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WO2025012358A1 - Production cellulaire d'oligosaccharides contenant du lacto-n-biose (ln3) - Google Patents

Production cellulaire d'oligosaccharides contenant du lacto-n-biose (ln3) Download PDF

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Publication number
WO2025012358A1
WO2025012358A1 PCT/EP2024/069604 EP2024069604W WO2025012358A1 WO 2025012358 A1 WO2025012358 A1 WO 2025012358A1 EP 2024069604 W EP2024069604 W EP 2024069604W WO 2025012358 A1 WO2025012358 A1 WO 2025012358A1
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lacto
beta
gal
seq
glcnac
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WO2025012358A9 (fr
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Sofie AESAERT
Joeri Beauprez
Annelies VERCAUTEREN
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Inbiose NV
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Inbiose NV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01094—Protein N-acetylglucosaminyltransferase (2.4.1.94)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of cultivation of genetically engineered cells.
  • the present invention describes (i) a method for the production of a lacto-N-triose (LN3)-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide by cultivating a genetically modified cell comprising a transporter protein; as well as (ii) the genetically engineered cell used in the method.
  • LN3 lacto-N-triose
  • oligosaccharides are gaining more and more attention as these diverse molecules exert a range of important biological activities and are widely distributed in all living organisms.
  • An example of such oligosaccharides are the milk oligosaccharides (MOs) (Usashima T. et al., 2011, Nova Biomedical Books, New York ISBN 978-1-61122-831-1).
  • MOs milk oligosaccharides
  • These oligosaccharides play important roles in a variety of normal physiological and pathological processes, such as cell metastasis, signal transduction, intercellular adhesion, inflammation and immune response.
  • An example of such saccharides are milk saccharides (Urashima T.
  • milk oligosaccharides i.e. (oligo)saccharides which are found in milk of animals such as mammals and humans (Urashima et al, 2011; Coppa et al, 2013).
  • MOs milk oligosaccharides
  • a replete amount of milk saccharide structures have been elucidated so far.
  • the majority of milk oligosaccharides found in animals such as mammals and humans comprise lactose at the reducing end (Urashima et al, 2011).
  • milk oligosaccharides comprise N-acetyllactosamine (Gal-pi,4- GIcNAc) or lacto-N-biose (Gal-pi,3-GlcNAc) at the reducing end (Urashima et al, 2011; Wrigglesworth et al, 2020, PLoS ONE 15(12); Urashima et al, 2013, Biosci. Biotechnol. Biochem 77(3): p. 455-466; Wei et al, 2018, Sci. Rep. 8:4688).
  • N-acetyllactosamine Gal-pi,4- GIcNAc
  • lacto-N-biose Gal-pi,3-GlcNAc
  • Examples hereof are 3-FLN (Gal-pi,4-(Fuc-al,3-)GlcNAc; also known as Lewis x antigen), 3'-SLN (Neu5Ac-a2,3-Gal-pi,4-GlcNAc), 6'-SLN (Neu5Ac-a2,6-Gal-pi,4-GlcNAc) (Urashima et al, 2011; Wrigglesworth et al, 2020; Wei et al, 2018).
  • Such milk more specifically, human milk is to date considered as the best food for newborns and infants. It is composed of several fractions of which milk oligosaccharides are the fourth largest fraction. Besides lactose, human milk, as well as milk of other mammals, contains various structurally diverse oligosaccharides which are also known as human milk oligosaccharides (HMOs) or mammalian milk oligosaccharides (MMOs), respectively (Urashima T. et al., 2011). The importance of MOs for mammalian and human infant nutrition is directly linked to their biological activities including protection of the neonate from pathogens, supporting development of the infant's immune system and cognitive abilities.
  • HMOs human milk oligosaccharides
  • MMOs mammalian milk oligosaccharides
  • HMOs and MMOs are further known to act as decoys to reduce the risk of infections by bacterial and viral pathogens which adhere to human cells by binding to these cells' surface glycoproteins. Additionally, various HMOs and MMOs possess an anti-inflammatory effect and act as immunomodulators (e.g. reducing the risk of developing food allergies). Altogether, these beneficial effects make milk oligosaccharides, especially mammalian (MMOs) and human milk oligosaccharides (HMOs), attractive components in the nutritional industry for the production of infant formulas or as dietary supplements for children and adults.
  • MMOs mammalian
  • HMOs human milk oligosaccharides
  • Saccharides such as milk saccharides may be chemically synthesized but is not attractive for several reasons including stereo-specificity issues, product impurities and high production cost. Therefore, over the last years serious efforts have been made to produce milk oligosaccharides in genetically engineered cells such as bacteria (e.g. Escherichia coli). While several milk oligosaccharides have been produced by genetically engineered host cells in the field, one is constantly looking for further improving the production yield of these oligosaccharides, i.e. reaching a higher titer (gram saccharide per liter cultivation medium) and/or a better cell performance index (gram saccharide per gram biomass).
  • titer gram saccharide per liter cultivation medium
  • cell performance index gram saccharide per gram biomass
  • LN3 lacto-N-triose
  • the invention provides a cell which is genetically engineered for the production of a lacto- N-triose (LN3)-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide, wherein said cell expresses, preferably overexpresses, a novel transporter protein.
  • LN3 lacto- N-triose
  • said transporter protein is able to transport a LN3-containing oligosaccharide and has a positive effect on the cellular, preferably fermentative, production of said LN3-containing oligosaccharide, providing a better yield, productivity, specific productivity and/or growth speed when used to genetically engineer a cell producing said LN3-containing oligosaccharide or oligosaccharide mixture comprising a LN3-containing oligosaccharide.
  • the invention provides a method for the production of a lacto-N-triose (LN3)- containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide, the method comprising the step of cultivating a cell according to the first aspect.
  • LN3 lacto-N-triose
  • the invention provides the use of a cell according to the first aspect for the production of a LN3-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide.
  • the invention provides the use of said novel transporter protein in the production of a
  • LN3-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide.
  • the invention provides a cell which is genetically engineered for the production of a lacto- N-triose (LN3)-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide, characterized in that said cell expresses, preferably overexpresses, a transporter protein comprising an amino acid sequence:
  • SEQ ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06 is preferably replaced with "SEQ ID NO 01, 13, 15 or 16", more preferably replaced with "SEQ ID NO 01, 13 or 15", even more preferably replaced with "SEQ ID NO 01 or 13", most preferably replaced with "SEQ ID NO 01".
  • SEQ ID NO 16 is preferably replaced with "SEQ ID NO 17, 18 or 19", more preferably replaced with “SEQ ID NO 17 or 18”, most preferably replaced with "SEQ ID NO 17”.
  • a protein e.g.
  • a transporter protein comprising an amino acid sequence having at least 80.0% sequence identity to the full-length amino acid sequence of a reference protein (indicated with a SEQ ID NO or Uniprot ID) preferably comprises an amino acid sequence having at least 85.0% 86.0% 87.0°% 87.5°% 88.0°% 89.0°% 90.0% 91.0°% 92.0°% 92.5°% 93.0°% 94.0°% 95.0% 96.0%, 97.0%, 97.5%, 98.0% or 99.0%, more preferably at least 85.0%, even more preferably at least 87.5%, even more preferably at least 90.0%, even more preferably at least 92.5%, even more preferably at least 95.0%, even more preferably at least 97.5%, even more preferably at least 98.0%, most preferably at least 99.0 %, sequence identity to the full length reference sequence.
  • a functional fragment of a polypeptide having at least 80.0% sequence identity to the full-length amino acid sequence of a reference protein preferably has at least 85.0%, 86.0%, 87.0%, 87.5%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 92.5%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 97.5%, 98.0% or 99.0%, more preferably at least 85.0%, even more preferably at least 87.5%, even more preferably at least 90.0%, even more preferably at least 92.5%, even more preferably at least 95.0%, even more preferably at least 97.5%, even more preferably at least 98.0%, most preferably at least 99.0 %, sequence identity to the full length reference sequence.
  • sequence identity of a protein is preferably determined by the program EMBOSS Needle 5.0 (https://galaxy-iuc.github.io/emboss-5.0-docs/needle.html), preferably with default parameters (the substitution matrix EBLOSUM62, the gap opening penalty 10, and the gap extension penalty 0.5).
  • a "conservative amino acid substitution” is the substitution of an amino acid by another structurally-related amino acid, i.e. having a similar sidechain.
  • a conservative amino acid substitution is preferably the substitution of: a hydrophobic amino acid (A, I, L, M, F, W, Y, V and G) by another hydrophobic amino acid (A, I, L, M, F, W, Y, V and G), a hydrophilic amino acid (S, T, C, N, Q and Y) by another hydrophilic amino acid (S, T, C, N, Q and Y), an amino acid with a positively charged side chain (R, H and K) by another amino acid with a positively charged side chain (R, H and K), an amino acid with a negatively charged side chain (D and E) by another amino acid with a negatively charged side chain (D and E), and/or an amino acid with a polar uncharged side chain (S, T, N and Q) by another amino acid with a polar uncharged side chain (S, T, N and Q).
  • a hydrophobic amino acid A, I, L, M, F, W, Y, V and G
  • a functional fragment of a reference protein or reference polypeptide refers to a polypeptide sequence which comprises or consists of an amount of consecutive amino acid residues from said reference protein or reference polypeptide and wherein said amount of consecutive amino acid residues is preferably at least 85.0% 86.0% 87.0% 87.5% 88.0% 89.0% 90.0% 91.0% 92.0% 92.5% 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 97.5%, 98.0% or 99.0%, more preferably at least 85.0%, even more preferably at least 87.5%, even more preferably at least 90.0%, even more preferably at least 92.5%, even more preferably at least 95.0%, even more preferably at least 97.5%, even more preferably at least 98.0%, most preferably at least 99.0 %
  • a reference transporter protein consists of 408 amino acid residues
  • a functional fragment of said reference transporter protein consists of at least 347 (i.e. at least 85.0 %) consecutive amino acids of said reference transporter protein.
  • a functional fragment of a reference protein or reference polypeptide refers to a polypeptide sequence that consists of the amino acid sequence of said reference protein or reference polypeptide, but wherein an amount of consecutive amino acid residues is missing and wherein said amount is preferably no more than 50.0%, 40.0 %, 30.0 %, 20.0%, 15.0%, 12.5%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.5%, 4.0%, 3.5%, 3.0 %, 2.5 %, 2.0 %, 1.5 %, 1.0 %, 0.5 % of the full-length of said protein represented by a SEQ ID NO, more preferably no more than 20.0 %, 15.0 %, 10.0 %, 9.0 %, 8.0 %, 7.0 %, 6.0 %, 5.0 %, 4.5 %, 4.0 %, 3.5 %, 3.0 %,
  • a functional fragment of a reference protein or reference polypeptide is functional, i.e. the fragment is able to transport a LN3-containing oligosaccharide (it is referred to the section "Oligosaccharide") and/or provides the cell according to the invention an improved production of said LN3-containing oligosaccharide compared to said cell with an identical genetic background but that lacks said transporter protein (as described later herein), preferably provides the cell according to the invention an improved production of said LN3- containing oligosaccharide compared to said cell with an identical genetic background but that lacks said transporter protein (as described later herein).
  • a transporter protein usually represented by a SEQ ID NO or Uniprot ID
  • said functional fragment retains at least 70.0%, more preferably at least 80.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, even more preferably at least 95.0%, most preferably at least 100.0 %, of the activity of the reference protein or reference polypeptide.
  • activity preferably refers to the transport of a LN3-containing oligosaccharide (it is referred to the section "Oligosaccharide”) and/or to an improved production of said LN3-containing oligosaccharide compared to said cell with an identical genetic background but that lacks said transporter protein (as described later herein), preferably refers to an improved production of said LN3-containing oligosaccharide compared to said cell with an identical genetic background but that lacks said transporter protein (as described later herein).
  • This can be assessed by the skilled person using his common general knowledge as exemplified in the present Examples.
  • the expression “at least 100.0% of the activity” refers to an activity which is equal or even higher than the activity of the reference protein or reference polypeptide.
  • a transporter protein according to the invention is functional, i.e. is able to transport a LN3-containing oligosaccharide (it is referred to the section "Oligosaccharide") and/or provides the cell according to the invention an improved production of said LN3-containing oligosaccharide compared to said cell with an identical genetic background but that lacks said transporter protein (as described later herein), preferably provides the cell according to the invention an improved production of said LN3-containing oligosaccharide compared to said cell with an identical genetic background but that lacks said transporter protein (as described later herein).
  • a transporter protein according to the invention has at least 70.0%, more preferably at least 80.0%, even more preferably at least 85.0%, even more preferably at least 90.0%, even more preferably at least 95.0%, most preferably at least 100.0 %, of the activity of the reference protein or reference polypeptide. Said "activity" is as defined earlier herein.
  • transporter protein is preferably replaced with “exporter protein”.
  • said transporter protein according to the invention is heterologous, i.e. said transporter protein originates from a source foreign to the particular cell.
  • said transporter protein according to the invention consists of at least 340, preferably at least 350, more preferably at least 360, even more preferably at least 370, even more preferably at least 380, even more preferably at least 390, most preferably at least 400, amino acids.
  • said transporter protein according to the invention consists of ⁇ 550, preferably ⁇ 525, more preferably ⁇ 500, even more preferably ⁇ 480, even more preferably ⁇ 470, even more preferably ⁇ 460, even more preferably ⁇ 450, even more preferably ⁇ 440, even more preferably ⁇ 430, most preferably ⁇ 420, amino acids.
  • a cell which is genetically engineered for the production of a lacto-N-triose (LN3)-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide is preferably replaced with the expression "a genetically engineered cell that is capable of producing a lacto-N-triose (LN3)-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide", more preferably replaced with the expression "a genetically engineered cell that produces a lacto-N-triose (LN3)-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide”.
  • said transporter protein according to the invention further: lacks one or more consecutive amino acids in its transmembrane domain 1 (TMl) compared to the TMl of the transporter protein represented by SEQ ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06, respectively, preferably lacks at least two consecutive amino acids in its TMl domain, more preferably lacks at least 8 consecutive amino acids in its TMl domain, most preferably lacks amino acids 1 to 16 of its TMl domain, compared to the TMl of the transporter protein represented by SEQ ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06, respectively; or comprises one or more non-conservative amino acid substitutions in its transmembrane domain 1 (TMl) compared to the TMl of the transporter protein represented by SEQ ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06, respectively, preferably at position: o 24, 28, 31 and/or 32 of TMl of the transporter protein represented by SEQ ID NO 01, o 26,
  • Such a transporter protein has a reduced ability to transport an antimicrobial agent compared to said transporter protein with an unmodified TMl (it is referred to WO2024/017987 in this regard).
  • said antimicrobial agent is selected from the list consisting of chloramphenicol, erythromycin, rifampin, tetracycline, puromycin, daunomycin, aminoglycosides and fluroquinolones, more preferably selected from the list consisting of chloramphenicol, erythromycin, rifampin, tetracycline, puromycin and daunomycin, even more preferably selected from the list consisting of chloramphenicol and tetracycline, most preferably chloramphenicol.
  • a reduced ability to transport an antimicrobial agent refers to a transporter which transports less of the antimicrobial agent than the unmodified transporter.
  • these agents are able to kill the cell if their concentrations in the cell's environment (i.e. medium) are at least the minimal inhibitory concentration (MIC), the skilled person can readily assess the transport of said agents by growing the cell at different concentrations of the antimicrobial agent.
  • a cell is not able to grow in a medium containing an antimicrobial agent if said cell is not able to grow in a medium containing 5 pg/mL, preferably 2 pg/mL, more preferably 1 pg/mL, of the antimicrobial agent.
  • transmembrane domain 1 transmembrane domain 1
  • TMl transporter protein
  • N-terminal transmembrane domain is interchangeably used herein.
  • the skilled person is well-aware that the transmembrane domains of a protein, such as a transporter protein according to the invention, can be predicted using publicly available algorithms.
