosaminyltransferase is chosen from the list comprising alpha-1,3-N-acetylgalactosaminyltransferase and beta-1,3-N-acetylgalactosaminyltransferase.

[0556] 51. Method according to any one of preferred embodiments 42 to 50 wherein the cell is modified in the expression or activity of at least one of the glycosyltransferases.

[0557] 52. Method according to any one of preferred embodiments 42 to 51 wherein any one of the additional glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-N-acetylneuraminic acid (CMP-Neu5Ac).

[0558] 53. Method according to any one of preferred embodiments 42 to 52 wherein any one of the additional glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-Fucose (GDP-Fuc).

[0559] 54. Method according to any one of preferred embodiments 42 to 53 wherein any one of the additional glycosyltransferases is an N-acetylglucosaminyltransferase and one of the donor nucleotide-sugars is UDP-N-acetylglucosamine (UDP-GlcNAc).

[0560] 55. Method according to any one of preferred embodiments 42 to 54 wherein any one of the additional glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is UDP-galactose (UDP-Gal).

[0561] 56. Method according to any one of preferred embodiments 42 to 55, wherein any one of the additional glycosyltransferases is an N-acetylgalactosaminyltransferase and one of the donor nucleotide-sugars is UDP-N-acetylgalactosamine (UDP-GalNAc).

[0562] 57. Method according to any one of preferred embodiments 42 to 56, wherein any one of the additional glycosyltransferases is an N-acetylmannosaminyltransferase and one of the donor nucleotide-sugars is UDP-N-acetylmannosamine (UDP-ManNAc).

[0563] 58. Method according to any one of preferred embodiments 42 to 57, wherein any one of the nucleotide-sugars is chosen from the list comprising GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5, 7(8,9)Ac2, UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose, UDP-2-acetamido-2,6-dideoxy--L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy--L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose.

[0564] 59. Method according to any one of preferred embodiments 42 to 58, wherein the cell expresses one or more polypeptides chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, GDP-fucose pyrophosphorylase, fucose-1-phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 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 uridylyltransferase, glucosamine-1-phosphate acetyltransferase, N-acetylneuraminate synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphate phosphatase, N-acylneuraminate cytidylyltransferase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridylyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein the cell is modified in the expression or activity of any one of the polypeptides.

[0565] 60. Method according to any one of preferred embodiments 42 to 59, 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.

[0566] 61. Method according to any one of preferred embodiments 42 to 60, wherein the oligosaccharide mixture comprises at least one neutral oligosaccharide in addition to three or more sialylated oligosaccharides.

[0567] 62. Method according to preferred embodiment 61, wherein the neutral oligosaccharide is chosen from the list comprising neutral fucosylated oligosaccharides and neutral non-fucosylated oligosaccharides.

[0568] 63. Method according to any one of preferred embodiments 42 to 62, wherein at least one of the sialylated oligosaccharides is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains an N-acetylglucosamine, contains an N-acetylneuraminate, contains an N-glycolylneuraminate, contains an N-acetylgalactosamine, contains a rhamnose, contains a glucuronate, contains a galacturonate, and/or contains an N-acetylmannosamine.

[0569] 64. Method according to any one of preferred embodiments 42 to 63, wherein the oligosaccharide mixture comprises at least one fucosylated oligosaccharide.

[0570] 65. Method according to any one of preferred embodiments 42 to 64, wherein the oligosaccharide mixture comprises at least one oligosaccharide that comprises an N-acetylglucosamine monosaccharide unit.

[0571] 66. Method according to any one of preferred embodiments 42 to 65, wherein the oligosaccharide mixture comprises at least one galactosylated oligosaccharide.

[0572] 67. Method according to any one of preferred embodiments 42 to 66, wherein the oligosaccharide mixture comprises at least one oligosaccharide that is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains an N-acetylglucosamine, contains an N-acetylneuraminate, contains an N-glycolylneuraminate, contains an N-acetylgalactosamine, contains a rhamnose, contains a glucuronate, contains a galacturonate, and/or contains an N-acetylmannosamine.

[0573] 68. Method according to any one of preferred embodiments 42 to 67, 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 precursors for the production of any one or more of the oligosaccharides, the precursor(s) being fed to the cell from the cultivation medium.

[0574] 69. Method according to any one of preferred embodiments 42 to 68, wherein the cell is producing at least one precursor for the production of any one of the oligosaccharides.

[0575] 70. Method according to any one of preferred embodiments 42 to 69, wherein the at least one precursor for the production of any one of the oligosaccharides is completely converted into any one of the oligosaccharides.

[0576] 71. Method according to any one of preferred embodiments 42 to 70, wherein the cell produces the oligosaccharides intracellularly and wherein a fraction or substantially all of the produced oligosaccharides remains intracellularly and/or is excreted outside the cell via passive or active transport.

[0577] 72. Method according to any one of preferred embodiments 42 to 71, wherein the cell is further genetically modified for [0578] i) modified expression of an endogenous membrane protein, and/or [0579] ii) modified activity of an endogenous membrane protein, and/or [0580] iii) expression of a homologous membrane protein, and/or [0581] iv) expression of a heterologous membrane protein, [0582] wherein the membrane protein is involved in the secretion of any one of the oligosaccharides from the mixture outside the cell, preferably wherein the membrane protein is involved in the secretion of all of the oligosaccharides from the mixture from the cell.

[0583] 73. Method according to any one of preferred embodiments 42 to 72, wherein the cell is further genetically modified for [0584] i) modified expression of an endogenous membrane protein, and/or [0585] ii) modified activity of an endogenous membrane protein, and/or [0586] iii) expression of a homologous membrane protein, and/or [0587] iv) expression of a heterologous membrane protein, [0588] wherein the membrane protein is involved in the uptake of a precursor and/or acceptor for the synthesis of any one of the oligosaccharides of the mixture, preferably wherein the membrane protein is involved in the uptake of all of the required precursors, more preferably wherein the membrane protein is involved in the uptake of all of the acceptors.

[0589] 74. Method according to any one of preferred embodiment 72 or 73, wherein the membrane protein is chosen from the list comprising porters, P-P-bond-hydrolysis-driven transporters, ?-barrel porins, auxiliary transport proteins, putative transport proteins and phosphotransfer-driven group translocators, [0590] preferably, the porters comprise MFS transporters, sugar efflux transporters and siderophore exporters, [0591] preferably, the P-P-bond-hydrolysis-driven transporters comprise ABC transporters and siderophore exporters.

[0592] 75. Method according to any one of preferred embodiment 72 to 74, wherein the membrane protein provides improved production and/or enabled and/or enhanced efflux of any one of the oligosaccharides.

[0593] 76. Method according to any one of preferred embodiment 42 to 75, wherein the cell resists the phenomenon of lactose killing when grown in an environment in which lactose is combined with one or more other carbon source(s).

[0594] 77. Method according to any one of preferred embodiment 42 to 76, wherein the cell comprises a modification for reduced production of acetate compared to a non-modified progenitor.

[0595] 78. Method according to preferred embodiment 77, wherein the cell comprises a lower or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the proteins comprising beta-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridylyltransferase, glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IcIR, lon protease, glucose-specific translocating phosphotransferase enzyme IIBC component ptsG, glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIA.sup.Glc, beta-glucoside specific PTS enzyme II, fructose-specific PTS multiphosphoryl transfer protein FruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, pyruvate decarboxylase compared to a non-modified progenitor.

[0596] 79. Method according to any one of preferred embodiment 42 to 78, wherein the cell is capable to produce phosphoenolpyruvate (PEP).

[0597] 80. Method according to any one of preferred embodiment 42 to 79, wherein the cell is modified for enhanced production and/or supply of phosphoenolpyruvate (PEP) compared to a non-modified progenitor.

[0598] 81. Method according to any one of preferred embodiments 42 to 80, wherein any one of the oligosaccharides is a mammalian milk oligosaccharide.

[0599] 82. Method according to any one of preferred embodiments 42 to 81, wherein all the oligosaccharides are mammalian milk oligosaccharides.

[0600] 83. Method according to any one of preferred embodiments 42 to 82, wherein any one of the oligosaccharide is an antigen of the human ABO blood group system.

[0601] 84. Method according to any one of preferred embodiment 42 to 83, wherein the conditions comprise: [0602] use of a culture medium comprising at least one precursor and/or acceptor for the production of any one of the oligosaccharides, and/or [0603] adding to the culture medium at least one precursor and/or acceptor feed for the production of any one of the oligosaccharides.

[0604] 85. Method according to any one of preferred embodiment 42 to 84, the method comprising at least one of the following steps: [0605] i) Use of a culture medium comprising at least one precursor and/or acceptor; [0606] ii) Adding to the culture medium in a reactor at least one precursor and/or acceptor feed wherein the total reactor volume ranges from 250 mL (milliliter) to 10,000 m.sup.3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the culture medium before the addition of the precursor and/or acceptor feed; [0607] iii) Adding to the culture medium in a reactor at least one precursor and/or acceptor feed wherein the total reactor volume ranges from 250 mL (milliliter) to 10,000 m.sup.3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the culture medium before the addition of the precursor and/or acceptor feed and wherein preferably, the pH of the precursor and/or acceptor feed is set between 3 and 7 and wherein preferably, the temperature of the precursor and/or acceptor feed is kept between 20? C. and 80? C.; [0608] iv) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; [0609] v) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein preferably, the pH of the feeding solution is set between 3 and 7 and wherein preferably, the temperature of the feeding solution is kept between 20? C. and 80? C.; [0610] the method resulting in any one of the oligosaccharides with a concentration of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L in the final cultivation.

[0611] 86. Method according to any one of preferred embodiment 42 to 84, the method comprising at least one of the following steps: [0612] i) Use of a culture medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per liter of initial reactor volume wherein the reactor volume ranges from 250 mL to 10,000 m.sup.3 (cubic meter); [0613] ii) Adding to the culture medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per liter of initial reactor volume wherein the reactor volume ranges from 250 mL to 10,000 m.sup.3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the culture medium before the addition of the lactose feed; [0614] iii) Adding to the culture medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per liter of initial reactor volume wherein the reactor volume ranges from 250 mL to 10,000 m.sup.3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the culture medium before the addition of the lactose feed and wherein preferably the pH of the lactose feed is set between 3 and 7 and wherein preferably the temperature of the lactose feed is kept between 20? C. and 80? C.; [0615] iv) Adding a lactose feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; [0616] v) Adding a lactose feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein the concentration of the lactose feeding solution is 50 g/L, preferably 75 g/L, more preferably 100 g/L, more preferably 125 g/L, more preferably 150 g/L, more preferably 175 g/L, more preferably 200 g/L, more preferably 225 g/L, more preferably 250 g/L, more preferably 275 g/L, more preferably 300 g/L, more preferably 325 g/L, more preferably 350 g/L, more preferably 375 g/L, more preferably, 400 g/L, more preferably 450 g/L, more preferably 500 g/L, even more preferably, 550 g/L, most preferably 600 g/L and wherein preferably the pH of the feeding solution is set between 3 and 7 and wherein preferably the temperature of the feeding solution is kept between 20? C. and 80? C.; [0617] the method resulting in any one of the oligosaccharides with a concentration of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L in the final cultivation.

[0618] 87. Method according to preferred embodiment 86, wherein the lactose feed is accomplished by adding lactose from the beginning of the cultivation in a concentration of at least 5 mM, preferably in a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably in a concentration>300 mM.

[0619] 88. Method according to any one of preferred embodiment 86 or 87, wherein the lactose feed is accomplished by adding lactose to the cultivation in a concentration, such, that throughout the production phase of the cultivation a lactose concentration of at least 5 mM, preferably 10 mM or 30 mM is obtained.

[0620] 89. Method according to any one of preferred embodiment 42 to 88, wherein the host cells are cultivated for at least about 60, 80, 100, or about 120 hours or in a continuous manner.

[0621] 90. Method according to any one of preferred embodiment 42 to 89, wherein the cell is cultivated in a culture medium comprising a carbon source comprising a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract; preferably, wherein the carbon source is chosen from the list comprising glucose, glycerol, fructose, sucrose, maltose, lactose, arabinose, malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvate.

[0622] 91. Method according to any one of preferred embodiment 42 to 90, wherein the culture medium contains at least one precursor selected from the group comprising lactose, galactose, fucose, GlcNAc, GalNAc, lacto-N-biose (LNB), N-acetyllactosamine (LacNAc).

[0623] 92. Method according to any one of preferred embodiment 42 to 91, wherein a first phase of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the culture medium before the precursor, preferably lactose, is added to the culture medium in a second phase.

[0624] 93. Method according to any one of preferred embodiment 42 to 92, wherein a first phase of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the culture medium comprising a precursor, preferably lactose, followed by a second phase wherein only a carbon-based substrate, preferably glucose or sucrose, is added to the culture medium.

[0625] 94. Method according to any one of preferred embodiment 42 to 93, wherein a first phase of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the culture medium comprising a precursor, preferably lactose, followed by a second phase wherein a carbon-based substrate, preferably glucose or sucrose, and a precursor, preferably lactose, are added to the culture medium.

[0626] 95. Method according to any one of preferred embodiments 42 to 94, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography.

[0627] 96. Method according to any one of preferred embodiments 42 to 95, further comprising purification of any one of the oligosaccharides from the cell.

[0628] 97. Method according to preferred embodiment 96, wherein the purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying or vacuum roller drying.

[0629] 98. Cell according to any one of preferred embodiments 1 to 41 or method according to any one of preferred embodiments 42 to 97, wherein the cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell, [0630] preferably the bacterium is an Escherichia coli strain, more preferably an Escherichia coli strain that is a K-12 strain, even more preferably the Escherichia coli K-12 strain is E. coli MG1655, [0631] preferably the fungus belongs to a genus chosen from the group comprising Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus, [0632] preferably the yeast belongs to a genus chosen from the group comprising Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces or Debaryomyces, [0633] preferably the plant cell is an algal cell or is derived from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant, [0634] preferably the animal cell is derived from non-human mammals, birds, fish, invertebrates, reptiles, amphibians or insects or is a genetically modified cell line derived from human cells excluding embryonic stem cells, more preferably the human and non-human mammalian cell is an epithelial cell, an embryonic kidney cell, a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell, an NIH-3T3 cell, a non-mammary adult stem cell or derivatives thereof, more preferably the insect cell is derived from Spodoptera frugiperda, Bombyx mori, Mamestra brassicae, Trichoplusia ni or Drosophila melanogaster, [0635] preferably the protozoan cell is a Leishmania tarentolae cell.

[0636] 99. Cell according to preferred embodiment 98, or method according to preferred embodiment 98, wherein the cell is a viable Gram-negative bacterium that comprises a reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose compared to a non-modified progenitor.

[0637] 100. Use of a cell according to any one of preferred embodiments 1 to 41, 98, 99, or method according to any one of preferred embodiment 42 to 99 for the production of a mixture of at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide.

[0638] The following examples will serve as further illustration and clarification of this disclosure and are not intended to be limiting.

EXAMPLES

Example 1. Materials and Methods Escherichia coli

Media

[0639] The Luria Broth (LB) medium comprised 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). The minimal medium used in the cultivation experiments in 96-well plates or in shake flasks contained 2.00 g/L NH4Cl, 5.00 g/L (NH4)2SO4, 2.993 g/L KH2PO4, 7.315 g/L K2HPO4, 8.372 g/L MOPS, 0.5 g/L NaCl, 0.5 g/L MgSO4.Math.7H2O, 30 g/L sucrose or 30 g/L glycerol, 1 ml/L vitamin solution, 100 ?l/L molybdate solution, and 1 mL/L selenium solution. As specified in the respective examples, 0.30 g/L sialic acid, 20 g/L lactose, 20 g/L LacNAc and/or 20 g/L LNB were additionally added to the medium as precursor(s). The minimal medium was set to a pH of 7 with 1M KOH. Vitamin solution comprised 3.6 g/L FeC12.Math.4H2O, 5 g/L CaCl2.Math.2H2O, 1.3 g/L MnC12.Math.2H2O, 0.38 g/L CuCl2.Math.2H2O, 0.5 g/L CoCl2.Math.6H2O, 0.94 g/L ZnCl2, 0.0311 g/L H3BO4, 0.4 g/L Na2EDTA.Math.2H2O and 1.01 g/L thiamine.Math.HCl. The molybdate solution contained 0.967 g/L NaMoO4.Math.2H2O. The selenium solution contained 42 g/L Seo2.

[0640] The minimal medium for fermentations contained 6.75 g/L NH4Cl, 1.25 g/L (NH4).sub.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 ?L/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above. As specified in the respective examples, 0.30 g/L sialic acid, 20 g/L lactose, 20 g/L LacNAc and/or 20 g/L LNB were additionally added to the medium as precursor(s).

[0641] Complex medium was sterilized by autoclaving (121? C., 21 min) and minimal medium by filtration (0.22 ?m Sartorius). When necessary, the medium was made selective by adding an antibiotic: e.g., chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L).

Plasmids

[0642] pKD46 (Red helper plasmid, Ampicillin resistance), pKD3 (contains an FRT-flanked chloramphenicol resistance (cat) gene), pKD4 (contains an FRT-flanked kanamycin resistance (kan) gene), and pCP20 (expresses FLP recombinase activity) plasmids were obtained from Prof. R. Cunin (Vrije Universiteit Brussel, Belgium in 2007). Plasmids were maintained in the host E. coli DH5alpha (F.sup.?, phi80dlacZ4M15, ?(lacZYA-argF) U169, deoR, recA1, endA1, hsdR17(rk.sup.?, mk.sup.+), phoA, supE44, lambda.sup.?, thi-1, gyrA96, relA1) bought from Invitrogen.

