Cellular production of lacto-n-triose (LN3)-containing oligosaccharides

Abstract

The present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of 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.

Description

[0001] Cellular production of lacto-N-biose (LN3)-containing oligosaccharides

[0002] Field of the invention

[0003] 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.

[0004] Background of the invention

[0005] To date 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). 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. et al., 2011, Milk Oligosaccharides, Nova Biomedical Books, New York ISBN 978-1-61122- 831-1; Coppa et al, 2013, Ital. J. Pediatr. 2013, 39(2)), in particular milk oligosaccharides (MOs), i.e. (oligo)saccharides which are found in milk of animals such as mammals and humans (Urashima et al, 2011; Coppa et al, 2013). 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). Other 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). 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).

[0006] 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 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.

[0007] 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). It is hence an object of the present invention to provide a genetically engineered cell for the production of a lacto-N-triose (LN3)-containing oligosaccharide in an efficient, time and cost-effective way and which yields higher amounts of the desired product.

[0008] Summary of the invention

[0009] In a first aspect, 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. It was surprisingly found that 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.

[0010] In a second aspect, 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.

[0011] In a third aspect, 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. In a fourth aspect, the invention provides the use of said novel transporter protein in the production of a

[0012] LN3-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide.

[0013] Detailed description of the invention

[0014] Cell for the production of an oligosaccharide

[0015] In a first aspect, 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:

[0016] - selected from SEQ ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06; or having at least 80.0 % sequence identity to the full-length amino acid sequence of SEQ ID NO 01, 13, 15, 16, 02, 03, 04, 05 or 06; or 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 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, 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.

[0017] Throughout the application and claims, the expression "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".

[0018] Throughout the application and claims, the expression "SEQ ID NO 02, 03, 04, 05 or 06" is preferably replaced with "SEQ ID NO 02".

[0019] Throughout the application and claims, "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". Throughout the application and claims, 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.

[0020] Likewise, throughout the application and claims, a functional fragment of a polypeptide 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 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. For the purpose of the present invention, the 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).

[0021] As understood by the skilled person, altering the amino acid sequence of a protein by introducing one or more conservative amino acid substitutions typically does not affect or only has a limited effect on the activity of said protein. In the context of the present invention, a "conservative amino acid substitution" is the substitution of an amino acid by another structurally-related amino acid, i.e. having a similar sidechain. In other words, 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).

[0022] In the context of the present invention, a functional fragment of a reference protein or reference polypeptide (e.g. a transporter protein; usually represented by a SEQ ID NO or Uniprot ID) 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 %, of the full-length of said reference protein or reference polypeptide. For example, if a reference transporter protein consists of 408 amino acid residues, then it is preferred that 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.

[0023] In other words, a functional fragment of a reference protein or reference polypeptide (e.g. a transporter protein; usually represented by a SEQ ID NO or Uniprot ID) 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 %, 2.5 %, 2.0 %, 1.5 %, 1.0 %, 0.5 %, more preferably no more than 15.0%, even more preferably no more than 10.0%, even more preferably no more than 7.5%, even more preferably no more than 5.0%, most preferably no more than 2.5%, of the full-length of said reference protein or reference polypeptide.

[0024] Further, a functional fragment of a reference protein or reference polypeptide (e.g. a transporter protein; usually represented by a SEQ ID NO or Uniprot ID) 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). It is preferred that 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. In this context, "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. As understood by the skilled person, 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.

[0025] Likewise, throughout the description and claims, 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). It is preferred that 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.

[0026] Throughout the application and claims, the feature "transporter protein" is preferably replaced with "exporter protein".

[0027] In a preferred embodiment, said transporter protein according to the invention is heterologous, i.e. said transporter protein originates from a source foreign to the particular cell.

[0028] In another preferred embodiment, 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. In an additional and / or alternative preferred embodiment, 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.

