PRODUCTION OF A MIXTURE OF MAMMALIAN MILK OLIGOSACCHARIDES BY A CELL

Abstract

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

Claims

1.-99. (canceled)

100. A cell that produces a mixture of at least three different mammalian milk oligosaccharides, wherein the cell is metabolically engineered for the production of the mixture, expresses at least two glycosyltransferases, and is capable of synthesizing one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for a glycosyltransferase.

101. The cell of claim 100, wherein the cell is capable of expressing at least three glycosyltransferases.

102. The cell of claim 100, wherein the mixture comprises neutral fucosylated and neutral non-fucosylated mammalian milk oligosaccharides and no charged mammalian milk oligosaccharides.

103. The cell of claim 100, wherein the mixture comprises at least four different mammalian milk oligosaccharides.

104. The cell of claim 100, wherein the cell is modified in the expression or activity of at least one of the glycosyltransferases.

105. The cell of claim 100, wherein: one of the glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-Fucose (GDP-Fuc), one of the glycosyltransferases is an N-acetylglucosaminyl transferase and one of the donor nucleotide-sugars is UDP-N-acetylglucosamine; one of the glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is UDP-galactose; one of the glycosyltransferases is an N-acetylgalactosaminyl transferase and one of the donor nucleotide-sugars is UDP-N-acetylgalactosamine; or one of the glycosyltransferases is an N-acetylmannosaminyl transferase and one of the donor nucleotide-sugars is UDP-N-acetylmannosamine.

106. The cell of claim 100, 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 (i) secretion of any one of the mammalian milk oligosaccharides from the mixture outside the cell or (ii) uptake of a precursor or acceptor for synthesis of any one of the mammalian milk oligosaccharides of the mixture.

107. The cell of claim 106, wherein the membrane protein provides improved production or enabled or enhanced efflux of any one of the mammalian milk oligosaccharides.

108. The cell of claim 100, wherein the cell is a bacterium, fungus, yeast, plant cell, animal cell, or protozoan cell.

109. The cell of claim 100, wherein the at least two glycosyltransferases are involved in producing the mixture.

110. The cell of claim 102, wherein the relative abundance of fucosylated MMOs in the mixture is at least 10%.

111. The cell of claim 102, wherein the relative abundance of fucosylated MMOs in the mixture is less than 90%.

112. The cell of claim 100, wherein the relative abundance of each mammalian milk oligosaccharide in the mixture is at least 5%.

113. The cell of claim 100, wherein the mammalian milk oligosaccharides are produced intracellularly.

114. A method of producing a mixture of at least three different mammalian milk oligosaccharides by a cell, the method comprising: a. providing a cell capable of expressing at least two glycosyltransferases and capable of synthesizing one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for the glycosyltransferases, and b. cultivating the cell under conditions permissive to express the glycosyltransferases and to synthesize the nucleotide-sugar(s), resulting in the cell producing at least three different mammalian milk oligosaccharides.

115. The method according to claim 114, wherein the mixture comprises neutral fucosylated and neutral non-fucosylated mammalian milk oligosaccharides and no charged mammalian milk oligosaccharides.

116. The method according to claim 114, wherein the cell is a cell that produces a mixture of at least three different mammalian milk oligosaccharides, wherein the cell is metabolically engineered for the production of the mixture, expresses at least two glycosyltransferases, and is capable of synthesizing one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for the glycosyltransferase.

117. The method according to claim 114, wherein the mixture comprises at least four different mammalian milk oligosaccharides.

118. The method according to claim 114, further comprising purification of any one of the mammalian milk oligosaccharides from the cell.

119. The method according to claim 114, further comprising separating at least one of the mammalian milk oligosaccharides from the cultivation.

120. A method of producing a mixture of at least three different mammalian milk oligosaccharides, the method comprising: cultivating the cell of claim 100.

121. The method according to claim 120, wherein the mixture comprises neutral fucosylated and neutral non-fucosylated mammalian milk oligosaccharides and no charged mammalian milk oligosaccharides.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0633] The disclosure will be described in more detail in the examples and the accompanying figures, in which:

[0634] 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 a-2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 with SEQ ID NO: 26 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).

[0635] 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 a-2,3-sialyltransferase from P. multocida with SEQ ID NO: 23 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

[0636] The following examples will serve as further illustration and clarification of the disclosure and are not intended to be limiting.

EXAMPLES

Example 1. Materials and Methods Escherichia coli

Media

[0637] The Luria Broth (LB) medium consisted of 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 NH.sub.4Cl, 5.00 g/L (NH.sub.4).sub.2SO.sub.4, 2.993 g/L KH.sub.2PO.sub.4, 7.315 g/L K.sub.2HIPO.sub.4, 8.372 g/L MOPS, 0.5 g/L NaCl, 0.5 g/L MgSO.sub.4.Math.7H.sub.2O, 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 consisted of 3.6 g/L FeCl.sub.2.Math.4H.sub.2O, 5 g/L CaCl.sub.2).Math.2H.sub.2O, 1.3 g/L MnCl.sub.2.Math.2H.sub.2O, 0.38 g/L CuCl.sub.2.Math.2H.sub.2O, 0.5 g/L COCl.sub.2.Math.6H.sub.2O, 0.94 g/L ZnCl.sub.2, 0.0311 g/L H.sub.3BO.sub.4, 0.4 g/L Na.sub.2EDTA.Math.2H.sub.2O and 1.01 g/L thiamine.Math.HCl. The molybdate solution contained 0.967 g/L NaMoO.sub.4.Math.2H.sub.2O. The selenium solution contained 42 g/L SeO.sub.2.

[0638] The minimal medium for fermentations contained 6.75 g/L NH.sub.4Cl, 1.25 g/L (NH.sub.4).sub.2SO.sub.4, 2.93 g/L KH.sub.2PO.sub.4 and 7.31 g/L KH.sub.2PO.sub.4, 0.5 g/L NaCl, 0.5 g/L MgSO.sub.4.Math.7H.sub.2O, 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).

[0639] 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

[0640] 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., phi80dlacZM15, (/acZYA-argF) U169, deoR, recA1, endA1, hsdR17(rk.sup., mk.sup.+), phoA, supE44, lambda.sup., thi-1, gyrA96, relA1) bought from Invitrogen.

Strains and Mutations

[0641] 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 Dpnl, re-purified from an agarose gel, and suspended in elution buffer (5 mM Tris, pH 8.0). Selected mutants were transformed with pCP20 plasmid, which is an ampicillin and chloramphenicol resistant plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis. The ampicillin-resistant transformants were selected at 30 C., after which a few were colony purified in LB at 42 C. and then tested for loss of all antibiotic resistance and of the FLP helper plasmid. The gene knock outs and knock ins are checked with control primers.

[0642] In an example for GDP-fucose production, the mutant strain was derived from E. coli K.sub.12 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., BaSPSP 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 selective marker like, e.g., the E. coli thyA with SEQ ID NO: 07. 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, icR, pgi and Ion 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., manA from E. coli with SEQ ID NO: 08, a phosphomannomutase like, e.g., manB from E. coli with SEQ ID NO: 09, a mannose-1-phosphate guanylyltransferase like, e.g., manC from E. coli with SEQ ID NO: 10, a GDP-mannose 4,6-dehydratase like, e.g., gmd from E. coli with SEQ ID NO: 11 and a GDP-L-fucose synthase like, e.g., fcl from E. coli with SEQ ID NO: 12. GDP-fucose production can also be obtained by genomic knock-outs of the E. coli fucK and fucI genes and genomic knock-ins of constitutive transcriptional units containing a fucose permease like, e.g., fucP from E. coli with SEQ ID NO: 13 and a bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase like, e.g., fkp from Bacteroides fragilis with SEQ ID NO: 14. 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: 15.

[0643] 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 AOA2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID POAEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207) or iceT from Citrobacter youngae (UniProt ID D4B8A6).

[0644] 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: 16, an N-acetylglucosamine 2-epimerase like, e.g., AGE from Bacteroides ovatus with SEQ ID NO: 17 and an N-acetylneuraminate (Neu5Ac) synthase like, e.g., NeuB from Neisseria meningitidis with SEQ ID NO: 18. 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, nanE, nanK, manX, manY and manZ as described in WO 2018122225 and/or genomic knock-outs of the E. coli genes comprising any one or more of nanT, poxB, ldhA, 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: 19 differing from the wild-type E. coli glmS by an A39T, an R250C and a G472S mutation and a phosphatase like any one or more of, e.g., yqaB from E. coli with SEQ ID NO: 20 or 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: 42, an N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase like, e.g., glmU from E. coli (UniProt ID POACC7), a UDP-N-acetylglucosamine 2-epimerase like, e.g., NeuC from Campylobacter jejuni with SEQ ID NO: 21 and an N-acetylneuraminate synthase like, e.g., NeuB from N. meningitidis with SEQ ID NO: 18. 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: 19 and a phosphatase like, e.g., yqaB from E. coli with SEQ ID NO: 20 or 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).

[0645] 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).

[0646] Alternatively, and/or additionally, sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like, e.g., glmM from E. coli (UniProt ID P31120), an N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase like, e.g., glmU from E. coli (UniProt ID POACC7), 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).

[0647] 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: 22, and a beta-galactoside alpha-2,3-sialyltransferase like, e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity like SEQ ID NO: 23, NmeniST3 from N. meningitidis (SEQ ID NO: 24) or PmultST2 from P. multocida subsp. multocida str. Pm70 (Genbank NO. AAK02592.1), a beta-galactoside alpha-2,6-sialyltransferase like, e.g., PdST6 from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity like SEQ ID NO: 25, P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 having beta-galactoside alpha-2,6-sialyltransferase activity like SEQ ID NO: 26 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: 15.

[0648] 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 O6:H1 (UniProt ID Q8FD59), nanT from E. coli O157:H7 (UniProt ID Q8X9G8) or nanT from E. albertii (UniProt ID B1EFH1) or a porter like, e.g., EntS from E. coli (UniProt ID P24077), EntS from Kluyvera ascorbata (UniProt ID A0A378GQ13) 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), lmrA from Lactococcus lactis subsp. lactis bv. diacetylactis (UniProt ID A0A1VONEL4), or Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4).

[0649] 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.

[0650] 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: 27. 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: 28 or an N-acetylglucosamine beta-1,4-galactosyltransferase like, e.g., LgtB from N. meningitidis with SEQ ID NO: 29, 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., glmS*54 from E. coli with SEQ ID NO: 19. In addition, the strains can optionally be modified for enhanced UDP-galactose production with genomic knock-outs of the E. coli ushA, galT, ldhA and agp genes. The mutant E. coli strains can also optionally be adapted with a genomic knock-in of a constitutive transcriptional unit for a UDP-glucose-4-epimerase like, e.g., galE from E. coli with SEQ ID NO: 30, a phosphoglucosamine mutase like, e.g., glmM from E. coli with SEQ ID NO: 42 and an N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase like, e.g., glmU from E. coli (UniProt ID P0ACC7). 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.

[0651] 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 AOA2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID POAEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207) or iceT from Citrobacter youngae (UniProt ID D4B8A6).

[0652] 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).

[0653] 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).

[0654] Optionally, the mutant E. coli strains are modified to create a glycominimized E. coli strain comprising genomic knock-out of any one or more of non-essential glycosyltransferase genes comprising pgaC, pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcaI, wcaJ, wcaL, waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbl, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA and yaiP.

[0655] 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 (UTROO10_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 the disclosure are summarized in Table 1.

[0656] All strains were stored in cryovials at 80 C. (overnight LB culture mixed in a 1:1 ratio with 7000 glycerol).

TABLE-US-00001 TABLE 1 Overview of SEQ ID NOs described in the disclosure. Country of origin SEQ of digital ID Name/ sequence 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 UA1234 Kingdom 05 HpFucT Helicobacter pylori Synthetic United UA1234 Kingdom 06 Mutant Helicobacter pylori Synthetic United HpFucT UA1234 Kingdom 07 thyA Escherichia coli K-12 Synthetic USA MG1655 08 manA Escherichia coli K-12 Synthetic USA MG1655 09 manB Escherichia coli K-12 Synthetic USA MG1655 10 manC Escherichia coli K-12 Synthetic USA MG1655 11 gmd Escherichia coli K-12 Synthetic USA MG1655 12 fcl Escherichia coli K-12 Synthetic USA MG1655 13 fucP Escherichia coli K-12 Synthetic USA MG1655 14 fkp Bacteroides fragilis Synthetic United Kingdom 15 LacY Escherichia coli K-12 Synthetic USA MG1655 16 GNA1 Saccharomyces cerevisiae Synthetic USA 17 AGE Bacteroides ovatus Synthetic USA 18 neuB Neisseria meningitidis Synthetic United Kingdom 19 glmS*54 Escherichia coli K-12 Synthetic USA MG1655 20 phosphatase Escherichia coli K-12 Synthetic USA MG1655 21 neuC Campylobacter jejuni Synthetic USA 22 neuA Pasteurella multocida Synthetic USA 23 alpha-2,3- Pasteurella multocida Synthetic USA sialyltransferase 24 alpha-2,3- Neisseria meningitidis Synthetic United sialyltransferase Kingdom 25 alpha-2,6- Photobacterium damselae Synthetic Japan sialyltransferase 26 alpha-2,6- Photobacterium sp. JT- Synthetic Japan sialyltransferase ISH-224 27 LgtA Neisseria meningitidis Synthetic United Kingdom 28 WbgO Escherichia coli O55:H7 Synthetic Germany 29 LgtB Neisseria meningitidis Synthetic United MC58 Kingdom 30 gale Escherichia coli K-12 Synthetic USA MG1655 31 Lac12 Kluyveromyces lactis Synthetic USA 32 GCNT2 Homo sapiens Synthetic Unknown 33 alpha-1,3- Bos taurus Synthetic Unknown galactosyl- transferase 34 WbpP Pseudomonas aeruginosa Synthetic USA 35 lgtD Haemophilus influenzae Synthetic Ireland 36 lgtC Neisseria gonorrhoeae Synthetic Japan 37 WbnI Escherichia coli K-12 Synthetic USA MG1655 38 BgtA Helicobacter mustelae Synthetic Unknown 39 Mutant a1, 3/4 Bifidobacterium longum Synthetic USA fucosidase subsp. infantis 40 fucT54 Sideroxydans Synthetic USA lithotrophicus ES-1 41 WbdO Salmonella enterica Synthetic United subsp. salamae Kingdom serovar Greenside 42 glmM Escherichia coli K-12 Synthetic USA MG1655 43 glmU Escherichia coli K-12 Synthetic USA MG1655

Cultivation Conditions

[0657] 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).