  • An example hereof is DeepTMHMM, a deep learning protein language model-based algorithm that can detect and predict the toplogy of both alpha helical and beta barrels proteins with high accuracy (Hallgren et al, 2022,
  • DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks
  • bioRxiv doi: https://doi.org/10.1101/2022.04.08.487609
  • This algorithm is accessible via the link https://dtu.biolib.com/DeepTMHMM.
  • version 1.0.24 as released on 30 March 2023 was used.
  • TM1 of the transporter protein with SEQ ID NO 01 is represented by
  • TM1 of the transporter protein with SEQ ID NO 13 is represented by
  • TM1 of the transporter protein with SEQ ID NO 02 is represented by
  • PLALVLFEFSVYIANDMIQPGM (SEQ ID NO 08).
  • TM1 of the transporter protein with SEQ ID NO 03 is represented by AGSLAVLLGALDTYVVVTIM
  • TM1 of the transporter protein with SEQ ID NO 04 is represented
  • PLALVLFEFAVYIANDMAQPAM SEQ ID NO 10
  • TM1 of the transporter protein with SEQ ID NO 05 is represented
  • TM1 of the transporter protein with SEQ ID NO 06 is represented
  • TM1 of the transporter protein with SEQ ID NO 15 is represented
  • TM1 of the transporter protein with SEQ ID NO 16 is represented
  • TM1 of the transporter protein with SEQ ID NO 17 is represented
  • TM1 of the transporter protein with SEQ ID NO 18 is represented
  • TM1 of the transporter protein with SEQ ID NO 19 is represented
  • a "non-conservative amino acid substitution” is an amino acid substitution which is not a conservative amino acid substitution as defined earlier herein.
  • Tyrosine (Y) is sometimes classified as a hydrophobic amino acid (due to the presence of an aromatic ring) and sometimes as a hydrophilic amino acid (due to the presence of the hydroxyl substituent).
  • a tyrosine (Y) is classified herein as a hydrophilic amino acid.
  • a conservative amino acid substitution is preferably the substitution of: a hydrophilic amino acid (S, T, C, N, Q. and Y), preferably S, T, N, Q.
  • Y preferably T, N and/or Y, even more preferably N and/or Y, most preferably Y, into a hydrophobic amino acid (A, I, L, M, F, W, Y, V and G), preferably into A, I, L or V, more preferably into A, I or L, even more preferably into A or L, most preferably into A; and vice versa; and/or an amino acid with a negatively charged side chain (D and E) into a hydrophobic amino acid (A, I, L, M, F, W, Y, V and G), preferably into A, I, L or V, more preferably into A, I or L, even more preferably into A or L, most preferably into A; and/or an amino acid with a positively charged side chain (R, H and K) into a hydrophobic amino acid (A, I, L, M, F, W, Y, V and G), preferably into A, I, L or V, more preferably into A, I or L,
  • said transporter protein according to the invention further lacks all amino acids N-terminally from its TMl domain compared to the transporter protein represented by SEQ ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06, respectively.
  • said cell is selected from the list consisting of a microorganism, a plant cell, an animal cell, an insect cell or a protozoan cell, more preferably said cell is a microorganism, more preferably said cell is a bacterium or a yeast, even more preferably said cell is a bacterium, even more preferably said cell is a bacterium belonging to the genus of Escherichia or Bacillus, even more preferably said cell is a bacterium belonging to the genus of Escherichia, even more preferably said cell is Escherichia coli, even more preferably said cell is an Escherichia coli K-12 strain, most preferably said cell is Escherichia coli MG1655.
  • a microorganism is preferably a bacterium, a yeast or a fungus, more preferably a bacterium or a yeast, most preferably a bacterium.
  • a bacterium preferably belongs to the phylum of the Proteobacteria or the phylum of the Firmicutes or the phylum of the Cyanobacteria or the phylum Deinococcus-Thermus.
  • Said bacterium belonging to the phylum Proteobacteria belongs preferably to the family Enterobacteriaceae, preferably to the species Escherichia coli.
  • Said bacterium preferably relates to any strain belonging to the species Escherichia coli such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, said bacterium relates to cultivated Escherichia coli strains - designated as E. coli K12 strains - which are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. Well-known examples of the E.
  • coli K12 strains are K12 Wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200.
  • the present invention specifically relates to an E. coli K12 strain, more preferably an E. coli MG1655 strain.
  • Said bacterium belonging to the phylum Firmicutes belongs preferably to the Bacilli, preferably from the species Bacillus, such as Bacillus subtilis or, B. amyloliquefaciens.
  • Said bacterium belonging to the phylum Actinobacteria preferably belonging to the family of the Corynebacteriaceae, with members Corynebacterium glutamicum or C. afermentans, or belonging to the family of the Streptomycetaceae with members Streptomyces griseus or S. fradiae.
  • a yeast cell preferably belongs to the phylum of the Ascomycota or the phylum of the Basidiomycota or the phylum of the Deuteromycota or the phylum of the Zygomycetes.
  • Said yeast cell belongs preferably to the genus Saccharomyces (with members like e.g. Saccharomyces cerevisiae, S. bayanus, S. boulardii), Pichia (with members like e.g. Pichia pastoris, P. anomala, P. kluyveri), Komagataella, Hansunella, Kluyveromyces (with members like e.g. Kluyveromyces lactis, K.
  • Said yeast cell is more preferably selected from Pichia pastoris, Yarrowia lipolitica, Saccharomyces cerevisiae and Kluyveromyces lactis.
  • a fungus preferably belongs to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus.
  • a plant cell includes cells of flowering and non-flowering plants, as well as algal cells, for example Chlamydomonas, Chlorella, etc.
  • said plant cell is a tobacco, alfalfa, rice, cotton, rapeseed, tomato, corn, maize or soybean cell.
  • an animal cell is preferably derived from non-human mammals (e.g. cattle, buffalo, pig, sheep, mouse, rat), birds (e.g. chicken, duck, ostrich, turkey, pheasant), fish (e.g. swordfish, salmon, tuna, sea bass, trout, catfish), invertebrates (e.g.
  • human and non-human mammalian cells are preferably chosen from the list consisting of an epithelial cell like e.g. a mammary epithelial cell, an embryonic kidney cell (e.g.
  • an insect cell is preferably derived from Spodoptera frugiperda like e.g. Sf9 or Sf21 cells, Bombyx mori, Mamestra brassicae,
  • Trichoplusia ni like e.g. BTI-TN-5B1-4 cells or Drosophila melanogaster like e.g. Drosophila S2 cells.
  • a protozoan cell is preferably a Leishmania tarentolae cell.
  • said cell according to the invention is a single cell. It is further preferred that said cell according to the invention is an isolated cell.
  • said cell according to the invention has an improved production of said LN3-containing oligosaccharide compared to said cell with an identical genetic background but that lacks said transporter protein.
  • said improved production comprises: better titer of said saccharide (gram saccharide per liter), and/or better production rate r (gram saccharide per liter per hour), and/or better cell performance index (gram saccharide per gram biomass), and/or better specific productivity (gram saccharide per gram biomass per hour), and/or better yield on sucrose (gram saccharide per gram sucrose), and/or better sucrose uptake/conversion rate (gram sucrose per gram per hour), and/or better lactose conversion/consumption rate (gram lactose per hour), and/or enhanced growth speed of the cell.
  • said improved production comprises: better titer of said saccharide (gram saccharide per liter), and/or better production rate r (gram saccharide per liter per hour), and/or better cell performance index (gram saccharide per gram biomass), and/or better specific productivity (gram saccharide per gram biomass per hour).
  • said cell according to the invention expresses a glycosyltransferase, wherein said glycosyltransferase is a galactoside beta-1, 3-N-acetylglucosaminyltransferase that is involved in the synthesis of said LN3-containing oligosaccharide.
  • said cell is modified (preferably genetically modified) in the expression or activity of said galactoside beta-1, 3-N-acetylglucosaminyltransferase.
  • galactoside beta-1, 3-N-acetylglucosaminyltransferases are well-known to be suitable for the synthesis of said LN3-containing oligosaccharide.
  • said galactoside beta-1, 3-N- acetylglucosaminyltransferase is selected from the list consisting of LgtA (preferably from Neisseria meningitidis or Neisseria gonorrhoeae), P3GlcNAcT (preferably from Helicobacter pylori) and NagT (preferably from Pasteurella multocida), more preferably LgtA (preferably from Neisseria meningitidis or Neisseria gonorrhoeae).
  • LgtA preferably from Neisseria meningitidis or Neisseria gonorrhoeae
  • P3GlcNAcT preferably from Helicobacter pylori
  • NagT preferably from Pasteurella multocida
  • LgtA preferably from Neisseria meningitidis or Neisseria gonorrhoeae
  • said cell according to the invention further expresses one or more glycosyltransferases involved in the synthesis of said LN3-containing oligosaccharide, wherein said one or more glycosyltransferases is/are selected from the list consisting of a galactosyltransferase, a fucosyltransferare, a N-acetylglucosaminyltransferase, a N- acetylgalactosaminyltransferase and a sialyltransferase, preferably is/are selected from the list consisting of a galactosyltransferase, a fucosyltransferare, a N-acetylglucosaminyltransferase and a N- acetylgalactosaminyltransferase, more preferably is/are selected from a fucosyltransferase and a gal
  • said cell is modified (preferably genetically modified) in the expression or activity of at least one of said glycosyltransferases.
  • said galactosyltransferase is selected from the list consisting of a beta-1, 3- galactosyltransferase, a beta-1, 4-galactosyltransferase, an alpha-1, 3-galactosyltransferase and an alpha- 1,3-galactosyltransferase, more preferably a beta-1, 3-galactosyltransferase or a beta-1, 4- galactosyltransferase, most preferably a beta-1, 3-galactosyltransferase.
  • Said beta-1, 3- galactosyltransferase is preferably selected from the list consisting of WbgO (preferably from Escherichia coli) , FurA (preferable from Pseudogulbenkiania ferrooxidans) and WbdO (preferable from Salmonella enterica), more preferably wbgO (preferably from Escherichia coli).
  • Said beta-1, 4-galactosyltransferase is preferably selected from the list consisting of LgtB (preferably from Neisseria meningitidis), CpslaJ (preferably from Streptococcus agalactiae), GalT (preferably from Helicobacter pylori) and Lexl (preferably from Aggregatibacter aphrophilus), more preferably LgtB (preferably from Neisseria meningitidis).
  • LgtB preferably from Neisseria meningitidis
  • CpslaJ preferably from Streptococcus agalactiae
  • GalT preferably from Helicobacter pylori
  • Lexl preferably from Aggregatibacter aphrophilus
  • LgtB preferably from Neisseria meningitidis
  • said fucosyltransferase is selected from the list consisting of alpha-1, 2-fucosyltransferase, alpha-1, 3-fucosyltransferase, alpha-1, 4-fucosyltransferase and alpha-1, 6-fucosyltransferase, more preferably selected from the list consisting of alpha-1, 2-fucosyltransferase, alpha-1, 3-fucosyltransferase and alpha-1, 4-fucosyltransferase, most preferably alpha-1, 2-fucosyltransferase or alpha-1, 3- fucosyltransferase.
  • Said alpha-1, 2-fucosyltransferase is preferably FutC (preferable from Helicobacter pylori).
  • Said alpha-1, 3-fucosyltransferase is preferably FucT (preferable from Helicobacter pylori).
  • said N-acetylglucosaminyltransferase is a beta-1, 6-N-acetylglucosaminyltransferase.
  • said N-acetylgalactosaminyltransferase is a beta-1,3- or an alpha-1, 3-N- acetylgalactosaminyltransferase, more preferably an alpha-1, 3-N-acetylgalactoaminyltransferase, even more preferably selected from the list consisting of BgtA (preferable from Helicobacter mustelae), BoGT6a (preferable from Bacteroides ovatus) more preferably BgtA (preferable from Helicobacter mustelae).
  • BgtA preferable from Helicobacter mustelae
  • BoGT6a preferable from Bacteroides ovatus
  • BgtA preferable from Helicobacter mustelae
  • said sialyltransferase is chosen from the list consisting of alpha-2, 3-sialyltransferase, alpha- 2,6-sialyltransferase and alpha-2, 8-sialyltransferase, more preferably chosen from the list consisting of alpha-2, 3-sialyltransferase and alpha-2, 6-sialyltransferase.
  • Said alpha-2, 3-sialyltransferase is preferably ST3 (preferable from Pasteurella multocida).
  • Said alpha-2, 6-sialyltransferase is preferably ST6 (preferable from Photobacterium damselae).
  • said cell according to the invention comprises at least one metabolic pathway involved in the synthesis of said LN3-containing oligosaccharide or oligosaccharide mixture comprising said LN3-containing oligosaccharide.
  • Said at least one metabolic pathway is preferably one or more selected from the list consisting of: a) N-acetylglucosaminylation pathway comprising of (i) at least one N- acetylglucosaminyltransferase (preferably as defined herein) and (ii) UDP-GIcNAc which is donor for said N-acetylglucosaminyltransferase(s); b) fucosylation pathway comprising of (i) at least one fucosyltransferase (preferably as defined herein) and (ii) GDP-fucose which is donor for said fucosyltransferase(s); c) galactosylation pathway comprising of (i) at least one galactosyltransferase (preferably as defined herein) and (ii) UDP-galactose which is donor for said galactosyltransferase(s); d) N-acetylgalactosamin
  • Said at least one metabolic pathway is more preferably one or more selected from the list consisting of: a) N-acetylglucosaminylation pathway comprising of (i) at least one N- acetylglucosaminyltransferase (preferably as defined herein) and (ii) UDP-GIcNAc which is donor for said N-acetylglucosaminyltransferase(s); b) fucosylation pathway comprising of (i) at least one fucosyltransferase (preferably as defined herein) and (ii) GDP-fucose which is donor for said fucosyltransferase(s); c) galactosylation pathway comprising of (i) at least one galactosyltransferase (preferably as defined herein) and (ii) UDP-galactose which is donor for said galactosyltransferase(s); and d) N-acetylgalactos
  • Said at least one metabolic pathway is even more preferably one or more selected from: a) N-acetylglucosaminylation pathway comprising of (i) at least one N- acetylglucosaminyltransferase (preferably as defined herein) and (ii) UDP-GIcNAc which is donor for said N-acetylglucosaminyltransferase(s); and b) fucosylation pathway comprising of (i) at least one fucosyltransferase (preferably as defined herein) and (ii) GDP-fucose which is donor for said fucosyltransferase(s); and c) galactosylation pathway comprising of (i) at least one galactosyltransferase (preferably as defined herein) and (ii) UDP-galactose which is donor for said galactosyltransferase(s).
  • Said at least one metabolic pathway is most preferably one or more selected from: a) N-acetylglucosaminylation pathway comprising of (i) at least one N- acetylglucosaminyltransferase (preferably as defined herein) and (ii) UDP-GIcNAc which is donor for said N-acetylglucosaminyltransferase(s); and b) galactosylation pathway comprising of (i) at least one galactosyltransferase (preferably as defined herein) and (ii) UDP-galactose which is donor for said galactosyltransferase(s).
  • Said N-acetylglucosaminylation pathway optionally further comprises one or more enzymes and their respective genes selected from the list consisting of L-glutamine— D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N- acetylglucosamine-6-phosphate deacetylase, glucosamine 6-phosphate N-acetyltransferase, N- acetylglucosamine-l-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase and glucosamine-l-phosphate acetyltransferase.
  • enzymes and their respective genes selected from the list consisting of L-glutamine— D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase,
  • Said fucosylation pathway optionally further comprises one or more enzymes and their respective genes selected from the list consisting of mannose-6-phosphate isomerase, phosphomannomutase, mannose- 1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase and salvage pathway L-fucokinase/GDP-fucose pyrophosphorylase.
  • Said galactosylation pathway optionally further comprises one or more enzymes and their respective genes selected from the list consisting of galactose-l-epimerase, galactokinase, glucokinase, galactose-1- phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase and glucophosphomutase.
