PRODUCTION OF A MIXTURE OF NEUTRAL FUCOSYLATED OLIGOSACCHARIDES BY A CELL

Abstract

Production of a mixture of neutral fucosylated oligosaccharides by a cell. 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 neutral mixture of at least four different neutral fucosylated oligosaccharides. Furthermore, the disclosure provides a method for the production of a neutral mixture of at least four different neutral fucosylated oligosaccharides by a cell as well as the purification of at least one of the neutral oligosaccharides from the cultivation.

Claims

1.-89. (canceled)

90. A cell that produces a mixture of at least four different neutral fucosylated oligosaccharides, wherein the cell is metabolically engineered for producing the mixture, expresses a glycosyltransferase, which glycosyltransferase is a fucosyltransferase, is capable of synthesizing the nucleotide-sugar GDP-fucose (GDP-Fuc), expresses at least one additional glycosyltransferase, and is capable of synthesizing at least one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for the additional glycosyltransferase.

91. The cell of claim 90, wherein the mixture comprises at least five different neutral fucosylated oligosaccharides.

92. The cell of claim 90, wherein any one of the additional glycosyltransferases is selected from the group consisting of fucosyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucosaminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases, and fucosaminyltransferases.

93. The cell of claim 90, wherein the cell is capable of expressing at least two additional glycosyltransferases.

94. The cell of claim 90, wherein the cell is modified in expression or activity of at least one of the glycosyltransferases.

95. The cell of claim 90, wherein the cell uses at least one precursor for producing any one or more of the neutral fucosylated oligosaccharides, the at least one precursor being fed to the cell from a cultivation medium.

96. The cell of claim 90, wherein the cell produces at least one precursor for producing any one of the neutral fucosylated oligosaccharides.

97. The cell of claim 90, 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, and/or iv) expression of a heterologous membrane protein, wherein the membrane protein is (i) involved in the secretion of any one of the neutral fucosylated oligosaccharides from the mixture outside the cell or (ii) uptake of a precursor or acceptor for the synthesis of any one of the neutral fucosylated oligosaccharides of the mixture.

98. The cell of claim 97, wherein the membrane protein provides improved production, enabled efflux, or enhanced efflux of any one of the neutral fucosylated oligosaccharides.

99. The cell of claim 90, wherein at least one of the neutral fucosylated oligosaccharides is a mammalian milk oligosaccharide.

100. The cell of claim 90, wherein all of the neutral fucosylated oligosaccharides are mammalian milk oligosaccharides.

101. The cell of claim 90, wherein at least one of the neutral fucosylated oligosaccharides is an antigen of the human ABO blood group system.

102. The cell of claim 90, wherein three of the neutral fucosylated oligosaccharides are antigens of the human ABO blood group system.

103. The cell of claim 90, wherein the cell is a bacterium, fungus, yeast, plant cell, animal cell, or protozoan cell.

104. The cell of claim 90, wherein the neutral fucosylated oligosaccharides are produced intracellularly.

105. The cell of claim 99, wherein the mammalian milk oligosaccharide is a lactose-based mammalian milk oligosaccharide.

106. The cell of claim 100, wherein all of the neutral fucosylated oligosaccharides are lactose-based mammalian milk oligosaccharides.

107. The cell of claim 96, wherein the precursor is: lactose for producing a lactose-based oligosaccharide, lacto-N-biose for producing a LNB-based oligosaccharide, and/or N-acetyllactosamine (LacNAc) for producing a LacNAc-based oligosaccharide.

108. A method of producing a mixture of at least four different neutral fucosylated oligosaccharides by a cell, the method comprising: i) providing a cell that (a) is capable of expressing a glycosyltransferase being a fucosyltransferase and is capable of synthesizing a nucleotide-sugar GDP-fucose, (b) expresses at least one additional glycosyltransferase, and (c) is capable of synthesizing at least one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for additional glycosyltransferases, and ii) cultivating the cell under conditions permissive to express the glycosyltransferases and to synthesize the nucleotide-sugars resulting in the cell producing the mixture of at least four different neutral fucosylated oligosaccharides.

109. The method according to claim 108, wherein the cell: is metabolically engineered for producing the mixture, expresses a glycosyltransferase, which glycosyltransferase is a fucosyltransferase, is capable of synthesizing the nucleotide-sugar GDP-fucose (GDP-Fuc), expresses at least one additional glycosyltransferase, and is capable of synthesizing at least one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for the additional glycosyltransferase.

110. The method according to claim 108, wherein the mixture comprises at least five different neutral fucosylated oligosaccharides.

111. A method of using the cell of claim 90 to produce a mixture of at least four different neutral fucosylated oligosaccharides, the method comprising: cultivating the cell, and producing the mixture of at least four different neutral fucosylated oligosaccharides.

Description

DETAILED DESCRIPTION

[0578] The disclosure will be described in more detail in the examples.

[0579] 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

[0580] 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.2HPO.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, 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 1 M KOH. Vitamin solution consisted of 3.6 g/L FeCl.sub.2.Math.4H.sub.2O, 5 g/L CaCl.sub.2.Math.2H2O, 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 H3BO.sub.4, 0.4 g/L Na.sub.2EDTA.Math.2H.sub.2O and 1.01 g/L thiamine.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.

[0581] 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.40.7H.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 20 g/L lactose, 20 g/L LacNAc and/or 20 g/L LNB were additionally added to the medium.