[0658] 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 H.sub.2SO.sub.4 and 20% NH.sub.4OH. The exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.

Optical Density

[0659] Cell density of the cultures was frequently monitored by measuring optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or with a Spark 10 M microplate reader, Tecan, Switzerland).

Analytical Analysis

[0660] 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.

[0661] Neutral mammalian milk 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 (2.1100 mm; 130 ; 1.7 m) column with an Acquity UPLC BEH Amide VanGuard column, 130 , 2.15 mm. The column temperature was 50 C. The mobile phase consisted of 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.

[0662] Sialylated mammalian milk 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 (2.1100 mm; 130 ; 1.7 m). The column temperature was 50 C. The mobile phase consisted of 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.

[0663] 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 (2.1100 mm; 130 ; 1.7 m). The column temperature was 50 C. The mobile phase consisted of a mixture of 72% acetonitrile and 28% ammonium acetate buffer (100 mM) to which 0.1% triethylamine was added. The method was isocratic with a flow of 0.260 mL/min. The temperature of the RI detector was set at 35 C.

[0664] 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 mammalian milk oligosaccharides. Separation was performed on a Waters Acquity UPLC with a Thermo Hypercarb column (2.1100 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 mammalian milk 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.

[0665] 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 4250 mm with a Dionex CarboPac PA200 guard column 450 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

[0666] 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

[0667] 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

[0668] 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 2p 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: 31, a GDP-mannose 4,6-dehydratase like, e.g., gmd from E. coli with SEQ ID NO: 11 and a GDP-L-fucose synthase like, e.g., fcl from E. coli with SEQ ID NO: 12. 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 2p 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: 31, a fucose permease like, e.g., fucP from E. coli with SEQ ID NO: 13 and a bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase like, e.g., fkp from B. fragilis with SEQ ID NO: 14. 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.

[0669] 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: 19, a phosphatase like, e.g., YqaB from E. coli with SEQ ID NO: 20 or 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, an N-acetylglucosamine 2-epimerase like, e.g., AGE from B. ovatus with SEQ ID NO: 17, an N-acetylneuraminate synthase like, e.g., NeuB from N. meningitidis with SEQ ID NO: 18 and an N-acylneuraminate cytidylyltransferase like, e.g., NeuA from P. multocida with SEQ ID NO: 22. Optionally, a constitutive transcriptional unit for a glucosamine 6-phosphate N-acetyltransferase like, e.g., GNA1 from S. cerevisiae with SEQ ID NO: 16 was added as well. 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: 31, and a beta-galactoside alpha-2,3-sialyltransferase like, e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity like SEQ ID NO: 23, NmeniST3 from N. meningitidis (SEQ ID NO: 24) or PmultST2 from P. multocida subsp. multocida str. Pm70 (Genbank NO. AAK02592.1), a beta-galactoside alpha-2,6-sialyltransferase like, e.g., PdST6 from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity like SEQ ID NO: 25, P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 having beta-galactoside alpha-2,6-sialyltransferase activity like SEQ ID NO: 26 and/or an alpha-2,8-sialyltransferase like, e.g., from M. musculus (UniProt ID Q64689).

[0670] 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 a UDP-glucose-4-epimerase like, e.g., galE from E. coli with SEQ ID NO: 30. To produce LN3 this plasmid was further modified with constitutive transcriptional units for a lactose permease like, e.g., LAC12 from K. lactis with SEQ ID NO: 31, a galactoside beta-1,3-N-acetylglucosaminyltransferase (lgtA) from N. meningitidis with SEQ ID NO: 27. To produce LN3-derived oligosaccharides like LNT or LNnT, the plasmid was 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: 28 or an N-acetylglucosamine beta-1,4-galactosyltransferase like, e.g., lgtB from N. meningitidis with SEQ ID NO: 29, respectively.

[0671] 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.

[0672] 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).

[0673] Plasmids were maintained in the host E. coli DH5alpha (F.sup., phi80dlacZdeltaM15, delta(/acZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk.sup., mk.sup.+), phoA, supE44, lambda, thi-1, gyrA96, relA1) bought from Invitrogen.

Heterologous and Homologous Expression

[0674] 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

[0675] 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

[0676] Genes were expressed using synthetic constitutive promoters, as described by Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012).

Example 3. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL and DiFL with a Modified E. coli Host Expressing an alpha-1,2-fucosyltransferase and an alpha-1,3-fucosyltransferase from Two Compatible Plasmids

[0677] An E. coli K12 strain modified for GDP-fucose production as described in Example 1 was sequentially transformed with a first plasmid expressing a constitutive transcriptional unit for the H. pylori alpha-1,2-fucosyltransferase with SEQ ID NO: 04 and a second compatible plasmid expressing a constitutive transcriptional unit for the H. pylori alpha-1,3-fucosyltransferase with SEQ ID NO: 05. The novel strain was evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contained sucrose and lactose. Each strain was grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth was harvested, and the sugar mixtures were analyzed on UPLC. The experiment demonstrated the novel strain produced an oligosaccharide mixture comprising 2FL, 3-FL and difucosylated lactose (DiFL) in whole broth samples.

Example 4. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL and DiFL with a Modified E. coli Host Expressing an alpha-1,2-fucosyltransferase and an alpha-1,3-fucosyltransferase from One Plasmid

[0678] An E. coli strain modified for GDP-fucose production as described in Example 1 was further transformed with one expression plasmid having two constitutive transcriptional units, including one for the H. pylori alpha-1,2-fucosyltransferase with SEQ ID NO: 04 and another one for the H. pylori alpha-1,3-fucosyltransferase with SEQ ID NO: 05. The novel strain was evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contained sucrose and lactose. Each strain was grown in four biological replicates in a 96-well plate. After 72 h of incubation, the culture broth was harvested, and the sugar mixtures were analyzed on UPLC. The experiment demonstrated the novel strain produced an oligosaccharide mixture comprising 2FL, 3-FL and difucosylated lactose (DiFL) in whole broth samples.

Example 5. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL and DiFL with a Modified E. coli Host in Fed-Batch Fermentations

[0679] Mutant E. coli strains as described in Examples 3 and 4 were further evaluated in a fed-batch fermentation process. Fed-batch fermentations at bioreactor scale were performed as described in Example 1. In these examples, sucrose was used as a carbon source and lactose was added in the batch medium as a precursor. Regular broth samples were taken and the production of 2FL, 3-FL and DiFL was measured using UPLC as described in Example 1. The experiment demonstrated that broth samples taken at the end of batch phase comprised an oligosaccharide mixture of 2FL and 3-FL together with unmodified lactose, whereas broth samples taken at the end of the fed-batch phase comprised an oligosaccharide mixture of 2FL, 3-FL and DiFL. As the ratios of lactose, 2FL, 3-FL and DiFL changed over time during fed-batch, they could be manipulated during the fermentation process by discontinuation of the fermentation process at a desired time in fed-batch phase.

Example 6. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, 2FLacNAc, 3-FLacNAc and Di-FLacNAc with a Modified E. coli Host

[0680] An E. coli strain modified for the production of GDP-fucose as described in Example 1, was further transformed with an expression plasmid having two constitutive transcriptional units, one to express the H. pylori alpha-1,2-fucosyltransferase with SEQ ID NO: 04 and the other one to express the H. pylori alpha-1,3-fucosyltransferase with SEQ ID NO: 05. Since lactose and N-acetyllactosamine (LacNAc, Gal-b1,4-GlcNAc) are suitable acceptors for both H. pylori fucosyltransferases, the novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL and fucosylated LacNAc (i.e., 2FLacNAc and 3-FLacNAc) in whole broth samples 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 and LacNAc as precursors. Since the enzyme with SEQ ID NO: 04 also shows fucosyltransferase activity on 2FL and the enzyme with SEQ ID NO: 05 also shows fucosyltransferase activity on 2FLacNAc, the novel strain is also evaluated for production of DiFL and Di-FLacNAc in the oligosaccharide mixture.

Example 7. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FLacNAc, 3-FLacNAc and Di-FLacNAc with a Modified E. coli Host Expressing Different Fucosyltransferases

[0681] An E. coli K-12 MG1655 strain optimized for GDP-fucose production as described in Example 1 is further modified for the production of GlcNAc and LacNAc with a knock-out of the E. coli N-acetylglucosamine-6-phosphate deacetylase (nagA) gene and the E. coli glucosamine-6-phosphate deaminase (nagB) gene together with genomic knock-ins of constitutive transcriptional units for the N-acetylglucosamine 1,4-galactosyltransferase (LgtB) of N. meningitidis with SEQ ID NO: 29, the mutant L-glutamine-D-fructose-6-phosphate aminotransferase (glmS*54) from E. coli with SEQ ID NO: 19, and the glucosamine 6-phosphate N-acetyltransferase (GNA1) of S. cerevisiae with SEQ ID NO: 16. In a next step, the novel strain is additionally transformed with two compatible expression plasmids wherein the first plasmid contains a constitutive transcriptional unit for the H. pylori alpha-1,2-fucosyltransferase with SEQ ID NO: 04 and the second compatible plasmid contains a constitutive transcriptional unit for the H. pylori alpha-1,3-fucosyltransferase with SEQ ID NO: 05. Alternatively, the novel strain can be transformed with one expression plasmid containing a constitutive transcriptional unit for both H. pylori fucosyltransferases with SEQ ID NO: 04 and 05. All novel strains are evaluated for production of a mixture of 2-fucosylated LacNAc (2FLacNAc), 3-fucosylated LacNAc (3-FLacNAc) and di-fucosylated LacNAc (Di-FLacNAc) in whole broth samples in a growth experiment according to the culture conditions provided in Example 1, in which the cultivation contains sucrose as carbon source.

Example 8. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, 2FLNB, 4-FLNB and Di-FLNB with a Modified E. coli Host

[0682] An E. coli strain modified for the production of GDP-fucose as described in Example 1, is further transformed with an expression plasmid having two constitutive transcriptional units, one to express the H. pylori alpha-1,2-fucosyltransferase with SEQ ID NO: 04 and one to express the H. pylori alpha-1,3-fucosyltransferase with SEQ ID NO: 05. Since lactose and lacto-N-biose (LNB, Gal-b1,3-GlcNAc) are suitable acceptors for both H. pylori fucosyltransferases, the novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL and fucosylated LNB (i.e., 2FLNB and 4-FLNB) in whole broth samples 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 and LNB as precursors. Since the enzyme with SEQ ID NO: 04 also shows fucosyltransferase activity on 2FL and the enzyme with SEQ ID NO: 05 also shows fucosyltransferase activity on 2FLNB, the novel strain is also evaluated for production of DiFL and Di-FLNB in the oligosaccharide mixture.

Example 9. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, 2FLNB, 4-FLNB, and Di-FLNB with a Modified E. coli Host

[0683] An E. coli strain modified for the production of GDP-fucose as described in Example 1 is further adapted for intracellular lactose synthesis by genomic knock-outs of lacZ, glk and the galETKM operon, together with genomic knock-ins of constitutive transcriptional units for lgtB from N. meningitidis with SEQ ID NO: 29 and the UDP-glucose 4-epimerase (galE) from E. coli with SEQ ID NO: 30. In a next step, the mutant E. coli strain was transformed with an expression plasmid having two constitutive transcriptional units, one to express the H. pylori alpha-1,2-fucosyltransferase with SEQ ID NO: 04 and one to express the H. pylori alpha-1,3-fucosyltransferase with SEQ ID NO: 05. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL and fucosylated LNB (i.e., 2FLNB, 4-FLNB and DiFLNB) in whole broth samples in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contains sucrose as carbon source and LNB as precursor.

Example 10. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FLNB, 4-FLNB and Di-Fucosylated LNB with a Modified E. coli Host

[0684] An E. coli K-12 MG1655 strain optimized for GDP-fucose production as exemplified in Example 1, is further modified for the production of GlcNAc and LNB with a knock-out of the E. coli nagA and nagB genes, together with genomic knock-ins of constitutive transcriptional units for the N-acetylglucosamine 1,3-galactosyltransferase (WbgO) from E. coli 055:H7 with SEQ ID NO: 28, the mutant glmS*54 from E. coli with SEQ ID NO: 19, and GNA1 from S. cerevisiae with SEQ ID NO: 16. In a next step, the novel strain is additionally transformed with two compatible expression plasmids, one plasmid containing a constitutive transcriptional unit for the H. pylori alpha-1,2-fucosyltransferase with SEQ ID NO: 04 and the other plasmid containing a constitutive transcriptional unit for the H. pylori alpha-1,3-fucosyltransferase with SEQ ID NO: 05. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2-fucosylated LNB (2FLNB), 4-fucosylated LNB (4-FLNB) and di-fucosylated LNB (Di-FLNB) in whole broth samples in a growth experiment according to the culture conditions provided in Example 1, in which the cultivation contains sucrose as carbon source.

Example 11. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FLNB, 4-FLNB and Di-Fucosylated LNB with a Modified E. coli Host

[0685] An E. coli K-12 MG1655 strain optimized for GDP-fucose production as exemplified in Example 1, is further modified for the production of GlcNAc and LNB with a knock-out of the E. coli nagA and nagB genes, together with genomic knock-ins of constitutive transcriptional units for WbgO from E. coli 055:H7 with SEQ ID NO: 28, the mutant glmS*54 from E. coli with SEQ ID NO: 19, and GNA1 from S. cerevisiae with SEQ ID NO: 16. In a next step, the novel strain is additionally transformed with one expression plasmid containing a constitutive transcriptional unit for the H. pylori alpha-1,2-fucosyltransferase with SEQ ID NO: 04 and a constitutive transcriptional unit for the H. pylori alpha-1,3-fucosyltransferase with SEQ ID NO: 05. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2-fucosylated LNB (2FLNB), 4-fucosylated LNB (4-FLNB) and di-fucosylated LNB (Di-FLNB) in whole broth samples in a growth experiment according to the culture conditions provided in Example 1, in which the cultivation contains sucrose as carbon source.

Example 12. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, DiFL, 2FLNB and Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a Modified E. coli Host

[0686] An E. coli strain modified for the production of GDP-fucose as described in Example 1, is transformed with an expression plasmid having constitutive transcriptional units for the alpha-1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and the alpha-1,3-galactosyltransferase WbnI from E. coli with SEQ ID NO: 37. Since lactose and LNB are suitable acceptors for the H. pylori alpha-1,2-fucosyltransferase, the novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL and 2-fucosylated LNB (2FLNB) 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 and LNB as precursors. In this experiment the novel strain is additionally evaluated for production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc in the oligosaccharide mixture since 2FLNB is an acceptor of the E. coli alpha-1,3-galactosyltransferase.