  • Said N-acetylgalactosaminylation pathway optionally further comprises one or more enzymes and their respective genes selected from the list consisting of L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, UDP-N-acetylglucosamine 4-epimerase, UDP-glucose 4- epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase.
  • enzymes and their respective genes selected from the list consisting of L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine 1-phosphate uri
  • Said sialylation pathway optionally further comprises one or more enzymes and their respective genes selected from the list consisting of L-glutamine— D-fructose-6-phosphate aminotransferase, glucosamine- 6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acetylglucosamine epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylglucosamine-6P 2- epimerase, Glucosamine 6-phosphate N-acetyltransferase, N-AcetylGlucosamine-6-phosphate phosphatase, N-acetylmannosamine-6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-l-phosphate
  • CMP-sialic acid is preferably “CMP-Neu5Ac”.
  • sialic acid is preferably replaced with “Neu5Ac” (N- acetylneuraminate and N-acetylneuraminic acid are interchangeably used for Neu5Ac).
  • At least one gene of said at least one metabolic pathway is genetically engineered.
  • a glycosyltransferase of said at least one metabolic pathway is genetically engineered.
  • a metabolic pathway for the production of UDP-GIcNAC, GDP-fucose, UDP-galactose, UDP-GalNAc and/or CMP-sialic acid is present in said cell according to the invention.
  • a metabolic pathway for the production of UDP-GIcNAc comprises one or more enzymes and their respective genes selected from (i) the list consisting of glucosamine 6-phosphate N- acetyltransferase, phosphatase (preferably a HAD-like phosphatase), L-glutamine— D-fructose-6- phosphate aminotransferase and UDP-glucose 4-epimerase, more preferably a glucosamine 6-phosphate N-acetyltransferase and a phosphatase (preferably a HAD-like phosphatase).
  • a cell according to the invention preferably comprises any one or more modification(s) selected from the list consisting of knock-out of an N-acetylglucosamine-6-phosphate deacetylase, overexpression of an L-glutamine— D-fructose-6-phosphate aminotransferase, over-expression of a phosphoglucosamine mutase and over-expression of an N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase.
  • a metabolic pathway for the production of GDP-fucose comprises one or more enzymes and their respective genes selected from a bifunctional fucose kinase/fucose-l-phosphate guanylyltransferase or the combination of a fucose kinase a fucose-l-phosphate guanylyltransferase.
  • a cell according to the invention preferably comprises any one or more modification(s) selected from the list consisting of a knock-out of an N-acetylglucosamine-6-phosphate deacetylase, over-expression of an L-glutamine— D-fructose-6-phosphate aminotransferase, overexpression of a phosphoglucosamine mutase and over-expression of an N-acetylglucosamine-1- phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase.
  • a metabolic pathway for the production of UDP-galactose comprises UDP-glucose-4- epimerase.
  • a cell according to the invention preferably comprises any one or more modification(s) selected from the list consisting of a knock-out of an bifunctional 5'-nucleotidase/UDP-sugar hydrolase encoding gene, knock-out of a galactose-l-phosphate uridylyltransferase encoding gene and over-expression of an UDP-glucose-4-epimerase encoding gene, preferably over-expression of an UDP-glucose-4-epimerase encoding gene.
  • a metabolic pathway for the production of UDP-GalNAc comprises an UDP-glucose-4- epimerase.
  • a cell according to the invention preferably comprises any one or more modification(s) selected from the list consisting of a knock-out of a bifunctional 5'-nucleotidase/UDP-sugar hydrolase encoding gene, knock-out of a galactose-l-phosphate uridylyltransferase encoding gene and over-expression of an UDP-glucose-4-epimerase encoding gene, preferably over-expression of an UDP-glucose-4-epimerase encoding gene.
  • said cell according to the invention produces a precursor saccharide for the synthesis of said LN3-containing oligosaccharide and/or wherein said cell takes up a precursor saccharide for the synthesis of said LN3-containing oligosaccharide, preferably wherein said precursor saccharide is lactose, optionally wherein said lactose further comprises a fucose.
  • said fucose is linked to a monosaccharide (preferably selected from the list consisting of glucose, N- acetylglucosamine and galactose) in an alpha-1,2-, alpha-1,3- or alpha-1, 4-linkage, preferably an alpha- 1,2- or an alpha-1, 3-linkage, more preferably an alpha-1, 3-linkage.
  • a monosaccharide preferably selected from the list consisting of glucose, N- acetylglucosamine and galactose
  • precursor saccharide refers to a saccharide which lacks at least one monosaccharide compared to the corresponding saccharide (i.e. LN3-containing oligosaccharide).
  • saccharide refers to a molecule comprising at least one monosaccharide, preferably it refers to a molecule consisting of one or more monosaccharide residue(s).
  • monosaccharide refers to a sugar that is not decomposable into simpler sugars by hydrolysis, is classed either an aldose or ketose, and contains one or more hydroxyl groups per molecule. Monosaccharides are hence saccharides containing only one simple sugar.
  • the cell according to the invention is genetically modified with one or more expression modules, preferably for the expression of a transporter protein according to the invention, a glycosyltransferase as described herein and/or an enzyme involved in a metabolic pathway as described herein.
  • the expression module(s) can be integrated in the genome of said cell or can be presented to said cell on a vector.
  • Said vector is preferably a plasmid.
  • said cell according to the invention is genetically engineered for the production of a lacto-N-triose (LN3)-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide.
  • LN3 lacto-N-triose
  • oligosaccharide preferably refers to a saccharide containing 2 up to and including 20 monosaccharides, i.e. the degree of polymerization (DP) is 2-20.
  • An oligosaccharide can be a linear structure or can include branches.
  • the linkage e.g. glycosidic linkage, galactosidic linkage, glucosidic linkage, etc.
  • Each monosaccharide can be in the cyclic form (e.g. pyranose or furanose form).
  • oligosaccharide can contain both alpha- and beta-glycosidic bonds or can contain only beta-glycosidic bonds. More preferably, the term "oligosaccharide” refers to a saccharide consisting of 3-12, preferably 3-11, more preferably 3-10, even more preferably 3-9, even more preferably 3-8, even more preferably 3-7, even more preferably 3-6, most preferably 3-5, monosaccharides.
  • x-y refers to a range from and including x to and including y.
  • 3-5 monosaccharides means that 3, 4 or 5 monosaccharides are present.
  • lacto-N-triose (LN3)-containing oligosaccharide refers to an oligosaccharide that comprises LN3, i.e. it refers to LN3 (GlcNAc-betal,3-Gal-betal,4-Glc) or an oligosaccharide that comprises LN3 and one or more additional monosaccharide(s).
  • Said monosaccharide(s) is/are preferably selected from the list consisting of galactose, fucose, N- acetylglucosamine and N-acetylgalactosamine, more preferably selected from the list consisting of galactose, fucose and N-acetylglucosamine.
  • said LN3-containing oligosaccharide according to the invention consists of at least 3, preferably at least 4, monosaccharides.
  • said LN3-containing oligosaccharide according to the invention preferably has a DP of at least 3, more preferably a DP of at least 4.
  • said LN3-containing oligosaccharide according to the invention consists of ⁇ 10, preferably ⁇ 9, more preferably ⁇ 8, even more preferably ⁇ 7, even more preferably ⁇ 6, most preferably ⁇ 5, monosaccharides.
  • said LN3-containing oligosaccharide according to the invention consists of 3-12, preferably 3-11, more preferably 3-10, even more preferably 3-9, even more preferably 3-8, even more preferably 3-7, even more preferably 3-6, most preferably 3-5, monosaccharides.
  • said LN3-containing oligosaccharide according to the invention preferably has a DP of 3-12, more preferably 3-11, even more preferably 3-10, even more preferably 3-9, even more preferably 3-8, even more preferably 3-7, even more preferably 3-6, most preferably 3-5.
  • oligosaccharide mixture and “mixture comprising different oligosaccharides” are used interchangeably herein.
  • the term “different” oligosaccharides preferably means “structurally different” or “structurally distinct”. These terms are hence preferably interchangeably used in the context of the present invention.
  • said oligosaccharide mixture is a mixture comprising at least two, preferably at least three, more preferably at least four, most preferably at least five, different oligosaccharides. At least one, preferably at least two, more preferably at least three, most preferably all, of said different oligosaccharides is/are a LN3-containing oligosaccharide as described herein.
  • said LN3-containing oligosaccharide or any one, preferably at least two, more preferably at least three, even more preferably at least four, most preferably all, of the oligosaccharides in said mixture is/are a milk oligosaccharide (MO), more preferably a mammalian milk oligosaccharide (MMO), most preferably a human milk oligosaccharide (HMO).
  • MMOs mammalian milk oligosaccharides
  • MMOs comprise oligosaccharides present in milk found in any phase during lactation including colostrum milk from humans (i.e.
  • human milk oligosaccharides or HMOs human milk oligosaccharides or HMOs
  • mammals including but not limited to cows (Bos Taurus), sheep (Ovis aries), goats (Capra aegagrus hircus), bactrian camels (Camelus bactrianus), horses (Eguusferus caballus), pigs (Sus scropha), dogs (Canis lupus familiaris), ezo brown bears (Ursus arctos yesoensis), polar bear (Ursus maritimus), Japanese black bears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asian elephants (Elephas maximus), African elephant (Loxodonta africana), giant anteater (Myrmecophaga tridactyla), common bottlenose dolphins (T
  • milk oligosaccharides comprise N- acetyllactosamine (Gal-pi,4-GlcNAc) or lacto-N-biose (Gal-pi,3-GlcNAc) at the reducing end (Urashima et al, 2011; Wrigglesworth et al, 2020, PLoS ONE 15(12); Urashima et al, 2013, Biosci. Biotechnol. Biochem 77(3): p. 455-466; Wei et al, 2018, Sci. Rep. 8:4688).
  • N- acetyllactosamine Gal-pi,4-GlcNAc
  • lacto-N-biose Gal-pi,3-GlcNAc
  • milk saccharides comprise milk glycosaminoglycans (GAGs; Coppa et al, 2013; Rai et al, 2021, Int. J. Biol. Macromolecules, 193(A): p. 137-144).
  • said LN3-containing oligosaccharide according to the invention is not a glycosaminoglycan.
  • said LN3-containing oligosaccharide or any one, preferably at least two, more preferably at least three, even more preferably at least four, most preferably all, of the oligosaccharides in said mixture comprises a lactose at its reducing end, more preferably LN3 at its reducing end.
  • said LN3-containing oligosaccharide is selected from the list consisting of Lacto-N-triose II (LN3, LNT-II), GlcNAc-beta-l,6-(GlcNAc-beta-l,3-)Gal-beta-l,4-Glc, Lacto-N- neotetraose (LNnT), Lacto-N-tetraose (LNT), Gal-alpha-1, 3-Gal-beta-l,4-GlcNAc-beta-l,3-Gal-beta-l, 4- Glc, Gal-alpha-1, 3-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-Glc, GlcNAc-beta-l,6-(Gal-beta-l,4- GlcNAc-beta-bet,6
  • said LN3-containing oligosaccharide is selected from the list consisting of Lacto-N-triose II (LN3, LNT-II), GlcNAc-beta-l,6-(GlcNAc-beta-l,3-)Gal-beta-l,4-Glc, Lacto-N-neotetraose (LNnT), Lacto-N-tetraose (LNT), Gal-alpha-1, 3-Gal-beta-l,4-GlcNAc-beta-l,3-Gal- beta-l,4-Glc, Gal-alpha-1, 3-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-Glc, GlcNAc-beta-l,6-(Gal-beta-l,
  • said "sialic acid” preferably has a nine-carbon backbone (i.e. a nine-carbon sialic acid) or an eight-carbon backbone (i.e. an eight-carbon sialic acid), more preferably a nine-carbon backbone (preferably selected from the list consisting of Neu5Ac; Neu4Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4, Neu4,5,7,8,9Ac5 and Neu5Gc; more preferably said nine-carbon sialic acid is Neu5Ac, i.e.
  • N-acetylneuraminic acid A sialic acid having a nine-carbon backbone is well-known to the skilled person and refers to a group of monosaccharides that are derived from an acidic, nine-carbon parent compound being either N-acetylneuraminic acid (Neu5Ac) or 2-keto-3-deoxynononic acid (Kdn; a desamino form of N-acetylneuraminic acid), by modification such as addition of acetyl, phosphate, methyl, sulfate and/or lactyl groups. Further, the N-acetylgroup of Neu5Ac can be hydroxylated giving rise to N- glycolylneuraminic acid (Neu5Gc).
  • sialic acid having a nine-carbon backbone More than 50 different examples of a sialic acid having a nine-carbon backbone are known (Essentials of Glycobiology, 2 nd edition, 2009, Chapter 14, Varki and Schauer).
  • a sialic acid having an eight-carbon backbone is structurally related to a sialic acid having a nine-carbon backbone, in particular related to Kdn (Essentials of Glycobiology, 2 nd edition, 2009, Chapter 14, Varki and Schauer).
  • the term "sialic acid having an eight-carbon backbone” is preferably replaced with "eight-carbon 2-keto-3-deoxyoctonic acid".
  • said LN3-containing oligosaccharide according to the invention is LN3, a LNT-containing oligosaccharide or a LNnT-containing oligosaccharide, preferably a LNT-containing oligosaccharide or a LNnT-containing oligosaccharide.
  • LNnT- containing oligosaccharide refers to an oligosaccharide that comprises LNnT and optionally one or more additional monosaccharide(s).
  • Said monosaccharide(s) is/are preferably selected from the list consisting of galactose, fucose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid, more preferably selected from the list consisting of galactose, fucose, N-acetylglucosamine and N-acetylgalactosamine, even more preferably selected from the list consisting of galactose, fucose and N-acetylglucosamine, most preferably fucose.
  • said LNnT-containing oligosaccharide is on oligosaccharide comprising LNnT at its reducing end.
  • said LNnT-containing oligosaccharide (and throughout the description and claims) is selected from the list consisting of Lacto-N-neotetraose (LNnT), Gal-alpha-1, 3-Gal-beta-l, 4- GlcNAc-beta-l,3-Gal-beta-l,4-Glc, GlcNAc-beta-l,6-(Gal-beta-l,4-GlcNAc-beta-l,3-)Gal-beta-l,4-Glc, lacto-N-neopentaose, para-Lacto-N-neopentaose, para-lacto-N-hexaose (pLNH), lacto-N-neohexaose (LNnH), para-Lacto-N-neohexaose (pLNnH), lacto-N
  • said LNnT-containing oligosaccharide (and throughout the description and claims) is selected from the list consisting of Lacto-N-neotetraose (LNnT), Gal-alpha-1, 3- Gal-beta-l,4-GlcNAc-beta-l,3-Gal-beta-l,4-Glc, GlcNAc-beta-l,6-(Gal-beta-l,4-GlcNAc-beta-l,3-)Gal- beta-l,4-Glc, lacto-N-neopentaose, para-Lacto-N-neopentaose, para-lacto-N-hexaose (pLNH), lacto-N- neohexaose (LNnH), para-Lacto-N-neohexaose (pLNnH), lacto-N-
  • Lacto-N-neotetraose LNnT
  • para-lacto-N-hexaose pLNH
  • lacto-N-neohexaose LNnH
  • para-Lacto-N-neohexaose pLNnH
  • Lacto-N-neofucopentaose I LNnFP I
  • lacto-N-fucopentaose III lacto-N-neofucopentaose V
  • lacto-N-neodifucohexaose LNnDFH
  • lacto-N-neodifucohexaose LNnDFH
  • LNnT Lacto-N-neofucopentaosetraose
  • pLNH para-lacto-N-hexaose
  • lacto-N-nH lacto-N-neohexaose
  • LNT- containing oligosaccharide refers to an oligosaccharide that comprises LNT and optionally one or more additional monosaccharide(s).