Strains and Mutations

[0643] Escherichia coli K12 MG1655 [?.sup.?, F.sup.?; rph-1] was obtained from the Coli Genetic Stock Center (US), CGSC Strain #: 7740, in March 2007. Gene disruptions, gene introductions and gene replacements were performed using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). This technique is based on antibiotic selection after homologous recombination performed by lambda Red recombinase. Subsequent catalysis of a flippase recombinase ensures removal of the antibiotic selection cassette in the final production strain. Transformants carrying a Red helper plasmid pKD46 were grown in 10 mL LB media with ampicillin, (100 mg/L) and L-arabinose (10 mM) at 30? ? C. to an OD.sub.600 nm of 0.6. The cells were made electrocompetent by washing them with 50 mL of ice-cold water, a first time, and with 1 mL ice cold water, a second time. Then, the cells were resuspended in 50 ?L of ice-cold water. Electroporation was done with 50 ?L of cells and 10-100 ng of linear double-stranded-DNA product by using a Gene Pulser? (BioRad) (600 ?, 25 ?FD, and 250 volts). After electroporation, cells were added to 1 mL LB media incubated 1 h at 37? C., and finally spread onto LB-agar containing 25 mg/L of chloramphenicol or 50 mg/L of kanamycin to select antibiotic resistant transformants. The selected mutants were verified by PCR with primers upstream and downstream of the modified region and were grown in LB-agar at 42?C for the loss of the helper plasmid. The mutants were tested for ampicillin sensitivity. The linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and their derivates as template. The primers used had a part of the sequence complementary to the template and another part complementary to the side on the chromosomal DNA where the recombination must take place. For the genomic knock-out, the region of homology was designed 50-nt upstream and 50-nt downstream of the start and stop codon of the gene of interest. For the genomic knock-in, the transcriptional starting point (+1) had to be respected. PCR products were PCR-purified, digested with Dpn1, re-purified from an agarose gel, and suspended in elution buffer (5 mM Tris, pH 8.0). Selected mutants were transformed with pCP20 plasmid, which is an ampicillin and chloramphenicol resistant plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis. The ampicillin-resistant transformants were selected at 30? C., after which a few were colony purified in LB at 42? C. and then tested for loss of all antibiotic resistance and of the FLP helper plasmid. The gene knock outs and knock ins are checked with control primers.

[0644] In an example for GDP-fucose production, the mutant strain was derived from E. coli K12 MG1655 comprising knock-outs of the E. coli wcaJ and thyA genes and genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g., CscB from E. coli W with SEQ ID NO: 01, a fructose kinase like e.g., Frk originating from Zymomonas mobilis with SEQ ID NO: 02 and a sucrose phosphorylase like e.g., BaSP originating from Bifidobacterium adolescentis with SEQ ID NO: 03. For production of fucosylated oligosaccharides, the mutant GDP-fucose production strain was additionally modified with expression plasmids comprising constitutive transcriptional units for an alpha-1,2-fucosyltransferase like e.g., HpFutC from H. pylori with SEQ ID NO: 04 and/or an alpha-1,3-fucosyltransferase like e.g., HpFucT from H. pylori with SEQ ID NO: 05 and with a constitutive transcriptional unit for a selection marker like e.g. E. coli thyA with SEQ ID NO: 06. The constitutive transcriptional units of the fucosyltransferase genes could also be present in the mutant E. coli strain via genomic knock-ins. GDP-fucose production can further be optimized in the mutant E. coli strain by genomic knock-outs of the E. coli genes comprising glgC, agp, pfkA, pfkB, pgi, arcA, iclR, pgi and lon as described in WO 2016075243 and WO 2012007481. GDP-fucose production can additionally be optimized comprising genomic knock-ins of constitutive transcriptional units for a mannose-6-phosphate isomerase like e.g., the E. coli manA with SEQ ID NO: 07, a phosphomannomutase like e.g., manB from E. coli with SEQ ID NO: 08, a mannose-1-phosphate guanylyltransferase like e.g., manC from E. coli with SEQ ID NO: 09, a GDP-mannose 4,6-dehydratase like e.g., gmd from E. coli with SEQ ID NO: 10 and a GDP-L-fucose synthase like e.g., fcl from E. coli with SEQ ID NO: 11. GDP-fucose production can also be obtained by genomic knock-outs of the E. coli fucK and fuel genes and genomic knock-ins of constitutive transcriptional units containing a fucose permease like e.g., fucP from E. coli with SEQ ID NO: 12 and a bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase like e.g., fkp from Bacteroides fragilis with SEQ ID NO: 13. If the mutant strains producing GDP-fucose were intended to make fucosylated lactose structures, the strains were additionally modified with genomic knock-outs of the E. coli LacZ, LacY and LacA genes and with a genomic knock-in of a constitutive transcriptional unit for a lactose permease like e.g., the E. coli LacY with SEQ ID NO: 14.

[0645] Alternatively, and/or additionally, production of GDP-fucose and/or fucosylated structures can further be optimized in the mutant E. coli strains with genomic knock-ins of a constitutive transcriptional unit comprising a membrane transporter protein like e.g., MdfA from Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID POAEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207) or iceT from Citrobacter youngae (UniProt ID D4B8A6).

[0646] In an example for sialic acid production, the mutant strain was derived from E. coli K12 MG1655 comprising knock-outs of the E. coli nagA and nagB genes and genomic knock-ins of constitutive transcriptional units containing a glucosamine 6-phosphate N-acetyltransferase like e.g., GNA1 from Saccharomyces cerevisiae with SEQ ID NO: 15, an N-acetylglucosamine 2-epimerase like e.g., AGE from Bacteroides ovatus with SEQ ID NO: 16 and an N-acetylneuraminate (Neu5Ac) synthase like e.g., NeuB from Neisseria meningitidis with SEQ ID NO: 17. Sialic acid production can further be optimized in the mutant E. coli strain with genomic knock-outs of any one or more of the E. coli genes comprising nagC, nagD, nagE, nanA, nank, nanK, manX, manY and manZ as described in WO 2018122225 and/or genomic knock-outs of the E. coli genes comprising any one or more of nanT, poxB, IdhA, adhE, aldB, pflA, pflC, ybiY, ackA and/or pta and with genomic knock-ins of constitutive transcriptional units comprising an L-glutamine-D-fructose-6-phosphate aminotransferase like e.g., the mutant glmS*54 from E. coli with SEQ ID NO: 18 (differing from the wild-type E. coli glmS protein by an A39T, an R250C and an G472S mutation) and a phosphatase like e.g., yqaB from E. coli with SEQ ID NO: 19 or any one of the E. coli genes 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 or PsMupP from Pseudomonas putida, ScDOG1 from S. cerevisiae and BsAraL from Bacillus subtilis as described in WO 2018122225 and an acetyl-CoA synthetase like e.g., acs from E. coli (UniProt ID P27550). Sialic acid production can also be obtained by knock-outs of the E. coli nagA and nagB genes and genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g., glmM from E. coli with SEQ ID NO: 31, an N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase like e.g., glmU from E. coli with SEQ ID NO: 32, an UDP-N-acetylglucosamine 2-epimerase like e.g., NeuC from Campylobacter jejuni with SEQ ID NO: 20 and an N-acetylneuraminate synthase like e.g., NeuB from N. meningitidis with SEQ ID NO: 17. Also in this mutant strain, sialic acid production can further be optimized with genomic knock-ins of constitutive transcriptional units comprising an L-glutamine-D-fructose-6-phosphate aminotransferase like e.g., the mutant glmS*54 from E. coli with SEQ ID NO: 18 and a phosphatase like e.g., yqaB from E. coli with SEQ ID NO: 19 or any one of the E. coli genes 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 or PsMupP from Pseudomonas putida, ScDOG1 from S. cerevisiae and BsAraL from Bacillus subtilis as described in WO 2018122225 and an acetyl-CoA synthetase like e.g., acs from E. coli (UniProt ID P27550).

[0647] Alternatively, and/or additionally, sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a bifunctional UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase like e.g., from Mus musculus (strain C57BL/6J) (UniProt ID Q91WG8), an N-acylneuraminate-9-phosphate synthetase like e.g., from Pseudomonas sp. UW4 (UniProt ID K9NPH9) and an N-acylneuraminate-9-phosphatase like e.g., from Candidatus magnetomorum sp. HK-1 (UniProt ID KPA15328.1) or from Bacteroides thetaiotaomicron (UniProt ID Q8A712).

[0648] Alternatively, and/or additionally, sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g., glmM from E. coli (SEQ ID NO: 31), an N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase like e.g., glmU from E. coli (SEQ ID NO: 32), a bifunctional UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase like e.g., from M. musculus (strain C57BL/6J) (UniProt ID Q91WG8), an N-acylneuraminate-9-phosphate synthetase like e.g., from Pseudomonas sp. UW4 (UniProt ID K9NPH9) and an N-acylneuraminate-9-phosphatase like e.g., from Candidatus magnetomorum sp. HK-1 (UniProt ID KPA15328.1) or from Bacteroides thetaiotaomicron (UniProt ID Q8A712).

[0649] In an example for sialylated oligosaccharide production, the sialic acid production strains further need to express an N-acylneuraminate cytidylyltransferase like e.g., NeuA from Pasteurella multocida with SEQ ID NO: 21, and a beta-galactoside alpha-2,3-sialyltransferase like e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide comprising amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity like SEQ ID NO: 22, NmeniST3 from N. meningitidis with SEQ ID NO: 23, PmultST2 from P. multocida subsp. multocida str. Pm70 (GenBank NO. AAK02592.1), a beta-galactoside alpha-2,6-sialyltransferase like e.g., PdST6 from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide comprising amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity like SEQ ID NO: 24, P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide comprising amino acid residues 18 to 514 of UniProt ID A8QYL1 having beta-galactoside alpha-2,6-sialyltransferase activity like SEQ ID NO: 25 and/or an alpha-2,8-sialyltransferase like e.g., from M. musculus (UniProt ID Q64689). Constitutive transcriptional units of PmNeuA and the sialyltransferases can be delivered to the mutant strain either via genomic knock-in or via expression plasmids. If the mutant strains producing sialic acid and CMP-sialic acid were intended to make sialylated lactose structures, the strains were additionally modified with genomic knock-outs of the E. coli LacZ, LacY and LacA genes and with a genomic knock-in of a constitutive transcriptional unit for a lactose permease like e.g., the E. coli LacY with SEQ ID NO: 14.

[0650] Alternatively, and/or additionally, sialic acid and/or sialylated oligosaccharide production can further be optimized in the mutant E. coli strains with a genomic knock-in of a constitutive transcriptional unit comprising a membrane transporter protein like e.g., a sialic acid transporter like e.g., nanT from E. coli K-12 MG1655 (UniProt ID P41036), nanT from E. coli 06:H1 (UniProt ID Q8FD59), nanT from E. coli O157:H7 (UniProt ID Q8X9G8) or nanT from E. albertii (UniProt ID BIEFH1) or a porter like e.g., EntS from E. coli (UniProt ID P24077), EntS from Kluyvera ascorbata (UniProt ID A0A378GQ13) or EntS from Salmonella enterica subsp. arizonae (UniProt ID A0A6Y2K4E8), MdfA from Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID POAEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207), iceT from Citrobacter youngae (UniProt ID D4B8A6), SetA from E. coli (UniProt ID P31675), SetB from E. coli (UniProt ID P33026) or SetC from E. coli (UniProt ID P31436) or an ABC transporter like e.g., oppF from E. coli (UniProt ID P77737), ImrA from Lactococcus lactis subsp. lactis bv. diacetylactis (UniProt ID AOAIVONEL4), or Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4).

[0651] All mutant strains producing sialic acid, CMP-sialic acid and/or sialylated oligosaccharides could optionally be adapted for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g., CscB from E. coli W with SEQ ID NO: 01, a fructose kinase like e.g., Frk originating from Z. mobilis with SEQ ID NO: 02 and a sucrose phosphorylase like e.g., BaSP originating from B. adolescentis with SEQ ID NO: 03.

[0652] In an example to produce LN3 (GlcNAc-b1,3-Gal-b1,4-Glc) and oligosaccharides originating thereof comprising lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), the mutant strain was derived from E. coli K12 MG1655 and modified with a knock-out of the E. coli LacZ and nagB genes and with a genomic knock-in of a constitutive transcriptional unit for a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g., LgtA from N. meningitidis with SEQ ID NO: 26. For LNT or LNnT production, the mutant strain is further modified with constitutive transcriptional units for an N-acetylglucosamine beta-1,3-galactosyltransferase like e.g., WbgO from E. coli 055:H7 with SEQ ID NO: 27 or an N-acetylglucosamine beta-1,4-galactosyltransferase like e.g., LgtB from N. meningitidis with SEQ ID NO: 28, respectively, that can be delivered to the strain either via genomic knock-in or from an expression plasmid. Optionally, multiple copies of the galactoside beta-1,3-N-acetylglucosaminyltransferase, the N-acetylglucosamine beta-1,3-galactosyltransferase and/or the N-acetylglucosamine beta-1,4-galactosyltransferase genes could be added to the mutant E. coli strains. Also, LNT and/or LNnT production can be enhanced by improved UDP-GlcNAc production by modification of the strains with one or more genomic knock-ins of a constitutive transcriptional unit for an L-glutamine-D-fructose-6-phosphate aminotransferase like e.g., the mutant glmS*54 from E. coli with SEQ ID NO: 18. In addition, the strains can optionally be modified for enhanced UDP-galactose production with genomic knock-outs of the E. coli ushA, galT, IdhA and agp genes. The mutant E. coli strains can also optionally be adapted with a genomic knock-in of a constitutive transcriptional unit for an UDP-glucose-4-epimerase like e.g., galE from E. coli with SEQ ID NO: 29, a phosphoglucosamine mutase like e.g., glmM from E. coli with SEQ ID NO: 31 and an N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase like e.g., glmU from E. coli with SEQ ID NO: 32. The mutant strains could also optionally be adapted for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g., CscB from E. coli W with SEQ ID NO: 01, a fructose kinase like e.g., Frk originating from Z. mobilis with SEQ ID NO: 02 and a sucrose phosphorylase like e.g., BaSP originating from B. adolescentis with SEQ ID NO: 03.

[0653] Alternatively, and/or additionally, production of LN3, LNT, LNnT and oligosaccharides derived thereof can further be optimized in the mutant E. coli strains with a genomic knock-in of a constitutive transcriptional unit comprising a membrane transporter protein like e.g., MdfA from Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID POAEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207) or iceT from Citrobacter youngae (UniProt ID D4B8A6).

[0654] Preferably but not necessarily, the glycosyltransferases, the proteins involved in nucleotide-activated sugar synthesis and/or membrane transporter proteins were N- and/or C-terminally fused to a solubility enhancer tag like e.g., a SUMO-tag, an MBP-tag, His, FLAG, Strep-II, Halo-tag, NusA, thioredoxin, GST and/or the Fh8-tag to enhance their solubility (Costa et al., Front. Microbiol. 2014, doi.org/10.3389/fmicb.2014.00063; Fox et al., Protein Sci. 2001, 10(3), 622-630; Jia and Jeaon, Open Biol. 2016, 6: 160196).

[0655] Optionally, the mutant E. coli strains were modified with a genomic knock-in of a constitutive transcriptional unit encoding a chaperone protein like e.g., DnaK, DnaJ, GrpE, or the GroEL/ES chaperonin system (Baneyx F., Palumbo J. L. (2003) Improving Heterologous Protein Folding via Molecular Chaperone and Foldase Co-Expression. In: Vaillancourt P. E. (eds) E. coli Gene Expression Protocols. Methods in Molecular Biology?, vol 205. Humana Press).

[0656] Optionally, the mutant E. coli strains are modified to create a glycominimized E. coli strain comprising genomic knock-out of any one or more of non-essential glycosyltransferase genes comprising pgaC, pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcal, 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.

[0657] All constitutive promoters, UTRs and terminator sequences originated from the libraries described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360) and Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148): the genes were expressed using promoters MutalikP5 (PROM0005_MutalikP5) and apFAB82 (PROM0050_apFAB82) as described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360), UTRs used comprised 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 sequence used was ilvGEDA (TER0007_ilvGEDA) as described by Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148). All genes were ordered synthetically at Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com) and the codon usage was adapted using the tools of the supplier. The SEQ ID NOs described in this disclosure are summarized in Table 1.

[0658] All strains were stored in cryovials at ?80? C. (overnight LB culture mixed in a 1:1 ratio with 70% glycerol).