[0582] 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

[0583] 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, (lacZYA-argF) U169, deoR, recA1, endA1, hsdR17(rk.sup., mk.sup.+), phoA, supE44, lambda.sup., thi-1, gyrA96, relA1) bought from Invitrogen.

Strains and Mutations

[0584] 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.

[0585] In an example for GDP-fucose production, the mutant strain was derived from E. coli K12 MG1655 comprising knock-outs of the E. coli wcaJ and thyA genes and genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like, e.g., CscB from E. coli W with SEQ ID NO: 01, a fructose kinase like, e.g., Frk originating from Zymomonas mobilis with SEQ ID NO: 02 and a sucrose phosphorylase like, e.g., BaSP originating from Bifidobacterium adolescentis with SEQ ID NO: 03. For production of fucosylated oligosaccharides, the mutant GDP-fucose production strain was additionally modified with expression plasmids comprising constitutive transcriptional units for an alpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori with SEQ ID NO: 04 and/or an alpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori with SEQ ID NO: 05 and with a constitutive transcriptional unit for a 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 glg C, agp, pfkA, pfkB, pgi, arcA, icIR, 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 fuel genes and genomic knock-ins of constitutive transcriptional units containing a fucose permease like, e.g., fucP from E. coli with SEQ ID NO: 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.

[0586] Alternatively, and/or additionally, production of GDP-fucose and/or fucosylated structures can further be optimized in the mutant E. coli strains with genomic knock-ins of a constitutive transcriptional unit comprising a membrane transporter protein like, e.g., MdfA from Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID POAEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207) or iceT from Citrobacter youngae (UniProt ID D48A6).

[0587] 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: 18. 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: 19 or an N-acetylglucosamine beta-1,4-galactosyltransferase like, e.g., LgtB from N. meningitidis with SEQ ID NO: 20, 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: 17. In addition, the strains can optionally be modified for enhanced UDP-galactose production with genomic knock-outs of the E. coli ushA, galT, IdhA and agp genes. The mutant E. coli strains can also optionally be adapted with a genomic knock-in of a constitutive transcriptional unit for an UDP-glucose-4-epimerase like, e.g., galE from E. coli with SEQ ID NO: 21, a phosphoglucosamine mutase like, e.g., glmM from E. coli with SEQ ID NO: 26 and an N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase like, e.g., glmU from E. coli with SEQ ID NO: 27. 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.

[0588] Alternatively, and/or additionally, production of LN3, LNT, LNnT and oligosaccharides derived thereof can further be optimized in the mutant E. coli strains with a genomic knock-in of a constitutive transcriptional unit comprising a membrane transporter protein like, e.g., MdfA from Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID POAEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207) or iceT from Citrobacter youngae (UniProt ID D48A6).

[0589] 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).

[0590] 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).

[0591] 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, wbbI, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA and yaiP.

[0592] 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). 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 disclosure are summarized in Table 1.

[0593] All strains were stored in cryovials at 80 C. (overnight LB culture mixed in a 1:1 ratio with 70% glycerol).

TABLE-US-00001 TABLE 1 Overview of SEQ ID NOs: described in the disclosure. Country of origin of SEQ digital ID sequence NO: Name/identifier Organism Origin information 01 CscB Escherichia coli W Synthetic USA 02 Frk Zymomonas Synthetic United mobilis 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 Synthetic USA K-12 MG1655 08 manA Escherichia coli Synthetic USA K-12 MG1655 09 manB Escherichia coli Synthetic USA K-12 MG1655 10 manC Escherichia coli Synthetic USA K-12 MG1655 11 gmd Escherichia coli Synthetic USA K-12 MG1655 12 fcl Escherichia coli Synthetic USA K-12 MG1655 13 fucP Escherichia coli Synthetic USA K-12 MG1655 14 fkp Bacteroides fragilis Synthetic United Kingdom 15 LacY Escherichia coli Synthetic USA K-12 MG1655 16 GNA1 Saccharomyces Synthetic USA cerevisiae 17 glmS*54 Escherichia coli Synthetic USA K-12 MG1655 18 LgtA Neisseria meningitidis Synthetic United Kingdom 19 WbgO Escherichia coli Synthetic Germany O55:H7 20 lgtB Neisseria meningitidis Synthetic United MC58 Kingdom 21 galE Escherichia coli Synthetic USA K-12 MG1655 22 Lac12 Kluyveromyces lactis Synthetic USA 23 wbnI Escherichia coli Synthetic USA K-12 MG1655 24 BgtA Helicobacter mustelae Synthetic USA 25 Mutant a1, 3/4 Bifidobacterium Synthetic USA fucosidase longum subsp. infantis 26 glmM Escherichia coli Synthetic USA K-12 MG1655 27 glmU Escherichia coli Synthetic USA K-12 MG1655

Cultivation Conditions

[0594] 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).

[0595] 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

[0596] 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

[0597] Standards such as but not limited to sucrose, lactose, LacNAc, lacto-N-biose (LNB), fucosylated LacNAc (2FLacNAc, 3-FLacNAc), fucosylated LNB (2FLNB, 4FLNB), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neo-tetraose (LNnT), LNFP-I, LNFP-II, LNFP-III and LNFP-V were purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analyzed with in-house made standards.

[0598] 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 1/4 water and 3/4 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.