  • Said monosaccharide(s) is/are preferably selected from the list consisting of galactose, fucose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid, more preferably selected from the list consisting of galactose, fucose, N-acetylglucosamine and N-acetylgalactosamine, even more preferably selected from the list consisting of galactose, fucose and N-acetylglucosamine, most preferably fucose.
  • said LNT-containing oligosaccharide is on oligosaccharide comprising LNT at its reducing end.
  • said LNT-containing oligosaccharide (and throughout the description and claims) is selected from the list consisting of Lacto-N-tetraose (LNT), Gal-alpha-1, 3-Gal-beta-l, 3-GlcNAc- beta-l,3-Gal-beta-l,4-Glc, GlcNAc-beta-l,6-(Gal-beta-l,3-GlcNAc-beta-l,3-)Gal-beta-l,4-Glc, Lacto-N- pentaose, para-Lacto-N-pentaose, GlcNAc-beta-1, 3-Gal-beta-l, 3-GlcNAc-beta-l, 3-Gal-beta-l, 4-Glc, GalNAc-beta-l,3-LNT, Gal-beta-1, 3-GalNAc-bet
  • said LNT-containing oligosaccharide (and throughout the description and claims) is selected from the list consisting of Lacto-N-tetraose (LNT), Gal-alpha-1, 3-Gal- beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-Glc, GlcNAc-beta-l,6-(Gal-beta-l,3-GlcNAc-beta-l,3-)Gal-beta- 1,4-Glc, Lacto-N-pentaose, para-Lacto-N-pentaose, GlcNAc-beta-l,3-Gal-beta-l,3-GlcNAc-beta-l,3-Gal- beta-l,4-Glc, GalNAc-beta-l,3-LNT, Gal-beta-1, 3-GalNAc
  • LNT Lac
  • said LN3-containing oligosaccharide according to the invention is a neutral oligosaccharide.
  • a "neutral" oligosaccharide as used herein and as generally understood in the state of the art is an oligosaccharide that has no negative charge originating from a carboxylic acid group.
  • said LN3-containing oligosaccharide according to the invention does not comprise a fucose.
  • a transporter protein comprising an amino acid: selected from SEQ ID NO 01 or 13 (preferably SEQ ID NO 01); or having at least 80.0 % sequence identity to the full-length amino acid sequence of SEQ ID NO 01 or 13 (preferably SEQ ID NO 01); or that is a functional fragment of SEQ ID NO 01 or 13 (preferably SEQ ID NO 01), preferably wherein said fragment retains at least 70.0 %, more preferably at least 80.0 %, even more preferably at least 85.0 %, even more preferably at least 90.0 %, even more preferably at least 95.0 %, most preferably at least 100.0 %, of the activity of the full-length sequence represented by SEQ ID NO 01 or 13 (preferably SEQ ID NO 01), respectively; or that is a functional fragment of a polypeptide having at least 80.0 % sequence identity to the full- length amino acid sequence of SEQ ID NO 01 or 13 (preferably SEQ ID NO 01), preferably wherein said fragment retains at least 70.0 %, more preferably at least
  • a transporter protein comprising an amino acid: represented by SEQ ID NO 13; or having at least 80.0 % sequence identity to the full-length amino acid sequence of SEQ ID NO 13; or that is a functional fragment of SEQ ID NO 13, preferably wherein said fragment retains at least 70.0 %, more preferably at least 80.0 %, even more preferably at least 85.0 %, even more preferably at least 90.0 %, even more preferably at least 95.0 %, most preferably at least 100.0 %, of the activity of the full-length sequence represented by SEQ ID NO 13; or that is a functional fragment of a polypeptide having at least 80.0 % sequence identity to the full- length amino acid sequence of SEQ ID NO 13, preferably wherein said fragment retains at least 70.0 %, more preferably at least 80.0 %, even more preferably at least 85.0 %, even more preferably at least 90.0 %, even more preferably at least 95.0 %, most preferably at least 100.0 %, of
  • a transporter protein comprising an amino acid: represented by SEQ ID NO 15 or 16, preferably SEQ ID NO 15; or having at least 80.0 % sequence identity to the full-length amino acid sequence of SEQ ID NO 15 or 16, preferably SEQ ID NO 15; or that is a functional fragment of SEQ ID NO 15 or 16 (preferably SEQ ID NO 15), preferably wherein said fragment retains at least 70.0 %, more preferably at least 80.0 %, even more preferably at least 85.0 %, even more preferably at least 90.0 %, even more preferably at least 95.0 %, most preferably at least 100.0 %, of the activity of the full-length sequence represented by SEQ ID NO 15 or 16 (preferably SEQ ID NO 15), respectively; or that is a functional fragment of a polypeptide having at least 80.0 % sequence identity to the full- length amino acid sequence of SEQ ID NO 15 or 16 (preferably SEQ ID NO 15), preferably wherein said fragment retains at least 70.0 %, more preferably at least
  • a transporter protein comprising an amino acid:
  • SEQ ID NO 02, 03, 04, 05 or 06 preferably SEQ ID NO 03, 04, 05 or 06
  • SEQ ID NO 02, 03, 04, 05 or 06 amino acid sequence of SEQ ID NO 02, 03, 04, 05 or 06
  • SEQ ID NO 03, 04, 05 or 06 preferably SEQ ID NO 03, 04, 05 or 06
  • said fragment retains at least 70.0 %, more preferably at least 80.0 %, even more preferably at least 85.0 %, even more preferably at least 90.0 %, even more preferably at least 95.0 %, most preferably at least 100.0 %, of the activity of the full-length sequence represented by SEQ ID NO 02, 03, 04, 05 or 06 (preferably SEQ ID NO 03, 04, 05 or 06), respectively; or that is a functional fragment of a polypeptide having at least 80.0 % sequence identity to the full- length
  • the invention provides a method for the production of a lacto-N-triose (LN3)- containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide, the method comprising the step of:
  • Said LN3-containing saccharide and said oligosaccharide mixture are preferably as described in the first aspect of the invention (it is referred to the Section "Oligosaccharide” in this regard).
  • permissive conditions are understood to be conditions relating to physical or chemical parameters including but not limited to temperature, pH, pressure, osmotic pressure and product/precursor/acceptor concentration.
  • the permissive conditions include a temperature-range of 30 +/- 20 degrees centigrade and/or a pH-range of 7 +/- 3.
  • the method according to the invention as described herein results in the production of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, of the LN3- containing oligosaccharide or oligosaccharide mixture according to the invention in the whole broth or in the supernatant.
  • said cell according to the invention is cultivated in a suitable cultivation medium to form a cultivation broth and under conditions permissive for the production of said LN3-containing oligosaccharide or said oligosaccharide mixture.
  • said cell according to the invention is cultivated in a minimal salt medium with a carbon source on which said cell grows.
  • the minimal salt medium contains sulphate, phosphate, chloride, ammonium, calcium ion, magnesium ion, sodium ion, potassium ion, iron ion, copper ion, zinc ion, manganese ion, cobalt ion, and/or selenium ion.
  • said cell according to the invention grows on a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, a complex medium or a mixture thereof as the main carbon source.
  • main is meant the most important carbon source for the bioproducts of interest, biomass formation, carbon dioxide and/or by-products formation (such as acids and/or alcohols, such as acetate, lactate, and/or ethanol), i.e.
  • said carbon source is the sole carbon source for said organism, i.e. 100 % of all the required carbon is derived from the above-indicated carbon source.
  • Common main carbon sources comprise but are not limited to glucose, glycerol, fructose, maltose, lactose, arabinose, malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, sucrose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, corn-steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvate.
  • complex medium is meant a medium for which the exact constitution is not determined. Examples are molasses, corn steep liquor, peptone, tryptone or yeast extract. It is preferred in the context of the present invention, that said energy source is added, preferably continuously added, to the culture medium, preferably together with said lactose as described herein.
  • said carbon source comprises one or more of glucose, fructose, mannose, sucrose, maltose, corn steep liquor, lactose, galactose, high fructose syrup, starch, cellulose, hemi-cellulose, malto-oligosaccharides, trehalose, glycerol, acetate, citrate, lactate and pyruvate.
  • step (a) comprises at least one of the following steps: i) Adding to the culture medium a lactose feed comprising at least 50, preferably at least 75, more preferably at least 100, even more preferably at least 120, most preferably at least 150 gram, of lactose per liter of initial reactor volume wherein the total reactor volume ranges from 250 mL (milliliter) to 10.000 m 3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, even more preferably less than 2-fold, of the volume of the culture medium before the addition of said lactose feed; ii) Adding a lactose feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days or 5 days by means of a feeding solution; iii) Adding a lactose feed in a continuous manner to the culture medium over the course
  • a lactose feed is accomplished by adding lactose from the beginning of the cultivating in a concentration of at least 5mM, preferably in a concentration of 30, 40, 50, 60, 70, 80, 90, 100 or 150 mM, more preferably in a concentration of > 300 mM.
  • a lactose feed is accomplished by adding lactose to the cultivation medium in a concentration, such that throughout the production phase of the cultivation a lactose concentration of at least 5 mM, preferably at last 10 mM, more preferably at least 15 mM, even more preferably at least 20 mM, even more preferably at least 25 mM, most preferably at least 30 mM is obtained.
  • said cell according to the invention is cultivated for at least 24 hours, preferably at least 36 hours, more preferably at least 48 hours, even more preferably at least 60 hours, even more preferably at least 72 hours, even more preferably at least 84 hours, even more preferably at least 96 hours, most preferably at least 120 hours.
  • a carbon-based substrate is provided, preferably sucrose, in the culture medium for 3 or more days, preferably up to 7 days; and/or a carbon-based substrate, preferably sucrose, is provided in the culture medium at a concentration of at least 100, advantageously at least 105, more advantageously at least 110, even more advantageously at least 120 grams, per liter of initial culture volume, preferably in a continuous manner, so that the final volume of the culture medium is not more than three-fold, advantageously not more than two-fold, more advantageously less than two-fold of the volume of the culturing medium before the cultivation.
  • a first phase of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the culture medium before the lactose is added to the culture medium in a second phase.
  • a carbon-based substrate preferably glucose or sucrose
  • the lactose is added already in the first phase of exponential growth together with the carbon-based substrate.
  • a step of separating, preferably purifying i.e. purification step
  • said LN3-containing oligosaccharide from the cultivation broth or separating, preferably purifying, any one, preferably at least two, more preferably at least three, even more preferably at least four, most preferably all, of the oligosaccharides in said mixture from the cultivation broth is present.
  • Said separation step preferably purification step, provides a solution, preferably an aqueous solution, comprising a separated/purified LN3-containing oligosaccharide or a mixture of at least one, preferably at least two, more preferably at least three, even more preferably at least four, most preferably all, separated/purified oligosaccharides of said mixture.
  • the term "separating from said cultivation broth” means harvesting, collecting or retrieving said LN3-containing oligosaccharide or at least one, preferably at least two, more preferably at least three, even more preferably at least four, most preferably all, oligosaccharides of said mixture from the cell and/or the medium of its growth.
  • the terms "at least one oligosaccharide”, “at least two oligosaccharide”, “at least three oligosaccharide”, “at least four oligosaccharide” and “all oligosaccharides” in the context of the oligosaccharide mixture according to the invention preferably comprises at least the LN3-containing oligosaccharide according to the invention.
  • said LN3-containing oligosaccharide or at least one, preferably at least two, more preferably at least three, even more preferably at least four, most preferably all, oligosaccharides of said mixture can be separated, preferably purified, in a conventional manner from the aqueous culture medium in which the cell was grown.
  • said separation step comprises at least one step selected from the list consisting of clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography (such as but not limited to cation exchange, anion exchange, mixed bed ion exchange), hydrophobic interaction chromatography, gel filtration (i.e. size exclusion chromatography) and ligand exchange chromatography. With the exception of size exclusion chromatography, proteins and related impurities are retained by a chromatography medium or a selected membrane.
  • said method according to the invention further comprises the step of purifying said LN3-containing oligosaccharide or any one, preferably at least two, more preferably at least three, even more preferably at least four, most preferably all, of the saccharides in said mixture.
  • said purifying comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying or lyophilization.
  • said method comprises a further separation step, preferably a purification step, which is at least one step selected from the list consisting of use of (activated) charcoal or carbon, nanofiltration, ultrafiltration and ion exchange, to remove any remaining DNA, protein, LPS, endotoxins, or other impurity.
  • a purification step which is at least one step selected from the list consisting of use of (activated) charcoal or carbon, nanofiltration, ultrafiltration and ion exchange, to remove any remaining DNA, protein, LPS, endotoxins, or other impurity.
  • Alcohols such as ethanol, and aqueous alcohol mixtures can also be used.
  • said separation step, preferably purification step, of a method according to the invention comprises:
  • the clarification is combined with the removal of salts and/or medium components.
  • the clarification is combined with the step of concentrating the LN3-containing oligosaccharide or oligosaccharide mixture in the clarified cultivation.
  • the clarification is combined with the removal of salts and/or medium components and further combined with the step of concentrating the LN3-containing oligosaccharide or oligosaccharide mixture resulting from the step of removal of salts and/or medium components.
  • the clarification is combined with the step of concentrating the LN3-containing oligosaccharide or oligosaccharide mixture and further combined with the removal of salts and/or medium components of the LN3-containing oligosaccharide or oligosaccharide mixture resulting from the step of concentrating.
  • said LN3-containing oligosaccharide or said mixture of oligosaccharides are obtained in large quantities and at high purity.
  • step (iii) preferably comes before step (ii).
  • the separation preferably purification, preferably involves clarifying [i.e. step (i)] the LN3-containing oligosaccharide or oligosaccharide mixture containing cultivation broth to remove suspended particulates and contaminants, particularly cells, cell components, insoluble metabolites and debris produced by culturing said cell.
  • the cultivation broth containing the produced LN3-containing oligosaccharide or oligosaccharide mixture can be clarified in a conventional manner.
  • clarification is done by centrifugation, flocculation, decantation, ultrafiltration and/or filtration.
  • the step i) of clarifying the cultivation broth comprises one or more of clarification, clearing, filtration, microfiltration, centrifugation, decantation and ultrafiltration, preferably said step i) further comprising use of a filter aid and/or flocculant.
  • step i) comprises subjecting the cultivation broth to two membrane filtration steps using different membranes (i.e. different cut-offs).
  • step i) of clarifying the cultivation broth further comprises use of a filtration aid, preferably an adsorbing agent, more preferably active carbon.
  • step (i) comprises a first step of clarification by microfiltration.
  • step i) comprises a first step of clarification by centrifugation.
  • step i) comprises a first step of clarification by flocculation.
  • step i) comprises a first step of clarification by ultrafiltration.
  • step (i) comprises ultrafiltration.
  • the ultrafiltration in step i) has a molecular weight cut-off equal to or higher than 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10 kDa, 11 kDa, 12kDa, 13 kDa, 14 kDa, 15 kDa.
  • step i) comprises two consecutive ultrafiltrations, and wherein the membrane molecular weight cut-off of the first ultrafiltration is higher than that of the second ultrafiltration.
  • step i) is preceded by an enzymatic treatment.
  • the enzymatic treatment comprises incubation of the cultivation broth with one or more enzymes selected from the group consisting of: glycosidase, lactase, b-galactosidase, fucosidase, sialidase, maltase, amylase, hexaminidase, glucuronidase, trehalase, and invertase.
  • the enzymatic treatment converts lactose and/or sucrose to monosaccharides.
  • the separation, preferably purification preferably involves removing salts and/or medium components [i.e.
  • step (ii)] comprising proteins, as well as peptides, amino acids, RNA and DNA and any endotoxins and glycolipids that could influence purity, from the cultivation broth containing the LN3-containing oligosaccharide or oligosaccharide mixture, preferably after it has been clarified.
  • proteins, salts, by-products, color and other related impurities are removed from the LN3-containing oligosaccharide or oligosaccharide mixture containing (clarified) cultivation broth by ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange (such as but not limited to cation exchange, anion exchange, mixed bed ion exchange), hydrophobic interaction chromatography and/or gel filtration (i.e., size exclusion chromatography), particularly by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography.