TABLE-US-00001 TABLE 1 Overview of SEQ ID NOs described in this disclosure Country of origin of digital SEQ Name/ sequence ID NO: identifier Organism Origin information 01 CscB Escherichia coli W Synthetic USA 02 Frk Zymomonas mobilis Synthetic United Kingdom 03 BaSP Bifidobacterium Synthetic Germany adolescentis 04 HpFutC Helicobacter pylori Synthetic United Kingdom UA1234 05 HpFucT Helicobacter pylori Synthetic United Kingdom UA1234 06 thyA Escherichia coli K-12 Synthetic USA MG1655 07 manA Escherichia coli K-12 Synthetic USA MG1655 08 manB Escherichia coli K-12 Synthetic USA MG1655 09 manC Escherichia coli K-12 Synthetic USA MG1655 10 gmd Escherichia coli K-12 Synthetic USA MG1655 11 fcl Escherichia coli K-12 Synthetic USA MG1655 12 fucP Escherichia coli K-12 Synthetic USA MG1655 13 fkp Bacteroides fragilis Synthetic United Kingdom 14 LacY Escherichia coli K-12 Synthetic USA MG1655 15 GNA1 Saccharomyces cerevisiae Synthetic USA 16 AGE Bacteroides ovatus Synthetic USA 17 neuB Neisseria meningitidis Synthetic United Kingdom 18 glmS*54 Escherichia coli K-12 Synthetic USA MG1655 19 phosphatase Escherichia coli K-12 Synthetic USA MG1655 20 neuC Campylobacter jejuni Synthetic USA 21 neuA Pasteurella multocida Synthetic USA 22 alpha-2,3- Pasteurella multocida Synthetic USA sialyltransferase 23 alpha-2,3- Neisseria meningitidis Synthetic United Kingdom sialyltransferase 24 alpha-2,6- Photobacterium damselae Synthetic Japan sialyltransferase 25 alpha-2,6- Photobacterium sp. JT- Synthetic Japan sialyltransferase ISH-224 26 LgtA Neisseria meningitidis Synthetic United Kingdom 27 WbgO Escherichia coli O55:H7 Synthetic Germany 28 LgtB Neisseria meningitidis Synthetic United Kingdom MC58 29 galE Escherichia coli K-12 Synthetic USA MG1655 30 Lac12 Kluyveromyces lactis Synthetic USA 31 glmM Escherichia coli K-12 Synthetic USA MG1655 32 glmU Escherichia coli K-12 Synthetic USA MG1655

Cultivation Conditions

[0659] A preculture of 96-well microtiter plate experiments was started from a cryovial, in 150 ?L LB and was incubated overnight at 37? ? C. on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96-well square microtiter plate, with 400 ?L minimal medium by diluting 400?. These final 96-well culture plates were then incubated at 37? ? C. on an orbital shaker at 800 rpm for 72 h, or shorter, or longer. To measure sugar concentrations at the end of the cultivation experiment whole broth samples were taken from each well by boiling the culture broth for 15 min at 60? ? C. before spinning down the cells (=average of intra- and extracellular sugar concentrations).

[0660] A preculture for the bioreactor was started from an entire 1 mL cryovial of a certain strain, inoculated in 250 mL or 500 mL minimal medium in a 1 L or 2.5 L shake flask and incubated for 24 h at 37? C. on an orbital shaker at 200 rpm. A 5 L bioreactor was then inoculated (250 mL inoculum in 2 L batch medium); the process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). Culturing conditions were set to 37? C., and maximal stirring; pressure gas flow rates were dependent on the strain and bioreactor. The pH was controlled at 6.8 using 0.5 M H2SO4 and 20% NH4OH. The exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.

Optical Density

[0661] Cell density of the cultures was frequently monitored by measuring optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or with a Spark 10M microplate reader, Tecan, Switzerland).

Analytical Analysis

[0662] Standards such as but not limited to sucrose, lactose, LacNAc, lacto-N-biose (LNB), fucosylated LacNAc (2FLacNAc, 3-FLacNAc), sialylated LacNAc, (3SLacNAc, 6SLacNAc), fucosylated LNB (2FLNB, 4FLNB), 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 made standards.

[0663] Neutral oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Evaporative Light Scattering Detector (ELSD) or a Refractive Index (RI) detection. A volume of 0.7 ?L sample was injected on a Waters Acquity UPLC BEH Amide column (2.1?100 mm; 130 ?; 1.7 ?m) column with an Acquity UPLC BEH Amide VanGuard column, 130 ?, 2.1?5 mm. The column temperature was 50? C. The mobile phase comprised a ? water and ? acetonitrile solution to which 0.2% triethylamine was added. The method was isocratic with a flow of 0.130 mL/min. The ELS detector had a drift tube temperature of 50? C. and the N2 gas pressure was 50 psi, the gain 200 and the data rate 10 pps. The temperature of the RI detector was set at 35? C.

[0664] Sialylated oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Refractive Index (RI) detection. A volume of 0. 5 ?L sample was injected on a Waters Acquity UPLC BEH Amide column (2.1?100 mm; 130 ?; 1.7 ?m). The column temperature was 50? C. The mobile phase comprised a mixture of 70% acetonitrile, 26% ammonium acetate buffer (150 mM) and 4% methanol to which 0.05% pyrrolidine was added. The method was isocratic with a flow of 0.150 mL/min. The temperature of the RI detector was set at 35? C.

[0665] Both neutral and sialylated sugars were analyzed on a Waters Acquity H-class UPLC with Refractive Index (RI) detection. A volume of 0.5 ?L sample was injected on a Waters Acquity UPLC BEH Amide column (2.1?100 mm; 130 ?; 1.7 ?m). The column temperature was 50? C. The mobile phase comprised a mixture of 72% acetonitrile and 28% ammonium acetate buffer (100 mM) to which 0.1% triethylamine was added. The method was isocratic with a flow of 0.260 mL/min. The temperature of the RI detector was set at 35? C.

[0666] For analysis on a mass spectrometer, a Waters Xevo TQ-MS with Electron Spray Ionization (ESI) was used with a desolvation temperature of 450? C., a nitrogen desolvation gas flow of 650 L/h and a cone voltage of 20 V. The MS was operated in selected ion monitoring (SIM) in negative mode for all oligosaccharides. Separation was performed on a Waters Acquity UPLC with a Thermo Hypercarb column (2.1?100 mm; 3 ?m) on 35? C. A gradient was used wherein eluent A was ultrapure water with 0.1% formic acid and wherein eluent B was acetonitrile with 0.1% formic acid. The oligosaccharides were separated in 55 min using the following gradient: an initial increase from 2 to 12% of eluent B over 21 min, a second increase from 12 to 40% of eluent B over 11 min and a third increase from 40 to 100% of eluent B over 5 min. As a washing step 100% of eluent B was used for 5 min. For column equilibration, the initial condition of 2% of eluent B was restored in 1 min and maintained for 12 min.

[0667] Both neutral and sialylated sugars at low concentrations (below 50 mg/L) were analyzed on a Dionex HPAEC system with pulsed amperometric detection (PAD). A volume of 5 ?L of sample was injected on a Dionex CarboPac PA200 column 4?250 mm with a Dionex CarboPac PA200 guard column 4?50 mm. The column temperature was set to 30? C. A gradient was used wherein eluent A was deionized water, wherein eluent B was 200 mM Sodium hydroxide and wherein eluent C was 500 mM Sodium acetate. The oligosaccharides were separated in 60 min while maintaining a constant ratio of 25% of eluent B using the following gradient: an initial isocratic step maintained for 10 min of 75% of eluent A, an initial increase from 0 to 4% of eluent C over 8 min, a second isocratic step maintained for 6 min of 71% of eluent A and 4% of eluent C, a second increase from 4 to 12% of eluent C over 2.6 min, a third isocratic step maintained for 3.4 min of 63% of eluent A and 12% of eluent C and a third increase from 12 to 48% of eluent C over 5 min. As a washing step 48% of eluent C was used for 3 min. For column equilibration, the initial condition of 75% of eluent A and 0% of eluent C was restored in 1 min and maintained for 11 min. The applied flow was 0.5 mL/min.

Example 2. Materials and Methods Saccharomyces cerevisiae

Media

[0668] Strains were grown on Synthetic Defined yeast medium with Complete Supplement Mixture (SD CSM) or CSM drop-out (SD CSM-Ura, SD CSM-Trp, SD CSM-His) containing 6.7 g/L Yeast Nitrogen Base without amino acids (YNB w/o AA, Difco), 20 g/L agar (Difco) (solid cultures), 22 g/L glucose monohydrate or 20 g/L lactose and 0.79 g/L CSM or 0.77 g/L CSM-Ura, 0.77 g/L CSM-Trp, or 0.77 g/L CSM-His (MP Biomedicals).

Strains

[0669] S. cerevisiae BY4742 created by Brachmann et al. (Yeast (1998) 14:115-32) was used, available in the Euroscarf culture collection. All mutant strains were created by homologous recombination or plasmid transformation using the method of Gietz (Yeast 11:355-360, 1995).

Plasmids

[0670] In an example to produce GDP-fucose, the yeast expression plasmid p2a_2?_Fuc (Chan 2013, Plasmid 70, 2-17) was used for expression of foreign genes in S. cerevisiae. This plasmid contained an ampicillin resistance gene and a bacterial origin of replication to allow for selection and maintenance in E. coli and the 2? yeast ori and the Ura3 selection marker for selection and maintenance in yeast. This plasmid further contained constitutive transcriptional units for a lactose permease like e.g., LAC12 from Kluyveromyces lactis with SEQ ID NO: 30, a GDP-mannose 4,6-dehydratase like e.g., gmd from E. coli with SEQ ID NO: 10 and a GDP-L-fucose synthase like e.g., fcl from E. coli with SEQ ID NO: 11. In another example, the yeast expression plasmid p2a_2 ?_Fuc2 can be used as an alternative expression plasmid of the p2a_2 ?_Fuc plasmid comprising next to the ampicillin resistance gene, the bacterial ori, the 2? yeast ori and the Ura3 selection marker constitutive transcriptional units for a lactose permease like e.g., LAC12 from K. lactis with SEQ ID NO: 30, a fucose permease like e.g., fucP from E. coli with SEQ ID NO: 12 and a bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase like e.g., fkp from B. fragilis with SEQ NO: ID 13. To further produce fucosylated oligosaccharides, the p2a_2 ?_Fuc and its variant the p2a_2 ?_Fuc2, additionally contains a constitutive transcriptional unit for an alpha-1,2-fucosyltransferase like e.g., HpFutC from H. pylori with SEQ ID NO: 04 and/or an alpha-1,3-fucosyltransferase like e.g., HpFucT from H. pylori with SEQ ID NO: 05.

[0671] In an example to produce sialic acid and CMP-sialic acid, a yeast expression plasmid was derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the TRP1 selection marker and constitutive transcriptional units for an L-glutamine-D-fructose-6-phosphate aminotransferase like e.g., the mutant glmS*54 from E. coli with SEQ ID NO: 18, a phosphatase like e.g., yqaB from E. coli with SEQ ID NO: 19 or any one of the E. coli genes 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 or PsMupP from Pseudomonas putida, ScDOGI from S. cerevisiae and BsAraL from Bacillus subtilis as described in WO 2018122225, an N-acetylglucosamine 2-epimerase like e.g., AGE from B. ovatus with SEQ ID NO: 16, an N-acetylneuraminate synthase like e.g., NeuB from N. meningitidis with SEQ ID NO: 17 and an N-acylneuraminate cytidylyltransferase like e.g., NeuA from P. multocida with SEQ ID NO: 21. Optionally, a constitutive transcriptional unit for a glucosamine 6-phosphate N-acetyltransferase like e.g., GNA1 from S. cerevisiae with SEQ ID NO: 15 was added as well. In an example to produce sialylated oligosaccharides, the plasmid further comprised constitutive transcriptional units for a lactose permease like e.g., LAC12 from K. lactis with SEQ ID NO: 30, and a beta-galactoside alpha-2,3-sialyltransferase like e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide comprising amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity like SEQ ID NO: 22, NmeniST3 from N. meningitidis (SEQ ID NO: 23) or PmultST2 from P. multocida subsp. multocida str. Pm70 (GenBank NO. AAK02592.1), a beta-galactoside alpha-2,6-sialyltransferase like e.g., PdST6 from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide comprising amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity like SEQ ID NO: 24, P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide comprising amino acid residues 18 to 514 of UniProt ID A8QYL1 having beta-galactoside alpha-2,6-sialyltransferase activity like SEQ ID NO: 25 and/or an alpha-2,8-sialyltransferase like e.g., from M. musculus (UniProt ID Q64689).

[0672] In an example to produce UDP-galactose, a yeast expression plasmid was derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the HIS3 selection marker and a constitutive transcriptional unit for an UDP-glucose-4-epimerase like e.g., galE from E. coli with SEQ ID NO: 29. In an example to produce LN3 and LN3-derived oligosaccharides like LNT or LNnT, this plasmid was further modified with constitutive transcriptional units for a lactose permease like e.g., LAC12 from K. lactis with SEQ ID NO: 30, a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g., lgtA from N. meningitidis with SEQ ID NO: 26 and, an N-acetylglucosamine beta-1,3-galactosyltransferase like e.g., WbgO from E. coli 055:H7 with SEQ ID NO: 27 or an N-acetylglucosamine beta-1,4-galactosyltransferase like e.g., lgtB from N. meningitidis with SEQ ID NO: 28, respectively.

[0673] Preferably but not necessarily, any one or more of the glycosyltransferases, the proteins involved in nucleotide-activated sugar synthesis and/or membrane transporter proteins were N- and/or C-terminally fused to a SUMOstar tag (e.g., obtained from pYSUMOstar, Life Sensors, Malvern, PA) to enhance their solubility.

[0674] Optionally, the mutant yeast strains were modified with a genomic knock-in of a constitutive transcriptional unit encoding a chaperone protein like e.g., Hsp31, Hsp32, Hsp33, Sno4, Kar2, Ssb1, Sse1, Sse2, Ssa1, Ssa2, Ssa3, Ssa4, Ssb2, Ecm10, Ssc1, Ssq1, Ssz1, Lhs1, Hsp82, Hsc82, Hsp78, Hsp104, Tcp1, Cct4, Cct8, Cct2, Cct3, Cct5, Cct6, or Cct7 (Gong et al., 2009, Mol. Syst. Biol. 5: 275).

[0675] Plasmids were maintained in the host E. coli DH5alpha (F.sup.?, phi80dlacZdeltaM15, delta(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk.sup.?, mk.sup.+), phoA, supE44, lambda.sup.?, thi-1, gyrA96, relA1) bought from Invitrogen.

Heterologous and Homologous Expression

[0676] Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9, IDT or Twist Bioscience. Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.

Cultivations Conditions

[0677] In general, yeast strains were initially grown on SD CSM plates to obtain single colonies. These plates were grown for 2-3 days at 30? C. Starting from a single colony, a preculture was grown over night in 5 mL at 30? C., shaking at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL media. These shake flasks were incubated at 30? C. with an orbital shaking of 200 rpm.

Gene Expression Promoters

[0678] Genes were expressed using synthetic constitutive promoters, as described by Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012).

Example 3. Production of an Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LacNAc and 6-Sialylated LacNAc with a Modified E. coli Host

[0679] An E. coli K-12 MG1655 strain modified with a genomic knock-in of a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase (neuA) from P. multocida with SEQ ID NO: 21 and containing a knock-out of the E. coli lacZ gene is further transformed with an expression plasmid containing constitutive transcriptional units for the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 24. The novel strain is evaluated for production of an oligosaccharide mixture comprising 3SL, 6SL, 3-sialylated LacNAc (3SLacNAc) and 6-sialylated (6SLacNAc) in whole broth samples in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains glycerol as carbon source and sialic acid, lactose and LacNAc as precursors.

Example 4. Production of an Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LacNAc and 6-Sialylated LacNAc with a Modified E. coli Host

[0680] An E. coli K-12 MG1655 strain modified with a genomic knock-in of a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase (neuA) from P. multocida with SEQ ID NO: 21 is further mutated with a genomic knock-out of the E. coli nagA, nagB and lacZ genes together with genomic knock-ins of constitutive transcriptional units for the mutant glmS*54 with SEQ ID NO: 18 from E. coli, GNA1 with SEQ ID NO: 15 from S. cerevisiae, the phosphatase yqaB from E. coli with SEQ ID NO: 19 and LgtB with SEQ ID NO: 28 from N. meningitidis. In a next step, the novel strain is transformed with an expression plasmid containing constitutive transcriptional units for the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 24. The novel strain is evaluated for production of an oligosaccharide mixture comprising 3SL, 6SL, 3-sialylated LacNAc (3SLacNAc) and 6-sialylated LacNAc (6SLacNAc) in whole broth samples in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains glycerol as carbon source and sialic acid and lactose as precursors.

Example 5. Production of an Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LacNAc and 6-Sialylated LacNAc with a Modified E. coli Host

[0681] An E. coli K-12 MG1655 strain modified to produce sialic acid as described in Example 1 is further modified with a knock-out of the E. coli lacZ gene and transformed with an expression plasmid comprising constitutive transcriptional units for neuA from P. multocida with SEQ ID NO: 21, the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 24. The novel strain is evaluated for production of an oligosaccharide mixture comprising 3SL, 6SL, 3-sialylated LacNAc (3SLacNAc) and 6-sialylated LacNAc (6SLacNAc) in whole broth samples in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains glycerol as carbon source and lactose and LacNAc as precursors.

Example 6. Production of an Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LacNAc and 6-Sialylated LacNAc with a Modified E. coli Host

[0682] An E. coli K-12 MG1655 strain modified to produce sialic acid as described in Example 1 is further mutated with a genomic knock-out of E. coli lacZ together with a genomic knock-in of a constitutive transcriptional unit for LgtB with SEQ ID NO: 28 from N. meningitidis to produce LacNAc, and transformed with an expression plasmid containing constitutive transcriptional units for neuA from P. multocida with SEQ ID NO: 21, the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 24. The novel strain is evaluated for production of an oligosaccharide mixture comprising 3SL, 6SL, 3-sialylated LacNAc (3SLacNAc) and 6-sialylated LacNAc (6SLacNAc) in whole broth samples in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains glycerol as carbon source and lactose as precursor.