[0599] For analysis on a mass spectrometer, a Waters Xevo TQ-MS with Electron Spray Ionization (ESI) was used with a desolvation temperature of 450 C., a nitrogen desolvation gas flow of 650 L/h and a cone voltage of 20 V. The MS was operated in selected ion monitoring (SIM) in negative mode for all oligosaccharides. Separation was performed on a Waters Acquity UPLC with a Thermo Hypercarb column (2.1100 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 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.

[0600] 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

[0601] 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

[0602] 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

[0603] In an example to produce GDP-fucose, the yeast expression plasmid p2a_2_Fuc (Chan 2013, Plasmid 70, 2-17) was used for expression of foreign genes in S. cerevisiae. This plasmid contained an ampicillin resistance gene and a bacterial origin of replication to allow for selection and maintenance in E. coli and the 2 yeast ori and the Ura3 selection marker for selection and maintenance in yeast. This plasmid further contained constitutive transcriptional units for a lactose permease like, e.g., LAC12 from Kluyveromyces lactis with SEQ ID NO: 22, 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 2u 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: 22, 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 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.

[0604] In an example to produce UDP-galactose, a yeast expression plasmid was derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110:119-122) containing the HIS3 selection marker and a constitutive transcriptional unit for an UDP-glucose-4-epimerase like, e.g., galE from E. coli with SEQ ID NO: 21. 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: 22 and a galactoside beta-1,3-N-acetylglucosaminyltransferase like, e.g., IgtA from N. meningitidis with SEQ ID NO: 18. 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: 19 or and an N-acetylglucosamine beta-1,4-galactosyltransferase like, e.g., IgtB from N. meningitidis with SEQ ID NO: 20, respectively.

[0605] 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.

[0606] 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).

[0607] Plasmids were maintained in the host E. coli DH5alpha (F, phi80dlacZdeltaM15, delta (lacZYA-argF) U169, deoR, recAl, endAI, hsdR17 (rk.sup., mk.sup.+), phoA, supE44, lambda , thi-1, gyrA96, relA1) bought from Invitrogen.

Heterologous and Homologous Expression

[0608] 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

[0609] 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

[0610] Genes were expressed using synthetic constitutive promoters, as described by Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012).

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

[0611] 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 to express the H. pylori alpha-1,2-fucosyltransferase with SEQ ID NO: 04 and 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 4. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, 2FLacNAc, 3-FLacNAc and Di-FLacNAc with a Modified E. coli Host

[0612] 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 IgtB from N. meningitidis with SEQ ID NO: 20 and the UDP-glucose 4-epimerase (galE) from E. coli with SEQ ID NO: 21. In a next step, the mutant E. coli strain is transformed with an expression plasmid having two constitutive transcriptional units to express the H. pylori alpha-1,2-fucosyltransferase with SEQ ID NO: 04 and 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, Di-FL and fucosylated N-acetyllactosamine, i.e., 2FLacNAc, 3-FLacNAc and Di-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 LacNAc as precursor.

Example 5. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, 2FLacNAc, 3-FLacNAc and Di-FLacNAc with a Modified E. coli Host

[0613] 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: 20, the mutant L-glutamine-D-fructose-6-phosphate aminotransferase (glmS*54) from E. coli with SEQ ID NO: 17, 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 NOS: 04 and 05. All novel strains are evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 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 and lactose as precursor.

Example 6. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, 2FLNB, 4-FLNB and Di-FLNB with a Modified E. coli Host

[0614] 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 to express the H. pylori alpha-1,2-fucosyltransferase with SEQ ID NO: 04 and 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, DiFL and fucosylated LNB, i.e., 2FLNB, 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 culture medium contains sucrose as carbon source and lactose and LNB as precursors.

Example 7. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, 2FLNB, 4-FLNB and Di-FLNB with a Modified E. coli Host

[0615] An E. coli strain modified for the production of 2FL, 3-FL and DiFL as described in Example 4 is evaluated in another growth experiment for production of an oligosaccharide mixture comprising 2FL, 3-FL, Di-FL and fucosylated LNB, i.e., 2FLNB, 4-FLNB, and di-fucosylated LNB (Di-FLNB) in whole broth samples according to the culture conditions provided in Example 1, in which the culture medium contains sucrose as carbon source and LNB as precursor.

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

[0616] 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 LNB 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 1,3-galactosyltransferase (WbgO) of E. coli O55:H7 with SEQ ID NO: 19, the mutant glmS*54 from E. coli with SEQ ID NO: 17, and 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 NOS: 04 and 05. All novel strains are evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2FLNB, 4-FLNB and 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 and lactose as precursor.

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

[0617] An E. coli strain modified for the production of 2FL, 3-FL and DiFL as described in Example 4 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 of E. coli 055:H7 with SEQ ID NO: 19, the mutant glmS*54 from E. coli with SEQ ID NO: 17, and GNA1 of S. cerevisiae with SEQ ID NO: 16. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2FLNB, 4-FLNB and 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 and no precursors.

Example 10. Production of an oligosaccharide mixture comprising 2FL, DiFL, 2FLNB and Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a modified E. coli host

[0618] 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: 23. Since lactose and LNB are suitable acceptors for the H. pylori alpha-1,2-fucosyltransferase and 2FLNB is as an acceptor of the E. coli alpha-1,3-galactosyltransferase the novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL, 2-fucosylated LNB (2FLNB) 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 and LNB as precursors.