[0029] Throughout the application and claims, unless specifically stated otherwise, the expression "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". Optionally, 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, 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 26, 30, 33 and / or 34 of TMl of the transporter protein represented by SEQ ID NO 16, o 22, 26, 29 and / or 30 of TMl of the transporter protein represented by SEQ ID NO 02, o 22 of TMl of the transporter protein represented by SEQ ID NO 03, o 27 , 31, 34 and / or 35 of TMl of the transporter protein represented by SEQ ID NO 04, o 28, 32, 35 and / or 36 of TMl of the transporter protein represented by SEQ ID NO 05, o 26, 30, 33 and / or 34 of TMl of the transporter protein represented by SEQ ID NO 06.

[0030] 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). Preferably, 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.

[0031] In the context of the present invention, "a reduced ability to transport an antimicrobial agent" refers to a transporter which transports less of the antimicrobial agent than the unmodified transporter. As 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. Preferably, 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.

[0032] Throughout the application and claims, the features "transmembrane domain 1", "TMl" and "N-terminal transmembrane domain" are 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,

[0033] 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. For the purpose of the present invention, version 1.0.24 as released on 30 March 2023 was used.

[0034] Preferably, TM1 of the transporter protein with SEQ ID NO 01 is represented by

[0035] PLCLVLYEFSTYIANDMIQPGM (SEQ ID NO 07).

[0036] Preferably, TM1 of the transporter protein with SEQ ID NO 13 is represented by

[0037] PLCLVLYEFSTYIANDMIQPGM (SEQ ID NO 14).

[0038] Preferably, TM1 of the transporter protein with SEQ ID NO 02 is represented by

[0039] PLALVLFEFSVYIANDMIQPGM (SEQ ID NO 08).

[0040] Preferably, TM1 of the transporter protein with SEQ ID NO 03 is represented by AGSLAVLLGALDTYVVVTIM

[0041] (SEQ ID NO 09).

[0042] Preferably, TM1 of the transporter protein with SEQ ID NO 04 is represented

[0043] PLALVLFEFAVYIANDMAQPAM (SEQ ID NO 10).

[0044] Preferably, TM1 of the transporter protein with SEQ ID NO 05 is represented

[0045] PLALVLFEFATYISNDMILPGM (SEQ ID NO 11).

[0046] Preferably, TM1 of the transporter protein with SEQ ID NO 06 is represented

[0047] PLCLVLYEFTTYIGNDMIQPGM (SEQ ID NO 12).

[0048] Preferably, TM1 of the transporter protein with SEQ ID NO 15 is represented

[0049] PLCLVLFEFATYIANDMIQPGM (SEQ ID NO 20).

[0050] Preferably, TM1 of the transporter protein with SEQ ID NO 16 is represented

[0051] PLCLVLFEFATYIGNDMIQPGM (SEQ ID NO 21).

[0052] Preferably, TM1 of the transporter protein with SEQ ID NO 17 is represented

[0053] PLCLVLFEFATYIGNDMIQPGM (SEQ ID NO 22).

[0054] Preferably, TM1 of the transporter protein with SEQ ID NO 18 is represented

[0055] PLCLVLFEFATYIGNDMIQPGM (SEQ ID NO 23).

[0056] Preferably, TM1 of the transporter protein with SEQ ID NO 19 is represented

[0057] PLCLVLFEFATYIGNDMIQPGM (SEQ ID NO 24).

[0058] In the context of the invention, 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). For the purpose of the present invention, a tyrosine (Y) is classified herein as a hydrophilic amino acid. In other words, 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. and / or Y, more 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, even more preferably into A or L, most preferably into A.

[0059] Optionally, 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.

[0060] Cell

[0061] In a preferred embodiment of the first aspect of the invention, 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.

[0062] Throughout the application and claims, unless specifically stated otherwise, a microorganism is preferably a bacterium, a yeast or a fungus, more preferably a bacterium or a yeast, most preferably a bacterium.

[0063] Throughout the application and claims, unless specifically stated otherwise, 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. Hence, preferably 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.

[0064] Throughout the application and claims, unless specifically stated otherwise, 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. marxianus, K. thermotolerans), Yarrowia (like e.g. Yarrowia lipolytica), Eremothecium, Zygosaccharomyces, Starmerella l ike e.g. Starmerella bombicola) or Debaromyces. Said yeast cell is more preferably selected from Pichia pastoris, Yarrowia lipolitica, Saccharomyces cerevisiae and Kluyveromyces lactis.