Example 7. Production of Sialylated LacNAc and Poly-LacNAc Structures with a Modified E. coli Host

[0683] An E. coli K-12 MG1655 strain modified to produce sialic acid as described in Example 1 is further mutated with a genomic knock-in of a constitutive transcriptional unit for LgtB with SEQ ID NO: 28 from N. meningitidis to produce LacNAc, and transformed with an expression plasmid containing constitutive transcriptional units for neuA from P. multocida with SEQ ID NO: 21, the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 24. In a next step, the mutant strain is further transformed with a compatible expression plasmid containing a constitutive transcriptional unit for the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis with SEQ ID NO: 26. The novel strain is evaluated for production of an oligosaccharide mixture comprising LacNAc, poly-LacNAc structures i.e., (Gal-b1,4-GlcNAc) in which are built of repeated N-acetyllactosamine units that are beta1,3-linked to each other, 3-sialylated LacNAc, 6-sialylated LacNAc, and sialylated poly-LacNAc structures in which the Gal residue is sialylated, together with, in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains glycerol as carbon source and wherein no precursor needs to be supplied to the cultivation.

Example 8. Production of an Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LNB and 6-Sialylated LNB with a Modified E. coli Host

[0684] An E. coli K-12 MG1655 strain modified with a genomic knock-in of a constitutive transcriptional unit for neuA from P. multocida with SEQ ID NO: 21 and containing a knock-out of the E. coli lacZ gene is further transformed with an expression plasmid containing constitutive transcriptional units for the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 24. The novel strain is evaluated for production of an oligosaccharide mixture comprising 3SL, 6SL, 3-sialylated LNB (3SLNB) and 6-sialylated LNB (6SLNB) in whole broth samples in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains glycerol as carbon source and sialic acid, lactose and LNB as precursors.

Example 9. Production of an Oligosaccharide Mixture Comprising 3SL, 6SI, 3-Sialylated LNB and 6-Sialylated LNB with a Modified E. coli Host

[0685] An E. coli K-12 MG1655 strain modified with a genomic knock-in of a constitutive transcriptional unit for neuA from P. multocida with SEQ ID NO: 21 is further mutated with a genomic knock-out of the E. coli nagA, nagB and lacZ genes together with genomic knock-ins of constitutive transcriptional units for the mutant glmS*54 with SEQ ID NO: 18 from E. coli, GNA1 with SEQ ID NO: 15 from S. cerevisiae and WbgO with SEQ ID NO: 27 from E. coli 055:H7 to produce LNB. In a next step, the novel strain is transformed with an expression plasmid containing constitutive transcriptional units for the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 24. The novel strain is evaluated for production of an oligosaccharide mixture comprising 3SL, 6SL, 3-sialylated LNB (3SLNB) and 6-sialylated LNB (6SLNB) in whole broth samples in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains glycerol as carbon source and sialic acid and lactose as precursors.

Example 10. Production of an Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LNB and 6-Sialylated LNB with a Modified E. coli Host

[0686] An E. coli K-12 MG1655 strain modified to produce sialic acid as described in Example 1 is further modified with a knock-out of the E. coli lacZ gene and transformed with an expression plasmid comprising constitutive transcriptional units for neuA from P. multocida with SEQ ID NO: 21, the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 24. The novel strain is evaluated for production of an oligosaccharide mixture comprising 3SL, 6SL, 3-sialylated LNB (3SLNB) and 6-sialylated LNB (6SLNB) in whole broth samples in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains glycerol as carbon source and lactose and LNB as precursors.

Example 11. Production of an Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LNB and 6-Sialylated LNB with a Modified E. coli Host

[0687] An E. coli K-12 MG1655 strain modified to produce sialic acid as described in Example 1 is further mutated with a genomic knock-out of the E. coli lacZ gene together with a genomic knock-in of a constitutive transcriptional unit for WbgO with SEQ ID NO: 27 from E. coli 055:H7 to produce LNB, and transformed with an expression plasmid containing constitutive transcriptional units for neuA from P. multocida with SEQ ID NO: 21, the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 24. The novel strain is evaluated for production of an oligosaccharide mixture comprising 3SL, 6SL, 3-sialylated LNB (3SLNB) and 6-sialylated LNB (6SLNB) in whole broth samples in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains glycerol as carbon source and lactose as precursor.

Example 12. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Lactose Structures with a Modified E. coli Host

[0688] An E. coli strain adapted for GDP-fucose production as exemplified in Example 1 is further transformed with two compatible expression plasmids wherein a first plasmid contains constitutive expression units for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and the N-acylneuraminate cytidylyltransferase (NeuA) from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The strains are transformed to express 1) one fucosyltransferase combined with two sialyltransferases, 2) two fucosyltransferases combined with one sialyltransferase or 3) two fucosyltransferases combined with two sialyltransferases (Table 3). The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose structures in whole broth samples as shown in Table 3, in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and sialic acid and lactose as precursors.

TABLE-US-00002 TABLE 2 Overview of the plasmids cloned with constitutive transcriptional units for one or two fucosyltransferase gene(s) or for one or two sialyltransferase gene(s) Fucosyltransferase expressed Plasmid a1,2-linkage a1,3-linkage pMF_1A SEQ ID NO: 04 None pMF_1B None SEQ ID NO: 05 pMF_2 SEQ ID NO: 04 SEQ ID NO: 05 Sialyltransferase expressed Plasmid a2,3-linkage a2,6-linkage pMS_1A SEQ ID NO: 22 None pMS_1B None SEQ ID NO: 24 pMS_2 SEQ ID NO: 22 SEQ ID NO: 24

TABLE-US-00003 TABLE 3 Oligosaccharide production evaluated in the mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and sialic acid and lactose as precursors. Strain Plasmids* present Oligosaccharides SF1 pMF_1A, pMS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SF2 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SF3 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SF4 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SF5 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL *See Table 2 for plasmid info

Example 13. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Lactose Structures with a Modified E. coli Host

[0689] An E. coli strain adapted for GDP-fucose production as exemplified in Example 1 is further modified for sialic acid production with genomic knock-outs of the E. coli genes nagA, nagB, nanA, nanE and nanK together with genomic knock-ins of constitutive transcriptional units for the mutant glmS*54 from E. coli with SEQ ID NO: 18, GNA1 of S. cerevisiae with SEQ ID NO: 15, the N-acetylglucosamine 2-epimerase (AGE) of Bacteroides ovatus with SEQ ID NO: 16, and the N-acetylneuraminate synthase (neuB) of N. meningitidis with SEQ ID NO: 17. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The strains are transformed to express 1) one fucosyltransferase combined with two sialyltransferases, 2) two fucosyltransferases combined with one sialyltransferase or 3) two fucosyltransferases combined with two sialyltransferases (Table 4). The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose structures in whole broth samples as shown in Table 4, in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00004 TABLE 4 Oligosaccharide production evaluated in the mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF6 pMF_1A, pMS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SF7 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SF8 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SF9 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SF10 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL *See Table 2 for plasmid info

Example 14. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Lactose Structures with a Modified E. coli Host

[0690] An E. coli strain adapted for GDP-fucose production as exemplified in Example 1 is further modified for sialic acid production with genomic knock-outs of the E. coli genes nagA, nagB, nanA, nanE and nanK together with genomic knock-ins of constitutive transcriptional units for the mutant glmS*54 from E. coli with SEQ ID NO: 18, the UDP-N-acetylglucosamine 2-epimerase (neuC) of Campylobacter jejuni with SEQ ID NO: 20 and the N-acetylneuraminate synthase (neuB) of N. meningitidis with SEQ ID NO: 17. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The strains are transformed to express 1) one fucosyltransferase combined with two sialyltransferases, 2) two fucosyltransferases combined with one sialyltransferase or 3) two fucosyltransferases combined with two sialyltransferases (Table 5). The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose structures in whole broth samples as shown in Table 5, in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00005 TABLE 5 Oligosaccharide production evaluated in the mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF11 pMF_1A, pMS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SF12 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SF13 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SF14 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SF15 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL *See Table 2 for plasmid info

Example 15. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Lactose Structures with a Modified E. coli Host

[0691] An E. coli strain adapted for sialic acid production as exemplified in Example 1 is further modified via a genomic knock-out of the E. coli wca gene to increase the intracellular pool of GDP-fucose. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The strains are transformed to express 1) one fucosyltransferase combined with two sialyltransferases, 2) two fucosyltransferases combined with one sialyltransferase or 3) two fucosyltransferases combined with two sialyltransferases (Table 6). The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose structures in whole broth samples as shown in Table 6, in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00006 TABLE 6 Oligosaccharide production evaluated in the mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF16 pMF_1A, pMS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SF17 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SF18 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SF19 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SF20 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL *See Table 2 for plasmid info

Example 16. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Lactose Structures with a Modified E. coli Host

[0692] An E. coli strain adapted for sialic acid production as exemplified in Example 1 is further modified via genomic knock-outs of the E. coli wcaJ, fucK and fucI genes and genomic knock-ins of constitutive expression units for the fucose permease (fucP) from E. coli with SEQ ID NO: 12 and the bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase (fkp) from B. fragilis with SEQ NO: ID 13 to increase the intracellular pool of GDP-fucose. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The strains are transformed to express 1) one fucosyltransferase combined with two sialyltransferases, 2) two fucosyltransferases combined with one sialyltransferase or 3) two fucosyltransferases combined with two sialyltransferases (Table 7). The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose structures in whole broth samples as shown in Table 7, in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00007 TABLE 7 Oligosaccharide production evaluated in the mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF21 pMF_1A, pMS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SF22 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SF23 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SF24 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SF25 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL *See Table 2 for plasmid info

Example 17. Production of an Oligosaccharide Mixture Comprising Sialylated LN3, 3SL and LSTa with a Modified E. coli Host

[0693] An E. coli strain modified to produce LNT as described in Example 1 is further modified with a genomic knock-out of the E. coli lacZ gene and transformed with an expression plasmid containing constitutive expression cassettes for NeuA from P. multocida with SEQ ID NO: 21 and the ?-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22. The novel strain is evaluated for production of an oligosaccharide mixture comprising LN3, 3-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc), LNT, 3SL and LSTa in a growth experiment according to the culture conditions in a 96-well plate provided in Example 1, in which the culture medium contains glycerol as carbon source and both sialic acid and lactose as precursors.

Example 18. Production of an Oligosaccharide Mixture Comprising 6SL, Sialylated LN3 and LSTc with a Modified E. coli Host

[0694] An E. coli strain modified to produce LNnT as described in Example 1 was further modified with a genomic knock-out of the E. coli lacZ gene and transformed with an expression plasmid containing constitutive expression cassettes for NeuA from P. multocida with SEQ ID NO: 21 and one selected ?-2,6-sialyltransferase. In this experiment, the ?-2,6-sialyltransferase from P. damselae with SEQ ID NO: 24 and the ?-2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 with SEQ ID NO: 25 were tested. As such, three different strains were created each expressing a single ?-2,6-sialyltransferase in a specific transcriptional unit. Table 8 shows an overview of the transcriptional units used for the selected ?-2,6-sialyltransferase proteins. The novel strains were evaluated in a growth experiment in a 96-well plate according to the culture conditions provided in Example 1, in which the culture medium contained glycerol as carbon source and both sialic acid and lactose as precursors. After 72 h of incubation, the culture broth was harvested, and the sugar mixtures were analyzed as described in Example 1. All novel strains produced a mixture of oligosaccharides comprising 6SL, LN3, LNnT and LSTc (Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc). FIG. 1 shows the chromatogram obtained for the strain S2 analyzed via the Dionex method as described in Example 1. The compound 6-sialylated LN3 (Neu5Ac-a2,6-(GlcNAc-b1,3)-Gal-b1,4-Glc) could also be detected in these samples using an UPLC with RI detection as described in Example 1. The ratio of 6SL, LN3, sialylated LN3, LNnT and LSTc produced in the mixtures of the new strains could be accurately tuned by the choice of ?-2,6-sialyltransferase expressed and the transcriptional units used to express these sialyltransferases.

TABLE-US-00008 TABLE 8 Overview of mutant E. coli strains expressing a different a-2,6-sialyltransferase in a specific transcriptional unit. Transcriptional unit SEQ ID Strain Promoter UTR NO: CDS Terminator S1 PROM0005_MutalikP5 UTR0010_GalE_BCD12 24 TER0007_ilvGEDA S2 PROM0005_MutalikP5 UTR0010_GalE_BCD12 25 TER0007_ilvGEDA S3 PROM0050_apFAB82 UTR0014_GalE_LeuAB 25 TER0007_ilvGEDA

Example 19. Production of an Oligosaccharide Mixture Comprising Sialylated LN3, 3SL and LSTd with a Modified E. coli Host

[0695] An E. coli strain modified to produce LNnT as described in Example 1 was further modified with a genomic knock-out of the E. coli lacZ gene and transformed with an expression plasmid containing constitutive expression cassettes for NeuA from P. multocida with SEQ ID NO: 21 and one selected ?-2,3-sialyltransferase. In this experiment, the ?-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22 and the ?-2,3-sialyltransferase from N. meningitidis with SEQ ID NO: 23 were tested, and both ?-2,3-sialyltransferases were each cloned in two different transcriptional units. As such, four different strains were created each expressing a single ?-2,3-sialyltransferase in a specific transcriptional unit. Table 9 shows an overview of the transcriptional units used for the selected ?-2,3-sialyltransferase proteins. The novel strains were evaluated in a growth experiment in a 96-well plate according to the culture conditions provided in Example 1, in which the culture medium contained glycerol as carbon source and both sialic acid and lactose as precursors. After 72 h of incubation, the culture broth was harvested, and the sugar mixtures were analyzed as described in Example 1. All novel strains produced a mixture of oligosaccharides comprising 3SL, LN3, LNnT and LSTd (Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc). FIG. 2 shows the chromatogram obtained for the strain S5 analyzed via the Dionex method as described in Example 1. The compound 3-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc) could also be detected in these samples using an UPLC with RI detection as described in Example 1. The ratio of 3SL, LN3, sialylated LN3, LNnT and LSTd produced in the mixtures of the new strains could be accurately tuned by the choice of ?-2,3-sialyltransferase expressed and the transcriptional units used to express these sialyltransferases.

TABLE-US-00009 TABLE 9 Overview of mutant E. coli strains expressing a different ?-2,3-sialyltransferase in a specific transcriptional unit. Transcriptional unit SEQ ID Strain Promoter UTR NO: CDS Terminator S4 PROM0050_apFAB82 UTR0014_GalE_LeuAB NO 22 TER0007_ilvGEDA S5 PROM0005_MutalikP5 UTR0010_GalE_BCD12 NO 22 TER0007_ilvGEDA S6 PROM0050_apFAB82 UTR0014_GalE_LeuAB NO 23 TER0007_ilvGEDA S7 PROM0005_MutalikP5 UTR0010_GalE_BCD12 NO 23 TER0007_ilvGEDA

Example 20. Production of an Oligosaccharide Mixture Comprising Sialylated LN3, 3SL and LSTa with a Modified E. coli Host

[0696] An E. coli strain modified to produce sialic acid as described in Example 1 is further modified with a genomic knock-in of constitutive transcriptional units for the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis with SEQ ID NO: 26 and for the N-acetylglucosamine beta-1,3-galactosyltransferase (WbgO) from E. coli 055:H7 with SEQ ID NO: 27 to allow production of LNT. In a next step, the novel strain is further modified with a genomic knock-out of the E. coli lacZ gene and transformed with an expression plasmid having constitutive transcriptional units for NeuA from P. multocida with SEQ ID NO: 21 and the ?-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22. The novel strain is evaluated for production of an oligosaccharide mixture comprising LN3, 3-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc), LNT, 3SL and LSTa in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor.

Example 21. Production of an Oligosaccharide Mixture Comprising Sialylated LN3, 6SL and LSTc with a Modified E. coli Host

[0697] An E. coli strain modified to produce sialic acid as described in Example 1 is further modified with a genomic knock-in of constitutive transcriptional units for LgtA from N. meningitidis with SEQ ID NO: 26 and for LgtB from N. meningitidis with SEQ ID NO: 28 to allow production of LNnT. In a next step, the novel strain is further modified with a genomic knock-out of the E. coli lacZ gene and transformed with an expression plasmid having constitutive transcriptional units for NeuA from P. multocida with SEQ ID NO: 21 and the ?-2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 with SEQ ID NO: 25. The novel strain is evaluated for production of an oligosaccharide mixture comprising LN3, 6-sialylated LN3 (Neu5Ac-a2,6-(GlcNAc-b1,3)-Gal-b1,4-Glc), 6SL, LNnT and LSTc in a growth experiment in a 96-well plate according to the culture conditions provided in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor.

Example 22. Production of an Oligosaccharide Mixture Comprising Sialylated LN3, 3SL and LSTd with a Modified E. coli Host

[0698] An E. coli strain modified to produce sialic acid as described in Example 1 is further modified with a genomic knock-in of constitutive transcriptional units for LgtA from N. meningitidis with SEQ ID NO: 26 and for LgtB from N. meningitidis with SEQ ID NO: 28 to allow production of LNnT. In a next step, the novel strain is further modified with a genomic knock-out of the E. coli lacZ gene and transformed with an expression plasmid having constitutive transcriptional units for NeuA from P. multocida with SEQ ID NO: 21 and the ?-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22. The novel strain is evaluated for production of an oligosaccharide mixture comprising LN3, 3-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc), LNnT, 3SL and LSTd in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor.