Example 11. Production of an Oligosaccharide Mixture Comprising 2FL, DiFL, 2FLNB and Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a Modified E. coli Host

[0619] 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: 19, the mutant glmS*54 from E. coli with SEQ ID NO: 17, 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: 23. 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 12. Production of an Oligosaccharide Mixture Comprising 2FL, DiFL, 2FLNB and GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GleNAc with a Modified E. coli Host

[0620] 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: 24. Since lactose and LNB are suitable acceptors for the H. pylori alpha-1,2-fucosyltransferase and 2FLNB is an acceptor of the H. mustelae alpha-1,3-N-acetylgalactosaminyltransferase, the novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL, 2-fucosylated LNB (2FLNB) 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 and LNB as precursors.

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

[0621] 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: 19, the mutant glmS*54 from E. coli with SEQ ID NO: 17 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: 24. 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 14. 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

[0622] 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 nagA and nagB genes and genomic knock-ins of constitutive transcriptional units for the mutant glmS*54 from E. coli with SEQ ID NO: 17, the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. In a next step, the novel strain is further transformed with an expression plasmid containing a constitutive transcriptional unit for the -1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and with a second compatible expression plasmid containing a constitutive transcriptional unit for the alpha-1,3-galactosyltransferase WbnI from E. coli with SEQ ID NO: 23. An E. coli strain modified for GDP-fucose production and for LNFP-I production as described in Example 23 was further transformed. 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 15. 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

[0623] 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 nagA and nagB genes and genomic knock-ins of constitutive transcriptional units for the mutant glmS*54 from E. coli with SEQ ID NO: 17, the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. In a next step, the novel strain is further transformed with an expression plasmid containing a constitutive transcriptional unit for the -1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and with a second compatible expression plasmid containing a constitutive transcriptional unit for the alpha-1,3-N-acetylgalactosaminyltransferase BgtA from H. mustelae with SEQ ID NO: 24. 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 16. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LN3, LNT and LNFP-I with a Modified E. coli Host

[0624] 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 nagA and nagB genes and genomic knock-ins of constitutive transcriptional units for the mutant glmS*54 from E. coli with SEQ ID NO: 17, the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. In a next step, the novel strain is further transformed with an expression plasmid containing a constitutive transcriptional unit for the -1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and with a second compatible expression plasmid containing a constitutive transcriptional unit for the -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, 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 17. Production of an Oligosaccharide Mixture Comprising 2FL, DiFL, LNFP-I and LNFP-II with a Modified E. coli Host

[0625] 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 nagA and nagB genes and genomic knock-ins of constitutive transcriptional units for the glmS*54 from F. coli with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. In a next step to produce lacto-N-fucopentaose II (LNFP-II, Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc), the mutant 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: 25 and for the -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 18. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LN3, LNT, LNFP-I and LNFP-II with a Modified E. coli Host

[0626] An E. coli strain modified for GDP-fucose production and for LNFP-II production as described in Example 17 is further transformed with a compatible expression plasmid containing a constitutive transcriptional unit for the -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 19. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LN3, LNT, LNFP-I and LNFP-V with a Modified E. coli Host

[0627] 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 nagA and nagB genes and genomic knock-ins of constitutive transcriptional units for the mutant glmS*54 from E. coli with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. In a next step, the novel strain is further transformed with an expression plasmid containing constitutive transcriptional units for the a1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and 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 2FL, 3-FL, DiFL, LN3, LNT, LNFP-I and lacto-N-fucopentaose V (LNFP-V, Gal-b1,3-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 20. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LN3, LNnT, LNFP-III and LNFP-VI with a Modified E. coli Host

[0628] 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 nagA and nagB genes and genomic knock-ins of constitutive transcriptional units for the mutant glmS*54 from E. coli with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 18 and LgtB from N. meningitidis with SEQ ID NO: 20. In a next step, the novel strain is further transformed with an expression plasmid containing constitutive transcriptional units for the a1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and for the truncated -1,3-fucosyltransferase from H. pylori with SEQ ID NO: 06. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LN3, 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 21. Production of an Oligosaccharide Mixture Comprising Neutral Non-Fucosylated and Fucosylated Oligosaccharide Structures with a Modified E. coli Host

[0629] An E. coli strain modified as described in Example 16 is evaluated in another growth experiment for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2-FLNB, 4-FLNB, Di-FLNB, LN3, LNT and LNFP-I in whole broth samples 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.

Example 22. Production of an Oligosaccharide Mixture Comprising Neutral Non-Fucosylated and Fucosylated Oligosaccharide Structures with a Modified E. coli Host

[0630] An E. coli strain modified as described in Example 16 is evaluated in another growth experiment for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2-FLacNAc, 3-FLacNAc, Di-FLacNAc, LN3, LNT and LNFP-I in whole broth samples 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.

Example 23. Production of an Oligosaccharide Mixture Comprising Neutral Non-Fucosylated and Fucosylated Oligosaccharide Structures with a Modified E. coli Host

[0631] An E. coli strain modified as described in Example 16 is evaluated in another growth experiment for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2-FLacNAc, 3-FLacNAc, Di-FLacNAc, 2-FLNB, 4-FLNB, Di-FLNB, LN3, LNT and LNFP-I in whole broth samples according to the culture conditions provided in Example 1, in which the culture medium contains sucrose as carbon source and lactose, LacNAc and LNB as precursors.