[0065] Throughout the application and claims, unless specifically stated otherwise, a fungus preferably belongs to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus.

[0066] Throughout the application and claims, unless specifically stated otherwise, a plant cell includes cells of flowering and non-flowering plants, as well as algal cells, for example Chlamydomonas, Chlorella, etc. Preferably, said plant cell is a tobacco, alfalfa, rice, cotton, rapeseed, tomato, corn, maize or soybean cell. Throughout the application and claims, unless specifically stated otherwise, 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. lobster, crab, shrimp, clams, oyster, mussel, sea urchin), reptiles (e.g. snake, alligator, turtle), amphibians (e.g. frogs) or insects (e.g. fly, nematode) or is a genetically modified cell line derived from human cells excluding embryonic stem cells. Both human and non-human mammalian cells are preferably chosen from the list consisting of an epithelial cell like e.g. a mammary epithelial cell, an embryonic kidney cell (e.g. HEK293 or HEK 293T cell), a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell like e.g. an N20, SP2 / 0 or YB2 / 0 cell, an NIH-3T3 cell, a non-mammary adult stem cell or derivatives thereof such as described in WO21067641. Throughout the application and claims, unless specifically stated otherwise, an insect cell is preferably derived from Spodoptera frugiperda like e.g. Sf9 or Sf21 cells, Bombyx mori, Mamestra brassicae,

[0067] Trichoplusia ni like e.g. BTI-TN-5B1-4 cells or Drosophila melanogaster like e.g. Drosophila S2 cells.

[0068] Throughout the application and claims, unless specifically stated otherwise, a protozoan cell is preferably a Leishmania tarentolae cell.

[0069] In another preferred embodiment, 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.

[0070] In the context of the present invention, it is particularly preferred that 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.

[0071] Preferably, 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.

[0072] More preferably, 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).

[0073] Throughout the application and claims, the feature "growth speed" is preferably replaced with "maximum growth rate (pmax)".

[0074] In a preferred embodiment, 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. Preferably, said cell is modified (preferably genetically modified) in the expression or activity of said galactoside beta-1, 3-N-acetylglucosaminyltransferase. Several galactoside beta-1, 3-N-acetylglucosaminyltransferases are well-known to be suitable for the synthesis of said LN3-containing oligosaccharide. Preferably 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).

[0075] In an additional and / or alternative preferred embodiment, 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 galactosyltransferase, most preferably a galactosyltransferase. Several examples for each of the indicated glycosyltransferases are well-known to be suitable for the synthesis of said LN3-containing oligosaccharide.

[0076] Preferably, said cell is modified (preferably genetically modified) in the expression or activity of at least one of said glycosyltransferases.

[0077] Preferably, 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). Throughout the application and claims, the feature "galactosyltransferase" is preferably replaced with "N-acetylglucosamine-galactosyltransferase".

[0078] Preferably, 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). Preferably, said N-acetylglucosaminyltransferase is a beta-1, 6-N-acetylglucosaminyltransferase.

[0079] Preferably, 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).

[0080] Preferably, 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).

[0081] In another preferred embodiment of the invention, 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.

[0082] 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-acetylgalactosaminylation pathway comprising of (i) at least one N-acetylgalactosaminyl- transferase (preferably as defined herein) and (ii) UDP-GalNAc which is donor for said N- acetylgalactosaminyltransferase(s); and e) sialylation pathway comprising of (i) at least one sialyltransferase (preferably as defined herein) and (ii) CMP-sialic acid which is donor for said sialyltransferase(s);

[0083] 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-acetylgalactosaminylation pathway comprising of (i) at least one N-acetylgalactosaminyl- transferase (preferably as defined herein) and (ii) UDP-GalNAc which is donor for said N- acetylgalactosaminyltransferase(s).

[0084] 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).

[0085] 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).

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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. 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 uridyltransferase, glucosamine-1- phosphate acetyltransferase, sialic acid synthase, N-acetylneuraminate lyase, N-acylneuraminate-9- phosphate synthase, N-acylneuraminate-9-phosphate phosphatase and CMP-sialic acid synthase.