Example 13. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, DiFL, 2FLNB and GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a Modified E. coli Host

[0687] An E. coli strain modified for the production of GDP-fucose as described in Example 1, is transformed with an expression plasmid having constitutive transcriptional units for the alpha-1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and the alpha-1,3-N-acetylgalactosaminyltransferase BgtA from H. mustelae with SEQ ID NO: 38. Since lactose and LNB are suitable acceptors for the H. pylori alpha-1,2-fucosyltransferase the novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL and 2-fucosylated LNB (2FLNB) 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 and LNB as precursors. In this experiment the novel strain is additionally evaluated for production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc in the oligosaccharide mixture since 2FLNB is an acceptor of the H. mustelae alpha-1,3-N-acetylgalactosaminyltransferase.

Example 14. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, DiFL, 2FLNB and Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a Modified E. coli Host

[0688] An E. coli strain modified for the production of GDP-fucose as described in Example 1, is further modified for the production of GlcNAc and LNB with a knock-out of the E. coli nagA and nagB genes, together with genomic knock-ins of constitutive transcriptional units for the N-acetylglucosamine 1,3-galactosyltransferase (WbgO) from E. coli 055:H7 with SEQ ID NO: 28, the mutant glmS*54 from E. coli with SEQ ID NO: 19, and GNA1 from S. cerevisiae with SEQ ID NO: 16. In a next step, the novel strain is transformed with an expression plasmid containing constitutive transcriptional units for the alpha-1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and the alpha-1,3-galactosyltransferase WbnI from E. coli with SEQ ID NO: 37. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL, 2-fucosylated LNB and Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc 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 15. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, DiFL, 2FLNB and GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a Modified E. coli Host

[0689] An E. coli strain modified for the production of GDP-fucose as described in Example 1, is further modified for the production of GlcNAc and LNB with a knock-out of the E. coli nagA and nagB genes, together with genomic knock-ins of constitutive transcriptional units for WbgO from E. coli 055:H7 with SEQ ID NO: 28, the mutant glmS*54 from E. coli with SEQ ID NO: 19, and GNA1 from S. cerevisiae with SEQ ID NO: 16. In a next step, the novel strain is transformed with an expression plasmid containing constitutive transcriptional units for the alpha-1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and the alpha-1,3-N-acetylgalactosaminyltransferase BgtA from H. mustelae with SEQ ID NO: 38. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL, 2-fucosylated LNB and GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc 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 16. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LacNAc and 6-Sialylated LacNAc with a Modified E. coli Host

[0690] 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: 22 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: 23 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 25. 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 17. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LacNAc and 6-Sialylated LacNAc with a Modified E. coli Host

[0691] 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: 22 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: 19 from E. coli, GNA1 with SEQ ID NO: 16 from S. cerevisiae, the phosphatase yqaB from E. coli with SEQ ID NO: 20 and LgtB with SEQ ID NO: 29 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: 23 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 25. 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 18. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LacNAc and 6-Sialylated LacNAc with a Modified E. coli Host

[0692] 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 consisting of constitutive transcriptional units for neuA from P. multocida with SEQ ID NO: 22, the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 23 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 25. 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 19. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LacNAc and 6-Sialylated LacNAc with a Modified E. coli Host

[0693] 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 LgtB with SEQ ID NO: 29 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: 22, the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 23 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 25. 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 20. Production of Sialylated LacNAc and Poly-LacNAc Structures with a Modified E. coli Host

[0694] 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: 29 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: 22, the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 23 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 25. 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: 27. The novel strain is evaluated for production of LacNAc and an oligosaccharide mixture comprising poly-LacNAc structures, i.e., (Gal-b1,4-GlcNAc)n, which are built of repeated N-acetyllactosamine units that are beta1,3-linked to each other by alternate activity of both the transferases LgtB with SEQ ID NO: 29 and LgtA with SEQ ID NO: 27, together with 3-sialylated LacNAc and 6-sialylated LacNAc, and sialylated poly-LacNAc structures in which the Gal residue is sialylated, 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 21. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LNB and 6-Sialylated LNB with a Modified E. coli Host

[0695] 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: 22 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: 23 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 25. 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 22. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LNB and 6-Sialylated LNB with a Modified E. coli Host

[0696] 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: 22 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: 19 from E. coli, GNA1 with SEQ ID NO: 16 from S. cerevisiae, the phosphatase yqaB from E. coli with SEQ ID NO: 20 and WbgO with SEQ ID NO: 28 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: 23 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 25. 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 23. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LNB and 6-Sialylated LNB with a Modified E. coli Host

[0697] 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 consisting of constitutive transcriptional units for neuA from P. multocida with SEQ ID NO: 22, the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 23 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 25. 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 24. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3SL, 6SL, 3-Sialylated LNB and 6-Sialylated LNB with a Modified E. coli Host

[0698] 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: 28 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: 22, the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 23 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 25. 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 25. Production of Sialylated LNB with a Modified E. coli Host

[0699] 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 WbgO with SEQ ID NO: 28 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: 22, the alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 23 and the alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 25. The novel strain is evaluated for production of LNB together with 3-sialylated LNB and 6-sialylated LNB 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 26. Production of a Mammalian Milk Oligosaccharide Mixture Comprising GlcNAc-b1,3-Gal-b1,4-G/cNAc and Poly-LacNAc Structures with a Modified E. coli Host

[0700] An E. coli K-12 MG1655 strain is modified with a knock-out of the E. coli nagA and nagB genes together with genomic knock-ins of constitutive transcriptional units for the mutant glmS*54 with SEQ ID NO: 19 from E. coli, GNA1 with SEQ ID NO: 16 from S. cerevisiae and LgtB with SEQ ID NO: 29 from N. meningitidis to produce LacNAc. In a next step, the novel strain is additionally transformed with an expression plasmid containing a constitutive transcriptional unit for the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis with SEQ ID NO: 27. By subsequent action of the glmS*54 enzyme and the homologous EcGlmM and EcGlmU enzymes, the mutant strain is capable to produce UDP-GlcNAc, which is used by the heterologous LgtA protein to modify LacNAc into GlcNAc-b1,3-Gal-b1,4-GlcNAc. The novel strain is evaluated for production of the GlcNAc-b1,3-Gal-b1,4-GlcNAc 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. The novel strain is also evaluated for production of poly-LacNAc structures, i.e., (Gal-b1,4-GlcNAc)n, which are built of repeated N-acetyllactosamine units that are beta1,3-linked to each other by alternate activity of the heterologous LgtB and LgtA expressed in the strain.

Example 27. Production of a Mammalian Milk Oligosaccharide Mixture Comprising GlcNAc-b1,3-Gal-b1,4-GlcNAc, Beta-Gal-(1,4)-Beta-GlcNAc-(1,3)-[Beta-GlcNAc-(1,6)]-Beta-Gal-(1,4)-GlcNAc and Poly-LacNAc Structures with a Modified E. coli Host

[0701] In a next example, the mutant E. coli strain adapted to produce LacNAc, UDP-GlcNAc, GlcNAc-b1,3-Gal-b1,4-GlcNAc and (Gal-b1,4-GlcNAc)n structures as described in Example 26, is further modified with a second expression plasmid containing a constitutive transcriptional unit for the human N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase GCNT2 with SEQ ID NO: 32. The novel strain is evaluated for production of an oligosaccharide mixture comprising LacNAc, GlcNAc-b1,3-Gal-b1,4-GlcNAc, GlcNAc-b1,6-Gal-b1,4-GlcNAc, (Gal-b1,4-GlcNAc)n structures and beta-Gal-(1,4)-beta-GlcNAc-(1,3)-[beta-GlcNAc-(1,6)]-beta-Gal-(1,4)-GlcNAc 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.

Example 28. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Gal-a1,3-LacNAc, LN3, LNnT and Gal-a1,3-LNnT with a Modified E. coli Host

[0702] An E. coli strain modified to produce LacNAc as described in Example 26 is further modified with a genomic knock-out of the E. coli lacZ gene and transformed with a compatible expression plasmid containing a constitutive transcriptional unit for the C-terminal catalytic domain of the bovine alpha-1,3-galactosyltransferase (a3FTcd) with SEQ ID NO: 33. The novel strain is evaluated for production of an oligosaccharide mixture comprising Gal-a1,3-LacNAc (Gal-a1,3-Gal-b1,4-GlcNAc), LN3, LNnT and Gal-a1,3-LNnT (Gal-a1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc) 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 29. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LacNAc, GalNAc-b1,3-Lactose, Gal-b1,3-GalNAc-b1,3-Lactose, LN3, LNnT, GalNAc-b1,3-LacNAc, Gal-b1,3-GalNAc-b1,3-LacNAc, Poly-LacNAc (Gal-b1,4-GlcNAc)n and GalNAc-ylated Poly-LacNAc Structures with a Modified E. coli Host

[0703] An E. coli strain modified to produce LacNAc as described in Example 26 is further modified with a genomic knock-out of the E. coli lacZ gene and with genomic knock-ins of constitutive expression units for the 4-epimerase (WbpP) of Pseudomonas aeruginosa with SEQ ID NO: 34, the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis with SEQ ID NO: 27 and the 1,3-N-acetylgalactosaminyltransferase (LgtD) from Haemophilus influenzae with SEQ ID NO: 35. The novel strain is evaluated for production of an oligosaccharide mixture comprising LacNAc, LN3, LNnT, GalNAc-b1,3-lactose, Gal-b1,3-GalNAc-b1,3-lactose, GalNAc-b1,3-LacNAc, Gal-b1,3-GalNAc-b1,3-LacNAc, poly-LacNAc structures, i.e., (Gal-b1,4-GlcNAc)n, which are built of repeated N-acetyllactosamine units that are beta1,3-linked to each other by alternate activity of the N-acetylglucosamine beta-1,4-galactosyltransferase and the galactoside beta-1,3-N-acetylglucosaminyltransferase, and GalNAc-ylated poly-LacNAc structures 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 30. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LNB, LN3, LNT, GalNAc-b1,3-Lactose, Gal-b1,3-GalNAc-b1,3-Lactose, GalNAc-b1,3-LNB and Gal-b1,3-GalNAc-b1,3-LNB with a Modified E. coli Host

[0704] An E. coli strain is modified with a genomic knock-out of the E. coli nagB gene and genomic knock-ins of constitutive expression cassettes for the mutant glmS*54 with SEQ ID NO: 19 from E. coli, GNA1 with SEQ ID NO: 16 from S. cerevisiae and WbgO with SEQ ID NO: 28 from E. coli 055:H7 to produce LNB. In a next step, the LNB producing E. coli strain is further modified with a knock-out of the E. coli lacZ gene and with knock-ins of constitutive expression units for the 4-epimerase (WbpP) of P. aeruginosa with SEQ ID NO: 34, the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis with SEQ ID NO: 27 and the 1,3-N-acetylgalactosaminyltransferase (LgtD) from H. influenzae with SEQ ID NO: 35. The novel strain is evaluated for production of an oligosaccharide mixture comprising LNB, LN3, LNT, GalNAc-b1,3-lactose, Gal-b1,3-GalNAc-b1,3-lactose, GalNAc-b1,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, GalNAc-b1,3-LNB and Gal-b1,3-GalNAc-b1,3-LNB, in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium comprises glycerol as carbon source and lactose as precursor.

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

[0705] 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: 22. 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 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 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: 23 None pMS_1B None SEQ ID NO: 25 pMS_2 SEQ ID NO: 23 SEQ ID NO: 25

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_1A 2FL, 3SL, 3S-2FL SF2 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL SF3 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL SF4 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL SF5 pMF_1A, pMS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SF6 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SF7 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SF8 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SF9 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 32. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Lactose Structures with a Modified E. coli Host

[0706] 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: 19, GNA1 of S. cerevisiae with SEQ ID NO: 16, the N-acetylglucosamine 2-epimerase (AGE) of Bacteroides ovatus with SEQ ID NO: 17, and the N-acetylneuraminate synthase (neuB) of N. meningitidis with SEQ ID NO: 18. 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: 22. 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 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 SF10 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL SF11 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL SF12 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL SF13 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL SF14 pMF_1A, pMS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SF15 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SF16 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SF17 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SF18 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 33. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Lactose Structures with a Modified E. coli Host

[0707] 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: 19, the UDP-N-acetylglucosamine 2-epimerase (neuC) of Campylobacter jejuni with SEQ ID NO: 21 and the N-acetylneuraminate synthase (neuB) of N. meningitidis with SEQ ID NO: 18. 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: 22. 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 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 SF19 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL SF20 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL SF21 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL SF22 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL SF23 pMF_1A, pMS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SF24 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SF25 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SF26 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SF27 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 34. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Lactose Structures with a Modified E. coli Host

[0708] 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 wcaJ 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: 22. 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 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 SF28 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL SF29 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL SF30 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL SF31 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL SF32 pMF_1A, pMS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SF33 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SF34 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SF35 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SF36 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 35. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Lactose Structures with a Modified E. coli Host

[0709] 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: 13 and the bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase (fkp) from B. fragilis with SEQ ID NO: 14 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: 22. 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 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 SF37 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL SF38 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL SF39 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL SF40 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL SF41 pMF_1A, pMS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SF42 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SF43 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SF44 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SF45 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 36. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LN3, Sialylated LN3, LNT, 3SL and LSTa with a Modified E. coli Host

[0710] 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: 22 and the -2,3-sialyltransferase from P. multocida with SEQ ID NO: 23. The novel strain is evaluated for production of a mixture of oligosaccharides comprising LN3, 3-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc), LNT, 3SL and LSTa (Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) 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. After 72 h of incubation, the culture broth is harvested, and the sugar mixtures are analyzed as described in Example 1.