Example 24. Production of an Oligosaccharide Mixture Comprising Neutral Non-Fucosylated and Fucosylated Oligosaccharide Structures with a Modified E. coli Host

[0632] An E. coli strain modified for GFP-fucose production as described in Example 1 is further adapted by a genomic knock-out of the E. coli nagA and nagB genes and genomic knock-ins of constitutive transcriptional units for the mutant glmS*54 from E. coli with SEQ ID NO: 17, GNA1 of S. cerevisiae with SEQ ID NO: 16, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. In a next step, the novel strain is further transformed with an expression plasmid containing constitutive transcriptional units for the a1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and for the al, 3-fucosyltransferase from H. pylori with SEQ ID NO: 05. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2-FLNB, 4-FLNB, Di-FLNB, LN3, LNT and LNFP-I 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 25. Production of an Oligosaccharide Mixture Comprising Neutral Non-Fucosylated and Fucosylated Oligosaccharide Structures with a Modified E. coli Host

[0633] An E. coli strain modified for GFP-fucose production as described in Example 1 is further adapted by a genomic knock-out of the E. coli nagA and nagB genes and genomic knock-ins of constitutive transcriptional units for the mutant glmS*54 from E. coli with SEQ ID NO: 17, GNA1 of S. cerevisiae with SEQ ID NO: 16, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. In a next step, the novel strain is further transformed with an expression plasmid containing constitutive transcriptional units for the a1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and for the a1, 3-fucosyltransferase from H. pylori with SEQ ID NO: 05. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2-FLacNAc, 3-FLacNAc, Di-FLacNAc, 2-FLNB, 4-FLNB, Di-FLNB, LN3, LNT and LNFP-I 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.

Example 26. Production of an Oligosaccharide Mixture Comprising Neutral Non-Fucosylated and Fucosylated Oligosaccharide Structures with a Modified E. coli Host

[0634] An E. coli strain modified as described in Example 20 is evaluated in another growth experiment for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2-FLNB, 4-FLNB, Di-FLNB, LN3, LNnT, LNFP-III and LNFP-VI in whole broth samples 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.

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

[0635] An E. coli strain modified as described in Example 20 is evaluated in another growth experiment for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2-FLacNAc, 3-FLacNAc, Di-FLacNAc, LN3, LNnT, LNFP-III and LNFP-VI in whole broth samples 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.

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

[0636] An E. coli strain modified as described in Example 20 is evaluated in another growth experiment for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2-FLacNAc, 3-FLacNAc, Di-FLacNAc, 2-FLNB, 4-FLNB, Di-FLNB, LN3, LNnT, LNFP-III, and LNFP-VI in whole broth samples according to the culture conditions provided in Example 1, in which the culture medium contains sucrose as carbon source and lactose, LacNAc and LNB as precursors.

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

[0637] An E. coli strain modified for GFP-fucose production as described in Example 1 is further adapted by a genomic knock-out of the E. coli nagA and nagB genes and genomic knock-ins of constitutive transcriptional units for the mutant glmS*54 from E. coli with SEQ ID NO: 17, GNA1 of S. cerevisiae with SEQ ID NO: 16, LgtA from N. meningitidis with SEQ ID NO: 18 and LgtB from N. meningitidis with SEQ ID NO: 20. In a next step, the novel strain is further transformed with an expression plasmid containing constitutive transcriptional units for the a1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and for the a1, 3-fucosyltransferase from H. pylori with SEQ ID NO: 05. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2-FLacNAc, 3-FLacNAc, Di-FLacNAc, LN3, LNnT, LNFP-III and LNFP-VI 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 30. Production of an Oligosaccharide Mixture Comprising Neutral Non-Fucosylated and Fucosylated Oligosaccharide Structures with a Modified E. coli Host

[0638] An E. coli strain modified for GFP-fucose production as described in Example 1 is further adapted by a genomic knock-out of the E. coli nagA, nagB, ushA and galT genes and genomic knock-ins of constitutive transcriptional units for galE from E. coli with SEQ ID NO: 21, the mutant glmS*54 from E. coli with SEQ ID NO: 17, GNA1 of S. cerevisiae with SEQ ID NO: 16, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. In a next step, the novel strain is further transformed with an expression plasmid containing constitutive transcriptional units for the a1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and for the a1, 3-fucosyltransferase from H. pylori with SEQ ID NO: 05. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2-FLNB, 4-FLNB, Di-FLNB, LN3, LNT and LNFP-I 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 31. Production of an Oligosaccharide Mixture Comprising Neutral Non-Fucosylated and Fucosylated Oligosaccharide Structures with a Modified E. coli Host

[0639] An E. coli strain modified for GFP-fucose production as described in Example 1 is further adapted by a genomic knock-out of the E. coli nagA, nagB, ushA and galT genes and genomic knock-ins of constitutive transcriptional units for galE from E. coli with SEQ ID NO: 21, the mutant glmS*54 from E. coli with SEQ ID NO: 17, GNA1 of S. cerevisiae with SEQ ID NO: 16, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. In a next step, the novel strain is further transformed with an expression plasmid containing constitutive transcriptional units for the a1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and for the a1, 3-fucosyltransferase from H. pylori with SEQ ID NO: 05. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2-FLacNAc, 3-FLacNAc, Di-FLacNAc, 2-FLNB, 4-FLNB, Di-FLNB, LN3, LNT and LNFP-I 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.