  • ion exchange such as but not limited to cation exchange, anion exchange, mixed bed ion exchange
  • hydrophobic interaction chromatography and/or gel filtration i.e., size exclusion chromatography
  • step ii) of removing salts and/or medium components from the cultivation broth, preferably clarified cultivation broth comprises at least one or more of nanofiltration, dialysis, electrodialysis, use of activated charcoal or carbon, use of charcoal, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration, ligand exchange chromatography, column chromatography, cation exchange adsorbent resin, and use of ion exchange resin.
  • step ii) of removing salts and/or medium components from the (clarified) cultivation broth by ion exchange is any one or more of cation exchange, anion exchange, mixed bed ion exchange, simulated moving bed chromatography.
  • step ii) of removing salts and/or medium components from the (clarified) cultivation broth comprises anion exchange, preferably wherein said anion exchange resin has a moisture content of 30-48%, and preferably is a gel type anion exchanger.
  • anion exchanger is preferably selected from the group comprising Dowex 1-X8, XA4023, XA3112, DIAION SA20A, DIAION SA10A, preferably in OH- form.
  • Such anion exchange treatment is very performant for a saccharide mixture solution purification wherein the saccharide mixture comprises charged oligosaccharide, especially sialylated saccharides.
  • such anion exchange resin can be used in a pure anion exchange step combined with a cation exchange step or used in a mixed bed ion exchange setting.
  • step ii) comprises a step of cation exchange combined with a step of anion exchange, preferably wherein the anion exchange resin has a moisture content of 30-48% and preferably is a gel type anion exchanger, preferably as described herein.
  • the step of cation exchange precedes the step of anion exchange.
  • the anion exchange resin preferably having a moisture content of 30-48 percent, is preferably a gel type anion exchanger which desalts the (clarified) cultivation broth, though without thereby binding the charged, e.g. sialyl, group containing saccharides, which oligosaccharides are also present in salt form.
  • this involves an anion exchange resin which has selectivity for negatively charged minerals, but not for sialyllactose.
  • the moisture content that is, the water content
  • the desalting capacity starts to become too low to yield an effective process.
  • anion exchange resin mentioned is preferably and usually in the free base form (hydroxide form) because this results in a greatest possible desalting capacity.
  • Suitable anion exchange resins are strongly cross-linked polystyrene-divinylbenzene gels, such as Diaion SA20A, Diaion WA20A.
  • step ii) comprises a treatment with a mixed bed ion exchange resin.
  • a mixed bed ion exchange resin is a mixed bed column of Diaion SA20A and Amberlite FPC 22H mixed in a ratio 1,1:1 to 1,9:1.
  • such mixed bed ion exchange resin comprises an anion exchange resin, preferably having a moisture content of 30-48% and preferably being microporous or a gel type anion exchanger. As explained above, such anion exchange type is very useful in the purification of solutions comprising charged saccharide.
  • step ii) comprises nanofiltration and/or electrodialysis.
  • said nanofiltration and/or electrodialysis is performed twice. More preferably, said nanofiltration and/or electrodialysis steps are performed consecutively.
  • the ultrafiltration permeate of step i) is nanofiltered and/or electrodialysed in step ii).
  • the cationic ion exchanger treatment is a strongly acidic cation exchanger treatment, preferably treatment with a strong cation exchange resin in H+ form, K+ or Na+ form.
  • step (i) is ultrafiltration
  • step (ii) is nanofiltration and/or electrodialysis treatment combined with treatment with an ion exchange resin and/or chromatography.
  • the ion exchange resin is a strongly acidic cation exchange resin and/or a weakly basic anion exchange resin. More preferably, the ion exchange resin is a strongly acidic cation exchange resin and a weakly basic anion exchange resin.
  • step (ii) comprises treatment with a strong cation exchange resin in H+ form and a weak anion exchange resin in free base form, preferably in Cl- form, alternatively preferably in OH- form.
  • the treatment with a strong cation exchange resin in H+-form is directly followed by a treatment with a weak anion exchange resin in free base form.
  • the method does not comprise electrodialysis.
  • step (i) is ultrafiltration
  • step (ii) is nanofiltration and/or electrodialysis treatment combined with treatment with an ion exchange resin being strongly acidic cation exchange resin and/or a weakly basic anion exchange resin
  • the treatment with a strong cation exchange resin and/or a weak anion exchange resin is preceded by ultrafiltration followed by nanofiltration and/or electrodialysis.
  • the separation preferably purification, preferably involves concentrating the cultivation broth containing the LN3-containing oligosaccharide or oligosaccharide mixture [i.e. step (iii)].
  • step (iii) precedes the second step.
  • the step of concentrating precedes the second step and is once more applied after the second step as described above.
  • step (iii) of concentrating comprises one or more of nanofiltration, diafiltration, reverse osmosis, evaporation, wiped film evaporation and falling film evaporation.
  • the method further comprises decolorization.
  • a step of drying the cultivation broth obtained from step (a) and/or a step of drying the solution obtained from step (b) is present, preferably a step of drying the solution obtained from step (b) is present.
  • a drying step/technique is preferably selected from the list consisting of spray drying, freeze drying, spray freeze-drying, crystallization, lyophilization, band or belt drying, drum or roller drying, and agitated thin film drying, preferably selected from the list consisting of spray drying, drum or roller drying and agitated thin film drying, more preferably agitated thin film drying.
  • a method for the production of a LN3-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosacchraide according to the invention can comprise one or more drying steps.
  • the same or different drying techniques, preferably as disclosed herein, can be used as understood by the skilled person.
  • the invention provides the use of a cell according to the first aspect of the invention for the production of a lacto-N-triose (LN3)-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide.
  • LN3-containing saccharide and said oligosaccharide mixture are preferably as described in the first aspect of the invention (it is referred to the Section "Oligosaccharide" in this regard).
  • the invention further provides the use of a transporter protein as described in the first aspect of the invention in the production, preferably fermentative production, of a LN3-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide.
  • a transporter protein as described in the first aspect of the invention in the production, preferably fermentative production, of a LN3-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide.
  • Said LN3- containing saccharide and said oligosaccharide mixture are preferably as described in the first aspect of the invention (it is referred to the Section "Oligosaccharide" in this regard).
  • a cell which is genetically engineered for the production of a lacto-N-triose (LN3)-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide, characterized in that said cell expresses, preferably overexpresses, a transporter protein comprising an amino acid sequence:
  • SEQ ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06 having at least 80.0 %, preferably at least 85.0 %, more preferably at least 87.5 %, even more preferably at least 90.0 %, even more preferably at least 92.5 %, even more preferably at least 95.0 %, even more preferably at least 97.5 %, sequence identity to the full-length amino acid sequence of SEQ ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06; that is a functional fragment of SEQ ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06, preferably wherein said fragment retains at least 70.0 %, more preferably at least 80.0 %, even more preferably at least 85.0 %, even more preferably at least 90.0 %, even more preferably at least 95.0 %, most preferably at least 100.0 %, of the activity of the full-length sequence represented by SEQ ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06, respectively; or
  • Cell according to embodiment 1 or 2 wherein said transporter protein consists of ⁇ 550, preferably ⁇ 525, more preferably ⁇ 500, even more preferably ⁇ 480, even more preferably ⁇ 470, even more preferably ⁇ 460, even more preferably ⁇ 450, even more preferably ⁇ 440, even more preferably ⁇ 430, most preferably ⁇ 420, amino acids.
  • LN3-containing oligosaccharide is a milk oligosaccharide, preferably a mammalian milk oligosaccharide, most preferably a human milk oligosaccharide.
  • said LN3-containing oligosaccharide comprises a lactose at its reducing end.
  • LN3-containing oligosaccharide is selected from the list consisting of Lacto-N-triose II (LN3, LNT-II), GlcNAc-beta-l,6-(GlcNAc-beta-l,3- )Gal-beta-l,4-Glc, Lacto-N-neotetraose (LNnT), Lacto-N-tetraose (LNT), Gal-alpha-1, 3-Gal-beta-l, 4- GlcNAc-beta-l,3-Gal-beta-l,4-Glc, Gal-alpha-1, 3-Gal-beta-l, 3-GlcNAc-beta-l, 3-Gal-beta-l, 4-Glc,
  • LN3-containing oligosaccharide is selected from the list consisting of Lacto-N-triose II (LN3, LNT-II), GlcNAc-beta-l,6-(GlcNAc-beta-l,3- )Gal-beta-l,4-Glc, Lacto-N-neotetraose (LNnT), Lacto-N-tetraose (LNT), Lacto-N-hexaose (LNH), para- lacto-N-hexaose (pLNH), lacto-N-neohexaose (LNnH) and para-Lacto-N-neohexaose (pLNnH); optionally wherein said LN3-containing oligosaccharide further comprises: a fucose, preferably wherein said fucose is linked to a monos
  • LN3-containing oligosaccharide is selected from the list consisting of Lacto-N-triose II (LN3, LNT-II), GlcNAc-beta-l,6-(GlcNAc-beta-l,3- )Gal-beta-l,4-Glc, Lacto-N-neotetraose (LNnT), Lacto-N-tetraose (LNT), Gal-alpha-1, 3-Gal-beta-l, 4- GlcNAc-beta-l,3-Gal-beta-l,4-Glc, Gal-alpha-1, 3-Gal-beta-l, 3-GlcNAc-beta-l, 3-Gal-beta-l, 4-Glc, GlcNAc-beta-l,6-(Gal-beta-l,4-GlcNAc,
  • LN3-containing oligosaccharide further comprises a sialic acid, preferably wherein said sialic acid is linked to a monosaccharide (preferably selected from the list consisting of galactose, N-acetylglucosamine and sialic acid, more preferably galactose or N- acetylglucosamine, most preferably galactose) in an alpha-2,3
  • LN3-containing oligosaccharide is selected from the list consisting of LN3, LNT, LNnT, Lacto-N-neofucopentaose I (LNnFP I), lacto-N- fucopentaose III (LNFP III), lacto-N-neofucopentaose V (LNnFP V, LNFP VI), lacto-N-neodifucohexaose (LNnDFH), lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose V (LNFP V), lacto-N-difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), lewis b-lewis
  • LN3-containing oligosaccharide is LN3, a LNT-containing oligosaccharide or a LNnT-containing oligosaccharide.
  • LNT-containing oligosaccharide is selected from the list consisting of Lacto-N-tetraose (LNT), Gal-alpha-1, 3-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l, 4- Glc, GlcNAc-beta-l,6-(Gal-beta-l,3-GlcNAc-beta-l,3-)Gal-beta-l,4-Glc, Lacto-N-pentaose, para- Lacto-N-pentaose, GlcNAc-beta-l,3-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-Glc, GalNAc-beta-
  • LNT Lacto-N-tetraose
  • Gal-alpha-1 3-Gal-beta-
  • LNT Lacto-N-hexaose
  • PLLNnH II para-lacto-N-hexaose II
  • pLNH II para-lacto-N-hexaose II
  • LNO lacto-N-octaose
  • para lacto-N-octaose iso lacto-N-nonaose, novo lacto-N- nonaose, lacto-N-nonaose, lacto-N-decaose, iso lacto-N-decaose and novo lacto-N-decaose
  • said LNT-containing oligosaccharide further comprises
  • LNT-containing oligosaccharide is selected from the list consisting of Lacto-N-tetraose (LNT), Gal-alpha-1, 3-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l, 4- Glc, GlcNAc-beta-l,3-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-Glc, GalNAc-beta-l,3-LNT, Gal- beta-1, 3-GalNAc-beta-l,3-LNT and Lacto-N-hexaose (LNH); optionally wherein said LNT-containing oligosaccharide further comprises: a fucose, preferably wherein said fucose is linked to a monosaccharide (preferably selected from the list consisting of glucose, N-acety
  • LNT-containing oligosaccharide is selected from the list consisting of Lacto-N-tetraose (LNT), Gal-alpha-1, 3-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l, 4- Glc, GlcNAc-beta-l,6-(Gal-beta-l,3-GlcNAc-beta-l,3-)Gal-beta-l,4-Glc, Lacto-N-pentaose, para- Lacto-N-pentaose, GlcNAc-beta-l,3-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-Glc, GalNAc-beta- 1,3-LNT, Gal-beta-1, 3-GalNAc-beta-
  • LNnT-containing oligosaccharide is selected from the list consisting of Lacto-N-neotetraose (LNnT), Gal-alpha-1, 3-Gal-beta-l, 4-GlcNAc- beta-1, 3-Gal-beta-l, 4-Glc, para-lacto-N-hexaose (pLNH), lacto-N-neohexaose (LNnH) and para-Lacto- N-neohexaose (pLNnH); optionally wherein said LNnT-containing oligosaccharide further comprises: a fucose, preferably wherein said fucose is linked to a monosaccharide (preferably selected from the list consisting of glucose, N-acetylglucosamine and galactose) in an alpha-1,2-, alpha- 1,3- or alpha-1, 4-
  • LNnT-containing oligosaccharide is selected from the list consisting of Lacto-N-neotetraose (LNnT), Gal-alpha-1, 3-Gal-beta-l, 4-GlcNAc- beta-l,3-Gal-beta-l,4-Glc, GlcNAc-beta-l,6-(Gal-beta-l,4-GlcNAc-beta-l,3-)Gal-beta-l,4-Glc, lacto- N-neopentaose, para-Lacto-N-neopentaose, para-lacto-N-hexaose (pLNH), lacto-N-neohexaose (LNnH), para-Lacto-N-neohexaose (pLNnH), lacto-N-ne
  • LNnT-containing oligosaccharide is selected from the list consisting of Lacto-N-neotetraose (LNnT), para-lacto-N-hexaose (pLNH), lacto- N-neohexaose (LNnH), para-Lacto-N-neohexaose (pLNnH), Lacto-N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose III (LNFP III), lacto-N-neofucopentaose V (LNnFP V, LNFP VI), Fuc-alphal,2-Gal- beta-l,4-GlcNAc-beta-l, 3-Gal-beta-l, 4-(Fuc-alphal,3-)Glc, Fuc-alphal,2-Gal-beta-l, 3-Gal-beta-l,
  • LN3-containing oligosaccharide is a neutral oligosaccharide.
  • N-acetylglucosaminyltransferase is a beta-1, 6-N-acetylglucosaminyltransferase
  • N-acetylgalactosaminyltransferase is a beta-1,3- or an alpha-1, 3-N-acetyl- galactosaminyltransferase, preferably an alpha-1, 3-N-acetylgalactoaminyltransferase; and Sialyltransferase is selected from the list consisting of alpha-2, 3-sialyltransferase, alpha-2, 6- sialyltransferase and alpha-2, 8-sialyltransferase, more preferably an alpha-2, 3- sialyltransferase or an alpha-2, 6-sialyltransferase.
  • ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06 respectively, preferably lacks at least two consecutive amino acids in its TMl domain, more preferably lacks at least 8 consecutive amino acids in its TMl domain, most preferably lacks amino acids 1 to 16 of its TMl domain, compared to the TMl of the transporter protein represented by SEQ ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06, respectively; or comprises one or more non-consecutive amino acid substitutions in its transmembrane domain 1 (TMl) compared to the TMl of the transporter protein represented by SEQ ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06, respectively, preferably at position: o 24, 28, 31 and/or 32 of TMl of the transporter protein represented by SEQ ID NO 01, o 26, 30, 33 and/or 34 of TMl of the transporter protein represented by SEQ ID NO 13, o 16, 20, 23 and/or 24 of TMl of the transporter protein represented by SEQ ID NO 15, o
  • TMl of SEQ ID NO 16 is represented by SEQ ID NO 213.
  • said separating comprises at least one step selected from the list consisting of clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography (such as but not limited to cation exchange, anion exchange, mixed bed ion exchange), hydrophobic interaction chromatography, gel filtration (i.e. size exclusion chromatography) and ligand exchange chromatography.
  • Method according to embodiment 43 or 44 further comprising the step of purifying said LN3- containing oligosaccharide or any one, preferably at least two, more preferably at least three, even more preferably at least four, most preferably all, of the saccharides in said mixture.