Example 37. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 6SL, LN3, Sialylated LN3, LNnT and LSTc with a Modified E. coli Host

[0711] 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: 22 and one selected -2,6-sialyltransferase. In this experiment, the -2,6-sialyltransferase from P. damselae with SEQ ID NO: 25 and the -2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 with SEQ ID NO: 26 were tested, wherein the -2,6-sialyltransferase with SEQ ID NO: 26 was expressed from two different transcriptional units. 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 a UPLC with RI detection as described in Example 1. The ratio of 6SL, 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 NO: Strain Promoter UTR CDS Terminator S1 PROM0005_MutalikP5 UTR0010_GalE_BCD12 25 TER0007_ilvGEDA S2 PROM0005_MutalikP5 UTR0010_GalE_BCD12 26 TER0007_ilvGEDA S3 PROM0050_apFAB82 UTR0014_GalE_LeuAB 26 TER0007_ilvGEDA

Example 38. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LN3, LNnT, Sialylated LN3, 3SL and LSTd with a Modified E. coli Host

[0712] 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: 22 and one selected -2,3-sialyltransferase. In this experiment, the -2,3-sialyltransferase from P. multocida with SEQ ID NO: 23 and the -2,3-sialyltransferase from N. meningitidis with SEQ ID NO: 24 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 a UPLC with RI detection as described in Example 1. The ratio of 3SL, 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 NO: Strain Promoter UTR CDS Terminator S4 PROM0050_apFAB82 UTR0014_GalE_LeuAB 23 TER0007_ilvGEDA S5 PROM0005_MutalikP5 UTR0010_GalE_BCD12 23 TER0007_ilvGEDA S6 PROM0050_apFAB82 UTR0014_GalE_LeuAB 24 TER0007_ilvGEDA S7 PROM0005_MutalikP5 UTR0010_GalE_BCD12 24 TER0007_ilvGEDA

Example 39. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LN3, Sialylated LN3, LNT, 3SL and LSTa with a Modified E. coli Host

[0713] 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: 27 and for the N-acetylglucosamine beta-1,3-galactosyltransferase (WbgO) from E. coli 055:H7 with SEQ ID NO: 28 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: 22 and the -2,3-sialyltransferase from P. multocida with SEQ ID NO: 23. 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 40. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LN3, Sialylated LN3, LNnT, 6SL and LSTc with a Modified E. coli Host

[0714] An E. coli strain modified to produce sialic acid as described in Example 1 was further modified with a genomic knock-in of constitutive transcriptional units for LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29 to allow production of LNnT. In a next step, the novel strain was 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: 22 and the -2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 with SEQ ID NO: 26. The novel strain was 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 sucrose as carbon source and lactose as precursor. After 72 h of incubation, the culture broth was harvested, and the sugar mixtures were analyzed on UPLC. The experiment showed that the novel strain produced a mixture of oligosaccharides comprising LN3, 6-sialylated LN3 (Neu5Ac-a2,6-(GlcNAc-b1,3)-Gal-b1,4-Glc), 6SL, LNnT and LSTc.

Example 41. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LN3, Sialylated LN3, LNnT, 3SL and LSTd with a Modified E. coli Host

[0715] 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: 27 and for LgtB from N. meningitidis with SEQ ID NO: 29 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: 22 and the -2,3-sialyltransferase from P. multocida with SEQ ID NO: 23. 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 42. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LN3, Sialylated LN3, LNT, 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

[0716] The mutant E. coli strain able to produce LN3, LNT, 3SL and LSTa as described in Example 36 is selected for further evaluation in a fed-batch fermentation process in a 5 L 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. In contrast to the cultivation experiments that are described herein and wherein only end samples were taken at the end of cultivation (i.e., 72 hours as described herein), regular broth samples are taken at several time points during the fermentation process and 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 43. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LN3, Sialylated LN3, LNT, LSTa and 3SL in Fermentation Broth of Mutant E. coli Strains when Evaluated in a Fed-Batch Fermentation Process with Sucrose and Lactose

[0717] The mutant E. coli strain able to produce LN3, LNT, 3SL and LSTa as described in Example 39 is selected for further evaluation in a fed-batch fermentation process in a 5 L 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. In contrast to the cultivation experiments that are described herein and wherein only end samples were taken at the end of cultivation (i.e., 72 hours as described herein), regular broth samples are taken at several time points during the fermentation process and 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 44. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LN3, Sialylated LN3, LNnL Para-Lacto-N-Neohexaose, Di-Sialylated LNnL 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

[0718] 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: 26 as described in Example 37 were selected for further evaluation in a fed-batch fermentation process in a 5 L bioreactor. Fed-batch fermentations at bioreactor scale were performed as described in Example 1. In these examples, glycerol is 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 contains lactose, LN3 and LNnT, whereas fermentation broth of the selected strain taken after the fed-batch phase comprises 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 comprises para-lacto-N-neohexaose (pLNnH), sialylated para-lacto-N-neohexaose, and di-sialylated LNnT, i.e., structures that were not detected in growth experiment assays due to limited detection levels and overall smaller production levels.

Example 45. Production of an Oligosaccharide Mixture Comprising LN3, Sialylated LN3, LNnL Para-Lacto-N-Neohexaose, Di-Sialylated LNnL LSTc and 6SL in Fermentation Broth of Mutant E. coli Strains when Evaluated in a Fed-Batch Fermentation Process with Sucrose and Lactose

[0719] The mutant E. coli strain able to produce LN3, sialylated LN3, LNnT, 6SL and LSTc as described in Example 40 was selected for further evaluation in a fed-batch fermentation process in a 5 L bioreactor. Fed-batch fermentations at bioreactor scale were performed as described in Example 1. In these examples, sucrose was used as a carbon source and lactose was added in the batch medium as precursor. 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 contains lactose, LN3, 6SL, and LNnT, whereas fermentation broth of the selected strain taken after the fed-batch phase comprises an oligosaccharide mixture comprising LN3, 6-sialylated LN3 (Neu5Ac-a-2,6-(GlcNAc-b-1,3)-Gal-b-1,4-Glc), LNnT, LSTc and 6SL. At end of fed-batch, the mixture also comprises para-lacto-N-neohexaose, 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 46. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LNT, LNnT and Poly-Galactosylated Structures in a Modified E. coli Host when Evaluated in Fed-Batch Fermentations

[0720] An E. coli strain modified to produce LNnT as described in Example 1, is further modified with genomic knock-ins of constitutive transcriptional units for WbgO from E. coli 055:H7 with SEQ ID NO: 28. In a next step, the novel strain is evaluated in a fed-batch fermentation process in a 5 L bioreactor as described in Example 1. In this example sucrose is used as a carbon source and lactose is added in the batch medium as precursor. In contrast to the cultivation experiments that are described herein and wherein only end samples were taken at the end of cultivation (i.e., 72 hours as described herein), regular broth samples are taken at several time points during the fermentation process and evaluated for production of an oligosaccharide mixture 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 and beta-(1,4)Galactosyl-para-Lacto-N-pentaose.

Example 47. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Galactosylated and GalNAc-Ylated Lactose Structures with a Modified E. coli Host

[0721] An E. coli strain optimized for UDP-galactose as described in Example 1, is further modified with a knock-out of the E. coli lacZ gene and a knock-in of a constitutive expression unit for the a1,4-galactosyltransferase (LgtC) from Neisseria gonorrhoeae with SEQ ID NO: 36 to produce alpha-1,4-galactosylated lactose (Gal-a1,4-Gal-b1,4-Glc) when grown on glycerol and lactose. In a next step, the mutant strain is further modified with a knock-out of the E. coli nagB gene together with a knock-in of a constitutive expression unit for the mutant glmS*54 of E. coli with SEQ ID NO: 19, and transformed with an expression plasmid comprising constitutive expression units for the 4-epimerase (WbpP) of P. aeruginosa with SEQ ID NO: 34 and the 1,3-N-acetylgalactosaminyltransferase (LgtD) from H. influenzae with SEQ ID NO: 35. Besides its 1,3-N-Acetyl-galactosaminyl transferase activity the LgtD enzyme also has a 1,3-galactose transferase activity and can add a galactose to a non-reducing terminal GalNAc molecule resulting in a terminal Gal-b1,3-GalNAc at the non-reducing end of a glycan. The novel strain is evaluated for production of an oligosaccharide mixture comprising Gal-a1,4-Gal-b1,4-Glc (Gal-a1,4-lactose), Gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, Gal-a1,4-Gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, GalNAc-b1,3-Gal-b1,4-Glc (GalNAc-b1,3-Lactose), Gal-b1,3-GalNAc-b1,3-lactose, GalNAc-b1,3-Gal-a1,4-Gal-b1,4-Glc (globo-N-tetraose) and Gal-b1,3-GalNAc-b1,3-Gal-a1,4-Gal-b1,4-Glc 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 48. Production of an Oligosaccharide Mixture Comprising LN3, LNT, GalNAc-b1, 3-LNT, Gal-b1,3-GaNAc-b1,3-LNT, Ga/NAc-b1, 3-Lactose and Gal-b1,3-GalNAc-b1,3-Lactose with a Modified E. coli Host

[0722] An E. coli strain modified to produce LNT as described in Example 1, is further modified with knock-ins of constitutive expression units for the 4-epimerase (WbpP) from P. aeruginosa with SEQ ID NO: 34 and the 1,3-N-acetylgalactosaminyltransferase (LgtD) from H. influenzae with SEQ ID NO: 35. The novel strain is evaluated for production of an oligosaccharide mixture comprising LN3, LNT, GalNAc-b1,3-LNT, Gal-b1,3-GalNAc-b1,3-LNT, GalNAc-b1,3-lactose and Gal-b1,3-GalNAc-b1,3-lactose in whole broth samples in a growth experiment, according to the culture conditions in Example 1, in which the culture medium contains glycerol as carbon source and lactose as precursor.

Example 49. Production an Oligosaccharide Mixture Comprising LN3, LNnT, GalNAc-b1, 3-LNnT, Gal-b1,3-GalNAc-b1,3-LNnT, GalNAc-b1,3-Lactose, Gal-b1,3-GalNAc-b1,3-Lactose and (GalNAc-)Poly-LNnT Structures with a Modified E. coli Host

[0723] An E. coli strain modified to produce LNnT as described in Example 1, is further modified with knock-ins of constitutive expression units for WbpP from P. aeruginosa with SEQ ID NO: 34 and LgtD from H. influenzae with SEQ ID NO: 35. The novel strain is evaluated for production of an oligosaccharide mixture comprising LN3, LNnT, GalNAc-b1,3-LNnT, Gal-b1,3-GalNAc-b1,3-LNnT, GalNAc-b1,3-lactose and Gal-b1,3-GalNAc-b1,3-lactose as well as poly-LNnT structures (by alternate activity of NmlgtA and NmLgtB) and GalNAc-ylated poly-LNnT structures (by additional activity of HiLgtD) in whole broth samples in a growth experiment, according to the culture conditions in Example 1, in which the culture medium contains glycerol as carbon source and lactose as precursor.

Example 50. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, DiFL, LN3, LNT and LNFP-I with a Modified E. coli Host

[0724] An E. coli strain modified for GDP-fucose production as described in Example 1 was further adapted for LN3 and LNT production by genomic knock-ins of constitutive transcriptional units for the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis with SEQ ID NO: 27 and the N-acetylglucosamine beta-1,3-galactosyltransferase (WbgO) from E. coli 055:H7 with SEQ ID NO: 28. In a next step, the novel strain was further transformed with an expression plasmid containing a constitutive transcriptional unit for the a-1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 that accepts both LNT and lactose as acceptors for its fucosyltransferase activity. The novel strain produced an oligosaccharide mixture comprising 2FL, DiFL, LN3, LNT and lacto-N-fucopentaose I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples when evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contained sucrose as carbon source and lactose as precursor.

Example 51. Production of an Oligosaccharide Mixture Comprising LN3, LNT and LNFP-I with a Modified E. coli Host

[0725] An E. coli K-12 MG1655 strain modified for GDP-fucose production as described in Example 1 is further adapted for LN3 and LNT production by genomic knock-outs of the E. coli nagB and arabinose isomerase (araA) genes and genomic knock-ins of constitutive transcriptional units for galE from E. coli with SEQ ID NO: 30, the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis with SEQ ID NO: 27 and the N-acetylglucosamine beta-1,3-galactosyltransferase (WbdO) from Salmonella enterica subsp. salamae serovar Greenside with SEQ ID NO: 41. In a next step, the novel strain is further transformed with an expression plasmid containing a constitutive transcriptional unit for the a-1,2-fucosyltransferase (fucT54) from Sideroxydans lithotrophicus ES-1 with SEQ ID NO: 40. The novel strain is evaluated for production of an oligosaccharide mixture comprising LN3, LNT and lacto-N-fucopentaose I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples 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 52. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, DiFL, Ga/NAc-b1, 3-Lactose, LN3, LNT, Ga/NAc-b1, 3-LNT, LNFP-I and Ga/NAc-b1,3-LNFP-I with a Modified E. coli Host

[0726] An E. coli strain modified for GDP-fucose production and for LNFP-I production as described in Example 50 is further transformed with a compatible expression plasmid containing a constitutive transcriptional unit for the b1,3-N-acetylgalactosaminyltransferase (LgtD) from H. influenzae with SEQ ID NO: 35. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL, GalNAc-b1,3-Lactose (GalNAc-b1,3-Gal-b1,4-Glc), LN3, LNT, GalNAc-b1,3-LNT (GalNAc-b1,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), LNFP-I (Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and GalNAc-b1,3-LNFP-I (GalNAc-b1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples 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 53. Production of an Oligosaccharide Mixture Comprising 2FL, DiFL, LN3, LNT, LNFP-I and Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified E. coli Host

[0727] An E. coli strain modified for GDP-fucose production and for LNFP-I production as described in Example 50 is further transformed with a compatible expression plasmid containing a constitutive transcriptional unit for the alpha-1,3-galactosyltransferase WbnI from E. coli with SEQ ID NO: 37. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples 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 54. Production of an Oligosaccharide Mixture Comprising 2FL, DiFL, LN3, LNT, LNFP-I and GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a Modified E. coli Host

[0728] An E. coli strain modified for GDP-fucose production and for LNFP-I production as described in Example 50 is further transformed with a compatible expression plasmid containing a constitutive transcriptional unit for the alpha-1,3-N-acetylgalactosaminyltransferase BgtA from H. mustelae with SEQ ID NO: 38. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples 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 55. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LN3, LNT and LNFP-I with a Modified E. coli Host

[0729] An E. coli strain modified for GDP-fucose and LNFP-I production comprising expression of glmS*54 from E. coli with SEQ ID NO: 19, LgtA from N. meningitidis with SEQ ID NO: 27, WbgO from E. coli 055:H7 with SEQ ID NO: 28 and the a-1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 as described in Example 50, is further transformed with a compatible expression plasmid containing a constitutive transcriptional unit for the a-1,3-fucosyltransferase (HpFucT) from H. pylori with SEQ ID NO: 05. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LN3, LNT and LNFP-I (Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples 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 56. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LN3, LNT and LNFP-II with a Modified E. coli Host