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

[0640] An E. coli strain modified for GFP-fucose production as described in Example 1 is further adapted by a genomic knock-out of the E. coli nagA, nagB, ushA and gall genes and genomic knock-ins of constitutive transcriptional units for galE from E. coli with SEQ ID NO: 21, the mutant glmS*54 from E. coli with SEQ ID NO: 17, GNA1 of S. cerevisiae with SEQ ID NO: 16, LgtA from N. meningitidis with SEQ ID NO: 18 and LgtB from N. meningitidis with SEQ ID NO: 20. In a next step, the novel strain is further transformed with an expression plasmid containing constitutive transcriptional units for the a1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and for the a1, 3-fucosyltransferase from H. pylori with SEQ ID NO: 05. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2-FLacNAc, 3-FLacNAc, Di-FLacNAc, LN3, LNnT, LNFP-III and LNFP-VI 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 33. Production of an Oligosaccharide Mixture Comprising 2FL, DiFL, LNFP-I and LNFP-II with a Modified E. coli Host

[0641] 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 nagA, nagB, ushA and gall genes and genomic knock-ins of constitutive transcriptional units for galE from E. coli with SEQ ID NO: 21, the glmS*54 from E. coli with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. In a next step to produce lacto-N-fucopentaose II (LNFP-II, Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc), the mutant strain is further transformed with an expression plasmid containing a constitutive transcriptional unit for a mutant a1,3/4 fucosidase from B. longum subsp. infantis ATCC 15697 with SEQ ID NO: 25. In a final step, the mutant strain is transformed with a compatible expression plasmid containing a constitutive transcriptional unit for the -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 34. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LNFP-I and LNFP-V with a Modified E. coli Host

[0642] 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 nagA, nagB, ushA and gall genes and genomic knock-ins of constitutive transcriptional units for galE from E. coli with SEQ ID NO: 21, mutant glmS*54 from E. coli with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. In a next step, the novel strain is further transformed with an expression plasmid containing constitutive transcriptional units for the a1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and 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 2FL, 3-FL, DiFL, LN3, LNT, LNFP-I and lacto-N-fucopentaose V (LNFP-V, Gal-b1,3-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 35. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LNFP-III and LNFP-VI with a Modified E. coli Host

[0643] 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 nagA, nagB, ushA and galT genes and genomic knock-ins of constitutive transcriptional units for galE from E. coli with SEQ ID NO: 21, the mutant glmS*54 from E. coli with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 18 and LgtB from N. meningitidis with SEQ ID NO: 20. In a next step, the novel strain is further transformed with an expression plasmid containing constitutive transcriptional units for the a1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and for the truncated -1,3-fucosyltransferase from H. pylori with SEQ ID NO: 06. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LN3, 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 36. Production of an Oligosaccharide Mixture Comprising 2FL, DiFL, LNFP-I and LNFP-II with a Modified E. coli Host

[0644] 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 fucK, fucI, nagA, nagB, ushA and galT genes and genomic knock-ins of constitutive transcriptional units for galE from E. coli with SEQ ID NO: 21, fucP from E. coli with SEQ ID NO: 13, fkp from B. fragilis with SEQ ID NO: 14, glmS*54 from E. coli with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. In a next step to produce lacto-N-fucopentaose II (LNFP-II, Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc), the mutant strain is further transformed with an expression plasmid containing a constitutive transcriptional unit for a mutant a1,3/4 fucosidase from B. longum subsp. infantis ATCC 15697 with SEQ ID NO: 25. In a final step, the mutant strain is transformed with a compatible expression plasmid containing a constitutive transcriptional unit for the -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 37. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LNFP-I and LNFP-V with a Modified E. coli Host

[0645] 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 fucK, fuel, nagA, nagB, ushA and galT genes and genomic knock-ins of constitutive transcriptional units for galE from E. coli with SEQ ID NO: 21, fucP from E. coli with SEQ ID NO: 13, fkp from B. fragilis with SEQ ID NO: 14, glmS*54 from E. coli with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. In a next step, the novel strain is further transformed with an expression plasmid containing constitutive transcriptional units for the a1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and 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 2FL, 3-FL, DiFL, LN3, LNT, LNFP-I and lacto-N-fucopentaose V (LNFP-V, Gal-b1,3-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 38. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LNFP-III and LNFP-VI with a Modified E. coli Host

[0646] 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 fucK, fucI, nagA, nagB, ushA and gall genes and genomic knock-ins of constitutive transcriptional units for galE from E. coli with SEQ ID NO: 21, fucP from E. coli with SEQ ID NO: 13, fkp from B. fragilis with SEQ ID NO: 14, the mutant glmS*54 from E. coli with SEQ ID NO: 17, LgtA from N. meningitidis with SEQ ID NO: 18 and LgtB from N. meningitidis with SEQ ID NO: 20. In a next step, the novel strain is further transformed with an expression plasmid containing constitutive transcriptional units for the a1, 2-fucosyltransferase from H. pylori with SEQ ID NO: 04 and for the truncated -1,3-fucosyltransferase from H. pylori with SEQ ID NO: 06. The novel strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LN3, 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 39. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, 2FLNB and 4-FLNB with a Modified S. cerevisiae Host

[0647] 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: 22, 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, DiFL, 2FLNB and 4-FLNB in a growth experiment according to the culture conditions described in Example 2, using SD CSM-Ura drop-out medium comprising lactose and LNB as precursors.