Example 23. Production of an Oligosaccharide Mixture Comprising Sialylated LN3, LSTa and 3SL in Fermentation Broth of Mutant E. coli Strains when Evaluated in a Fed-Batch Fermentation Process with Glycerol, Sialic Acid and Lactose

[0699] The mutant E. coli strain able to produce LN3, LNT, 3SL and LSTa as described in Example 17 is selected for further evaluation in a fed-batch fermentation process in a 5L bioreactor. Fed-batch fermentations at bioreactor scale are performed as described in Example 1. In these examples, glycerol is used as a carbon source and lactose is added in the batch medium as precursor. During fed-batch, also sialic acid is added via an additional feed. Regular broth samples are taken, and sugars produced are measured as described in Example 1. Fermentation broth of the selected strain taken after the fed-batch phase is evaluated for production of an oligosaccharide mixture comprising LN3, 3-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc), LNT, LSTa and 3SL.

Example 24. Production of an Oligosaccharide Mixture Comprising Sialylated LN3, LSTa and 3SL. In Fermentation Broth of Mutant E. coli Strains when Evaluated in a Fed-Batch Fermentation Process with Sucrose and Lactose

[0700] The mutant E. coli strain able to produce LN3, LNT, 3SL and LSTa as described in Example 20 is selected for further evaluation in a fed-batch fermentation process in a 5L bioreactor. Fed-batch fermentations at bioreactor scale are performed as described in Example 1. In these examples, sucrose is used as a carbon source and lactose is added in the batch medium as precursor. Regular broth samples are taken, and sugars produced are measured as described in Example 1. Fermentation broth of the selected strain taken after the fed-batch phase is evaluated for production of an oligosaccharide mixture comprising LN3, 3-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc), LNT, LSTa and 3SL.

Example 25. Production of an Oligosaccharide Mixture Comprising Sialylated LN3, Para-Lacto-N-Neohexaose, Di-Sialylated LNnT, LSTc and 6SL in Fermentation Broth of Mutant E. coli Strains when Evaluated in a Fed-Batch Fermentation Process with Glycerol, Sialic Acid and Lactose

[0701] The mutant E. coli strains with a constitutive transcriptional unit for the ?-2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 with SEQ ID NO: 25 as described in Example 18 were selected for further evaluation in a fed-batch fermentation process in a 5L bioreactor. Fed-batch fermentations at bioreactor scale were performed as described in Example 1. In these examples, glycerol was used as a carbon source and lactose was added in the batch medium as precursor. During fed-batch, also sialic acid was added via an additional feed. Regular broth samples were taken, and sugars produced were measured as described in Example 1. UPLC analysis shows that fermentation broth of the selected strain taken after the batch phase contained lactose, LN3 and LNnT, whereas fermentation broth of the selected strain taken after the fed-batch phase comprised an oligosaccharide mixture comprising LN3, 6-sialylated LN3 (Neu5Ac-a2,6-(GlcNAc-b1,3)-Gal-b1,4-Glc), LNnT, LSTc and 6SL. At end of fed-batch, the mixture also comprised para-lacto-N-neohexaose (pLNnH), sialylated para-lacto-N-neohexaose and di-sialylated LNnT, two structures that were not detected in growth experiment assays due to limited detection levels and overall smaller production levels.

Example 26. Production of an Oligosaccharide Mixture Comprising Sialylated LN3, Para-Lacto-N-Neohexaose, Di-Sialylated LNnT, LSTc and 6SL in Fermentation Broth of Mutant E. coli Strains when Evaluated in a Fed-Batch Fermentation Process with Sucrose and Lactose

[0702] The mutant E. coli strain able to produce LN3, sialylated LN3, LNnT, 6SL and LSTc as described in Example 21 is selected for further evaluation in a fed-batch fermentation process in a 5L bioreactor. Fed-batch fermentations at bioreactor scale are performed as described in Example 1. In these examples, sucrose is used as a carbon source and lactose is added in the batch medium as precursor. Regular broth samples are taken, and sugars produced are measured as described in Example 1. Fermentation broth of the selected strain taken after the fed-batch phase is evaluated for production of an oligosaccharide mixture comprising LN3, 6-sialylated LN3 (Neu5Ac-?-2,6-(GlcNAc-b-1,3)-Gal-b-1,4-Glc), LNnT, LSTc, 6SL, para-lacto-N-neohexaose, sialylated para-lacto-N-neohexaose and di-sialylated LNnT.

Example 27. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0703] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with genomic knock-outs of the E. coli nagA and nagB genes and genomic knock-ins of constitutive expression cassettes for the mutant glmS*54 from E. coli with SEQ ID NO: 18, LgtA from N. meningitidis with SEQ ID NO: 26 and WbgO from E. coli 055:H7 with SEQ ID NO: 27. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (Table 10), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and sialic acid and lactose as precursors.

TABLE-US-00010 TABLE 10 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta- oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and sialic acid and lactose as precursors. Strain Plasmids* present Oligosaccharides SF26 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF27 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LN3, LNT, 3S-LN3, LSTa SF28 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LN3, 6S-LN3, LNT, LNFP-I SF29 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LN3, 6S-LN3, LNT SF30 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa SF31 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNT, LSTa SF32 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF33 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNT, LNFP-I SF34 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa *See Table 2 for plasmid info

Example 28. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0704] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with genomic knock-outs of the E. coli nagA and nagB genes and genomic knock-ins of constitutive expression cassettes for the mutant glmS*54 from E. coli with SEQ ID NO: 18, LgtA from N. meningitidis with SEQ ID NO: 26 and LgtB from N. meningitidis with SEQ ID NO: 28. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 1 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 11), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and sialic acid and lactose as precursors.

TABLE-US-00011 TABLE 11 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta- oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and sialic acid and lactose as precursors. Strain Plasmids* present Oligosaccharides SF35 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LN3, 3S-LN3, LNnT, LSTd SF36 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF37 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LN3, 6S-LN3, LNnT, LSTc SF38 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF39 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S-LN3, LNnT, LSTc, LSTd SF40 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd SF41 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF42 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF43 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plasmid info

Example 29. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0705] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with genomic knock-outs of the E. coli nagA, nagB, nanA, nanE and nanK genes and genomic knock-ins of constitutive expression cassettes for the mutant glmS*54 from E. coli with SEQ ID NO: 18, GNA1 from S. cerevisiae with SEQ ID NO: 15, the phosphatase yqaB from E. coli with SEQ ID NO: 19, AGE of B. ovatus with SEQ ID NO: 16, NeuB of N. meningitidis with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 26 and WbgO from E. coli O55:H7 with SEQ ID NO: 27. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (Table 12), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00012 TABLE 12 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF44 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF45 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF46 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, 6S-LN3, LNT, LNFP-I SF47 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF48 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LNB, 2FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa SF49 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LNB, 4-FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LSTa SF50 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF51 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 6SLNB, LN3, 6S-LN3, LNT, LNFP-I SF52 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa *See Table 2 for plasmid info

Example 30. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0706] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with genomic knock-outs of the E. coli nagA, nagB, nanA, nanE and nanK genes and genomic knock-ins of constitutive expression cassettes for the mutant glmS*54 from E. coli with SEQ ID NO: 18, GNA1 from S. cerevisiae with SEQ ID NO: 15, the phosphatase yqaB from E. coli with SEQ ID NO: 19, AGE of B. ovatus with SEQ ID NO: 16, neuB of N. meningitidis with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 26 and LgtB from N. meningitidis with SEQ ID NO: 28. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 13), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00013 TABLE 13 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF53 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LSTd SF54 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF55 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF56 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF57 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LacNAc, 2FLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LSTc, LSTd SF58 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LacNAc, 3FlacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd SF59 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF60 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF61 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plasmid info

Example 31. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0707] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with genomic knock-outs of the E. coli nagA, nagB, nanA, nanE and nanK genes and genomic knock-ins of constitutive expression cassettes for the mutant glmS*54 from E. coli with SEQ ID NO: 18, the UDP-N-acetylglucosamine 2-epimerase (neuC) from C. jejuni with SEQ ID 20, neuB of N. meningitidis with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 26 and WbgO from E. coli 055:H7 with SEQ ID NO: 27. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples as shown in Table 14, in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00014 TABLE 14 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF62 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF63 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LN3, 3S-LN3, LNT, LSTa SF64 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LN3, 6S-LN3, LNT, LNFP-I SF65 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LN3, 6S-LN3, LNT SF66 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa SF67 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNT, LSTa SF68 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF69 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNT, LNFP-I SF70 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LN3, LNT, 3S-LN3, 6S-LN3, LNFP-I, LSTa *See Table 2 for plasmid info

Example 32. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0708] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with genomic knock-outs of the E. coli nagA, nagB, nanA, nanE and nanK genes and genomic knock-ins of constitutive expression cassettes for the mutant glmS*54 from E. coli with SEQ ID NO: 18, neuC from C. jejuni with SEQ ID 20, neuB of N. meningitidis with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 26 and LgtB from N. meningitidis with SEQ ID NO: 28. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 15), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00015 TABLE 15 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF71 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LN3, 3S-LN3, LNnT, LSTd SF72 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF73 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LN3, 6S-LN3, LNnT, LSTc SF74 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF75 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S-LN3, LNnT, LSTc, LSTd SF76 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd SF77 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF78 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF79 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plasmid info

Example 33. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0709] An E. coli strain adapted for sialic acid production as described in Example 1 is further modified with a genomic knock-out of the E. coli wcaJ gene to increase the intracellular pool of GDP-fucose and genomic knock-ins of constitutive expression cassettes for LgtA from N. meningitidis with SEQ ID NO: 26 and WbgO from E. coli 055:H7 with SEQ ID NO: 27. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (Table 16), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00016 TABLE 16 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF80 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF81 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF82 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, LNT, 6S-LN3, LNFP-I SF83 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF84 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LNB, 2FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa SF85 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LNB, 4-FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LSTa SF86 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, LN3, LNT, 3S-LN3, LNFP-I, LSTa SF87 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 6SLNB, LN3, 6S-LN3, LNT, LNFP-I SF88 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa *See Table 2 for plasmid info

Example 34. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0710] An E. coli strain adapted for sialic acid production as described in Example 1 is further modified with a genomic knock-out of the E. coli wcaJ gene to increase the intracellular pool of GDP-fucose and genomic knock-ins of constitutive expression cassettes for LgtA from N. meningitidis with SEQ ID NO: 26 and LgtB from N. meningitidis with SEQ ID NO: 28. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 17), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00017 TABLE 17 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF89 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LSTd SF90 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF91 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF92 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF93 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LacNAc, 2FLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LSTc, LSTd SF94 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LacNAc, 3FlacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd SF95 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF96 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF97 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plasmid info

Example 35. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0711] An E. coli strain adapted for sialic acid production as described in Example 1 is further modified with genomic knock-outs of the E. coli wcaJ, fucK and fucI genes and genomic knock-ins of constitutive expression cassettes for the fucose permease (fucP) from E. coli with SEQ ID NO: 12, the bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase (fkp) from B. fragilis with SEQ NO: ID 13, LgtA from N. meningitidis with SEQ ID NO: 26 and WbgO from E. coli 055:H7 with SEQ ID NO: 27. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (Table 18), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00018 TABLE 18 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF98 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF99 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF100 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, 6S-LN3, LNT, LNFP-I SF101 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF102 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LNB, 2FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa SF103 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LNB, 4-FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LSTa SF104 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF105 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 6SLNB, LN3, 6S-LN3, LNT, LNFP-I SF106 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, 6SLNB, LN3, LNT, 3S-LN3, 6S-LN3, LNFP-I, LSTa *See Table 2 for plasmid info

Example 36. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0712] An E. coli strain adapted for sialic acid production as described in Example 1 is further modified with genomic knock-outs of the E. coli wcaJ, fucK and fucI genes and genomic knock-ins of constitutive expression cassettes for fucP from E. coli with SEQ ID NO: 12, fkp from B. fragilis with SEQ NO: ID 13, LgtA from N. meningitidis with SEQ ID NO: 26 and LgtB from N. meningitidis with SEQ ID NO: 28. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 19), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00019 TABLE 19 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF107 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LSTd SF108 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF109 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF110 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF111 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LacNAc, 2FLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LSTc, LSTd SF112 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LacNAc, 3FlacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd SF113 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF114 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF115 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plasmid info

Example 37. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0713] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with genomic knock-outs of the E. coli nagA, nagB, ushA and galT genes and genomic knock-ins of constitutive expression cassettes for galE from E. coli with SEQ ID NO: 29, the mutant glmS*54 from E. coli with SEQ ID NO: 18, GNA1 from S. cerevisiae with SEQ ID NO: 15, the phosphatase yqaB from E. coli with SEQ ID NO: 19, LgtA from N. meningitidis with SEQ ID NO: 26 and WbgO from E. coli 055:H7 with SEQ ID NO: 27. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (Table 20), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and sialic acid and lactose as precursors.

TABLE-US-00020 TABLE 20 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta- oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and sialic acid and lactose as precursors. Strain Plasmids* present Oligosaccharides SF116 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF117 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF118 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, 6S-LN3, LNT, LNFP-I SF119 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF120 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LNB, 2FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa SF121 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LNB, 4-FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LSTa SF122 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF123 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 6SLNB, LN3, 6S-LN3, LNT, LNFP-I SF124 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, 6SLNB, LN3, LNT, 3S-LN3, 6S-LN3, LNFP-I, LSTa *See Table 2 for plasmid info

Example 38. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0714] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with genomic knock-outs of the E. coli nagA, nagB, ushA and galT genes and genomic knock-ins of constitutive expression cassettes for galE from E. coli with SEQ ID NO: 29, the mutant glmS*54 from E. coli with SEQ ID NO: 18, GNA1 from S. cerevisiae with SEQ ID NO: 15, the phosphatase yqaB from E. coli with SEQ ID NO: 19, LgtA from N. meningitidis with SEQ ID NO: 26 and LgtB from N. meningitidis with SEQ ID NO: 28. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 21), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and sialic acid and lactose as precursors.

TABLE-US-00021 TABLE 21 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta- oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and sialic acid and lactose as precursors. Strain Plasmids* present Oligosaccharides SF125 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3SLN, LNnT, LSTd SF126 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF127 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF128 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF129 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LacNAc, 2FLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LSTc, LSTd SF130 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LacNAc, 3FlacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd SF131 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF132 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF133 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plasmid info

Example 39. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0715] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with genomic knock-outs of the E. coli nagA, nagB, nanA, nanE, nanK, ushA and galT genes and genomic knock-ins of constitutive expression cassettes for galE from E. coli with SEQ ID NO: 29, the mutant glmS*54 from E. coli with SEQ ID NO: 18, GNA1 from S. cerevisiae with SEQ ID NO: 15, the phosphatase yqaB from E. coli with SEQ ID NO: 19, the N-acetylglucosamine 2-epimerase (AGE) of B. ovatus with SEQ ID NO: 16, neuB of N. meningitidis with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 26 and WbgO from E. coli O55:H7 with SEQ ID NO: 27. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (Table 22), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00022 TABLE 22 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF134 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF135 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF136 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, 6S-LN3, LNT, LNFP-I SF137 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF138 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LNB, 2FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa SF139 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LNB, 4-FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LSTa SF140 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF141 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 6SLNB, LN3, 6S-LN3, LNT, LNFP-I SF142 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa *See Table 2 for plasmid info

Example 40. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0716] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with genomic knock-outs of the E. coli nagA, nagB, nanA, nanE, nanK, ushA and galT genes and genomic knock-ins of constitutive expression cassettes for galE from E. coli with SEQ ID NO: 29, the mutant glmS*54 from E. coli with SEQ ID NO: 18, GNA1 from S. cerevisiae with SEQ ID NO: 15, the phosphatase yqaB from E. coli with SEQ ID NO: 19, AGE of B. ovatus with SEQ ID NO: 16, neuB of N. meningitidis with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 26 and LgtB from N. meningitidis with SEQ ID NO: 28. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 23), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00023 TABLE 23 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF143 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LSTd SF144 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF145 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF146 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF147 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LacNAc, 2FLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LSTc, LSTd SF148 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LacNAc, 3FlacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd SF149 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF150 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF151 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plasmid info

Example 41. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0717] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with genomic knock-outs of the E. coli nagA, nagB, nanA, nanE, nanK, ushA and galT genes and genomic knock-ins of constitutive expression cassettes for galE from E. coli with SEQ ID NO: 29, the mutant glmS*54 from E. coli with SEQ ID NO: 18, neuC from C. jejuni with SEQ ID 20, neuB of N. meningitidis with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 26 and WbgO from E. coli 055:H7 with SEQ ID NO: 27. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (Table 24), when evaluated in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00024 TABLE 24 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF152 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF153 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LN3, 3S-LN3, LNT, LSTa SF154 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LN3, 6S-LN3, LNT, LNFP-I SF155 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LN3, 6S-LN3, LNT SF156 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa SF157 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNT, LSTa SF158 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF159 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNT, LNFP-I SF160 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa *See Table 2 for plasmid info

Example 42. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0718] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with genomic knock-outs of the E. coli nagA, nagB, nanA, nanE, nanK, ushA and galT genes and genomic knock-ins of constitutive expression cassettes for galE from E. coli with SEQ ID NO: 29, the mutant glmS*54 from E. coli with SEQ ID NO: 18, neuC from C. jejuni with SEQ ID 20, neuB of N. meningitidis with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 26 and LgtB from N. meningitidis with SEQ ID NO: 28. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 25), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00025 TABLE 25 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF161 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LN3, 3S-LN3, LNnT, LSTd SF162 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF163 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LN3, 6S-LN3, LNnT, LSTc SF164 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF165 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S-LN3, LNnT, LSTc, LSTd SF166 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd SF167 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF168 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF169 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plasmid info