[0730] An E. coli strain modified for GDP-fucose production as described in Example 1 is further adapted for LN3 and LNT production by a genomic knock-out of the E. coli nagB gene and genomic knock-ins of constitutive transcriptional units for LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 with SEQ ID NO: 28. In a next step, the novel strain is further transformed with an expression plasmid containing a constitutive transcriptional unit for a mutant a1,3/4 fucosidase from Bifidobacterium longum subsp. infantis ATCC 15697 with SEQ ID NO: 39. The novel strain is evaluated for production of an oligosaccharide mixture comprising LN3, LNT and lacto-N-fucopentaose II (LNFP-II, Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples 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 57. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, DiFL, LN3, LNT, LNFP-I and LNFP-II with a Modified E. coli Host

[0731] An E. coli strain modified for GDP-fucose production and for LNFP-II production as described in Example 56 is further transformed with n compatible expression plasmid containing a constitutive transcriptional unit for the a-1,2-fucosyltransferase (HpFutC) from H. pylori with SEQ ID NO: 04. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL, LN3, LNT, LNFP-I and lacto-N-fucopentaose II (LNFP-II, Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples 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 58. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3-FL, LN3, LNT and LNFP-II with a Modified E. coli Host

[0732] An E. coli strain modified for GDP-fucose production and for LNFP-II production as described in Example 56 is further transformed with a compatible expression plasmid containing a constitutive transcriptional unit for the a-1,3-fucosyltransferase (HpFucT) from H. pylori with SEQ ID NO: 05. The novel strain is evaluated for production of an oligosaccharide mixture comprising 3-FL, LN3, LNT and lacto-N-fucopentaose II (LNFP-II, Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples 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 59. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LN3, LNT, LNFP-I and LNFP-II with a Modified E. coli Host

[0733] An E. coli strain modified for GDP-fucose production and for LNFP-II production as described in Example 56 is further transformed with a compatible expression plasmid containing constitutive transcriptional units for the a-1,2-fucosyltransferase (HpFutC) from H. pylori with SEQ ID NO: 04 and for the a-1,3-fucosyltransferase (HpFucT) from H. pylori with SEQ ID NO: 05. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LN3, LNT, LNFP-I and lacto-N-fucopentaose II (LNFP-II, Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc) in whole broth samples 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 60. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3-FL, LN3, LNT and LNFP-V with a Modified E. coli Host

[0734] An E. coli strain modified for GDP-fucose production as described in Example 1 is further adapted for LN3 and LNT production by a genomic knock-out of the E. coli nagB gene and genomic knock-ins of constitutive transcriptional units for LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 with SEQ ID NO: 28. In a next step, the novel strain is further transformed with an expression plasmid containing a constitutive transcriptional unit for the truncated a1,3-fucosyltransferase from H. pylori with SEQ ID NO: 06. The novel strain is evaluated for production of an oligosaccharide mixture comprising 3-FL, LN3, LNT and lacto-N-fucopentaose V (LNFP-V, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuca1,3)-Glc) in whole broth samples 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 61. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3-FL, LN3, LNnT, LNFP-III and LNFP-VI with a Modified E. coli Host

[0735] An E. coli strain modified for GDP-fucose production as described in Example 1 is further adapted for LN3 and LNnT production by a genomic knock-out of the E. coli nagB gene and genomic knock-ins of constitutive transcriptional units for LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. In a next step, the novel strain is further transformed with an expression plasmid containing a constitutive transcriptional unit for the truncated a-1,3-fucosyltransferase from H. pylori with SEQ ID NO: 06. The novel strain is evaluated for production of an oligosaccharide mixture comprising 3-FL, LN3 and LNnT, lacto-N-fucopentaose III (LNFP-III, Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1,3-Gal-b1,4-Glc) and lacto-N-fucopentaose VI (LNFP-VI, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc) in whole broth samples 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 62. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0736] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with a genomic knock-out of the E. coli nagB gene and genomic knock-ins of constitutive expression cassettes for LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 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: 22. 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 a 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 SF46 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF47 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LN3, LNT, 3S-LN3, LSTa SF48 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LN3, 6S-LN3, LNT, LNFP-I SF49 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LN3, 6S-LN3, LNT SF50 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S- LN3, LNT, LNFP-I, LSTa SF51 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNT, LSTa SF52 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF53 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNT, LNFP-I SF54 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 63. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0737] An E. coli strain adapted for GDP-fucose production as described in Example 1 is further modified with a genomic knock-out of the E. coli nagB gene and genomic knock-ins of constitutive expression cassettes for LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. 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: 22. 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 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 a 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 SF55 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LN3, 3S-LN3, LNnT, LSTd SF56 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF57 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LN3, 6S-LN3, LNnT, LSTc SF58 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF59 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S- LN3, LNnT, LSTc, LSTd SF60 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd SF61 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF62 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF63 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 64. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0738] 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: 19, GNA1 from S. cerevisiae with SEQ ID NO: 16, AGE of B. ovatus with SEQ ID NO: 17, neuB of N. meningitidis with SEQ ID NO: 18, LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 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: 22. 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 a 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 SF64 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S- LN3, LNT, LNFP-I, LSTa SF65 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF66 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, 6S- LN3, LNT, LNFP-I SF67 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF68 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 SF69 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 SF70 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 SF71 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 SF72 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 65. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0739] 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: 19, GNA1 from S. cerevisiae with SEQ ID NO: 16, AGE of B. ovatus with SEQ ID NO: 17, neuB of N. meningitidis with SEQ ID NO: 18, LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. 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: 22. 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 a 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 SF73 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LSTd SF74 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S- LN3, LNnT, LNFP-III, LSTd SF75 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF76 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S- LN3, LNnT, LNFP-III, LSTc SF77 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 SF78 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 SF79 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 SF80 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 SF81 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 66. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0740] 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: 19, the UDP-N-acetylglucosamine 2-epimerase (neuC) from C. jejuni with SEQ ID NO: 21, neuB of N. meningitidis with SEQ ID NO: 18, LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 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: 22. 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 a 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 SF82 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF83 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LN3, 3S-LN3, LNT, LSTa SF84 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LN3, 6S-LN3, LNT, LNFP-I SF85 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LN3, 6S-LN3, LNT SF86 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S- LN3, LNT, LNFP-I, LSTa SF87 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNT, LSTa SF88 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF89 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNT, LNFP-I SF90 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 67. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0741] 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: 19, neuC from C. jejuni with SEQ ID NO: 21, neuB of N. meningitidis with SEQ ID NO: 18, LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. 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: 22. 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 a 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 SF91 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LN3, 3S-LN3, LNnT, LSTd SF92 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF93 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LN3, 6S-LN3, LNnT, LSTc SF94 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF95 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S- LN3, LNnT, LSTc, LSTd SF96 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd SF97 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF98 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF99 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 68. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0742] 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: 27 and WbgO from E. coli 055:H7 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: 22. Table 2 presents an overview of the six plasmids used. The novel strains are evaluated for production of an oligosacharide mixture comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylatedLN3, 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 a 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 SF100 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S- LN3, LNT, LNFP-I, LSTa SF101 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF102 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, LNT, 6S-LN3, LNFP-I SF103 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF104 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 SF105 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 SF106 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 SF107 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 SF108 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 69. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0743] 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 TD NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. 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: 22. 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 a 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 SF109 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LSTd SF110 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S- LN3, LNnT, LNFP-III, LSTd SF111 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF112 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S- LN3, LNnT, LNFP-III, LSTc SF113 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 SF114 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 SF115 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 SF116 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 SF117 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 70. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0744] 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: 13, the bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase (fkp) from B. fragilis with SEQ ID NO: 14, LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 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: 22. 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 a 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 SF118 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S- LN3, LNT, LNFP-I, LSTa SF119 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF120 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, 6S- LN3, LNT, LNFP-I SF121 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF122 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 SF123 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 SF124 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 SF125 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 SF126 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 71. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0745] 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: 13, fkp from B. fragilis with SEQ NO: ID 14, LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. 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: 22. 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 a 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 SF127 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LSTd SF128 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S- LN3, LNnT, LNFP-III, LSTd SF129 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF130 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S- LN3, LNnT, LNFP-III, LSTc SF131 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 SF132 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 SF133 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 SF134 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 SF135 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 72. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0746] 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: 30, the mutant glmS*54 from E. coli with SEQ TD NO: 19, GNA1 from S. cerevisiae with SEQ ID NO: 16, LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 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: 22. 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 a 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 SF136 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S- LN3, LNT, LNFP-I, LSTa SF137 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF138 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, 6S- LN3, LNT, LNFP-I SF139 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF140 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 SF141 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 SF142 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 SF143 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 SF144 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 73. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0747] 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: 30, the mutant glmS*54 from E. coli with SEQ ID NO: 19, GNA1 from S. cerevisiae with SEQ ID NO: 16, LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. 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: 22. 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 a 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 SF145 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3SLN, LNnT, LSTd SF146 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S- LN3, LNnT, LNFP-III, LSTd SF147 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF148 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S- LN3, LNnT, LNFP-III, LSTc SF149 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 SF150 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 SF151 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 SF152 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 SF153 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 74. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0748] 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: 30, the mutant glmS*54 from E. coli with SEQ ID NO: 19, GNA1 from S. cerevisiae with SEQ ID NO: 16, the N-acetylglucosamine 2-epimerase (AGE) of B. ovatus with SEQ ID NO: 17, neuB of N. meningitidis with SEQ ID NO: 18, LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 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: 22. 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 a 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 SF154 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF155 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF156 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, 6S-LN3, LNT, LNFP-I SF157 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF158 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 SF159 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 SF160 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 SF161 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 SF162 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 75. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0749] 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: 30, the mutant glmS*54 from E. coli with SEQ ID NO: 19, GNA1 from S. cerevisiae with SEQ ID NO: 16, AGE of B. ovatus with SEQ ID NO: 17, neuB of N. meningitidis with SEQ ID NO: 18, LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. 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: 22. 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 a 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 SF163 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LSTd SF164 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S- LN3, LNnT, LNFP-III, LSTd SF165 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF166 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S- LN3, LNnT, LNFP-III, LSTc SF167 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 SF168 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 SF169 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 SF170 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 SF171 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 76. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0750] 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: 30, the mutant glmS*54 from E. coli with SEQ ID NO: 19, neuC from C. jejuni with SEQ ID NO: 21, neuB of N. meningitidis with SEQ ID NO: 18, LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 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: 22. 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), 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 a 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 SF172 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF173 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LN3, 3S-LN3, LNT, LSTa SF174 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LN3, 6S-LN3, LNT, LNFP-I SF175 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LN3, 6S-LN3, LNT SF176 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S- LN3, LNT, LNFP-I, LSTa SF177 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNT, LSTa SF178 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF179 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNT, LNFP-I SF180 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 77. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0751] 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: 30, the mutant glmS*54 from E. coli with SEQ ID NO: 19, neuC from C. jejuni with SEQ ID NO: 21, neuB of N. meningitidis with SEQ ID NO: 18, LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. 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: 22. 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 a 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 Oligosaccharide mixture comprising SF181 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LN3, 3S-LN3, LNnT, LSTd SF182 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF183 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LN3, 6S-LN3, LNnT, LSTc SF184 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF185 pMF_1A, pMS_2 2FL, 3SL, DiFL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S- LN3, LNnT, LSTc, LSTd SF186 pMF_1B, pMS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd SF187 pMF_2, pMS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd SF188 pMF_2, pMS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc SF189 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 78. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. Coli Host

[0752] 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: 30, LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 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: 22. 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 a 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 SF190 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S-LN3, LNT, LNFP-I, LSTa SF191 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF192 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, 6S-LN3, LNT, LNFP-I SF193 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF194 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 SF195 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 SF196 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 SF197 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 SF198 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 79. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0753] 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: 30, LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. 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: 22. 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 a 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 SF199 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LSTd SF200 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S- LN3, LNnT, LNFP-III, LSTd SF201 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF202 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S- LN3, LNnT, LNFP-III, LSTc SF203 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 SF204 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 SF205 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 SF206 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 SF207 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 80. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0754] 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: 30, fucP from E. coli with SEQ ID NO: 13, fkp from B. fragilis with SEQ ID NO: 14, LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 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: 22. 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 a 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 SF208 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LNB, 2FLNB, 3SLNB, LN3, 3S- LN3, LNT, LNFP-I, LSTa SF209 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LNB, 4-FLNB, 3SLNB, LN3, 3S-LN3, LNT, LSTa SF210 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LNB, 2FLNB, 6SLNB, LN3, 6S- LN3, LNT, LNFP-I SF211 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LNB, 4-FLNB, 6SLNB, LN3, 6S-LN3, LNT SF212 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 SF213 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 SF214 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 SF215 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 SF216 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 81. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified E. coli Host

[0755] 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: 30, fucP from E. coli with SEQ ID NO: 13, fkp from B. fragilis with SEQ NO: ID 14, LgtA from N. meningitidis with SEQ ID NO: 27 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: 22. 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 a 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 SF217 pMF_1A, pMS_1A 2FL, 3SL, 3S-2FL, DiFL, LacNAc, 2FLacNAc, 3SLacNAc, LN3, 3S-LN3, LNnT, LSTd SF218 pMF_1B, pMS_1A 3-FL, 3SL, 3S-3-FL, LacNAc, 3FlacNAc, 3SLacNAc, LN3, 3S- LN3, LNnT, LNFP-III, LSTd SF219 pMF_1A, pMS_1B 2FL, 6SL, 6S-2FL, DiFL, LacNAc, 2FLacNAc, 6SLacNAc, LN3, 6S-LN3, LNnT, LSTc SF220 pMF_1B, pMS_1B 3-FL, 6SL, 6S-3-FL, LacNAc, 3FlacNAc, 6SlacNAc, LN3, 6S- LN3, LNnT, LNFP-III, LSTc SF221 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 SF222 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 SF223 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 SF224 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 SF225 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 82. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL and DiFL with a Modified S. cerevisiae Host

[0756] An S. cerevisiae strain is adapted for GDP-fucose production and fucosyltransferase expression as described in Example 2 with a yeast expression plasmid (a variant of p2a_2_Fuc) comprising constitutive transcriptional units for the lactose permease (LAC12) from K. lactis with SEQ ID NO: 31, the GDP-mannose 4,6-dehydratase (gmd) from E. coli with SEQ ID NO: 11, the GDP-L-fucose synthase (fcl) from E. coli with SEQ ID NO: 12, the alpha-1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and the alpha-1,3-fucosyltransferase from H. pylori with SEQ ID NO: 05. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL and DiFL in a growth experiment according to the culture conditions in Example 2 using SD CSM-Ura drop-out medium comprising lactose as precursor.