  • Method according to embodiment 45 wherein said purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying or lyophilization.
  • transporter protein as defined in any one of embodiments 1 to 3 and 33 to 42 in the production, preferably fermentative production, of a LN3-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide.
  • LN3-containing oligosaccharide is LN3, a lacto-N-tetraose (LNT)-containing oligosaccharide or a lacto-N-neotetraose (LNnT)-containing oligosaccharide: selected from SEQ ID NO 01 or 13; or having at least 85.0 % sequence identity to the full-length amino acid sequence of SEQ ID NO 01 or 13; or that is a functional fragment of SEQ ID NO 01 or 13, wherein said functional fragment consists of an amount of consecutive amino acid residues from SEQ ID NO 01 or 13, respectively, and wherein said amount is at least 85.0% of the full-length of SEQ ID NO 01 or 13, respectively; or that is a functional fragment of a polypeptide having at least 85.0 sequence identity to the full-length amino acid sequence of SEQ ID NO 01 or 13, wherein said functional fragment consists of an amount of consecutive amino acid residues from said polypeptide and wherein said amount is at least 85.0% of the
  • LN3-containing oligosaccharide is a lacto-N-tetraose (LNT)-containing oligosaccharide: represented by SEQ ID NO 13; or having at least 85.0 % sequence identity to the full-length amino acid sequence of SEQ ID NO 13; or that is a functional fragment of SEQ ID NO 13, wherein said functional fragment consists of an amount of consecutive amino acid residues from SEQ ID NO 13, and wherein said amount is at least 85.0% of the full-length of SEQ ID NO 13; or that is a functional fragment of a polypeptide having at least 85.0 sequence identity to the full-length amino acid sequence of SEQ ID NO 13, wherein said functional fragment consists of an amount of consecutive amino acid residues from said polypeptide and wherein said amount is at least 85.0% of the full-length of said polypeptide;
  • LNT lacto-N-tetraose
  • LN3-containing oligosaccharide is a lacto-N-neotetraose (LNnT)-containing oligosaccharide: selected from SEQ ID NO 15 or 16; or having at least 85.0 % sequence identity to the full-length amino acid sequence of SEQ ID NO 15 or 16; or that is a functional fragment of SEQ ID NO 15 or 16, wherein said functional fragment consists of an amount of consecutive amino acid residues from SEQ ID NO 15 or 16, respectively, and wherein said amount is at least 85.0% of the full-length of SEQ ID NO 15 or 16, respectively; or that is a functional fragment of a polypeptide having at least 85.0 sequence identity to the full-length amino acid sequence of SEQ ID NO 15 or 16, wherein said functional fragment consists of an amount of consecutive amino acid residues from said polypeptide and wherein said amount is at least 85.0% of the full-length of said polypeptide;
  • LN3-containing oligosaccharide is LN3: selected from SEQ ID NO 02, 03, 04, 05 or 06; or having at least 85.0 % sequence identity to the full-length amino acid sequence of SEQ ID NO 02, 03, 04, 05 or 06; or that is a functional fragment of SEQ ID NO 02, 03, 04, 05 or 06, wherein said functional fragment consists of an amount of consecutive amino acid residues from SEQ ID NO 02, 03, 04, 05 or 06, respectively, and wherein said amount is at least 85.0% of the full- length of SEQ ID NO 02, 03, 04, 05 or 06, respectively; or that is a functional fragment of a polypeptide having at least 85.0 sequence identity to the full-length amino acid sequence of SEQ ID NO 02, 03, 04, 05 or 06, wherein said functional fragment consists of an amount of consecutive amino acid residues from said polypeptide and wherein said amount is at least 85.0% of the full-length of said polypeptide.
  • a cell according to embodiment 11, wherein said improved production comprises: better titer of said saccharide (gram saccharide per liter), and/or better production rate r (gram saccharide per liter per hour), and/or better cell performance index (gram saccharide per gram biomass), and/or better specific productivity (gram saccharide per gram biomass per hour), and/or better yield on sucrose (gram saccharide per gram sucrose), and/or better sucrose uptake/conversion rate (gram sucrose per gram per hour), and/or better lactose conversion/consumption rate (gram lactose per hour), and/or enhanced growth speed of the cell.
  • Cell according to embodiment 11, wherein said improved production comprises: better titer of said saccharide (gram saccharide per liter), and/or better production rate r (gram saccharide per liter per hour), and/or better cell performance index (gram saccharide per gram biomass), and/or better specific productivity (gram saccharide per gram biomass per hour).
  • a method for the production of a lacto-N-triose (LN3)-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide comprising the step of:
  • Method according to embodiment 15, wherein said separating comprises at least one step selected from the list consisting of clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration and ligand exchange chromatography.
  • Method according to embodiment 15 or 16 further comprising the step of purifying said LN3- containing oligosaccharide or any one, preferably all, of the oligosaccharides in said mixture.
  • Method according to embodiment 17, wherein said purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying or lyophilization.
  • the verbs "to comprise”, “to have” and “to contain”, and their conjugations are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • the verb "to consist essentially of” means that e.g. a mixture as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • the verbs "to comprise”, “to have” and “to contain”, and their conjugations may be preferably replaced by "to consist” (and its conjugations) or “to consist essentially of” (and its conjugations).
  • indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements.
  • the indefinite article “a” or “an” thus usually means “at least one”.
  • the word “about” or “approximately” or “around” when used in association with a numerical value, parameter or numerical range such as amounts, volumes, volume ratios, volume percentages, weight ratios, weight percentages, or application rates of ingredients of a composition; means an amount, a volume, a volume ratio, a volume percentage, a weight ratio, a weight percentage, or an application rate that is recognized by those of ordinary skill in the art to provide a desired effect equivalent to that obtained from the specified amount, volume, volume ratios, volume percentages, weight ratio, weight percentage, or application rate; and is encompassed herein and should be construed in light of the number of reported significant digits and applying ordinary rounding techniques.
  • the word “about” or “approximately” or “around” when used in association with a numerical value preferably means that the value may be the given value (of 10) more or less 15%, preferably 10%, more preferably 5%, even more preferably 1%, of the value.
  • the expressions “capable of... ⁇ verb>” and “capable to... ⁇ verb>” are preferably replaced with the active voice of said verb and vice versa.
  • the expression “capable of expressing” is preferably replaced with “expresses” and vice versa, i.e. "expresses” is preferably replaced with "capable of expressing”.
  • isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • modified expression of a gene relates to a change in expression compared to the wild type expression of said gene. Said modified expression is either a lower or higher expression compared to the wild type, wherein the term “higher expression” is also defined as “overexpression” of said gene in the case of an endogenous gene or “expression” in the case of a heterologous gene that is not present in the wild type strain.
  • Lower expression is obtained by means of common well-known technologies for a skilled person (such as the usage of siRNA, CrispR, CrispRi, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutating genes, knocking-out genes, transposon mutagenesis, etc. which are used to change the genes in such a way that they are less-able (i.e. statistically significantly 'less-able' compared to a functional wild-type gene) or completely unable (such as knocked-out genes) to produce functional final products.
  • Overexpression or expression is obtained by means of common well- known technologies for a skilled person, wherein said gene is part of an "expression cassette" which relates to any sequence in which a promoter sequence, untranslated region sequence (containing either a ribosome binding sequence or Kozak sequence), a coding sequence (for instance a membrane protein gene sequence) and optionally a transcription terminator is present, and leading to the expression of a functional active protein.
  • Said expression is either constitutive or conditional or regulated or tuneable.
  • “Expression” of a transporter protein is defined as “overexpression” of the gene encoding said transporter protein in the case said gene is an endogenous gene or “expression” in the case the gene encoding said transporter protein is a heterologous gene that is not present in the wild type strain.
  • cell productivity index refers to the mass of the product (i.e. saccharide or saccharides according to the invention) produced by the cells divided by the mass of said cells in the culture.
  • the terms "LNT 11”, “LNT-II”, “LN3”, “lacto-N-triose 1 I”, “lacto-N-triose 1 I”, “lacto-N-triose”, “lacto-N-triose” and “GlcNAc-pi,3-Gal-pi,4-Glc" are used interchangeably.
  • LNT lacto-N-tetraose
  • lacto-/V-tetraose lacto-/V-tetraose
  • Gal-pi,3-GlcNAc-pi,3-Gal-pi,4Glc are used interchangeably.
  • LNnT lacto-N-neotetraose
  • lacto-/V-neotetraose lacto-/V-neotetraose
  • Gaipi-4GlcNAcpi- 3Gaipi-4Glc are used interchangeably.
  • lacto-N-pentaose and "LN5" are used interchangeably and refer to GlcNAC-bl,3-Gal-bl,4- GlcNAC-bl,3-Gal-bl,4-Glc.
  • lacto-N-neohexaose and “LNnH” are used interchangeably and refer to Gal-bl,4-GlcNAC- bl,6-(Gal-bl,4-GlcNAC-bl,3)-Gal-bl,4Glc.
  • pLNnH refers to Gal-bl,4-GlcNAC-bl,3-Gal-bl,4-GlcNAC-bl,3-Gal-bl,4-Glc.
  • para-lacto-N-neohexaose II and "pLNnH-l I” are used interchangeably and refer to Gal-bl,4- GlcNAC-bl,3-Gal-bl,3-GlcNAC-bl,3-Gal-bl,4-Glc.
  • pLNH refers to Gal-bl,3-GlcNAC-bl,3-Gal-bl,4-GlcNAC-bl,3-Gal-bl,4-Glc.
  • para-lacto-N-hexaose II and “pLNH-ll” are used interchangeably and refer to Gal-bl,3- GlcNAC-bl,3-Gal-bl,3-GlcNAC-bl,3-Gal-bl,4-Glc.
  • pLNnO refers to Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4- GlcNAc-bl,3-Gal-bl,4- Glc.
  • pLNnD refers to Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4- GlcNAC-bl,3-Gal-bl,4-Glc.
  • LNH refers to Gal-bl,4-GlcNAC-bl,6-(Gal-bl,3-GlcNAc-bl,3)-Gal-bl,4-Glc.
  • lacto-N-biose and “LNB” are used interchangeably and refer to Gal-bl,3-GlcNAc.
  • N-acetyllactosamine and “LacNAc” are used interchangeably and refer to Gal-bl,4-GlcNAc.
  • iso-LNO and iso-lacto-N-octaose are used interchangeably and refer to Gal-bl,3-GlcNAc- bl,3-(Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-GlcNAc-bl,6-)Gal-bl,4-Glc.
  • LND lacto-N-decaose
  • LNnD lacto-N-neodecaose
  • LNFP-I lacto-N-fucopentaose I
  • LNFP I lacto-N-fucopentaose I
  • LNF I OH type I determinant "LNF I”, “LNF1”, “LNF 1”
  • Bood group H antigen pentaose type 1 and "Fuc-al,2-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc" are used interchangeably.
  • GalNAc-LNFP-l blood group A antigen hexaose type I
  • GalNAc-al,3-(Fuc-al,2)-Gal- pi,3-GlcNAc- pi,3-Gal-pi,4-Glc are used interchangeably.
  • Gal-LNFP-I blood group B antigen hexaose type I
  • Gal-al,3-(Fuc-al,2)-Gal-pi,3- GlcNAc-pi,3-Gal-pi,4-Glc are used interchangeably.
  • LNFP-II lacto-N-fucopentaose II
  • Gal-pi,3-(Fuc-al,4)-GlcNAc-pi,3-Gal-pi,4-Glc are used interchangeably.
  • LNFP-III lacto-N-fucopentaose III
  • Gal-pi,4-(Fuc-al,3)-GlcNAc-pi,3-Gal-pi,4-Glc are used interchangeably.
  • LNFP-V lacto-N-fucopentaose V
  • Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-(Fuc-al,3)-Glc are used interchangeably.
  • LNDFH I Lacto-N-difucohexaose I
  • LNDFH-I LNDFH I
  • LNDFH I Lacto-N-difucohexaose I
  • LNDFH-I LNDFH I
  • LNDFH I LNDFH I
  • LNDFH I LNDFH I
  • LNDFH I LNDFH I
  • LNDFH I lactose
  • Lewis-b hexasaccharide LNDFH I
  • Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-Glc are used interchangeably.
  • LNDFH II Lacto-N-difucohexaose II
  • LDFH II Lacto-N-difucohexaose II
  • LDFH II Lacto-N-difucohexaose II
  • LDFH II Lacto-N-difucohexaose II
  • LDFH II Lacto-N-difucohexaose II
  • LDFH II LDFH II
  • Fuc-al,4-(Gal-pi,3)- GlcNAc-pi,3-Gal-pi,4-(Fuc-al,3)-Glc are used interchangeably.
  • lewis b-lewis x and "Fucal,4-[Fuc-al,2-Gaipi,3]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-Glc are used interchangeably.
  • MFLNH III "monofucosyllacto-N-hexaose-lll” and "Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6-[Gal- pi,3-GlcNAc-pi,3]-Gal-pi,4-Glc" are used interchangeably.
  • DFLNH (a) "difucosyllacto-N-hexaose (a)” and "Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6-[Fuc-al,2- Gal-pi,3-GlcNAc-pi,3]-Gal-pi,4-Glc" are used interchangeably.
  • DFLNH "difucosyllacto-N-hexaose” and "Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6-[Fuc-al,4-[Gal- pi,3]-GlcNAc-pi,3]-Gal-pi,4-Glc" are used interchangeably.
  • TFLNH "trifucosyllacto-N-hexaose” and "Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6-[Fuc-al,4-[Fuc- al,2-Gal-pi,3]-GlcNAc-pi,3]-Gal-pi,4-Glc" are used interchangeably.
  • LNnFP I Lacto-N-neofucopentaose I
  • Fuc-al,2-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc are used interchangeably.
  • LNFP-VI LNnFP V
  • lacto-N-neofucopentaose V lacto-N-neofucopentaose V
  • Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-(Fuc- al,3)-Glc are used interchangeably.
  • LNnDFH Lacto-N-neoDiFucohexaose
  • Lewis x hexaose Gal-pi,4-(Fuc-al,3)-GlcNAc-pi,3- Gal-pi,4-(Fuc-al,3)-Glc
  • LSTa LS-Tetrasaccharide a
  • Sialyl-lacto-N-tetraose a sialyllacto-N-tetraose a
  • Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc are used interchangeably.
  • LSTb LS-Tetrasaccharide b
  • Sialyl-lacto-N-tetraose b sialyllacto-N-tetraose b
  • Gal- bl,3-(Neu5Ac-a2,6)-GlcNAc-bl,3-Gal-bl,4-Glc are used interchangeably.
  • LSTc "LS-Tetrasaccharide c", "Sialyl-lacto-N-tetraose c", “sialyllacto-N-tetraose c”, “sialyllacto-N-neotetraose c" and "Neu5Ac-a2,6-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc" are used interchangeably.
  • LSTd LS-Tetrasaccharide d
  • Sialyl-lacto-N-tetraose d sialyl-lacto-N-tetraose d
  • sialyllacto-N-tetraose d sialyllacto-N-neotetraose d
  • Neu5Ac-a2,3-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc are used interchangeably.
  • 6'-sialyllacto-N-biose "6'SLNB” and "Neu5Ac-a2,6-Gal-bl,3-GlcNAc” are used interchangeably.
  • 6'-sialyllactosamine "6'SLacNAc” and "Neu5Ac-a2,6-Gal-bl,4-GlcNAc” are used interchangeably.
  • sialyl Lewis x , "sialyl Lex”, "5-acetylneuraminyl-(2-3)-galactosyl-(l-4)-(fucopyranosyl-(l-3))- N-acetylglucosamine” and "Neu5Ac-a2,3-Gal-pi,4-[Fuc-al,3-]GlcNAc" are used interchangeably.
  • Neuronon-2-ulopyranosonic acid and “4-O-acetyl neuraminic acid” are used interchangeably and have C11H19NO9 as molecular formula.