Example 40. Production of an Oligosaccharide Mixture Comprising 2FL, DiFL, 2FLacNAc, LN3, LNT and LNFP-I with a Modified S. cerevisiae Host

[0648] An S. cerevisiae strain is adapted for production of GDP-fucose and LNT and for expression of an -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: 22, gmd from E. coli with SEQ ID NO: 11, fcl from E. coli with SEQ ID NO: 12 and the -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: 21, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL, LN3, LNT, lacto-N-fucopentaose I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and 2FLacNAc in a growth experiment according to the culture conditions described in Example 2, using SD CSM-Ura-His drop-out medium comprising lactose and LacNAc as precursors.

Example 41. Production of an Oligosaccharide Mixture Comprising 2FL, DiFL, LNB, 2FLNB, LN3, LNT and LNFP-II with a Modified S. cerevisiae Host

[0649] An S. cerevisiae strain is adapted for production of GDP-fucose and LNT and for expression of an a1,3/4-fucosidase and an -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: 22, 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: 25 and the -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: 21, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising 2FL, DiFL, LNB, 2FLNB, LN3, LNT and lacto-N-fucopentaose II (LNFP-II, Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1,4-Glc) when evaluated in a growth experiment according to the culture conditions described in Example 2, using SD CSM-Ura-His drop-out medium comprising lactose and N-acetylglucosamine as precursors.

Example 42. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LNFP-I and LNFP-V with a Modified S. cerevisiae Host

[0650] An S. cerevisiae strain is adapted for production of GDP-fucose and LNT and for expression of an -1,2-fucosyltransferase and an -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: 22, gmd from E. coli with SEQ ID NO: 11, fcl from E. coli with SEQ ID NO: 12, the -1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 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: 21, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LN3, LNT, LNFP-I, and LNFP-V when evaluated in a growth experiment according to the culture conditions described in Example 2, using SD CSM-Ura-His drop-out medium comprising lactose as precursor.

Example 43. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LNFP-III and LNFP-VI with a Modified S. cerevisiae Host

[0651] An S. cerevisiae strain is adapted for production of GDP-fucose and LNnT and for expression of an -1,2-fucosyltransferase and an -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: 22, gmd from E. coli with SEQ ID NO: 11, fcl from E. coli with SEQ ID NO: 12, the -1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04 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: 21, LgtA from N. meningitidis with SEQ ID NO: 18 and LgtB from N. meningitidis with SEQ ID NO: 20. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LN3, LNnT, LNFP-III and lacto-N-fucopentaose VI (LNFP-VI, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-) Glc) and lacto-N-fucopentaose VI (LNFP-VI, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-) Glc) when evaluated in a growth experiment according to the culture conditions described in Example 2, using SD CSM-Ura-His drop-out medium comprising lactose as precursor.

Example 44. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LNFP-I and LNFP-V with a Modified S. cerevisiae Host

[0652] An S. cerevisiae strain is adapted for production of GDP-fucose and LNT and for expression of an -1,2-fucosyltransferase and an -1,3-fucosyltransferase as described in Example 2 with a yeast artificial chromosome (YAC) comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 22, gmd from E. coli with SEQ ID NO: 11, fcl from E. coli with SEQ ID NO: 12, the -1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04, the truncated a1,3-fucosyltransferase from H. pylori with SEQ ID NO: 06, galE from E. coli with SEQ ID NO: 21, LgtA from N. meningitidis with SEQ ID NO: 18 and WbgO from E. coli 055:H7 with SEQ ID NO: 19. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LN3, LNT, LNFP-I and LNFP-V when evaluated in a growth experiment according to the culture conditions described in Example 2, using SD CSM-Ura-His drop-out medium comprising lactose as precursor.

Example 45. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LNFP-III and LNFP-VI with a Modified S. cerevisiae Host

[0653] An S. cerevisiae strain is adapted for production of GDP-fucose and LNnT and for expression of an -1,2-fucosyltransferase and an -1,3-fucosyltransferase as described in Example 2 with a yeast artificial chromosome (YAC) comprising constitutive transcriptional units for LAC12 from K. lactis with SEQ ID NO: 22, gmd from E. coli with SEQ ID NO: 11, fcl from E. coli with SEQ ID NO: 12, the -1,2-fucosyltransferase from H. pylori with SEQ ID NO: 04, the truncated a1,3-fucosyltransferase from H. pylori with SEQ ID NO: 06, galE from E. coli with SEQ ID NO: 21, LgtA from N. meningitidis with SEQ ID NO: 18 and LgtB from N. meningitidis with SEQ ID NO: 20. The mutant yeast strain is evaluated for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LN3, LNnT, LNFP-III and lacto-N-fucopentaose VI (LNFP-VI, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-) Glc) and lacto-N-fucopentaose VI (LNFP-VI, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-) Glc) when evaluated in a growth experiment according to the culture conditions described in Example 2, using SD CSM-Ura-His drop-out medium comprising lactose as precursor.

Example 46. Material and methods Bacillus subtilis

Media

[0654] 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.

[0655] Trace element mix consisted of 0.735 g/L CaCl.sub.2.Math.2H.sub.2O, 0.1 g/L MnCl.sub.2.Math.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.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).

[0656] 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.

[0657] 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 K.sub.2HPO.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 1 M KOH. Depending on the experiment lactose, LNB or LacNAc could be added.