Example 83. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Lactose Structures with a Modified S. cerevisiae Host

[0757] An S. cerevisiae strain is adapted for production of GDP-fucose and CMP-sialic acid and for expression of one or more fucosyltransferases and one or more 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: 31, gmd from E. coli with SEQ ID NO: 11, fcl from E. coli with SEQ ID NO: 12 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: 19, the phosphatase yqaB from E. coli with SEQ ID NO: 20, AGE from B. ovatus with SEQ ID NO: 17, neuB from N. meningitidis with SEQ ID NO: 18, neuA from P. multocida with SEQ ID NO: 22 and one or two selected sialyltransferase(s). Table 30 shows the fucosyltransferases and sialyltransferases selected in the plasmids cloned 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 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: 23 None pYS_1B None SEQ ID NO: 25 pYS_2 SEQ ID NO: 23 SEQ ID NO: 25

TABLE-US-00031 TABLE 31 Oligosaccharide production evaluated by mutant S. cerevisiae strains expressing selected fucosyltransferase and sialyltransferase genes and cultivated in SD CSM-Ura-Trp drop-out medium comprising lactose as precursor. Strain Plasmids* present Oligosaccharides SY1 p YF_1A, p YS_1A 2FL, 3SL, 3S-2FL SY2 p YF_1B, p YS_1A 3-FL, 3SL, 3S-3-FL SY3 p YF_1A, p YS_1B 2FL, 6SL, 6S-2FL SY4 p YF_1B, p YS_1B 3-FL, 6SL, 6S-3-FL SY5 p YF_1A, p YS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SY6 p YF_1B, p YS_2 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SY7 p YF_2, p YS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SY8 p YF_2, p YS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SY9 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 84. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LN3, 3-Sialylated LN3, LNT, LNB, 3SL and LSTa with a Modified S. cerevisiae Host

[0758] 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: 31, the mutant glmS*54 from E. coli with SEQ ID NO: 19, the phosphatase yqaB from E. coli with SEQ ID NO: 20, AGE from B. ovatus with SEQ ID NO: 17, NeuB from N. meningitidis with SEQ ID NO: 18, NeuA from P. multocida with SEQ ID NO: 22, and the beta-galactoside alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 23 and a second yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli with SEQ ID NO: 30, LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 with SEQ ID NO: 28. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising LN3, 3-sialylated LN3, LNT, 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 85. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LN3, 6-Sialylated LN3, LNnT, LacNAc, 6SL and LSTc with a Modified S. cerevisiae Host

[0759] 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: 31, the mutant glmS*54 from E. coli with SEQ ID NO: 19, the phosphatase yqaB from E. coli with SEQ ID NO: 20, AGE from B. ovatus with SEQ ID NO: 17, NeuB from N. meningitidis with SEQ ID NO: 18, NeuA from P. multocida with SEQ ID NO: 22, and the beta-galactoside alpha-2,6-sialyltransferase from P. damselae with SEQ ID NO: 25 and a second yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli with SEQ ID NO: 30, LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising LN3, 6-sialylated LN3, LNnT, 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 86. Production of a Mammalian Milk Oligosaccharide Mixture Comprising LN3, 3-Sialylated LN3, LNnT, LacNAc, 3SL and LSTd with a Modified S. cerevisiae Host

[0760] 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: 31, the mutant glmS*54 from E. coli with SEQ ID NO: 19, the phosphatase yqaB from E. coli with SEQ ID NO: 20, AGE from B. ovatus with SEQ ID NO: 17, NeuB from N. meningitidis with SEQ ID NO: 18, NeuA from P. multocida with SEQ ID NO: 22, and the beta-galactoside alpha-2,3-sialyltransferase from P. multocida with SEQ ID NO: 23 and a second yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli with SEQ ID NO: 30, LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising LN3, 3-sialylated LN3, LNnT, 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 87. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, DiFL, LN3, LNT and LNFP-I with a Modified S. cerevisiae Host

[0761] An S. cerevisiae strain is adapted for production of GDP-fucose and LNT and for expression of an a-1,2-fucosyltransferase as described in Example 2 with a first yeast expression plasmid comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 31, gmd from E. coli with SEQ ID NO: 11, fcl from E. coli with SEQ ID NO: 12 and the a-1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and with a second yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli with SEQ ID NO: 30, LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 with SEQ ID NO: 28. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL, LN3, LNT and lacto-N-fucopentaose I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experiment according to the culture conditions in Example 2 using SD CSM-Ura-His drop-out medium comprising lactose as precursor.

Example 88. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, DiFL, LN3, LNT and LNFP-H with a Modified S. cerevisiae Host

[0762] An S. cerevisiae strain is adapted for production of GDP-fucose and LNT and for expression of an a1,3/4-fucosidase and an a-1,2-fucosyltransferase as described in Example 2 with a first yeast expression plasmid comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 31, gmd from E. coli with SEQ ID NO: 11, fcl from E. coli with SEQ ID NO: 12, a mutant a1,3/4-fucosidase from B. longum subsp. infantis ATCC 15697 with SEQ ID NO: 39 and the a-1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and with a second yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli with SEQ ID NO: 30, LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H.sub.7 with SEQ ID NO: 28. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL, LN3, LNT and lacto-N-fucopentaose II (LNFP-II, Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experiment according to the culture conditions in Example 2 using SD CSM-Ura-His drop-out medium comprising lactose as precursor.

Example 89. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3-FL, LN3, LNT and LNFP-V with a Modified S. cerevisiae Host

[0763] An S. cerevisiae strain is adapted for production of GDP-fucose and LNT and for expression of an a-1,3-fucosyltransferase as described in Example 2 with a first yeast expression plasmid comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 31, gmd from E. coli with SEQ ID NO: 11, fcl from E. coli with SEQ ID NO: 12 and the truncated a1,3-fucosyltransferase from H. pylori with SEQ ID NO: 06 and with a second yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli with SEQ ID NO: 30, LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 with SEQ ID NO: 28. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising 3-FL, LN3, LNT and lacto-N-fucopentaose V (LNFP-V, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuca 1,3)-Glc) in a growth experiment according to the culture conditions in Example 2 using SD CSM-Ura-His drop-out medium comprising lactose as precursor.

Example 90. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3-FL, LN3, LNnT and LNFP-III with a Modified S. cerevisiae Host

[0764] An S. cerevisiae strain is adapted for production of GDP-fucose and LNnT and for expression of an a-1,3-fucosyltransferase in Example 2 with a first yeast expression plasmid comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 31, gmd from E. coli with SEQ ID NO: 11, fcl from E. coli with SEQ ID NO: 12 and the truncated a1,3-fucosyltransferase from H. pylori with SEQ ID NO: 06 and with a second yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli with SEQ ID NO: 30, LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising 3-FL, LN3, LNnT and lacto-N-fucopentaose III (LNFP-III, Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experiment according to the culture conditions in Example 2 using SD CSM-Ura-His drop-out medium comprising lactose as precursor.

Example 91. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified S. cerevisiae Host

[0765] 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: 31, gmd from E. coli with SEQ ID NO: 11, fcl from E. coli with SEQ ID NO: 12 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: 19, the phosphatase yqaB from E. coli with SEQ ID NO: 20, AGE from B. ovatus with SEQ ID NO: 17, neuB from N. meningitidis with SEQ ID NO: 18, neuA from P. multocida with SEQ ID NO: 22 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: 30, LgtA from N. meningitidis with SEQ ID NO: 27 and WbgO from E. coli 055:H7 with SEQ ID NO: 28. The mutant yeast strains 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 a mixture comprising tri-, tetra- and penta- oligosaccharides in whole broth of mutant S. cerevisiae strains expressing selected fucosyltransferase and sialyltransferase genes and cultivated in SD CSM-Ura- Trp-His drop-out medium comprising lactose as precursor. Strain Plasmids* present Oligosaccharides SY10 pYF_1C, pYS_1A 2FL, DiFL, 3SL, 3S-2FL, LN3, 3S-LN3, LNT, LNFP-I, LSTa, 3SLNB SY11 pYF_1D, pYS_1A 3-FL, 3SL, 3S-3-FL, LN3, 3S-LN3, LNT, LSTa, 3SLNB SY12 pYF_1C, pYS_1B 2FL, DiFL, 6SL, 6S-2FL, LN3, 6S-LN3, LNT, LNFP-I, 6SLNB SY13 pYF_1D, pYS_1B 3-FL, 6SL, 6S-3-FL, LN3, LNT, 6S-LN3, 6SLNB SY14 pYF_1C, pYS_2 2FL, DiFL, 3SL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S- LN3, LNT, LNFP-I, LSTa, 3SLNB, 6SLNB SY15 pYF_1D, pYS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S-LN3, LNT, LSTa, 3SLNB, 6SLNB SY16 pYF_2CD, pYS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNT, LNFP-I, LSTa, 3SLNB SY17 pYF_2CD, pYS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNT, LNFP-I, 6SLNB SY18 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, 3SLNB, 6SLNB *See Table 30 for plasmid info

Example 92. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified S. cerevisiae Host

[0766] 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: 31, gmd from E. coli with SEQ ID NO: 11, fcl from E. coli with SEQ ID NO: 12 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: 19, the phosphatase yqaB from E. coli with SEQ ID NO: 20, AGE from B. ovatus with SEQ ID NO: 17, neuB from N. meningitidis with SEQ ID NO: 18, neuA from P. multocida with SEQ ID NO: 22 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: 30, LgtA from N. meningitidis with SEQ ID NO: 27 and LgtB from N. meningitidis with SEQ ID NO: 29. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising fucosylated and sialylated lactose, LacNAc, sialylated 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 a mixture comprising tri-, tetra- and penta- oligosaccharides in whole broth of mutant S. cerevisiae strains expressing selected fucosyltransferase and sialyltransferase genes and cultivated in SD CSM-Ura- Trp-His drop-out medium comprising lactose as precursor. Strain Plasmids* present Oligosaccharides SY19 pYF_1C, pYS_1A 2FL, DiFL, 3SL, 3S-2FL, LN3, 3S-LN3, LNnT, LSTd, 3SLacNAc SY20 pYF_1D, pYS_1A 3-FL, 3SL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd, 3SLacNAc SY21 pYF_1C, pYS_1B 2FL, DiFL, 6SL, 6S-2FL, LN3, 6S-LN3, LNnT, LSTc, 6SLacNAc SY22 pYF_1D, pYS_1B 3-FL, 6SL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc, 6SLacNAc SY23 pYF_1C, pYS_2 2FL, DiFL, 3SL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S- LN3, LNnT, LSTc, LSTd, 3SLacNAc, 6SLacNAc SY24 pYF_1D, pYS_2 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL, LN3, 3S-LN3, 6S-LN3, LNnT, LNFP-III, LSTc, LSTd, 3SLacNAc, 6SLacNAc SY25 pYF_2CD, pYS_1A 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL, LN3, 3S-LN3, LNnT, LNFP-III, LSTd, 3SLacNAc SY26 pYF_2CD, pYS_1B 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL, LN3, 6S-LN3, LNnT, LNFP-III, LSTc, 6SLacNAc SY27 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, 3SLacNAc, 6SLacNAc *See Table 30 for plasmid info

Example 93. Production of a Mammalian Milk Oligosaccharide Mixture Comprising Fucosylated and Sialylated Oligosaccharide Structures with a Modified S. cerevisiae Host

[0767] An S. cerevisiae strain is adapted for production of GDP-fucose and CMP-sialic acid and for expression of one or more fucosyltransferases and one or more sialyltransferases with a yeast artificial chromosome (YAC) comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 31, gmd from E. coli with SEQ ID NO: 11, fcl from E. coli with SEQ ID NO: 12, the mutant glmS*54 from E. coli with SEQ ID NO: 19, the phosphatase yqaB from E. coli with SEQ ID NO: 20, AGE from B. ovatus with SEQ ID NO: 17, neuB from N. meningitidis with SEQ ID NO: 18, neuA from P. multocida with SEQ ID NO: 22, one or two selected fucosyltransferase(s) and one or two selected sialyltransferases(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 None SEQ ID None NO: 04 NO: 23 YAC2 None SEQ ID SEQ ID None NO: 05 NO: 23 YAC3 SEQ ID None None SEQ ID NO: 04 NO: 25 YAC4 None SEQ ID None SEQ ID NO: 05 NO: 25 YAC5 SEQ ID None SEQ ID SEQ ID NO: 04 NO: 23 NO: 25 YAC6 None SEQ ID SEQ ID SEQ ID NO: 05 NO: 23 NO: 25 YAC7 SEQ ID SEQ ID SEQ ID None NO: 04 NO: 05 NO: 23 YAC8 SEQ ID SEQ ID None SEQ ID NO: 04 NO: 05 NO: 25 YAC9 SEQ ID SEQ ID SEQ ID SEQ ID NO: 04 NO: 05 NO: 23 NO: 25

TABLE-US-00035 TABLE 35 Evaluation of oligosaccharide production in whole broth of mutant S. cerevisiae strains expressing selected fucosyltransferase and sialyltransferase genes from yeast artificial chromosomes and cultivated in SD CSM medium comprising lactose as precursor. YACs* Strain present Oligosaccharides SY28 YAC1 2FL, 3SL, 3S-2FL SY29 YAC2 3-FL, 3SL, 3S-3-FL SY30 YAC3 2FL, 6SL, 6S-2FL SY31 YAC4 3-FL, 6SL, 6S-3-FL SY32 YAC5 2FL, 3SL, 3S-2FL, 6SL, 6S-2FL SY33 YAC6 3-FL, 3SL, 3S-3-FL, 6SL, 6S-3-FL SY34 YAC7 2FL, 3-FL, DiFL, 3SL, 3S-2FL, 3S-3-FL SY35 YAC8 2FL, 3-FL, DiFL, 6SL, 6S-2FL, 6S-3-FL SY36 YAC9 2FL, 3-FL, DiFL, 3SL, 6SL, 3S-2FL, 3S-3-FL, 6S- 2FL, 6S-3-FL *See Table 34 for the YAC overview

Example 94. Material and Methods Bacillus subtilis

Media

[0768] 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.

[0769] Trace element mix consisted of 0.735 g/L CaCl.sub.2).Math.2H.sub.2O, 0.1 g/L MnCl.sub.2.2H.sub.2O, 0.033 g/L CuCl.sub.2.Math.2H.sub.2O, 0.06 g/L COCl.sub.2.Math.6H.sub.2O, 0.17 g/L ZnCl.sub.2, 0.0311 g/L H.sub.3BO.sub.4, 0.4 g/L Na.sub.2EDTA.Math.2H.sub.2O and 0.06 g/L Na.sub.2MoO.sub.4. The Fe-citrate solution contained 0.135 g/L FeCl.sub.3.Math.6H.sub.2O, 1 g/L Na-citrate (Hoch 1973 PMC1212887).