[0090] Throughout the application and claims, unless specifically stated otherwise, said "CMP-sialic acid" is preferably "CMP-Neu5Ac". Likewise, "sialic acid" is preferably replaced with "Neu5Ac" (N- acetylneuraminate and N-acetylneuraminic acid are interchangeably used for Neu5Ac).

[0091] In an additional and / or alternative preferred embodiment, at least one gene of said at least one metabolic pathway is genetically engineered. Preferably a glycosyltransferase of said at least one metabolic pathway is genetically engineered.

[0092] In an additional and / or alternative preferred embodiment, 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.

[0093] Preferably, 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). For an enhanced production of UDP-GIcNAc, 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.

[0094] Preferably, 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. For an enhanced production of GDP-fucose, 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.

[0095] Preferably, a metabolic pathway for the production of UDP-galactose comprises UDP-glucose-4- epimerase. For an enhanced production of UDP-galactose, 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.

[0096] Preferably, a metabolic pathway for the production of UDP-GalNAc comprises an UDP-glucose-4- epimerase. For an enhanced production of UDP-GalNAc, 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.

[0097] In another preferred embodiment, 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. 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-linkage, preferably an alpha- 1,2- or an alpha-1, 3-linkage, more preferably an alpha-1, 3-linkage.

[0098] In the context of the present invention, the term "precursor saccharide" refers to a saccharide which lacks at least one monosaccharide compared to the corresponding saccharide (i.e. LN3-containing oligosaccharide).

[0099] As understood by the skilled person, the term "saccharide" refers to a molecule comprising at least one monosaccharide, preferably it refers to a molecule consisting of one or more monosaccharide residue(s). The term "monosaccharide" as used herein 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.

[0100] In another preferred embodiment of the present invention, 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.

[0101] In an embodiment of the first aspect of the invention, 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.

[0102] In the context of the present invention, the term "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.) between two sugar units can be expressed, for example, as 1,4, l->4, or (1-4), used interchangeably herein. Each monosaccharide can be in the cyclic form (e.g. pyranose or furanose form). An 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. For the sake of clarity, throughout the application and claims, the expression "x-y" refers to a range from and including x to and including y. For example, 3-5 monosaccharides means that 3, 4 or 5 monosaccharides are present.

[0103] In the context of the present invention, the term "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.

[0104] In a preferred embodiment, said LN3-containing oligosaccharide according to the invention consists of at least 3, preferably at least 4, monosaccharides. In other words, said LN3-containing oligosaccharide according to the invention preferably has a DP of at least 3, more preferably a DP of at least 4.

[0105] In an additional and / or alternative preferred embodiment, 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.

[0106] In a more preferred embodiment, 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. In other words, 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.

[0107] Throughout the description and claims, the terms "oligosaccharide mixture" and "mixture comprising different oligosaccharides" are used interchangeably herein. In the context of the present invention, 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.

[0108] Preferably, 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.

[0109] In another preferred embodiment, 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). As understood by the skilled person, 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) and 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 (Tursiops truncates), northern minke whales (Balaenoptera acutorostrata), tammar wallabies (Macropus eugenii), red kangaroos (Macropus rufus), common brushtail possum (Trichosurus Vulpecula), koalas (Phascolarctos cinereus), eastern quolls (Dasyurus viverrinus), platypus (Ornithorhynchus anatinus) (Urashima T. et al., 2011, Milk Oligosaccharides, Nova Biomedical Books, New York ISBN 978-1-61122-831-1; Coppa et al, 2013, Ital. J. Pediatr. 2013, 39(2)). 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). Other 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). Examples hereof are 3'-SLN (Neu5Ac-a2,3-Gal-pi,4- GIcNAc) and 6'-SLN (Neu5Ac-a2,6-Gal-pi,4-GlcNAc) (Urashima et al, 2011; Wrigglesworth et al, 2020; Wei et al, 2018). Further, milk saccharides comprise milk glycosaminoglycans (GAGs; Coppa et al, 2013; Rai et al, 2021, Int. J. Biol. Macromolecules, 193(A): p. 137-144). In the context of the invention, it is preferred that said LN3-containing oligosaccharide according to the invention is not a glycosaminoglycan.