Example 43. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0719] An E. coli strain adapted for sialic acid production as described in Example 1 is further modified with genomic knock-outs of the E. coli wcaJ, ushA and galT genes and genomic knock-ins of constitutive expression cassettes for galE from E. coli with SEQ ID NO: 29, LgtA from N. meningitidis with SEQ ID NO: 26 and WbgO from E. coli 055:H7 with SEQ ID NO: 27. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (Table 26), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00026 TABLE 26 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF170 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF171 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF172 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, 6S-LN3, LNT, LNFP-I SF173 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF174 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LNB, 2FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa SF175 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LNB, 4-FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LSTa SF176 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF177 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 6SLNB, LN3, 6SLN, LNT, LNFP-I SF178 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa *See Table 2 for plasmid info

Example 44. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0720] An E. coli strain adapted for sialic acid production as described in Example 1 is further modified with genomic knock-outs of the E. coli wcaJ, ushA and galT genes and genomic knock-ins of constitutive expression cassettes for galE from E. coli with SEQ ID NO: 29, LgtA from N. meningitidis with SEQ ID NO: 26 and LgtB from N. meningitidis with SEQ ID NO: 28. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 27), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00027 TABLE 27 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF179 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LSTd SF180 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF181 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF182 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF183 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LacNAc, 2FLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LSTc, LSTd SF184 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LacNAc, 3FlacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd SF185 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF186 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF187 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plasmid info

Example 45. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0721] An E. coli strain adapted for sialic acid production as described in Example 1 is further modified with genomic knock-outs of the E. coli wcaJ, fucK, fucI, ushA and galT genes and genomic knock-ins of constitutive expression cassettes for galE from E. coli with SEQ ID NO: 29, fucP from E. coli with SEQ ID NO: 12, fkp from B. fragilis with SEQ NO: ID 13, LgtA from N. meningitidis with SEQ ID NO: 26 and WbgO from E. coli 055:H7 with SEQ ID NO: 27. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (Table 28), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00028 TABLE 28 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF188 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF189 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF190 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, 6S-LN3, LNT, LNFP-I SF191 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF192 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LNB, 2FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa SF193 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LNB, 4-FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LSTa SF194 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF195 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 6SLNB, LN3, 6SLN, LNT, LNFP-I SF196 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LNB, 2FLNB, 4-FLNB, Di-FLNB, 3SLNB, 6SLNB, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa *See Table 2 for plasmid info

Example 46. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0722] An E. coli strain adapted for sialic acid production as described in Example 1 is further modified with genomic knock-outs of the E. coli wcaJ, fucK, fucI, ushA and galT genes and genomic knock-ins of constitutive expression cassettes for galE from E. coli with SEQ ID NO: 29, fucP from E. coli with SEQ ID NO: 12, fkp from B. fragilis with SEQ NO: ID 13, LgtA from N. meningitidis with SEQ ID NO: 26 and LgtB from N. meningitidis with SEQ ID NO: 28. In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contains (a) constitutive expression unit(s) for one or two selected fucosyltransferase(s) and wherein a second plasmid contains constitutive expression units for one or two selected sialyltransferase(s) and NeuA from P. multocida with SEQ ID NO: 21. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 29), in a growth experiment according to the culture conditions provided in Example 1 in which the cultivation contains sucrose as carbon source and lactose as precursor.

TABLE-US-00029 TABLE 29 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant E. coli strains in a growth experiment according the cultivation conditions as described in Example 1, in which the culture medium contains sucrose as carbon source and lactose as precursor. Strain Plasmids* present Oligosaccharides SF197 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LSTd SF198 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF199 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF200 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF201 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, di-SL, LacNAc, 2FLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LSTc, LSTd SF202 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, di-SL, LacNAc, 3FlacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd SF203 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF204 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF205 pMF_2, pMS_2 2FL, 3-FL, DiFL, 3SL, 6SL, di-SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LacNAc, 2FLacNAc, 3FLacNAc, DiFLacNAc, 3SLacNAc, 6SLacNAc, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd *See Table 2 for plasmid info

Example 47. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Lactose Structures with a Modified S. cerevisiae Host

[0723] An S. cerevisiae strain is adapted for production of GDP-fucose and CMP-sialic acid and for expression of one or more fucosyltransferase(s) and one or more sialyltransferase(s) as described in Example 2 with a first yeast expression plasmid (a variant of p2a_2 ?_Fuc) comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 30, gmd from E. coli with SEQ ID NO: 10, fcl from E. coli with SEQ ID NO: 11 and one or two selected fucosyltransferase(s) and with a second yeast expression plasmid (a pRS420-plasmid variant) comprising constitutive transcriptional units for the mutant glmS*54 from E. coli with SEQ ID NO: 18, the phosphatase yqaB from E. coli with SEQ ID NO: 19, AGE from B. ovatus with SEQ ID NO: 16, neuB from N. meningitidis with SEQ ID NO: 17, neuA from P. multocida with SEQ ID NO: 21 and one or two selected sialyltransferase(s). Table 30 shows the fucosyltransferases and sialyltransferases selected in the plasmids cloned in this experiment. The strains are transformed to express 1) one fucosyltransferase combined with two sialyltransferases, 2) two fucosyltransferases combined with one sialyltransferase or 3) two fucosyltransferases combined with two sialyltransferases (Table 31). The mutant yeast strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose structures, as shown in Table 31, in a growth experiment according to the culture conditions in Example 2 using SD CSM-Ura-Trp drop-out medium comprising lactose as precursor.

TABLE-US-00030 TABLE 30 Overview of the plasmids cloned with constitutive transcriptional units for one or two fucosyltransferase gene(s) or for one or two sialyltransferase gene(s) Fucosyltransferase(s) cloned in the p2a_2?_Fuc plasmid variants Plasmid nr a1,2-linkage a1,3-linkage pYF 1A SEQ ID NO: 04 None pYF_1B None SEQ ID NO: 05 pYF_2 SEQ ID NO: 04 SEQ ID NO: 05 Sialyltransferase(s) cloned in the pRS420-plasmid variant Plasmid nr a2,3-linkage a2,6-linkage pYS_1A SEQ ID NO: 22 None PYS_1B None SEQ ID NO: 24 pYS_2 SEQ ID NO: 22 SEQ ID NO: 24

TABLE-US-00031 TABLE 31 Evaluation of production of an oligosaccharide mixture by mutant S. cerevisiae strains expressing selected fucosyltransferase and sialyltransferase genes when cultivated in SD CSM-Ura- Trp drop-out medium comprising lactose as precursor. Strain Plasmids* present Oligosaccharides SY1 p YF_1A, p YS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SY2 p YF_1B, p YS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SY3 p YF_2, p YS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SY4 p YF_2, p YS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SY5 p YF_2, p YS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL *See Table 30 for plasmid info

Example 48. Production of an Oligosaccharide Mixture Comprising 3-Sialylated LN3, LNB, 3SL and LSTa with a Modified S. cerevisiae Host

[0724] An S. cerevisiae strain is adapted for production of CMP-sialic acid and LNT and for expression of a beta-galactoside alpha-2,3-sialyltransferase as described in Example 2 with a first yeast expression plasmid comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 30, the mutant glmS*54 from E. coli with SEQ ID NO: 18, the phosphatase yqaB from E. coli with SEQ ID NO: 19, AGE from B. ovatus with SEQ ID NO: 16, NeuB from N. meningitidis with SEQ ID NO: 17, NeuA from P. multocida with SEQ ID NO: 21, and the beta-galactoside alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22 and a second yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli with SEQ ID NO: 29, LgtA from N. meningitidis with SEQ ID NO: 26 and WbgO from E. coli 055:H7 with SEQ ID NO: 27. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising LN3, 3-sialylated LN3, LNT, LNB, sialylated LNB, 3SL and LSTa in a growth experiment according to the culture conditions in Example 2 using SD CSM-Trp-His drop-out medium comprising lactose as precursor.

Example 49. Production of an Oligosaccharide Mixture Comprising 6-Sialylated LN3, LacNAc, 6SL and LSTc with a Modified S. cerevisiae Host

[0725] An S. cerevisiae strain is adapted for production of CMP-sialic acid and LNnT and for expression of a beta-galactoside alpha-2,6-sialyltransferase as described in Example 2 with a first yeast expression plasmid comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 30, the mutant glmS*54 from E. coli with SEQ ID NO: 18, the phosphatase yqaB from E. coli with SEQ ID NO: 19, AGE from B. ovatus with SEQ ID NO: 16, NeuB from N. meningitidis with SEQ ID NO: 17, NeuA from P. multocida with SEQ ID NO: 21, and the beta-galactoside alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 24 and a second yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli with SEQ ID NO: 29, LgtA from N. meningitidis with SEQ ID NO: 26 and LgtB from N. meningitidis with SEQ ID NO: 28. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising LN3, 6-sialylated LN3, LNnT, LacNAc, sialylated LacNAc, 6SL and LSTc in a growth experiment according to the culture conditions in Example 2 using SD CSM-Trp-His drop-out medium comprising lactose as precursor.

Example 50. Production of an Oligosaccharide Mixture Comprising 3-Sialylated LN3, LacNAc, 3SL and LSTd with a Modified S. cerevisiae Host

[0726] An S. cerevisiae strain is adapted for production of CMP-sialic acid and LNnT and for expression of a beta-galactoside alpha-2,3-sialyltransferase as described in Example 2 with a first yeast expression plasmid comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 30, the mutant glmS*54 from E. coli with SEQ ID NO: 18, the phosphatase yqaB from E. coli with SEQ ID NO: 19, AGE from B. ovatus with SEQ ID NO: 16, NeuB from N. meningitidis with SEQ ID NO: 17, NeuA from P. multocida with SEQ ID NO: 21, and the beta-galactoside alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 22 and a second yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli with SEQ ID NO: 29, LgtA from N. meningitidis with SEQ ID NO: 26 and LgtB from N. meningitidis with SEQ ID NO: 28. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising LN3, 3-sialylated LN3, LNnT, LacNAc, sialylated LacNAc, 3SL and LSTd in a growth experiment according to the culture conditions in Example 2 using SD CSM-Trp-His drop-out medium comprising lactose as precursor.

Example 51. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified S. cerevisiae Host

[0727] An S. cerevisiae strain is adapted for production of GDP-fucose, CMP-sialic acid and LNT and for expression of selected fucosyltransferases and sialyltransferases as described in Example 2 with a first yeast expression plasmid (a variant of p2a_2 ?_Fuc) comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 30, gmd from E. coli with SEQ ID NO: 10, fcl from E. coli with SEQ ID NO: 11 and one or two selected fucosyltransferase(s) (see Table 30), and with a second yeast expression plasmid (a pRS420-plasmid variant) comprising constitutive transcriptional units for the mutant glmS*54 from E. coli with SEQ ID NO: 18, the phosphatase yqaB from E. coli with SEQ ID NO: 19, AGE from B. ovatus with SEQ ID NO: 16, neuB from N. meningitidis with SEQ ID NO: 17, neuA from P. multocida with SEQ ID NO: 21 and one or two selected sialyltransferase(s) (see Table 30), and with a third yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli with SEQ ID NO: 29, LgtA from N. meningitidis with SEQ ID NO: 26 and WbgO from E. coli 055:H7 with SEQ ID NO: 27. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LNB, sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures (Table 32) in a growth experiment according to the culture conditions in Example 2 using SD CSM-Ura-Trp-His drop-out medium comprising lactose as precursor.

TABLE-US-00032 TABLE 32 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides in whole broth of mutant S. cerevisiae strains expressing selected fucosyltransferase and sialyltransferase genes whencultivated in SD CSM-Ura-Trp-His drop-out medium comprising lactose as precursor. Strain Plasmids* present Oligosaccharides SY6 pYF_1C, pYS_1A 2FL, DiFL, 3SL, 3S-2FL, 3S-LNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SY7 pYF_1D, pYS_1A 3-FL, 3SL, 3S-3-FL, 3S-LNB, LN3, 3S-LN3, LNT, LSTa SY8 pYF_1C, pYS_1B 2FL, DiFL, 6SL, 6S-2FL, 6S-LNB, LN3, 6S-LN3, LNT, LNFP-I SY9 pYF_1D, pYS_1B 3-FL, 6SL, 6S-3-FL, 6S-LNB, LN3, LNT, 6S-LN3 SY10 pYF_1C, pYS_2 2FL, DiFL, 3SL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa, 3S-LNB, 6S-LNB SY11 pYF_1D, pYS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S-LN3, LNT, LSTa, 3S-LNB, 6S-LNB SY12 pYF_2CD, pYS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNT, LNFP-I, LSTa, 3S-LNB SY13 pYF_2CD, pYS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNT, LNFP-I, 6S-LNB SY14 pYF_2CD, pYS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNT, LNFP-I, LSTa, 3S-LNB, 6S-LNB *See Table 30 for plasmid info

Example 52. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified S. cerevisiae Host

[0728] An S. cerevisiae strain is adapted for production of GDP-fucose, CMP-sialic acid and LNnT and for expression of selected fucosyltransferases and sialyltransferases as described in Example 2 with a first yeast expression plasmid (a variant of p2a_2 ?_Fuc) comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 30, gmd from E. coli with SEQ ID NO: 10, fcl from E. coli with SEQ ID NO: 11 and one or two selected fucosyltransferase(s) (see Table 30), and with a second yeast expression plasmid (a pRS420-plasmid variant) comprising constitutive transcriptional units for the mutant glmS*54 from E. coli with SEQ ID NO: 18, the phosphatase yqaB from E. coli with SEQ ID NO: 19, AGE from B. ovatus with SEQ ID NO: 16, neuB from N. meningitidis with SEQ ID NO: 17, neuA from P. multocida with SEQ ID NO: 21 and one or two selected sialyltransferase(s) (see Table 30), and with a third yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli with SEQ ID NO: 29, LgtA from N. meningitidis with SEQ ID NO: 26 and LgtB from N. meningitidis with SEQ ID NO: 28. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LacNAc, LN3, sialylated LN3, LNnT, and fucosylated and sialylated LNnT structures (Table 33) in a growth experiment according to the culture conditions in Example 2 using SD CSM-Ura-Trp-His drop-out medium comprising lactose as precursor.

TABLE-US-00033 TABLE 33 Evaluation of production of an oligosaccharide mixture comprising tri-, tetra- and penta-oligosaccharides detectable in whole broth of mutant S. cerevisiae strains expressing selected fucosyltransferase and sialyltransferase genes when cultivated in SD CSM-Ura-Trp-His drop-out medium comprising lactose as precursor. Strain Plasmids* present Oligosaccharides SY15 pYF_1C, pYS_1A 2FL, DiFL, 3SL, 3S-2FL, LN3, 3S-LN3, 3S-LacNAc, LNnT, LSTd SY16 pYF_1D, pYS_1A 3-FL, 3SL, 3S-3-FL, LN3, 3S-LN3, 3S-LacNAc, LNnT, LNFP-III, LSTd SY17 pYF_1C, pYS_1B 2FL, DiFL, 6SL, 6S-2FL, 6S-LacNAc, LN3, 6S-LN3, LNnT, LSTc SY18 pYF_1D, pYS_1B 3-FL, 6SL, 6S-3-FL, 6S-LacNAc, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SY19 pYF_1C, pYS_2 2FL, DiFL, 3SL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S-LN3, LNnT, LSTc, LSTd, 3S-LacNAc, 6S-LacNAc SY20 pYF_1D, pYS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd,3S-LacNAc, 6S-LacNAc SY21 pYF_2CD, pYS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd, 3S-LacNAc SY22 pYF_2CD, pYS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc, 6S-LacNAc SY23 pYF_2CD, pYS_2 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd, 3S-LacNAc, 6S-LacNAc *See Table 30 for plasmid info

Example 53. Production of an Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified S. cerevisiae Host

[0729] An S. cerevisiae strain is adapted for production of GDP-fucose and CMP-sialic acid and for expression of one or more fucosyltransferase(s) and one or more sialyltransferase(s) with a yeast artificial chromosome (YAC) comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 30, gmd from E. coli with SEQ ID NO: 10, fcl from E. coli with SEQ ID NO: 11, the mutant glmS*54 from E. coli with SEQ ID NO: 18, the phosphatase yqaB from E. coli with SEQ ID NO: 19, AGE from B. ovatus with SEQ ID NO: 16, neuB from N. meningitidis with SEQ ID NO: 17, neuA from P. multocida with SEQ ID NO: 21, one or two selected fucosyltransferase(s) and one or two selected sialyltransferase(s). Table 34 shows the fucosyltransferases and sialyltransferases selected in the YACs created in this experiment. The mutant yeast strains are evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose structures, as shown in Table 35, in a growth experiment according to the culture conditions in Example 2 using SD CSM medium comprising lactose as precursor.

TABLE-US-00034 TABLE 34 Overview of the fucosyltransferase gene(s) and sialyltransferase gene(s) cloned in the different yeast artificial chromosomes Yeast artificial Fucosyltransferase(s) Sialyltransferase(s) chromosomes a1,2-linkage a1,3-linkage a2,3-linkage a2,6-linkage YAC1 SEQ ID NO: 04 None SEQ ID NO: 22 SEQ ID NO: 24 YAC2 None SEQ ID NO: 05 SEQ ID NO: 22 SEQ ID NO: 24 YAC3 SEQ ID NO: 04 SEQ ID NO: 05 SEQ ID NO: 22 None YAC4 SEQ ID NO: 04 SEQ ID NO: 05 None SEQ ID NO: 24 YAC5 SEQ ID NO: 04 SEQ ID NO: 05 SEQ ID NO: 22 SEQ ID NO: 24

TABLE-US-00035 TABLE 35 Evaluation of production of an oligosaccharide mixture in whole broth of mutant S. cerevisiae strains expressing selected fucosyltransferase and sialyltransferase genes from yeast artificial chromosomes when cultivated in SD CSM medium comprising lactose as precursor. Strain YACs* present Oligosaccharides SY24 YAC1 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SY25 YAC2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SY26 YAC3 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SY27 YAC4 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SY28 YAC5 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S-2FL, 6S-3-FL *See Table 34 for the YAC overview

Example 54. Material and Methods Bacillus subtilis

Media

[0730] Two different media are used, namely a rich Luria Broth (LB) and a minimal medium for shake flask (MMsf). The minimal medium uses a trace element mix.