[0770] The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). Luria Broth agar (LBA) plates consisted of the LB media, with 12 g/L agar (Difco, Erembodegem, Belgium) added.

[0771] The minimal medium for the shake flasks (MMsf) experiments contained 2.00 g/L (NH.sub.4).sub.2SO.sub.4, 7.5 g/L KH.sub.2PO.sub.4, 17.5 g/L K2HPO.sub.4, 1.25 g/L Na-citrate, 0.25 g/L MgSO.sub.4.Math.7H.sub.2O, 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.

[0772] 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

[0773] Bacillus subtilis 168, available at Bacillus Genetic Stock Center (Ohio, USA).

[0774] 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.

[0775] 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.

[0776] 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: 15). 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: 27) 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: 28). 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: 29).

[0777] 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: 16), an N-acetylglucosamine-2-epimerase like, e.g., from B. ovatus (SEQ ID NO: 17) and an N-acetylneuraminate synthase like, e.g., from N. meningitidis (SEQ ID NO: 18) 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: 22), and one or more copies of a beta-galactoside alpha-2,3-sialyltransferase like, e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity (SEQ ID NO: 23), or NmeniST3 from N. meningitidis (SEQ ID NO: 24) or PmultST2 from P. multocida subsp. multocida str. Pm70 (Genbank No. AAK02592.1), a beta-galactoside alpha-2,6-sialyltransferase like, e.g., PdST6 from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity (SEQ ID NO: 25) or P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 having beta-galactoside alpha-2,6-sialyltransferase activity (SEQ ID NO: 26), and/or an alpha-2,8-sialyltransferase like, e.g., from M. musculus (UniProt ID Q64689).

Heterologous and Homologous Expression

[0778] 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.

[0779] 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

[0780] 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).

[0781] 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 95. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL and DiFL with a Modified B. subtilis Host

[0782] AB. subtilis strain is modified as described in Example 94 by genomic knock-ins of constitutive transcriptional units for the lactose permease (LacY) from E. coli with SEQ ID NO: 15 and 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 and DiFL in a growth experiment on MMsf medium comprising lactose according to the culture conditions provided in Example 94. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 96. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, DiFL, LNFP-I, LNT and LN3 with a Modified B. subtilis Host

[0783] A B. subtilis strain is first modified for LN3 production and growth on sucrose by genomic knock-out of the nagB, glmS and gamA genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding the lactose permease (LacY) from E. coli (SEQ ID NO: 15), the native fructose-6-P-aminotransferase (UniProt ID P0CI73), the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (SEQ ID NO: 27), 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: 28) to produce LNT. In a subsequent step, the LNT producing strain is transformed with an expression plasmid comprising a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (SEQ ID NO: 04). The novel strain is evaluated for the production of an oligosaccharide mixture comprising 2FL, DiFL, LN3, LNT and LNFP-I in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 94. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 97. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3SL, LN3, LNT, Sialylated LN3, LSTa with a Modified B. subtilis Host

[0784] The mutant B. subtilis strain producing LNT as described in Example 96 is further modified with a genomic knock-out of the nagA gene and a second compatible expression plasmid comprising the TRP1 selection marker and constitutive transcriptional units for two copies of the mutant L-glutamine D-fructose-6-phosphate aminotransferase (glmS*54) from E. coli (SEQ ID NO: 19), 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, ScDOG1 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: 17), the N-acetylneuraminate synthase (NeuB) from N. meningitidis (SEQ ID NO: 18), the N-acylneuraminate cytidylyltransferase NeuA from P. multocida (SEQ ID NO: 22) and three copies of a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity like SEQ ID NO: 23. 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 94. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 98. Production of an Oligosaccharide Mixture Comprising 2FL, DiFL, LNFP-I, LNFP-II, LNT and LN3 with a Modified B. subtilis Host

[0785] A B. subtilis strain is first modified for LN3 production and growth on sucrose by genomic knock-out of the nagB, glmS and gamA genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding the lactose permease (LacY) from E. coli (SEQ ID NO: 15), the native fructose-6-P-aminotransferase (UniProt ID POCI73), the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (SEQ ID NO: 27), 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: 28) to produce LNT. In a subsequent step, the LNT producing strain is transformed with an expression plasmid comprising constitutive transcriptional units for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (SEQ ID NO: 04) and a mutant a1,3/4 fucosidase from B. longum subsp. infantis ATCC 15697 with SEQ ID NO: 39. The novel strain is evaluated for the production of an oligosaccharide mixture comprising LN3, LNT, LNFP-I, LNFP-II, 2FL and DiFL in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 94. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 99. 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

[0786] A B. subtilis strain is modified 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: 15, 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: 16), the mutant L-glutamine-D-fructose-6-phosphate aminotransferase (glmS*54) from E. coli (SEQ ID NO: 19), 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, ScDOG1 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: 17), the N-acetylneuraminate synthase (NeuB) from N. meningitidis (SEQ ID NO: 18) and the N-acylneuraminate cytidylyltransferase NeuA from P. multocida (SEQ ID NO: 22). In a next step, the strain is transformed with an expression plasmid comprising constitutive transcriptional units for three copies of a PmultST3-like polypeptide consisting of 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 consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity (SEQ ID NO: 25). 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 94. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 100. Production of an Oligosaccharide Mixture Comprising LN3, LNnT, GalNAc-b1,3-LNnT, Gal-b1,3-GalNAc-b1,3-LNnT, GalNAc-b1,3-Lactose, Gal-b1,3-GalNAc-b1,3-Lactose and (GalNAc)-Poly-LNnT with a Modified B. subtilis Strain

[0787] A B. subtilis strain is first modified by genomic knock-out of the lacZ, nagB, gamA, glk, galE, galT, galK and galM genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding the lactose permease (LacY) from E. coli (SEQ ID NO: 15), the native fructose-6-P-aminotransferase (UniProt ID P0CI73), 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). The thus obtained mutant strain is further modified with genomic knock-ins of constitutive transcriptional units comprising lgtB from N. meningitidis (SEQ ID NO: 29), galE from E. coli (SEQ ID NO: 30), lgtA from N. meningitidis (SEQ ID NO: 27), the mutant glmS*54 from E. coli (SEQ ID NO: 19), WbpP from P. aeruginosa (SEQ ID NO: 34) and LgtD from H. influenzae (SEQ ID NO: 35). The novel strain is evaluated for production of an oligosaccharide mixture comprising LN3, LNnT, GalNAc-b1,3-LNnT, Gal-b1,3-GalNAc-b1,3-LNnT, GalNAc-b1,3-lactose and Gal-b1,3-GalNAc-b1,3-lactose as well as poly-LNnT structures and GalNAc-ylated poly-LNnT structures in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 94.

Example 101. Production of an Oligosaccharide Mixture Comprising LacNAc, GalNAc-b1,3-Lactose, Gal-b1,3-GalNAc-b1,3-Lactose, LN3, LNnT, GalNAc-b1,3-LacNAc, Gal-b1,3-GalNAc-b1,3-LacNAc, Poly-LacNAc (Gal-b1,4-GlcNAc)n and GalNAc-Ylated Poly-LacNAc Structures with a Modified B. subtilis Strain

[0788] A B. subtilis strain is first modified by genomic knock-out of the lacZ, nagB, gamA, glk, galE, galT, galK and galM genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding the lactose permease (LacY) from E. coli (SEQ ID NO: 15), the native fructose-6-P-aminotransferase (UniProt ID P0CI73), the mutant glmS*54 from E. coli (SEQ ID NO: 19), GNA1 from S. cerevisiae (SEQ ID NO: 16), the phosphatase yqaB from E. coli (SEQ ID NO: 20), the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (SEQ ID NO: 27), LgtB from N. meningitidis (SEQ ID NO: 29), the 4-epimerase (WbpP) of Pseudomonas aeruginosa (SEQ ID NO: 34) and the 1,3-N-acetylgalactosaminyltransferase (LgtD) from Haemophilus influenzae (SEQ ID NO: 35). The novel strain is evaluated for production of an oligosaccharide mixture comprising LacNAc, LN3, LNnT, GalNAc-b1,3-lactose, Gal-b1,3-GalNAc-b1,3-lactose, GalNAc-b1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, GalNAc-b1,3-LacNAc, Gal-b1,3-GalNAc-b1,3-LacNAc, poly-LacNAc structures, i.e., (Gal-b1,4-GlcNAc)n and GalNAc-ylated poly-LacNAc structures, in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 94.

Example 102. Material and Methods Corynebacterium glutamicum

Media

[0789] 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.

[0790] Trace element mix consisted of 10 g/L CaCl.sub.2), 10 g/L FeSO.sub.4.Math.7H.sub.2O, 10 g/L MnSO.sub.4.Math.H.sub.2O, 1 g/L ZnSO.sub.4.Math.7H.sub.2O, 0.2 g/L CuSO4, 0.02 g/L NiCl.sub.2.Math.6H.sub.2O, 0.2 g/L biotin (pH 7.0) and 0.03 g/L protocatechuic acid.

[0791] The minimal medium for the shake flasks (MMsf) experiments contained 20 g/L (NH.sub.4).sub.2SO.sub.4, 5 g/L urea, 1 g/L KH.sub.2PO.sub.4, 1 g/L K.sub.2HPO.sub.4, 0.25 g/L MgSO.sub.4.Math.7H.sub.2O, 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.

[0792] The TY medium consisted of 1.6% tryptone (Difco, Erembodegem, Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). TY agar (TYA) plates consisted of the TY media, with 12 g/L agar (Difco, Erembodegem, Belgium) added.

[0793] 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

[0794] Corynebacterium glutamicum ATCC 13032, available at the American Type Culture Collection.

[0795] 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.

[0796] 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: 15). 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.

[0797] 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: 27) 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: 28). 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: 29).

[0798] 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: 16), an N-acetylglucosamine-2-epimerase like, e.g., from B. ovatus (SEQ ID NO: 17) and an N-acetylneuraminate synthase like, e.g., from N. meningitidis (SEQ ID NO: 18) 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: 22), and one or more copies of a beta-galactoside alpha-2,3-sialyltransferase like, e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity (SEQ ID NO: 23), or NmeniST3 from N. meningitidis (SEQ ID NO: 24) or PmultST2 from P. multocida subsp. multocida str. Pm70 (Genbank No. AAK02592.1), a beta-galactoside alpha-2,6-sialyltransferase like, e.g., PdST6 from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2,6-sialyltransferase activity (SEQ ID NO: 25) or P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 having beta-galactoside alpha-2,6-sialyltransferase activity (SEQ ID NO: 26), and/or an alpha-2,8-sialyltransferase like, e.g., from M. musculus (UniProt ID Q64689).

Heterologous and Homologous Expression

[0799] 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.

[0800] 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

[0801] 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).

[0802] 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 103. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, 3SL and 6SL with a Modified C. glutamicum Host

[0803] A C. glutamicum strain is modified as described in Example 102 by genomic knockouts of the C. glutamicum genes ldh, cgl2645, nagB, gamA and nagA, together with genomic knock-ins of constitutive transcriptional units for the lactose permease (LacY) from E. coli with SEQ ID NO: 15, the sucrose permease CscB from E. coli W with SEQ ID NO: 01, the fructose kinase Frk originating from Zymomonas mobilis with SEQ ID NO: 02, the sucrose phosphorylase BaSP originating from Bifidobacterium adolescentis with SEQ ID NO: 03, the native fructose-6-P-aminotransferase (UniProt ID Q8NND3), the glucosamine-6-P-aminotransferase from S. cerevisiae (SEQ ID NO: 16), the N-acetylglucosamine-2-epimerase from B. ovatus (SEQ ID NO: 17), and the N-acetylneuraminate synthase from N. meningitidis (SEQ ID NO: 18). 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: 22), the beta-galactoside alpha-2,3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3), the beta-galactoside alpha-2,6-sialyltransferase PdST6 from P. damselae (UniProt ID 066375), the alpha-1,2-fucosyltransferase HpFutC from H. pylori (SEQ ID NO: 04) and the alpha-1,3-fucosyltransferase HpFucT from H. pylori (SEQ ID NO: 05). The novel strain is evaluated for the production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 3SL and 6SL in a growth experiment on MMsf medium comprising lactose according to the culture conditions provided in Example 102. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 104. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 3-FL, LNFP-III, LNnT and LN3 with a Modified C. glutamicum Host

[0804] A C. glutamicum strain is modified as described in Example 102 for LN3 production and growth on sucrose by genomic knock-out of the nagB, glmS and gamA genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding the lactose permease (LacY) from E. coli (SEQ ID NO: 15), the native fructose-6-P-aminotransferase (UniProt ID Q8NND3), the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (SEQ ID NO: 27), 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: 29) to produce LNnT. In a subsequent step, the LNnT producing strain is transformed with an expression plasmid comprising a constitutive transcriptional unit for the alpha-1,3-fucosyltransferase HpFucT from H. pylori (SEQ ID NO: 05). The novel strain is evaluated for the production of an oligosaccharide mixture comprising 3-FL, LN3, LNnT and LNFP-III in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 102. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 105. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LN3, LNnT, LNFP-III and Lacto-N-Neohexaose with a Modified C. glutamicum Host

[0805] A C. glutamicum strain is modified for LN3 production and growth on sucrose by genomic knock-out of the nagB, glmS and gamA genes and genomic knock-ins of constitutive transcriptional units comprising genes encoding the lactose permease (LacY) from E. coli (SEQ ID NO: 15), the native fructose-6-P-aminotransferase (UniProt ID Q8NND3), the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (SEQ ID NO: 27), 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: 29) to produce LNnT. In a subsequent step, the LNnT producing strain is transformed with an expression plasmid comprising a constitutive transcriptional unit for the alpha-1,2-fucosyltransferase HpFutC from H. pylori (SEQ ID NO: 04) and the alpha-1,3-fucosyltransferase HpFucT from H. pylori (SEQ ID NO: 05). The novel strain is evaluated for the production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LN3, LNnT, LNFP-III and lacto-N-neohexaose in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 102. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 106. Production of an Oligosaccharide Mixture Comprising LN3, Sialylated LN3, 6SL, LNnT and LSTc with a Modified C. glutamicum Host

[0806] A C. glutamicum strain is modified as described in Example 102 for LN3 production and growth on sucrose by genomic knock-out of the ldh, 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: 15), the native fructose-6-P-aminotransferase (UniProt ID Q8NND3), the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (SEQ ID NO: 27), 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: 29) 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), GNA1 from S. cerevisiae (SEQ ID NO: 16), AGE from B. ovatus (SEQ ID NO: 17), and the N-acetylneuraminate synthase from N. meningitidis (SEQ ID NO: 18) 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: 22) 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 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 102. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 107. Production of an Oligosaccharide Mixture Comprising 3SL, 6SL, LNB, 3-Sialylated LNB and 6-Sialylated LNB with a Modified C. glutamicum Host