  • Neuronon-2-ulopyranosonic acid "5-acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid"
  • D- glycero-5-acetamido-3,5-dideoxy-D-galacto-non-2-ulo-pyranosonic acid "5-(acetylamino)-3,5-dideoxy- D-glycero-D-galacto-2-nonulopyranosonic acid”
  • 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2- nonulosonic acid "5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-non-2-nonulosonic acid” and "5- (acetylamino)-3,5-dideoxy-D-glycero-D-galacto-n
  • Neuronac2 N-acetyl-4-O-acetylneuraminic acid
  • 4-O-acetyl-N-acetylneuraminic acid 4- O-acetyl-N-acetylneuraminate
  • 4-acetate 5-acetamido-3,5-dideoxy-D-glycero-D-galacto- nonulosonate "4-acetate 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonate
  • 4-acetate 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonic acid and "4-acetate 5-(acetylamino)- 3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid” are used interchangeably and have C13H21NO10 as molecular formula.
  • Neuro5,7Ac2 "7-O-acetyl-N-acetylneuraminic acid", “N-acetyl-7-O-acetylneuraminic acid", “7- O-acetyl-N-acetylneuraminate", "7-acetate 5-acetamido-3,5-dideoxy-D-glycero-D-galacto- nonulosonate", "7-acetate 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonate", "7- acetate 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonic acid” and "7-acetate 5-(acetylamino)- 3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid" are used interchangeably herein and have C13H21NO10 as molecular formula
  • Neuro5,8Ac2 and “5-n-acetyl-8-o-acetyl neuraminic acid” are used interchangeably herein and have C13H21NO10 as molecular formula.
  • Neuro5,9Ac2 N-acetyl-9-O-acetylneuraminic acid
  • 9-anana 9-O-acetylsialic acid
  • 9-0- acetyl-N-acetylneuraminic acid
  • 5-n-acetyl-9-O-acetyl neuraminic acid "N,9-O-diacetylneuraminate” and “N,9-O-diacetylneuraminate”
  • Neuro4,5,9Ac3 and “5-N-acetyl-4,9-di-O-acetylneuraminic acid” are used interchangeably herein.
  • Neuro5,7,9Ac3 and “5-N-acetyl-7,9-di-O-acetylneuraminic acid” are used interchangeably herein.
  • Neuro5,8,9Ac3 and “5-N-acetyl-8,9-di-O-acetylneuraminic acid” are used interchangeably herein.
  • Neuronamic4,5,7,9Ac4" and “5-N-acetyl-4,7,9-tri-O-acetylneuraminic acid” are used interchangeably herein.
  • Neuro5,7,8,9Ac4 and “5-N-acetyl-7,8,9-tri-O-acetylneuraminic acid” are used interchangeably herein.
  • Neuron-glycolyl-neuraminic acid N-glycolylneuraminic acid
  • N-glycolylneuraminic acid N- glycolylneuraminate
  • N-glycoloyl-neuraminate N-glycoloyl-neuraminic acid
  • N-glycoloylneuraminic acid N-glycoloylneuraminic acid
  • 3,5-dideoxy-5-((hydroxyacetyl)amino)-D-glycero-D-galacto-2-nonulosonic acid 3,5-dideoxy-5- (glycoloylamino)-D-glycero-D-galacto-2-nonulopyranosonic acid
  • 3,5-dideoxy-5-(glycoloylamino)-D- glycero-D-galacto-non-2-ulopyranosonic acid 3,5-dideoxy-5-[(hydroxyacetyl)amino]-D-glycer
  • DSLNnT and “Disialyllacto-N-neotetraose” are used interchangeably and refer to Neu5Ac- a2,6-[Neu5Ac-a2,6-Gal-bl,4-GlcNAc-bl,3]-Gal-bl,4-Glc.
  • DSLNT and “Disialyllacto-N-tetraose” are used interchangeably and refer to Neu5Ac-a2,6- (Neu5Ac-a2,3-Gal-bl,3-)GlcNAc-bl,3-Gal-bl,4-Glc.
  • D'LNT and "disialyllacto-N-tetraose analog” are used interchangeably and refer to Neu5Ac- a2,6-(Neu5Ac-a2,6-Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc.
  • D'LNnT and “disialyllacto-N-neotetraose analog” are used interchangeably and refer to Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-bl,4-GlcNAc-bl,3-)Gal-bl,4-Glc.
  • Gal refers to galactose, "GIcNAc” to N-acetylglucosamine, “Neu5Ac” to N-acetylneuraminic acid, “Glc” to glucose, “ManNAc” to N-acetylmannosamine, “GalNAc” to N-acetylgalactosamine, “Fuc” to fucose, “LacNAc” to N-acetyllactosamine and "Fruc” to fructose. Examples
  • 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 4 CI, 5.00 g/L (NH 4 ) 2 SO 4 , 2.993 g/L KH 2 PO 4 , 7.315 g/L K 2 HPO 4 , 8.372 g/L MOPS, 0.5 g/L NaCI, 0.5 g/L MgSO 4 .7H2O, 30 g/L sucrose or another carbon source when specified in the examples, 1 ml/L vitamin solution, 100 pL/L molybdate solution, and 1 mL/L selenium solution.
  • Vitamin solution consisted of 3.6 g/L FeCI 2 .4H 2 O, 5 g/L CaCI 2 .2H 2 O, 1.3 g/L MnCI 2 .2H 2 O, 0.38 g/L CUCI 2 .2H 2 O, 0.5 g/L COCI 2 .6H 2 O, 0.94 g/L ZnCI 2 , 0.0311 g/L H 3 BO4, 0.4 g/L Na 2 EDTA.2H 2 O and 1.01 g/L thiamine. HCI.
  • the molybdate solution contained 0.967 g/L NaMoO4.2H2O.
  • the selenium solution contained 42 g/L SeO 2 .
  • the minimal medium for fermentations contained 6.75 g/L NH 4 CI, 1.25 g/L (NH 4 ) 2 SO 4 , 2.93 g/L KH 2 PO 4 and 7.31 g/L KH 2 PO 4 , 0.5 g/L NaCI, 0.5 g/L MgSO 4 .7H 2 O, 30 g/L sucrose, 1 mL/L vitamin solution, 100 pL/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above.
  • 100 g/L lactose was additionally added to the medium as precursor.
  • Complex medium was sterilized by autoclaving (121°C, 21') and minimal medium by filtration (0.22 pm Sartorius). When necessary, the medium was made selective by adding an antibiotic (e.g. chloramphenicol (20 mg/L), carbenicill in (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L)).
  • Plasmids 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", phi80d/ocZde/toM15, de ⁇ ta(lacZYAargF) U169, deoR, recAl, endAl, hsdR17(rk", mk + ), phoA, supE44, lambda", thi-1, gyrA96, relAl) bought from Invitrogen.
  • Escherichia coli K12 MG1655 [X", F", 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), p. 6640-6645) as described in e.g. W02022/034067.
  • Gene mutations were created via a PCR-based method described by Sanchis et al. (Appl. Microbiol. Biotechnol. (2008) 81(2), p. 387-397).
  • 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 constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid like e.g. a pSClOl-derived plasmid, for a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. IgtA with UniProt ID Q9JXQ6 from Neisseria meningitidis.
  • an expression plasmid like e.g. a pSClOl-derived plasmid, for a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. IgtA with UniProt ID Q9JXQ6 from Neisseria meningitidis.
  • the mutant LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g.
  • wbdO from Salmonella enterica (Uniprot ID Q5UHA8) and/or furA from Pseudogulbenkiania ferrooxidans (Uniprot ID B9YZ84), alternatively wbgO from E. coli 055:1-17 (Uniprot ID D3QY14) can be used.
  • the mutant LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. GalT7 (Uniprot ID F4ZLW1) from Pasteurella multocida or gatD (Uniprot ID D0EAD4) from Pasteurella multocida.
  • LgtB Uniprot ID Q51116, sequence version 02, 01 Dec 2000
  • N. meningitidis can be used.
  • the LN3, LNT and/or LNnT production can further be optimized in the mutant E. coli strains with genomic knock-out of the E. coli LacY gene and with a genomic knock-in of one or more constitutive transcriptional units for a lactose permease like e.g. the E. coli LacY (UniProt ID P02920).
  • LN3, LNT and/or LNnT production can further be optimized in the mutant E. coli strains with genomic knock-outs of the E. coli genes comprising any one or more of galT, ushA, IdhA and agp.
  • the mutant LN3, LNT and/or LNnT producing strains can also be optionally modified for enhanced UDP- GIcNAc production with a genomic knock-in of a constitutive transcriptional unit for an L-glutamine— D- fructose-6-phosphate aminotransferase like e.g. the mutant glmS*54 from E. coli (differing from the wildtype E. coli glmS protein, having UniProt ID P17169 (sequence version 04, 23 Jan 2007), by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 2006, 88: 419-429).
  • a genomic knock-in of a constitutive transcriptional unit for an L-glutamine— D- fructose-6-phosphate aminotransferase like e.g. the mutant glmS*54 from E. coli (differing from the wildtype E. coli glmS protein,
  • 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 (UniProt ID P09147), a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03, 23 Jan 2007) and an N-acetylglucosamine-l-phosphate uridylyltransferase / glucosamine-l-phosphate acetyltransferase like e.g. glmU from E. coli (UniProt ID P0ACC7).
  • UDP-glucose-4-epimerase like e.g. galE from E. coli (UniProt ID P09147)
  • a phosphoglucosamine mutase like e.g. gl
  • the mutant LN3, LNT and/or LNnT producing E. coli strains can 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 (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6).
  • a sucrose transporter like e.g. CscB from E. coli W (UniProt ID E0IXR1)
  • a fructose kinase like e.g. Frk originating from Zymomonas mobilis
  • a sucrose phosphorylase like e.g. Ba
  • the mutant strain was derived from E. coli K12 MG1655 as described e.g. in W02022/034067, W02022/034068 or W02022/034070.
  • the mutant E. coli strain producing CMP-sialic acid was further modified with one or more transcriptional unit(s) encoding one or more sialyltransferases.
  • the strain could additionally be modified to comprise a transcriptional unit for a lactose permease like e.g., E. coli LacY (UniProt ID P02920).
  • coli strain can 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 (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6).
  • a sucrose transporter like e.g. CscB from E. coli W (UniProt ID E0IXR1)
  • a fructose kinase like e.g. Frk originating from Zymomonas mobilis
  • a sucrose phosphorylase like e.g. BaSP originating from Bifidobacterium adolescentis
  • the mutant was derived from E. coli K12 MG1655 as described in Example 1 of e.g. W02022/034067, W02022/034068 or W02022/034069, i.e. knock-ins of manB, manC, gmd and fcl.
  • the mutant E. coli strain producing GDP-fucose was further modified with one or more transcriptional unit(s) encoding one or more fucosyltransferases.
  • the mutant E. coli strain can 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 ⁇ N (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6).
  • an E. coli K12 M1655 strain is modified for production of GDP-fucose, LN3, LNT and/or LNnT as described herein and for expression of one or more compatible fucosyltransferase(s) as further specified in the Examples below.
  • an E. coli K12 MG1655 strain is modified for production of CMP-sialic acid, LN3 and LNT as described herein and for expression of one or more compatible sialyltransferase(s).
  • an E. coli K12 MG1655 strain is modified for production of CMP-sialic acid, LN3 and LNnT as described herein and for expression of one or more compatible sialyltransferase(s).
  • any one or more of the glycosyltransferases and/or the proteins involved in nucleotide-activated sugar synthesis were N- and/or C-terminally fused to a solubility enhancer tag like e.g. a SUMO-tag, an MBP-tag, His, FLAG, Strep-ll, Halo-tag, NusA, thioredoxin, GST and/or the Fh8-tag to enhance their solubility (Costa et al., Front. Microbiol. 2014, https://doi.org/10.3389/fmicb.2014.00063; Fox et al., Protein Sci. 2001, 10(3), 622-630; Jia and Jeaon, Open Biol.
  • a solubility enhancer tag like e.g. a SUMO-tag, an MBP-tag, His, FLAG, Strep-ll, Halo-tag, NusA, thioredoxin, GST and/or the Fh8-tag to enhance their so
  • the modified E. coli strains were modified with a genomic knock-ins 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 BiologyTM, vol 205. Humana Press).
  • a chaperone protein like e.g. DnaK, DnaJ, GrpE or the GroEL/ES chaperonin system
  • a preculture for the bioreactor was started from an 250pl 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, Melsoder, Germany). Culturing condition 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 H2S04 and 20% NH 4 OH.
  • the exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.
  • Neutral oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Evaporative Light Scattering Detector (ELSD) or a Refractive Index (Rl) detection.
  • ELSD Evaporative Light Scattering Detector
  • Rl Refractive Index
  • a volume of 0.7 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 A;1.7 pm) column with an Acquity UPLC BEH Amide VanGuard column, 130 A, 2. lx 5 mm.
  • the column temperature was 50 °C.
  • the mobile phase consisted of a % water and % acetonitrile solution to which 0.2 % triethylamine was added.
  • the method was isocratic with a flow of 0.130 mL/min.
  • the ELS detector had a drift tube temperature of 50 °C and the N2 gas pressure was 50 psi, the gain 200
  • Sialylated oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Refractive Index (Rl) detection.
  • Rl Refractive Index
  • a volume of 0. 5 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 A;1.7 pm).
  • the column temperature was 50 °C.
  • the mobile phase consisted of a mixture of 70 % acetonitrile, 26 % ammonium acetate buffer (150 mM) and 4 % methanol to which 0.05 % pyrrolidine was added.
  • the method was isocratic with a flow of 0.150 mL/min.
  • the temperature of the Rl detector was set at 35 °C.
  • E. coli K12 MG1655 strain was modified as described in Example 1 comprising genomic knock-outs of the E. coli genes lacZ, nagB, galT, ushA and IdhA and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B.
  • CscB sucrose transporter
  • Frk fructose kinase
  • BaSP sucrose phosphorylase
  • adolescentis (UniProt ID A0ZZH6), the galactoside beta-1, 3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6) and the N-acetylglucosamine beta-1, 3-galactosyltransferase wbgO (Uniprot ID D3QY14) from E. coli 055:1-17.
  • the mutant strain was transformed with an expression plasmid containing a constitutive transcriptional unit for a transporter protein with SEQ ID 01, 02, 03, 04, 05 or 06.
  • the novel strain was evaluated in a growth experiment for production of LN3 and LNT according to the culture conditions provided in Example 1, in which the strains were cultivated in minimal medium supplemented with 15 g/L sucrose and 20 g/L Lactose.
  • a reference strain was used with the same genetic make-up as the novel mutant strains but lacking the transporter protein.
  • the strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed as described in Example 1.
  • a transporter protein with SEQ ID NO 03, 04, 05 or 06 enhanced the production of LN3 in a LNT production host expressing the galactoside beta-1, 3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6) and N-acetylglucosamine beta-1, 3-galactosyltransferase wbgO (Uniprot ID D3QY14) from E. coli 055:1-17 (Tables 2 and 3).
  • SD represents the standard deviation (4 replicates of the same strain tested).
  • the “reference strain (REF)” is identical to the tested strains, except that the indicated transporter protein (SEQ ID NO 01 , 02, 03, 04, 05 or 06) is not expressed in the reference strain.
  • Table 3 The percentage of LN3 and LNT g/L measured in the whole broth compared to the reference strain.
  • SD represents the standard deviation (4 replicates of the same strain tested).
  • the “reference strain (REF)” is identical to the tested strains, except that the indicated transporter protein (SEQ ID NO 01, 02, 03, 04, 05 or 06) is not expressed in the reference strain.
  • Example 3 Lacto-N-neotetraose (LNnT) production in an E. coli host cultivated 72 h in a growth experiment in minimal media supplemented with 20 g/L lactose
  • E. coli K12 MG1655 strain was modified as described in Example 1 comprising genomic knock-outs of the E. coli genes lacZ, nagB, galT, ushA and IdhA and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B.