[0658] 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

[0659] Bacillus subtilis 168, available at Bacillus Genetic Stock Center (Ohio, USA).

[0660] Plasmids for gene deletion via Cre/lox are constructed as described by Yan et al. (Appl. & Environm. Microbial., September 2008, pages 5556-5562). 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.

[0661] 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.

[0662] 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., IgtA from N. meningitidis (SEQ ID NO: 18) 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: 19). 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., IgtB from N. meningitidis (SEQ ID NO: 20).

Heterologous and Homologous Expression

[0663] 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.

[0664] 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

[0665] 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).

[0666] 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 1/3rd of the optical density measured at 600 nm.

Example 47. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, 2FLacNAc and 3-FLacNAc with a Modified B. subtilis Host

[0667] A B. subtilis strain is modified as described in Example 46 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 an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, 2FLacNAc and 3-FLacNAc in a growth experiment on MMsf medium comprising lactose and LacNAc according to the culture conditions provided in Example 46. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 48. Production of 2FL, DiFL, LNFP-I, LNFP-II, LNT and LN3 with a Modified B. subtilis Host

[0668] 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--aminotransferase (UniProt ID POCI73), the galactoside beta-1,3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (SEQ ID NO: 18), 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 O55:H7 (SEQ ID NO: 19) 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: 25. 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 46. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 49. Material and Methods Corynebacterium glutamicum

Media

[0669] 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 1000stock trace element mix.

[0670] 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 CuSO.sub.4, 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.

[0671] 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.

[0672] 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.

[0673] 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

[0674] Corynebacterium glutamicum ATCC 13032, available at the American Type Culture Collection.

[0675] Integrative plasmid vectors based on the Cre/loxP technique as described by Suzuki et al. (Appl. Microbiol. Biotechnol., 2005 Apr, 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.

[0676] 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.

[0677] In an example for LN3 production, a constitutive transcriptional unit comprising a galactoside beta-1,3-N-acetylglucosaminyltransferase like, e.g., IgtA from N. meningitidis (SEQ ID NO: 18) 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: 19). 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., IgtB from N. meningitidis (SEQ ID NO: 20).

Heterologous and Homologous Expression

[0678] 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.

[0679] 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

[0680] 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).

[0681] 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 1/3rd of the optical density measured at 600 nm.

Example 50. Production of 2FL, 3-FL, DiFL, LN3, LNnT, LNFP-III and Lacto-N-Neohexaose with a Modified C. glutamicum Host

[0682] 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: 18), 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: 20) 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 49. After 72 h of incubation, the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 51. Materials and Methods Chlamydomonas reinhardtii

Media

[0683] 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.40.7H.sub.2O, 1.6 g/L CoCl.sub.2.Math.6H.sub.2O, 1.6 g/L CuSO.sub.4.Math.5H.sub.2O and 1.1 g/L (NH.sub.4) 6MoO.sub.3.

[0684] 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.4C1, 4 g/L MgSO.sub.4.Math.7H.sub.2O and 2 g/L CaCl.sub.2).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

[0685] 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.

[0686] 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).

[0687] 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 (2, 50 uFD). 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.

[0688] In an example for production of UDP-galactose, C. reinhardtii cells are modified with transcriptional units comprising the genes encoding a galactokinase like, e.g., from Arabidopsis thaliana (KIN, UniProt ID Q9SEE5) and an UDP-sugar pyrophosphorylase like, e.g., USP from A. thaliana (UniProt ID Q9C511).

[0689] In an example for LN3 production, a constitutive transcriptional unit comprising a galactoside beta-1,3-N-acetylglucosaminyltransferase like, e.g., IgtA from N. meningitidis (SEQ ID NO: 18) 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: 19). 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., IgtB from N. meningitidis (SEQ ID NO: 20).

[0690] 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).

[0691] 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).

Heterologous and Homologous Expression

[0692] 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.

[0693] 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

[0694] 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.

[0695] 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 52. Production of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LacNAc, 2FLacNAc and 3-FLacNAc in Mutant C. reinhardtii Cells

[0696] C. reinhardtii cells are engineered as described in Example 51 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 Q9C511). 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: 20), 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 51. After 5 days of incubation, the cells are harvested, and the saccharide production is analyzed on UPLC.

Example 53. Production of an 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

[0697] The mutant C. reinhardtii cells as described in Example 52 are further adapted with a genomic knock-in of a constitutive transcriptional unit for the galactoside beta-1,3-N-acetylglucosaminyltransferase (IgtA) from N. meningitidis with SEQ ID NO: 18. 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 51. After 5 days of incubation, the cells are harvested, and the saccharide production is analyzed on UPLC.

Example 54. Materials and Methods Animal Cells

Isolation of Mesenchymal Stem Cells from Adipose Tissue of Different Mammals

[0698] 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

[0699] 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

[0700] 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.

[0701] 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.

[0702] 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

[0703] 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

[0704] 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 55. Making of an Oligosaccharide Mixture Comprising 2FL, 3-FL, DiFL, LN3, LNT and LNFP-I in a Non-Mammary Adult Stem Cell

[0705] Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 54 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: 18, the N-acetylglucosamine beta-1,4-galactosyltransferase LgtB from N. meningitidis with SEQ ID NO: 20, 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 54, cells are subjected to UPLC to analyze for production of an oligosaccharide mixture comprising 2FL, 3-FL, DiFL, LN3, LNT and LNFP-I.