[0807] A C. glutamicum strain is modified as described in Example 102 by genomic knock-out of the ldh, 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: 15), WbgO with SEQ ID NO: 28 from E. coli 055:H7, galE with SEQ ID NO: 30 from E. coli, the native fructose-6-P-aminotransferase (UniProt ID Q8NND3), glmS*54 with SEQ ID NO: 19, the glucosamine-6-P-aminotransferase from S. cerevisiae (SEQ ID NO: 16), the N-acetylglucosamine-2-epimerase from B. ovatus (SEQ ID NO: 17), and the N-acetylneuraminate synthase from N. meningitidis (SEQ ID NO: 18). 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: 22), 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 102. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 108. Production of an Oligosaccharide Mixture Comprising LNB, LN3, LNT, GalNAc-b1,3-Lactose, Gal-b1,3-GalNAc-b1,3-Lactose, GalNAc-b1,3-LNB and Gal-b1,3-GalNAc-b1,3-LNB in Mutant C. glutamicum Strains

[0808] A wild-type C. glutamicum strain is first modified with genomic knockouts of the C. glutamicum genes ldh, cgl2645, nagB, gamA and nagA, together with genomic knock-ins of constitutive transcriptional units comprising genes encoding the phosphatase yqaB from E. coli (SEQ ID NO: 20), GNA1 from S. cerevisiae (SEQ ID NO: 16) and WbgO from E. coli 055:H.sub.7 (SEQ ID NO: 28) to produce LNB. In a next step, the LNB producing is further modified with knock-ins of constitutive expression units for the 4-epimerase (WbpP) of P. aeruginosa (SEQ ID NO: 34), the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (SEQ ID NO: 27) and the 1,3-N-acetylgalactosaminyltransferase (LgtD) from H. influenzae (SEQ ID NO: 35). The novel strain is evaluated for production of an oligosaccharide mixture comprising LNB, LN3, LNT, GalNAc-b1,3-lactose, Gal-b1,3-GalNAc-b1,3-lactose, GalNAc-b1,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, GalNAc-b1,3-LNB and Gal-b1,3-GalNAc-b1,3-LNB in a growth experiment on MMsf medium comprising lactose as precursor according to the culture conditions provided in Example 102.

Example 109. Materials and Methods Chlamydomonas reinhardtii

Media

[0809] C. reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7.0). The TAP medium uses a 1000 stock Hutner's trace element mix. Hutner's trace element mix consisted of 50 g/L Na.sub.2EDTA.Math.H.sub.2O (Titriplex III), 22 g/L ZnSO.sub.4.Math.7H.sub.2O, 11.4 g/L H.sub.3BO.sub.3, 5 g/L MnCl.sub.2.Math.4H.sub.2O, 5 g/L FeSO.sub.4.Math.7H.sub.2O, 1.6 g/L COCl.sub.2.Math.6H.sub.2O, 1.6 g/L CuSO4.5H.sub.2O and 1.1 g/L (NH.sub.4).sub.6MoO.sub.3.

[0810] The TAP medium contained 2.42 g/L Tris (tris(hydroxymethyl)aminomethane), 25 mg/L salt stock solution, 0.108 g/L K.sub.2HPO.sub.4, 0.054 g/L KH.sub.2PO.sub.4 and 1.0 mL/L glacial acetic acid. The salt stock solution consisted of 15 g/L NH.sub.4Cl, 4 g/L MgSO.sub.4.Math.7H.sub.2O and 2 g/L CaCl.sub.2).Math.2H.sub.2O. 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/cm.sup.2).

Strains, Plasmids and Mutations

[0811] 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.

[0812] 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).

[0813] 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 1.0-2.010.sup.7 cells/mL. Then, the cells were inoculated into fresh liquid TAP medium in a concentration of 1.010.sup.6 cells/mL and grown under continuous light for 18-20 h until the cell density reached 4.010.sup.6 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 5.010.sup.7 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 Q, 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.

[0814] 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 a UDP-sugar pyrophosphorylase like, e.g., USP from A. thaliana (UniProt ID Q9C5I1).

[0815] 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: 27) 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: 28). 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: 29).

[0816] In an example for production of GDP-fucose, C. reinhardtii cells are modified with a transcriptional unit for a GDP-fucose synthase like, e.g., from Arabidopsis thaliana (GER1, UniProt ID 049213).

[0817] In an example for fucosylation, C. 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).

[0818] In an example for CMP-sialic acid synthesis, C. reinhardtii cells are modified with constitutive transcriptional units for a UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase like, e.g., GNE from Homo sapiens (UniProt ID Q9Y223) or a mutant form of the human GNE polypeptide comprising the R263L mutation, an N-acylneuraminate-9-phosphate synthetase like, e.g., NANS from Homo sapiens (UniProt ID Q9NR45) and an N-acylneuraminate cytidylyltransferase like, e.g., CMAS from Homo sapiens (UniProt ID Q8NFW8). In an example for production of sialylated oligosaccharides, C. reinhardtii cells are modified with a CMP-sialic acid transporter like, e.g., CST from Mus musculus (UniProt ID Q61420), and a Golgi-localized sialyltransferase chosen from species like, e.g., Homo sapiens, Mus musculus, Rattus norvegicus.

Heterologous and Homologous Expression

[0819] 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.

[0820] 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

[0821] 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.

[0822] 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 110. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LacNAc, 2FLacNAc and 3-FLacNAc in Mutant C. reinhardtii Cells

[0823] C. reinhardtii cells are engineered as described in Example 109 for production of UDP-Gal 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 Q9C5I1). In a next step, the cells are modified with genomic knock-ins of constitutive transcriptional units comprising the b1,4-galactosyltransferase LgtB from N. meningitidis (SEQ ID NO: 29), GDP-fucose synthase from Arabidopsis thaliana (GER1, UniProt ID 049213), the alpha-1,2-fucosyltransferase HpFutC from H. pylori (SEQ ID NO: 04) and the alpha-1,3-fucosyltransferase HpFucT from H. pylori (SEQ ID NO: 05). The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LacNAc, 2FLacNAc and 3-FLacNAc in a cultivation experiment on TAP-agar plates comprising galactose and N-acetylglucosamine as precursors according to the culture conditions provided in Example 109. After 5 days of incubation, the cells are harvested, and the saccharide production is analyzed on UPLC.

Example 111. Production of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LacNAc, 2FLacNAc, 3-FLacNAc, LN3, LNnT, LNFP-III and Difucosyl-Lacto-N-Neohexaose in Mutant C. reinhardtii Cells

[0824] The mutant C. reinhardtii cells as described in Example 110 are further adapted with a genomic knock-in of a constitutive transcriptional unit for the galactoside beta-1,3-N-acetylglucosaminyltransferase (lgtA) from N. meningitidis with SEQ ID NO: 27. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LacNAc, 2FLacNAc, 3-FLacNAc, LN3, LNnT, LNFP-III and difucosyl-lacto-N-neohexaose in a cultivation experiment on TAP-agar plates comprising galactose and N-acetylglucosamine as precursors according to the culture conditions provided in Example 109. After 5 days of incubation, the cells are harvested, and the saccharide production is analyzed on UPLC.

Example 112. Production of an Oligosaccharide Mixture Comprising Sialylated LNB and Sialylated LacNAc Structures in Mutant C. reinhardtii Cells

[0825] C. reinhardtii cells are engineered as described in Example 109 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 Q9C5I1), together with the N-acetylglucosamine beta-1,3-galactosyltransferase WbgO from E. coli 055:H7 with SEQ ID NO: 28 and the N-acetylglucosamine beta-1,4-galactosyltransferase LgtB from N. meningitidis with SEQ ID NO: 29. 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 109. After 5 days of incubation, the cells are harvested, and the saccharide production is analyzed on UPLC.

Example 113. Production of an Oligosaccharide Mixture Comprising GlcNAc-b1,3-Gal-b1,4-GlcNAc, Beta-Gal-(1,4)-Beta-GlcNAc-(1,3)-[Beta-GlcNAc-(1,6)]-Beta-Gal-(1,4)-GlcNAc and Poly-LacNAc Structures in Mutant C. reinhardtii Cells

[0826] C. reinhardtii cells are engineered as described in Example 109, comprising 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 Q9C5I1), the mutant glmS*54 from E. coli (differing from the wild-type glmS (UniProt ID P17169) by an A39T, an R250C and an G472S mutation, the phosphatase yqaB from E. coli (UniProt ID NP_417175.1), galE from E. coli (UniProt ID P09147), LgtA from N. meningitidis (UniProt ID Q9JXQ6), LgtB from N. meningitidis (UniProt ID Q51116) and the human N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase GCNT2 (UniProt ID Q8NOV5). The novel strains are evaluated for production of an oligosaccharide mixture comprising GlcNAc-b1,3-Gal-b1,4-GlcNAc, beta-Gal-(1,4)-beta-GlcNAc-(1,3)-[beta-GlcNAc-(1,6)]-beta-Gal-(1,4)-GlcNAc and poly-LacNAc structures in a cultivation experiment on TAP-agar plates comprising galactose and GlcNAc as precursors according to the culture conditions provided in Example 109.

Example 114. Materials and Methods Animal Cells

Isolation of Mesenchymal Stem Cells from Adipose Tissue of Different Mammals

[0827] 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

[0828] 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

[0829] 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.

[0830] 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 g/ml insulin. At confluence, cells were fed with growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U/ml penicillin, 100 g/ml streptomycin), and 5 g/ml insulin for 48 h. To induce differentiation, the cells were fed with complete growth medium containing 5 g/ml insulin, 1 g/ml hydrocortisone, 0.65 ng/ml triiodothyronine, 100 nM dexamethasone, and 1 g/ml prolactin. After 24 h, serum is removed from the complete induction medium.

[0831] 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 g/ml insulin. At confluence, cells were fed with growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U/ml penicillin, 100 g/ml streptomycin), and 5 g/ml insulin for 48 h. To induce differentiation, the cells were fed with complete growth medium containing 5 g/ml insulin, 1 g/ml hydrocortisone, 0.65 ng/ml triiodothyronine, 100 nM dexamethasone, and 1 g/ml prolactin. After 24 h, serum is removed from the complete induction medium.

Method of Making Mammary-Like Cells

[0832] Mammalian cells are brought to induced pluripotency by reprogramming with viral vectors encoding for Oct4, Sox2, Klf4, and c-Myc. The resultant reprogrammed cells are then cultured in Mammocult media (available from Stem Cell Technologies), or mammary cell enrichment media (DMEM, 3% FBS, estrogen, progesterone, heparin, hydrocortisone, insulin, EGF) to make them mammary-like, from which expression of select milk components can be induced. Alternatively, epigenetic remodeling is performed using remodeling systems such as CRISPR/Cas9, to activate select genes of interest, such as casein, a-lactalbumin to be constitutively on, to allow for the expression of their respective proteins, and/or to down-regulate and/or knock-out select endogenous genes as described, e.g., in WO 2021067641, which is incorporated herein by reference in its entirety for all purposes.

Cultivation

[0833] 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 g/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 g/ml hydrocortisone, and 1 g/ml prolactin (5 ug/ml in Hyunh 1991). Cells are seeded at a density of 20,000 cells/cm.sup.2 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 115. Making of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL and DiFL in a Non-Mammary Adult Stem Cell

[0834] Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 114 are modified via CRISPR-CAS to over-express the GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), the alpha-1,2-fucosyltransferase HpFutC from H. pylori (SEQ ID NO: 04) and the alpha-1,3-fucosyltransferase HpFucT from H. pylori (SEQ ID NO: 05). Cells are seeded at a density of 20,000 cells/cm.sup.2 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 114, cells are subjected to UPLC to analyze for production of an oligosaccharide mixture comprising 2FL, 3-FL and DiFL.

Example 116. Making of a Mammalian Milk Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LN3, LNT and LNFP-I in a Non-Mammary Adult Stem Cell

[0835] Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 114 are modified via CRISPR-CAS to over-express the GlcN6P synthase from Homo sapiens (UniProt ID Q06210), the glucosamine 6-phosphate N-acetyltransferase from Homo sapiens (UniProt ID Q96EK6), the phosphoacetylglucosamine mutase from Homo sapiens (UniProt ID 095394), the UDP-N-acetylhexosamine pyrophosphorylase (UniProt ID Q16222), the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis with SEQ ID NO: 27, the N-acetylglucosamine beta-1,4-galactosyltransferase LgtB from N. meningitidis with SEQ ID NO: 29, the GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), the alpha-1,2-fucosyltransferase HpFutC from H. pylori (SEQ ID NO: 04) and the alpha-1,3-fucosyltransferase HpFucT from H. pylori (SEQ ID NO: 05). All genes introduced are codon-optimized to the host cells. Cells are seeded at a density of 20,000 cells/cm.sup.2 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 114, cells are subjected to UPLC to analyze for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LN3, LNT and LNFP-I.

Example 117. Evaluation of Production of an Oligosaccharide Mixture Comprising LNB, LN3, LNT, GalNAc-b1,3-Lactose, Gal-b1,3-GalNAc-b1,3-Lactose, GalNAc-b1,3-LNB and Gal-b1,3-GalNAc-b1,3-LNB in a Non-Mammary Adult Stem Cell

[0836] Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 114 are modified via CRISPR-CAS to over-express the beta-1,4-galactosyltransferase 1 B4GalT1 from Homo sapiens (UniProt ID P15291), the phosphatase ScDOG1 from S. cerevisiae (UniProt ID P38774), GNA1 from S. cerevisiae (SEQ ID NO: 16) and WbgO from E. coli 055:H7 (SEQ ID NO: 28), the 4-epimerase (WbpP) of P. aeruginosa (SEQ ID NO: 34), the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (SEQ ID NO: 27) and the 1,3-N-acetylgalactosaminyltransferase (LgtD) from H. influenzae (SEQ ID NO: 35). All genes introduced in the cells are codon-optimized to the host. Cells are seeded at a density of 20,000 cells/cm.sup.2 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 114, cells are subjected to UPLC and evaluated for production of an oligosaccharide mixture comprising LNB, LN3, LNT, GalNAc-b1,3-lactose, Gal-b1,3-GalNAc-b1,3-lactose, GalNAc-b1,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, GalNAc-b1,3-LNB and Gal-b1,3-GalNAc-b1,3-LNB.