  • CscB sucrose transporter
  • Frk fructose kinase
  • BaSP sucrose phosphorylase
  • adolescentis (UniProt ID A0ZZH6), the galactoside beta-1, 3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6) and the N-acetylglucosamine beta-1, 4-galactosyltransferase LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis.
  • the mutant strain was transformed with an expression plasmid containing a constitutive transcriptional unit for a transporter protein with SEQ ID 01.
  • the novel strain was evaluated in a growth experiment for production of LN3 and LNnT according to the culture conditions provided in Example 1, in which the strains were cultivated in minimal medium supplemented with 15 g/L sucrose and 20 g/L Lactose.
  • a reference strain was used with the same genetic make-up as the novel mutant strains but lacking the transporter protein.
  • the strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed as described in Example 1.
  • SD represents the standard deviation (4 replicates of the same strain tested).
  • the “reference strain (REF)” is identical to the tested strains, except that the indicated transporter protein (SEQ ID NO 01) is not expressed in the reference strain.
  • SD represents the standard deviation (4 replicates of the same strain tested).
  • the “reference strain (REF)” is identical to the tested strains, except that the indicated transporter protein (SEQ ID NO 01) is not expressed in the reference strain.
  • Example 4 2F(4)-LNT (LNFPI) production in an E. coli host cultivated 72 h in a growth experiment in minimal media supplemented with 20 g/L lactose
  • E. coli K12 MG1655 strain was modified as described in Example 1 comprising genomic knock-outs of the E. coli genes lacZ, nagB, galT, ushA and IdhA and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B.
  • CscB sucrose transporter
  • Frk fructose kinase
  • BaSP sucrose phosphorylase
  • adolescentis (UniProt ID A0ZZH6), the galactoside beta-1, 3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6), the N-acetylglucosamine beta-1, 3-galactosyltransferase FurA (Uniprot ID B9YZ84) from P. ferrooxidans and the N-acetylglucosamine beta-1, 3-galactosyltransferase wbdO (Uniprot ID Q5UHA8) from S. enterica.
  • the mutant strain was further modified for production of GDP-fucose as described in Example 1 and transformed with an expression plasmid containing a constitutive transcriptional unit for the alpha-1, 2-fucosyltransferase from D. mossii (UniProt ID F8X274).
  • the mutant strain was transformed with an expression plasmid containing a constitutive transcriptional unit for a transporter protein with SEQ ID 01.
  • the novel strain was evaluated in a growth experiment for production of LNFPI according to the culture conditions provided in Example 1, in which the strains were cultivated in minimal medium supplemented with 15 g/L sucrose and 20 g/L Lactose.
  • a reference strain was used with the same genetic make-up as the novel mutant strain but lacking the transporter protein.
  • the strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed as described in Example 1.
  • the experiment demonstrated that expression of a transporter protein with SEQ ID NO 01 enhanced the production of LNFPI that is being produced in a LNFPI production host expressing the galactoside beta-1, 3-N- acetylglucosaminyltransferase (LgtA) from N.
  • LgtA galactoside beta-1, 3-N- acetylglucosaminyltransferase
  • meningitidis (UniProt ID Q9JXQ6), the N-acetylglucosamine beta-1, 3-galactosyltransferase FurA (Uniprot ID B9YZ84) from P. ferrooxidans and wbdO (Uniprot ID Q5UHA8) from S. enterica and the alpha-1, 2-fucosyltransferase from D. mossii (UniProt ID F8X274) (Tables 6 and 7).
  • SD represents the standard deviation (4 replicates of the same strain tested).
  • the “reference strain (REF)” is identical to the tested strains, except that the indicated transporter protein (SEQ ID NO 01) is not expressed in the reference strain.
  • SD represents the standard deviation (4 replicates of the same strain tested).
  • the “reference strain (REF)” is identical to the tested strains, except that the indicated transporter protein (SEQ ID NO 01) is not expressed in the reference strain.
  • Example 5 3F(3)-LNnT (LNFPIII) production in an E. coli host cultivated 72 h in a growth experiment in minimal media supplemented with 20 g/L lactose
  • E. coli K12 MG1655 strain was modified as described in Example 1 comprising genomic knock-outs of the E. coli genes iacZ, nagB, galT, ushA and IdhA and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B.
  • CscB sucrose transporter
  • Frk fructose kinase
  • BaSP sucrose phosphorylase
  • adolescentis (UniProt ID A0ZZH6), the galactoside beta-1, 3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6), the N-acetylglucosamine beta-1, 4-galactosyltransferase GatD (Uniprot ID D0EAD4) from P. multocida.
  • the mutant strains was further modified for production of GDP-fucose as described in Example 1 and transformed with an expression plasmid containing a constitutive transcriptional unit for the alpha-1, 3-fucosyltransferase from P.
  • the mutant strain was transformed with an expression plasmid containing a constitutive transcriptional unit for a transporter protein with SEQ ID 01.
  • the novel strain was evaluated in a growth experiment for production of LNFPIII according to the culture conditions provided in Example 1, in which the strains were cultivated in minimal medium supplemented with 15 g/L sucrose and 20 g/L Lactose.
  • a reference strain was used with the same genetic make-up as the novel mutant strain but lacking the transporter protein.
  • the strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed as described in Example 1.
  • SD represents the standard deviation (4 replicates of the same strain tested).
  • the “reference strain (REF)” is identical to the tested strains, except that the indicated transporter protein (SEQ ID NO 01) is not expressed in the reference strain.
  • Example 6 2F(4)-4F(3)-LNT (LNDFHI) production in an E. coli host cultivated 72 h in a growth experiment in minimal media supplemented with 20 g/L lactose
  • E. coli K12 MG1655 strain was modified as described in Example 1 comprising genomic knock-outs of the E. coli genes lacZ, nagB, galT, ushA and IdhA and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B.
  • CscB sucrose transporter
  • Frk fructose kinase
  • BaSP sucrose phosphorylase
  • adolescentis (UniProt ID A0ZZH6), the galactoside beta-1, 3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6), the N-acetylglucosamine beta-1, 3-galactosyltransferase wbdO (Uniprot ID Q5UHA8) from S. enterica.
  • the mutant strain was further modified for production of GDP-fucose as described in Example 1 and transformed with an expression plasmid containing a constitutive transcriptional unit for the alpha-1, 4-fucosyltransferase from B.
  • the mutant strain was transformed with an expression plasmid containing a constitutive transcriptional unit for a transporter protein with SEQ ID 01.
  • the novel strain was evaluated in a growth experiment for production of LNDFHI according to the culture conditions provided in Example 1, in which the strains were cultivated in minimal medium supplemented with 15 g/L sucrose and 20 g/L Lactose.
  • a reference strain was used with the same genetic make-up as the novel mutant strain but lacking the transporter protein.
  • the strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed as described in Example 1. The experiment demonstrated that expression of a transporter protein with SEQ ID NO 01 enhanced the production of LNDFHI that is being produced in a LNDFHI production host expressing the galactoside beta- 1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6), the beta-1, 3- galactosyltransferase wbdO (Uniprot ID Q5UHA8) from S.
  • LgtA galactoside beta- 1,3-N-acetylglucosaminyltransferase
  • wbdO Uniprot ID Q5UHA8
  • enterica the alpha-1, 4-fucosyltransferase from B. catarrhinii (UniProt ID A0A4V6F2S0) and the alpha-1, 2-fucosyltransferase from D. alaskensis (UniProt ID Q316B5) (Tables 9 and 10).
  • SD represents the standard deviation (4 replicates of the same strain tested).
  • the “reference strain (REF)” is identical to the tested strains, except that the indicated transporter protein (SEQ ID NO 01) is not expressed in the reference strain.
  • Example 7 3F(3)-3F(l)-LNnT (LNnDFH) production in an E. coli host cultivated 72 h in a growth experiment in minimal media supplemented with 20 g/L lactose
  • E. coli K12 MG1655 strain was modified as described in Example 1 comprising genomic knock-outs of the E. coli genes lacZ, nagB, galT, ushA and IdhA and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B.
  • CscB sucrose transporter
  • Frk fructose kinase
  • BaSP sucrose phosphorylase
  • adolescentis (UniProt ID A0ZZH6), the galactoside beta-1, 3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6), the N-acetylglucosamine beta-1, 4-galactosyltransferase GalT7 (Uniprot ID F4ZLW1) from P. multocida.
  • the mutant strain was further modified for production of GDP-fucose as described in Example 1 and transformed with an expression plasmid containing a constitutive transcriptional unit for the alpha-1, 3-fucosyltransferase from M.
  • the mutant strain was transformed with an expression plasmid containing a constitutive transcriptional unit for a transporter protein with SEQ ID 01.
  • the novel strain was evaluated in a growth experiment for production of LNnDFH according to the culture conditions provided in Example 1, in which the strains were cultivated in minimal medium supplemented with 15 g/L sucrose and 20 g/L Lactose.
  • a reference strain was used with the same genetic make-up as the novel mutant strain but lacking the transporter protein.
  • the strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed as described in Example 1.
  • SD represents the standard deviation (4 replicates of the same strain tested).
  • the “reference strain (REF)” is identical to the tested strains, except that the indicated transporter protein (SEQ ID NO 01) is not expressed in the reference strain.
  • Example 8 lacto-N-tetraose (LNT) production in an E. coli host cultivated 72 h in a growth experiment in minimal media supplemented with 20 g/L lactose
  • E. coli K12 MG1655 strain was modified as described in Example 1 comprising genomic knock-outs of the E. coli genes lacZ, nagB, galT, ushA and IdhA and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B.
  • CscB sucrose transporter
  • Frk fructose kinase
  • BaSP sucrose phosphorylase
  • adolescentis (UniProt ID A0ZZH6), the galactoside beta-1, 3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6) and the N-acetylglucosamine beta-1, 3-galactosyltransferase wbdO (Uniprot ID Q5UHA8) from Salmonella enterica.
  • the mutant strain thus obtained was further engineered to create two new strains (A and B) wherein each strain was modified by a genomic knock-in containing a different promotor (P) and 5' untranslated region (UTR) sequence combined with a common terminator (T4) sequence (Table 13) leading to different constitutive transcriptional units for a transporter protein with SEQ ID NO 13.
  • P promotor
  • UTR 5' untranslated region
  • T4 common terminator sequence leading to different constitutive transcriptional units for a transporter protein with SEQ ID NO 13.
  • the novel strains were evaluated in a growth experiment for production of LNT according to the culture conditions provided in Example 1, in which the strains were cultivated in minimal medium supplemented with 15 g/L sucrose and 20 g/L Lactose.
  • a reference strain was used with the same genetic make-up as the novel mutant strains but lacking the transporter protein.
  • the strains were grown in eight biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed as described in Example 1. The experiment demonstrated that expression of a transporter protein with SEQ ID NO 13 enhanced the production of LNT that is being produced in a LNT production host expressing the galactoside beta-1, 3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6) and N-acetylglucosamine beta-1, 3-galactosyltransferase wbdO (Uniprot ID Q5UHA8) from Salmonella enterica (Table 14). Table 13. Promoter (P), untranslated region (UTR) and terminator (T) sequences used to express the transporter protein with SEQ ID NO 13 integrated in the genome of the mutant E. coli strains A and B as given in Table 14.
  • P promoter
  • UTR untran
  • Table 14 The percentage of LNT cell performance index (CPI) and g/L measured in the whole broth compared to the REF strain without transporter protein.
  • the modified E. coli strains A and B each expressing the transporter protein with SEQ ID NO 13 from a different expression cassette integrated in the genome (see Table 13). Strains were evaluated in a growth experiment according to the cultivation conditions provided in Example 1, in which the cultivation medium contained 15 g/L sucrose and 20 g/L Lactose.
  • SD represents the standard deviation (8 replicates of the same strain tested).
  • the “reference strain (REF)” is identical to the tested strains, except that the indicated transporter protein (SEQ ID NO 13) is not expressed in the reference strain.
  • Example 9 Lacto-N-neotetraose (LNnT) production in an E. coli host cultivated 72 h in a growth experiment in minimal media supplemented with 20 g/L lactose
  • E. coli K12 MG1655 strain was modified as described in Example 1 comprising genomic knock-outs of the E. coli genes lacZ, nagB, galT, ushA and IdhA and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B.
  • CscB sucrose transporter
  • Frk fructose kinase
  • BaSP sucrose phosphorylase
  • adolescentis (UniProt ID A0ZZH6), the galactoside beta-1, 3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6) and the N-acetylglucosamine beta-1, 4-galactosyltransferase (GalT) from H. pylori (UniProt ID Q9RHG8).
  • the mutant strain was further modified by a genomic knock-in of a constitutive transcriptional unit containing the transporter protein with SEQ ID 13.
  • the novel strain was evaluated in a growth experiment for production of LNnT according to the culture conditions provided in Example 1, in which the strains were cultivated in minimal medium supplemented with 15 g/L sucrose and 20 g/L Lactose.
  • a reference strain was used with the same genetic make-up as the novel mutant strain but lacking the transporter protein.
  • the strains were grown in eight biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed as described in Example 1.
  • SD represents the standard deviation (8 replicates of the same strain tested).
  • the “reference strain (REF)” is identical to the tested strains, except that the indicated transporter protein (SEQ ID NO 13) is not expressed in the reference strain.
  • “SD” represents the standard deviation (8 replicates of the same strain tested).
  • the “reference strain (REF)” is identical to the tested strains, except that the indicated transporter protein (SEQ ID NO 13) is not expressed in the reference strain.
  • Example 10 Lacto-N-neotetraose (LNnT) production in an E. coli host cultivated 72 h in a growth experiment in minimal media supplemented with 20 g/L lactose
  • E. coli K12 MG1655 strain was modified as described in Example 1 comprising genomic knock-outs of the E. coli genes lacZ, nagB, galT, ushA and IdhA and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B.
  • CscB sucrose transporter
  • Frk fructose kinase
  • BaSP sucrose phosphorylase
  • adolescentis (UniProt ID A0ZZH6), the galactoside beta-1, 3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6), the N-acetylglucosamine beta-1, 4-galactosyltransferase (GalT) from H. pylori (UniProt ID Q9RHG8) and the N-acetylglucosamine beta-1, 4-galactosyltransferase (LgtB) from N. meningitidis (Uniprot ID Q51116, sequence version 02, 01 Dec 2000).
  • the mutant strain was transformed with an expression plasmid containing a constitutive transcriptional unit for a transporter protein with SEQ ID 15, 17, 18 or 19.
  • the novel strains were evaluated in a growth experiment for production of LNnT according to the culture conditions provided in Example 1, in which the strains were cultivated in minimal medium supplemented with 15 g/L sucrose and 20 g/L Lactose.
  • a reference strain was used with the same genetic make-up as the novel mutant strains but lacking the transporter protein.
  • the strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed as described in Example 1.
  • SD represents the standard deviation (4 replicates of the same strain tested).
  • the “reference strain (REF)” is identical to the tested strains, except that the indicated transporter protein (SEQ. ID NO 15, 17, 18 or 19) is not expressed in the reference strain.

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Abstract

La présente invention se rapporte au domaine technique de la biologie synthétique et de l'ingénierie métabolique. Plus particulièrement, la présente invention se rapporte au domaine technique de la culture de cellules génétiquement modifiées. La présente invention concerne (i) un procédé de production d'un oligosaccharide contenant du lacto-N-triose (LN3) ou d'un mélange d'oligosaccharides comprenant un oligosaccharide contenant du LN3 par culture d'une cellule génétiquement modifiée comprenant une protéine transporteuse ; ainsi que (ii) la cellule génétiquement modifiée utilisée dans le procédé.
PCT/EP2024/069604 2023-07-11 2024-07-11 Production cellulaire d'oligosaccharides contenant du lacto-n-biose (ln3) Pending WO2025012358A1 (fr)

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WO2022034067A1 (fr) 2020-08-10 2022-02-17 Inbiose N.V. Production d'un mélange d'oligosaccharides par une cellule
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