[0731] Trace element mix comprised 0.735 g/L CaCl2.Math.2H2O, 0.1 g/L MnCl2.Math.2H2O, 0.033 g/L CuCl2.Math.2H2O, 0.06 g/L CoCl2.Math.6H2O, 0.17 g/L ZnCl2, 0.0311 g/L H3BO4, 0.4 g/L Na2EDTA.Math.2H2O and 0.06 g/L Na2MoO4. The Fe-citrate solution contained 0.135 g/L FeC13.6H2O, 1 g/L Na-citrate (Hoch 1973 PMC1212887).

[0732] The Luria Broth (LB) medium comprised 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). Luria Broth agar (LBA) plates comprised the LB media, with 12 g/L agar (Difco, Erembodegem, Belgium) added.

[0733] The minimal medium for the shake flasks (MMsf) experiments contained 2.00 g/L (NH4).sub.2SO4, 7.5 g/L KH2PO4, 17.5 g/L K2HPO4, 1.25 g/L Na-citrate, 0.25 g/L MgSO4.7H2O, 0.05 g/L tryptophan, from 10 up to 30 g/L glucose or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose when specified in the examples, 10 ml/L trace element mix and 10 ml/L Fe-citrate solution. The medium was set to a pH of 7 with 1M KOH. Depending on the experiment lactose, LNB or LacNAc could be added.

[0734] Complex medium, e.g., LB, was sterilized by autoclaving (121? C., 21) and minimal medium by filtration (0.22 ?m Sartorius). When necessary, the medium was made selective by adding an antibiotic (e.g., zeocin (20 mg/L)).

Strains, Plasmids and Mutations

[0735] Bacillus subtilis 168, available at Bacillus Genetic Stock Center (Ohio, USA).

[0736] Plasmids for gene deletion via Cre/lox are constructed as described by Yan et al. (Appl. & Environm. Microbial., September 2008, p5556-5562). Gene disruption is done via homologous recombination with linear DNA and transformation via electroporation as described by Xue et al. (J. Microb. Meth. 34 (1999) 183-191). The method of gene knockouts is described by Liu et al. (Metab. Engine. 24 (2014) 61-69). This method uses 1000 bp homologies up- and downstream of the target gene.

[0737] Integrative vectors as described by Popp et al. (Sci. Rep., 2017, 7, 15158) are used as expression vector and could be further used for genomic integrations if necessary. A suitable promoter for expression can be derived from the part repository (iGem): sequence id: Bba_K143012, Bba_K823000, Bba_K823002 or Bba_K823003. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

[0738] In an example for the production of lactose-based oligosaccharides, Bacillus subtilis mutant strains are created to contain a gene coding for a lactose importer (such as the E. coli lacY with SEQ ID NO: 14). In an example for 2FL, 3FL and/or diFL production, an alpha-1,2- and/or alpha-1,3-fucosyltransferase expression construct is additionally added to the strains. In an example for LN3 production, a constitutive transcriptional unit comprising a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g., lgtA from N. meningitidis (SEQ ID NO: 26) is additionally added to the strain. In an example for LNT production, the LN3 producing strain is further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1,3-galactosyltransferase like e.g., WbgO from E. coli 055:H7 (SEQ ID NO: 27). In an example for LNnT production, the LN3 producing strain is further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1,4-galactosyltransferase like e.g., lgtB from N. meningitidis (SEQ ID NO: 28).

[0739] In an example for sialic acid production, a mutant B. subtilis strain is created by overexpressing a fructose-6-P-aminotransferase like the native fructose-6-P-aminotransferase (UniProt ID P0CI73) to enhance the intracellular glucosamine-6-phosphate pool. Further on, the enzymatic activities of the genes nagA, nagB and gamA are disrupted by genetic knockouts and a glucosamine-6-P-aminotransferase like e.g., from S. cerevisiae (SEQ ID NO: 15), an N-acetylglucosamine-2-epimerase like e.g., from B. ovatus (SEQ ID NO: 16) and an N-acetylneuraminate synthase like e.g., from N. meningitidis (SEQ ID NO: 17) are overexpressed on the genome. To allow sialylated oligosaccharide production, the sialic acid producing strain is further modified with a constitutive transcriptional unit comprising an N-acylneuraminate cytidylyltransferase like e.g., the NeuA enzyme from P. multocida (SEQ ID NO: 21), and one or more copies of a beta-galactoside alpha-2,3-sialyltransferase like e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide comprising amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity (SEQ ID NO: 22), or NmeniST3 from N. meningitidis (SEQ ID NO: 23) or PmultST2 from P. multocida subsp. multocida str. Pm70 (GenBank No. AAK02592.1), a beta-galactoside alpha-2,6-sialyltransferase like e.g., PdST6 from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide comprising amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity (SEQ ID NO: 24) or P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide comprising amino acid residues 18 to 514 of UniProt ID A8QYL1 having beta-galactoside alpha-2,6-sialyltransferase activity (SEQ ID NO: 25), and/or an alpha-2,8-sialyltransferase like e.g., from M. musculus (UniProt ID Q64689).

Heterologous and Homologous Expression

[0740] Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.

[0741] Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.

Cultivation Conditions

[0742] A preculture of 96-well microtiter plate experiments was started from a cryovial or a single colony from an LB plate, in 150 ?L LB and was incubated overnight at 37? C. on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96-well square microtiter plate, with 400 ?L MMsf medium by diluting 400?. Each strain was grown in multiple wells of the 96-well plate as biological replicates. These final 96-well culture plates were then incubated at 37? C. on an orbital shaker at 800 rpm for 72 h, or shorter, or longer. At the end of the cultivation experiment samples were taken from each well to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 15 min at 90? C. or for 60 min at 60? C. before spinning down the cells (=whole broth concentration, intra- and extracellular sugar concentrations, as defined herein).

[0743] Also, a dilution of the cultures was made to measure the optical density at 600 nm. The cell performance index or CPI was determined by dividing the oligosaccharide concentrations by the biomass, in relative percentages compared to a reference strain. The biomass is empirically determined to be approximately ?rd of the optical density measured at 600 nm.

Example 55. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, 3SL, 6SL, 3's-2FL, 3's-3-FL, 6's-2FL, 6's-3-FL with a Modified B. subtilis Host

[0744] A B. subtilis strain is modified as described in Example 54 by genomic knock-out of the nagA, nagB, glmS and gamA genes and genomic knock-ins of constitutive transcriptional units for the lactose permease (LacY) from E. coli with SEQ ID NO: 14, the sucrose transporter (CscB) from E. coli W (SEQ ID NO: 01), the fructose kinase (Frk) from Z. mobilis (SEQ ID NO: 02), the sucrose phosphorylase (BaSP) from B. adolescentis (SEQ ID NO: 03), the native fructose-6-P-aminotransferase (UniProt ID P0CI73), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae (SEQ ID NO: 15), the mutant L-glutamine-D-fructose-6-phosphate aminotransferase (glmS*54) from E. coli (SEQ ID NO: 18), a phosphatase like e.g., a phosphatase chosen from the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from P. putida, ScDOGI from S. cerevisiae or BsAraL from B. subtilis as described in WO 2018122225, the N-acetylglucosamine 2-epimerase (AGE) from B. ovatus (SEQ ID NO: 16), the N-acetylneuraminate synthase (NeuB) from N. meningitidis (SEQ ID NO: 17) and the N-acylneuraminate cytidylyltransferase NeuA from P. multocida (SEQ ID NO: 21). In a next step, the strain is transformed with an expression plasmid comprising constitutive transcriptional units for three copies of a PmultST3-like polypeptide comprising amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity like SEQ ID NO: 23 and three copies of a PdST6-like polypeptide comprising amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity (SEQ ID NO: 24). In a further step, the mutant strain is transformed with a second compatible expression plasmid comprising constitutive transcriptional units for the alpha-1,2-fucosyltransferase HpFutC with SEQ ID NO: 04 and the alpha-1,3-fucosyltransferase HpFucT with SEQ ID NO: 05. The novel strain is evaluated for the production of 2FL, 3-FL, DiFL, 3SL, 6SL, 3'S-2FL, 3'S-3-FL, 6'S-2FL, 6'S-3-FL in a growth experiment on MMsf medium comprising lactose according to the culture conditions provided in Example 54. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 56. Production of an Oligosaccharide Mixture Comprising 3SL, LN3, LNT, Sialylated LN3 and LSTa with a Modified B. subtilis Host

[0745] A B. subtilis strain is modified for LN3 production and growth on sucrose as described in Example 54 by genomic knock-out of the nagA, nagB, glmS and gamA genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding the lactose permease (LacY) from E. coli (SEQ ID NO: 14), the native fructose-6-P-aminotransferase (UniProt ID P0CI73), the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (SEQ ID NO: 26), the sucrose transporter (CscB) from E. coli W (SEQ ID NO: 01), the fructose kinase (Frk) from Z. mobilis (SEQ ID NO: 02) and the sucrose phosphorylase (BaSP) from B. adolescentis (SEQ ID NO: 03). In a next step, the mutant strain is further modified with a genomic knock-in of a constitutive transcriptional unit comprising the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 (SEQ ID NO: 27) to produce LNT. The mutant B. subtilis strain is further modified with genomic knock-ins of constitutive transcriptional units comprising the glucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae (SEQ ID NO: 15), two copies of the mutant L-glutamine-D-fructose-6-phosphate aminotransferase (glmS*54) from E. coli (SEQ ID NO: 18), a phosphatase like e.g., a phosphatase chosen from the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from P. putida, ScDOGI from S. cerevisiae or BsAraL from B. subtilis as described in WO 2018122225, the N-acetylglucosamine 2-epimerase (AGE) from B. ovatus (SEQ ID NO: 16), the N-acetylneuraminate synthase (NeuB) from N. meningitidis (SEQ ID NO: 17), the N-acylneuraminate cytidylyltransferase NeuA from P. multocida (SEQ ID NO: 21) and three copies of a PmultST3-like polypeptide comprising amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity like SEQ ID NO: 22. The novel strain is evaluated for the production of a mixture comprising 3SL, LN3, sialylated LN3, LNT, and LSTa (Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 54. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 57. Material and Methods Corynebacterium glutamicum

Media

[0746] Two different media are used, namely a rich tryptone-yeast extract (TY) medium and a minimal medium for shake flask (MMsf). The minimal medium uses a 1000? stock trace element mix.

[0747] Trace element mix comprised 10 g/L CaCl2, 10 g/L FeSO4.Math.7H2O, 10 g/L MnSO4.Math.H2O, 1 g/L ZnSO4.Math.7H2O, 0.2 g/L CuSO4, 0.02 g/L NiCl2.Math.6H2O, 0.2 g/L biotin (pH 7.0) and 0.03 g/L protocatechuic acid.

[0748] The minimal medium for the shake flasks (MMsf) experiments contained 20 g/L (NH4)2SO4, 5 g/L urea, 1 g/L KH2PO4, 1 g/L K2HPO4, 0.25 g/L MgSO4.7H2O, 42 g/L MOPS, from 10 up to 30 g/L glucose or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose when specified in the examples and 1 ml/L trace element mix. Depending on the experiment lactose, LNB, and/or LacNAc could be added to the medium.

[0749] The TY medium comprised 1.6% tryptone (Difco, Erembodegem, Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). TY agar (TYA) plates comprised the TY media, with 12 g/L agar (Difco, Erembodegem, Belgium) added.

[0750] Complex medium, e.g., TY, was sterilized by autoclaving (121? C., 21) and minimal medium by filtration (0.22 ?m Sartorius). When necessary, the medium was made selective by adding an antibiotic (e.g., kanamycin, ampicillin).

Strains and Mutations

[0751] Corynebacterium glutamicum ATCC 13032, available at the American Type Culture Collection.

[0752] Integrative plasmid vectors based on the Cre/loxP technique as described by Suzuki et al. (Appl. Microbiol. Biotechnol., 2005 April, 67(2):225-33) and temperature-sensitive shuttle vectors as described by Okibe et al. (Journal of Microbiological Methods 85, 2011, 155-163) are constructed for gene deletions, mutations and insertions. Suitable promoters for (heterologous) gene expression can be derived from Yim et al. (Biotechnol. Bioeng., 2013 November, 110(11):2959-69). Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

[0753] In an example for the production of lactose-based oligosaccharides, C. glutamicum mutant strains are created to contain a gene coding for a lactose importer (such as e.g., the E. coli lacY with SEQ ID NO: 14). In an example for 2FL, 3FL and/or diFL production, an alpha-1,2- and/or alpha-1,3-fucosyltransferase expression construct is additionally added to the strains.

[0754] In an example for LN3 production, a constitutive transcriptional unit comprising a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g., lgtA from N. meningitidis (SEQ ID NO: 26) is additionally added to the strain. In an example for LNT production, the LN3 producing strain is further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1,3-galactosyltransferase like e.g., WbgO from E. coli 055:H7 (SEQ ID NO: 27). In an example for LNnT production, the LN3 producing strain is further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1,4-galactosyltransferase like e.g., lgtB from N. meningitidis (SEQ ID NO: 28).

[0755] In an example for sialic acid production, a mutant C. glutamicum strain is created by overexpressing a fructose-6-P-aminotransferase like the native fructose-6-P-aminotransferase (UniProt ID Q8NND3) to enhance the intracellular glucosamine-6-phosphate pool. Further on, the enzymatic activities of the genes nagA, nagB and gamA are disrupted by genetic knockouts and a glucosamine-6-P-aminotransferase like e.g., from S. cerevisiae (SEQ ID NO: 15), an N-acetylglucosamine-2-epimerase like e.g., from B. ovatus (SEQ ID NO: 16) and an N-acetylneuraminate synthase like e.g., from N. meningitidis (SEQ ID NO: 17) are overexpressed on the genome. To allow sialylated oligosaccharide production, the sialic acid producing strain is further modified with a constitutive transcriptional unit comprising an N-acylneuraminate cytidylyltransferase like e.g., the NeuA enzyme from P. multocida (SEQ ID NO: 21), and one or more copies of a beta-galactoside alpha-2,3-sialyltransferase like e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide comprising amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity (SEQ ID NO: 22), or NmeniST3 from N. meningitidis (SEQ ID NO: 23) or PmultST2 from P. multocida subsp. multocida str. Pm70 (GenBank No. AAK02592.1), a beta-galactoside alpha-2,6-sialyltransferase like e.g., PdST6 from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide comprising amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity (SEQ ID NO: 24) or P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide comprising amino acid residues 18 to 514 of UniProt ID A8QYL1 having beta-galactoside alpha-2,6-sialyltransferase activity (SEQ ID NO: 25), and/or an alpha-2,8-sialyltransferase like e.g., from M. musculus (UniProt ID Q64689).

Heterologous and Homologous Expression

[0756] Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.

[0757] Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.

Cultivation Conditions

[0758] A preculture of 96-well microtiter plate experiments was started from a cryovial or a single colony from a TY plate, in 150 ?L TY and was incubated overnight at 37? C. on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96-well square microtiter plate, with 400 ?L MMsf medium by diluting 400?. Each strain was grown in multiple wells of the 96-well plate as biological replicates. These final 96-well culture plates were then incubated at 37? C. on an orbital shaker at 800 rpm for 72 h, or shorter, or longer. At the end of the cultivation experiment samples were taken from each well to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 15 min at 60? C. before spinning down the cells (=whole broth concentration, intra- and extracellular sugar concentrations, as defined herein).

[0759] Also, a dilution of the cultures was made to measure the optical density at 600 nm. The cell performance index or CPI was determined by dividing the oligosaccharide concentrations, measured in the whole broth by the biomass, in relative percentages compared to the reference strain. The biomass is empirically determined to be approximately ?rd of the optical density measured at 600 nm.

Example 58. Production of an Oligosaccharide Mixture Comprising LN3, Sialylated LN3, 6SL, LNnT and LSTc with a Modified C. glutamicum Host

[0760] A C. glutamicum strain is modified as described in Example 57 for LN3 production and growth on sucrose by genomic knock-out of the Idh, cgl2645, nagB, gamA and nagA genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding the lactose permease (LacY) from E. coli (SEQ ID NO: 14), the native fructose-6-P-aminotransferase (UniProt ID Q8NND3), the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (SEQ ID NO: 26), the sucrose transporter (CscB) from E. coli W (SEQ ID NO: 01), the fructose kinase (Frk) from Z. mobilis (SEQ ID NO: 02) and the sucrose phosphorylase (BaSP) from B. adolescentis (SEQ ID NO: 03). In a next step, the mutant strain is further modified with a genomic knock-in of a constitutive transcriptional unit comprising the N-acetylglucosamine beta-1,4-galactosyltransferase LgtB from N. meningitidis (SEQ ID NO: 28) to produce LNnT. In a next step, the mutant strain is further modified with a genomic knock-in of a constitutive transcriptional unit comprising the native fructose-6-P-aminotransferase (UniProt ID Q8NND3), the glucosamine-6-P-aminotransferase from S. cerevisiae (SEQ ID NO: 15), the N-acetylglucosamine-2-epimerase from B. ovatus (SEQ ID NO: 16), and the N-acetylneuraminate synthase from N. meningitidis (SEQ ID NO: 17) to produce sialic acid. In a next step, the novel strain is transformed with an expression plasmid comprising constitutive transcriptional units for the NeuA enzyme from P. multocida (SEQ ID NO: 21) and the beta-galactoside alpha-2,6-sialyltransferase PdST6 from P. damselae (UniProt ID O66375). The novel strain is evaluated for production of an oligosaccharide mixture comprising LN3, 6-sialylated LN3 (Neu5Ac-a2,6-(GlcNAc-b1,3)-Gal-b1,4-Glc), 6SL, LNnT and LSTc in a growth experiment on MMsf medium comprising lactose according to the culture conditions provided in Example 57. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 59. Production of an Oligosaccharide Mixture Comprising 3SL, 6SL, LNB, 3-Sialylated LNB and 6-Sialylated LNB with a Modified C. Glutamicum Host

[0761] A C. glutamicum strain is modified as described in Example 57 by genomic knock-out of the Idh, cgl2645, nagB, gamA and nagA genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding the lactose permease (LacY) from E. coli (SEQ ID NO: 14), WbgO with SEQ ID NO: 27 from E. coli 055:H7, galE with SEQ ID NO: 29 from E. coli, the native fructose-6-P-aminotransferase (UniProt ID Q8NND3), glmS*54 with SEQ ID NO: 18, the glucosamine-6-P-aminotransferase from S. cerevisiae (SEQ ID NO: 15), the N-acetylglucosamine-2-epimerase from B. ovatus (SEQ ID NO: 16), and the N-acetylneuraminate synthase from N. meningitidis (SEQ ID NO: 17). In a next step, the novel strain is transformed with an expression plasmid comprising constitutive transcriptional units for the NeuA enzyme from P. multocida (SEQ ID NO: 21), the beta-galactoside alpha-2,3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) and the beta-galactoside alpha-2,6-sialyltransferase PdST6 from P. damselae (UniProt ID 066375). The novel strain is evaluated for production of an oligosaccharide mixture comprising 3SL, 6SL, LNB, 3-sialylated LNB (3SLNB) and 6-sialylated LNB (6SLNB) in a growth experiment on MMsf medium comprising lactose and glucose according to the culture conditions provided in Example 57. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 60. Materials and Methods Chlamydomonas reinhardtii

Media

[0762] C. reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7.0). The TAP medium uses a 1000? stock Hutner's trace element mix. Hutner's trace element mix comprised 50 g/L Na2EDTA.Math.H2O (Titriplex III), 22 g/L ZnSO4.Math.7H2O, 11.4 g/L H3BO3, 5 g/L MnCl2.Math.4H2O, 5 g/L FeSO4.Math.7H2O, 1.6 g/L CoCl2.6H2O, 1.6 g/L CuSO4.Math.5H2O and 1.1 g/L (NH4)6MoO3.

[0763] The TAP medium contained 2.42 g/L Tris (tris(hydroxymethyl)aminomethane), 25 mg/L salt stock solution, 0.108 g/L K2HPO4, 0.054 g/L KH2PO4 and 1.0 mL/L glacial acetic acid. The salt stock solution comprised 15 g/L NH4CL, 4 g/L MgSO4.Math.7H2O and 2 g/L CaCl2.Math.2H2O. As precursor(s) and/or acceptor(s) for saccharide synthesis, compounds like e.g., galactose, glucose, fructose, fucose, lactose, LacNAc, LNB could be added. Medium was sterilized by autoclaving (121? C., 21). For stock cultures on agar slants TAP medium was used containing 1% agar (of purified high strength, 1000 g/cm2).

Strains, Plasmids and Mutations

[0764] C. reinhardtii wild-type strains 21 gr (CC-1690, wild-type, mt+), 6145C (CC-1691, wild-type, mt?), CC-125 (137c, wild-type, mt+), CC-124 (137c, wild-type, mt?) as available from Chlamydomonas Resource Center (www.chlamycollection.org), University of Minnesota, U.S.A.

[0765] Expression plasmids originated from pSI103, as available from Chlamydomonas Resource Center. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation. Suitable promoters for (heterologous) gene expression can be derived from e.g., Scranton et al. (Algal Res. 2016, 15: 135-142). Targeted gene modification (like gene knock-out or gene replacement) can be carried using the Crispr-Cas technology as described e.g., by Jiang et al. (Eukaryotic Cell 2014, 13(11): 1465-1469).

[0766] Transformation via electroporation was performed as described by Wang et al. (Biosci. Rep. 2019, 39: BSR2018210). Cells were grown in liquid TAP medium under constant aeration and continuous light with a light intensity of 8000 Lx until the cell density reached 1.0-2.0?107 cells/mL. Then, the cells were inoculated into fresh liquid TAP medium in a concentration of 1.0?106 cells/mL and grown under continuous light for 18-20 h until the cell density reached 4.0?106 cells/mL. Next, cells were collected by centrifugation at 1250 g for 5 min at room temperature, washed and resuspended with pre-chilled liquid TAP medium containing 60 mM sorbitol (Sigma, U.S.A.), and iced for 10 min. Then, 250 ?L of cell suspension (corresponding to 5.0?107 cells) were placed into a pre-chilled 0.4 cm electroporation cuvette with 100 ng plasmid DNA (400 ng/ml). Electroporation was performed with 6 pulses of 500 V each having a pulse length of 4 ms and pulse interval time of 100 ms using a BTX ECM830 electroporation apparatus (1575 ?, 50 ?FD). After electroporation, the cuvette was immediately placed on ice for 10 min. Finally, the cell suspension was transferred into a 50 ml conical centrifuge tube containing 10 mL of fresh liquid TAP medium with 60 mM sorbitol for overnight recovery at dim light by slowly shaking. After overnight recovery, cells were recollected and plated with starch embedding method onto selective 1.5% (w/v) agar-TAP plates containing ampicillin (100 mg/L) or chloramphenicol (100 mg/L). Plates were then incubated at 23+?0.5? C. under continuous illumination with a light intensity of 8000 Lx. Cells were analyzed 5-7 days later.

[0767] In an example for production of UDP-galactose, C. reinhardtii cells are modified with transcriptional units comprising the genes encoding a galactokinase like e.g., from Arabidopsis thaliana (KIN, UniProt ID Q9SEE5) and an UDP-sugar pyrophosphorylase like e.g., USP from A. thaliana (UniProt ID Q9C511).

[0768] In an example for LN3 production, a constitutive transcriptional unit comprising a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g., IgtA from N. meningitidis (SEQ ID NO: 26) is additionally added to the strain. In an example for LNT production, the LN3 producing strain is further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1,3-galactosyltransferase like e.g., WbgO from E. coli 055:H7 (SEQ ID NO: 27). In an example for LNnT production, the LN3 producing strain is further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1,4-galactosyltransferase like e.g., lgtB from N. meningitidis (SEQ ID NO: 28).

[0769] In an example for production of GDP-fucose, (. reinhardtii cells are modified with a transcriptional unit for a GDP-fucose synthase like e.g., from Arabidopsis thaliana (GER1, UniProt ID 049213).

[0770] In an example for fucosylation, (. reinhardtii cells can be modified with an expression plasmid comprising a constitutive transcriptional unit for an alpha-1,2-fucosyltransferase like e.g., HpFutC from H. pylori (SEQ ID NO: 04) and/or an alpha-1,3-fucosyltransferase like e.g., HpFucT from H. pylori (SEQ ID NO: 05).

[0771] In an example for CMP-sialic acid synthesis, C. reinhardtii cells are modified with constitutive transcriptional units for an UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase like e.g., GNE from Homo sapiens (UniProt ID Q9Y223) or a mutant form of the human GNE polypeptide comprising the R263L mutation, an N-acylneuraminate-9-phosphate synthetase like e.g., NANS from Homo sapiens (UniProt ID Q9NR45) and an N-acylneuraminate cytidylyltransferase like e.g., CMAS from Homo sapiens (UniProt ID Q8NFW8). In an example for production of sialylated oligosaccharides, C. reinhardtii cells are modified with a CMP-sialic acid transporter like e.g., CST from Mus musculus (UniProt ID Q61420), and a Golgi-localized sialyltransferase chosen from species like e.g., Homo sapiens, Mus musculus, Rattus norvegicus.

Heterologous and Homologous Expression

[0772] Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.

[0773] Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.

Cultivation Conditions

[0774] Cells of C. reinhardtii were cultured in selective TAP-agar plates at 23+/?0.5? C. under 14/10 h light/dark cycles with a light intensity of 8000 Lx. Cells were analyzed after 5 to 7 days of cultivation.

[0775] For high-density cultures, cells could be cultivated in closed systems like e.g., vertical or horizontal tube photobioreactors, stirred tank photobioreactors or flat panel photobioreactors as described by Chen et al. (Bioresour. Technol. 2011, 102: 71-81) and Johnson et al. (Biotechnol. Prog. 2018, 34: 811-827).

Example 61. Production of an Oligosaccharide Mixture Comprising Sialylated LNB and Sialylated LacNAc Structures in Mutant C. reinhardtii Cells

[0776] C. reinhardtii cells are engineered as described in Example 60 for production of CMP-sialic acid with genomic knock-ins of constitutive transcriptional units comprising a mutant form of the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase GNE from Homo sapiens (UniProt ID Q9Y223) differing from the native polypeptide with a R263L mutation, the N-acylneuraminate-9-phosphate synthetase NANS from Homo sapiens (UniProt ID Q9NR45) and the N-acylneuraminate cytidylyltransferase CMAS from Homo sapiens (UniProt ID Q8NFW8). In a next step, the cells are modified with genomic knock-ins of constitutive transcriptional units comprising the CMP-sialic acid transporter CST from Mus musculus (UniProt ID Q61420), the alpha-2,3-sialyltransferases (UniProt IDs P61943 and E9PSJ1) from Rattus norvegicus and the alpha-2,6-sialyltransferase (UniProt ID P13721) from Rattus norvegicus. In a final step, the cells are transformed with genomic knock-ins of constitutive transcriptional units comprising the Arabidopsis thaliana genes encoding the galactokinase (KIN, UniProt ID Q9SEE5) and the UDP-sugar pyrophosphorylase (USP) (UniProt ID Q9C511), together with the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 with SEQ ID NO: 27 and the N-acetylglucosamine beta-1,4-galactosyltransferase LgtB from N. meningitidis with SEQ ID NO: 28. The novel strain is evaluated for production of an oligosaccharide mixture comprising 3-sialyllacto-N-biose (3SLNB), 6-sialyllacto-N-biose (6SLNB), 3-sialyllactosamine (3SLacNAc) and 6-sialyllactosamine (6SLacNAc) in a cultivation experiment on TAP-agar plates comprising galactose, glucose and N-acetylglucosamine as precursors according to the culture conditions provided in Example 60. After 5 days of incubation, the cells are harvested, and the saccharide production is analyzed on UPLC.

Example 62. Materials and Methods Animal Cells

[0777] Isolation of Mesenchymal Stem Cells from Adipose Tissue of Different Mammals

[0778] Fresh adipose tissue is obtained from slaughterhouses (e.g., cattle, pigs, sheep, chicken, ducks, catfish, snake, frogs) or liposuction (e.g., in case of humans, after informed consent) and kept in phosphate buffer saline supplemented with antibiotics. Enzymatic digestion of the adipose tissue is performed followed by centrifugation to isolate mesenchymal stem cells. The isolated mesenchymal stem cells are transferred to cell culture flasks and grown under standard growth conditions, e.g., 37? C., 5% CO2. The initial culture medium includes DMEM-F12, RPMI, and Alpha-MEM medium (supplemented with 15% fetal bovine serum), and 1% antibiotics. The culture medium is subsequently replaced with 10% FBS (fetal bovine serum)-supplemented media after the first passage. For example, Ahmad and Shakoori (2013, Stem Cell Regen Med. 9(2): 29-36), which is incorporated herein by reference in its entirety for all purposes, describes certain variation(s) of the method(s) described herein in this example.

Isolation of Mesenchymal Stem Cells from Milk

[0779] This example illustrates isolation of mesenchymal stem cells from milk collected under aseptic conditions from human or any other mammal(s) such as described herein. An equal volume of phosphate buffer saline is added to diluted milk, followed by centrifugation for 20 min. The cell pellet is washed thrice with phosphate buffer saline and cells are seeded in cell culture flasks in DMEM-F12, RPMI, and Alpha-MEM medium supplemented with 10% fetal bovine serum and 1% antibiotics under standard culture conditions. For example, Hassiotou et al. (2012, Stem Cells. 30(10): 2164-2174), which is incorporated herein by reference in its entirety for all purposes, describes certain variation(s) of the method(s) described herein in this example.

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

[0780] The isolated mesenchymal cells can be differentiated into mammary-like epithelial and luminal cells in 2D and 3D culture systems. See, for example, Huynh et al. 1991. Exp Cell Res. 197(2): 191-199; Gibson et al. 1991, In Vitro Cell Dev Biol Anim. 27(7): 585-594; Blatchford et al. 1999; Animal Cell Technology: Basic & Applied Aspects, Springer, Dordrecht. 141-145; Williams et al. 2009, Breast Cancer Res 11(3): 26-43; and Arevalo et al. 2015, Am J Physiol Cell Physiol. 310(5): C348-C356; each of which is incorporated herein by reference in their entireties for all purposes.

[0781] For 2D culture, the isolated cells were initially seeded in culture plates in growth media supplemented with 10 ng/ml epithelial growth factor and 5 pg/ml insulin. At confluence, cells were fed with growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U/ml penicillin, 100 ug/ml streptomycin), and 5 pg/ml insulin for 48 h. To induce differentiation, the cells were fed with complete growth medium containing 5 pg/ml insulin, 1 pg/ml hydrocortisone, 0.65 ng/ml triiodothyronine, 100 nM dexamethasone, and 1 pg/ml prolactin. After 24 h, serum is removed from the complete induction medium.

[0782] For 3D culture, the isolated cells were trypsinized and cultured in Matrigel, hyaluronic acid, or ultra-low attachment surface culture plates for six days and induced to differentiate and lactate by adding growth media supplemented with 10 ng/ml epithelial growth factor and 5 pg/ml insulin. At confluence, cells were fed with growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U/ml penicillin, 100 ug/ml streptomycin), and 5 pg/ml insulin for 48 h. To induce differentiation, the cells were fed with complete growth medium containing 5 pg/ml insulin, 1 pg/ml hydrocortisone, 0.65 ng/ml triiodothyronine, 100 nM dexamethasone, and 1 pg/ml prolactin. After 24 h, serum is removed from the complete induction medium.

Method of Making Mammary-Like Cells

[0783] Mammalian cells are brought to induced pluripotency by reprogramming with viral vectors encoding for Oct4, Sox2, Klf4, and c-Myc. The resultant reprogrammed cells are then cultured in Mammocult media (available from Stem Cell Technologies), or mammary cell enrichment media (DMEM, 3% FBS, estrogen, progesterone, heparin, hydrocortisone, insulin, EGF) to make them mammary-like, from which expression of select milk components can be induced. Alternatively, epigenetic remodelling is performed using remodelling systems such as CRISPR/Cas9, to activate select genes of interest, such as casein, a-lactalbumin to be constitutively on, to allow for the expression of their respective proteins, and/or to down-regulate and/or knock-out select endogenous genes as described e.g., in WO 2021067641, which is incorporated herein by reference in its entirety for all purposes.

Cultivation

[0784] Completed growth media includes high glucose DMEM/F12, 10% FBS, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, and 5 pg/ml hydrocortisone. Completed lactation media includes high glucose DMEM/F12, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, 5 pg/ml hydrocortisone, and 1 pg/ml prolactin (5 ug/ml in Hyunh 1991). Cells are seeded at a density of 20,000 cells/cm2 onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media. Upon exposure to the lactation media, the cells start to differentiate and stop growing. Within about a week, the cells start secreting lactation product(s) such as milk lipids, lactose, casein and whey into the media. A desired concentration of the lactation media can be achieved by concentration or dilution by ultrafiltration. A desired salt balance of the lactation media can be achieved by dialysis, for example, to remove unwanted metabolic products from the media. Hormones and other growth factors used can be selectively extracted by resin purification, for example, the use of nickel resins to remove His-tagged growth factors, to further reduce the levels of contaminants in the lactated product.

Example 63. Evaluation of LacNAc, Sialylated LacNAc Structures and Sialyl-Lewis x Production in a Non-Mammary Adult Stem Cell

[0785] Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 62 are modified via CRISPR-CAS to over-express the beta-1,4-galactosyltransferase 4 B4GalT4 from Homo sapiens (UniProt ID 060513), the GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), the galactoside alpha-1,3-fucosyltransferase FUT3 from Homo sapiens (UniProt ID P21217), the N-acylneuraminate cytidylyltransferase from Mus musculus (UniProt ID Q99KK2), and the CMP-N-acetylneuraminate-beta-1,4-galactoside alpha-2,3-sialyltransferase ST3GAL3 from Homo sapiens (UniProt ID Q11203) and the alpha-2,6-sialyltransferase (UniProt ID P13721) from Rattus norvegicus. All genes introduced in the cells are codon-optimized to the host. Cells are seeded at a density of 20,000 cells/cm2 onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media for about 7 days. After cultivation as described in Example 62, cells are subjected to UPLC to analyze for production of LacNAc, 3-sialylated LacNAc, 6-sialylated LacNAc and sialyl-Lewis x.