[0110] In an additional and / or alternative preferred embodiment, 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.

[0111] In a more preferred embodiment, 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-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, lacto-N-neopentaose, para-Lacto-N-neopentaose, para-Lacto-N-pentaose, GIcNAc- 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, Lacto-N-hexaose (LNH), para-lacto-N-hexaose (pLNH), lacto-N-neohexaose (LNnH), para- Lacto-N-neohexaose (pLNnH), para-lacto-N-neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N- octaose (LNO), lacto-N-neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose, iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N- nonaose, lacto-N-decaose, iso lacto-N-decaose, novo lacto-N-decaose and lacto-N-neodecaose; preferably 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-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, lacto-N-neopentaose, para-Lacto-N-neopentaose, 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-l,3-LNT, Lacto-N-hexaose (LNH), para-lacto-N-hexaose (pLNH), lacto- N-neohexaose (LNnH), para-Lacto-N-neohexaose (pLNnH), para-lacto-N-neohexaose II (pLNnH II), para- lacto-N-hexaose II (pLNH II), lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose and para lacto-N-neooctaose; more preferably 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-1, 4-GlcNAc-beta-l,3-)Gal-beta-l,4-Glc, GlcNAc-beta-l,6-(Gal-beta-l,3-GlcNAc-beta-l,3-)Gal-beta-

[0112] 1.4-Glc, Lacto-N-pentaose, lacto-N-neopentaose, para-Lacto-N-neopentaose, 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-beta-l,3-LNT, Lacto-N-hexaose (LNH), para-lacto-N-hexaose (pLNH), lacto-N-neohexaose (LNnH), para-Lacto-N-neohexaose (pLNnH), para-lacto-N-neohexaose II (pLNnH II) and para-lacto-N-hexaose II (pLNH II); even more preferably selected from the list consistingof Lacto-N-triose II (LN3, LNT-II), GIcNAc- 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,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, Lacto-N-hexaose (LNH), para-lacto-N-hexaose (pLNH), lacto-N- neohexaose (LNnH), para-Lacto-N-neohexaose (pLNnH), para-lacto-N-neohexaose II (pLNnH II) and para- lacto-N-hexaose II (pLNH II); even more preferably 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), para- Lacto-N-neohexaose (pLNnH), para-lacto-N-neohexaose II (pLNnH II) and para-lacto-N-hexaose II (pLNH II); even more preferably selected from the list consisting of Lacto-N-triose II (LN3, LNT-II), GIcNAc-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); most preferably selected from the list consisting of LN3, LNT and LNnT; optionally wherein said LN3-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-linkage, preferably an alpha-1,2- or an alpha-1, 3-linkage, more preferably an alpha- 1,3-linkage; and / or 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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage.

[0113] In an alternative more preferred embodiment, 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-

[0114] 1.4-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, lacto-N-neopentaose, para-Lacto-N-neopentaose, 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-beta-l,3-LNT, Lacto-N-hexaose (LNH), para-lacto-N-hexaose (pLNH), lacto-N-neohexaose (LNnH), para-Lacto-N-neohexaose (pLNnH), para-lacto-N-neohexaose II (pLNnH 11), para-lacto-N-hexaose II (pLNH ll)7lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N- octaose (LNO), lacto-N-neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose, iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N- nonaose, lacto-N-decaose, iso lacto-N-decaose, novo lacto-N-decaose, lacto-N-neodecaose, 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,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-Glc, lacto-N-neodifucohexaose (LNnDFH), Fuc- alphal,2-Gal-beta-l,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N- fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose V (LNFP V), Gal-LNFP I, GalNAc-LNFP I, Fuc-alphal,2-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N- difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), lewis b-lewis x, GlcNAc-beta-l,3-Gal-beta- l,4-(Fuc-alpha-l,3-)Glc, GlcNAc-beta-l,6-(GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, GIcNAc- beta-l,6-(Gal-beta-l,4-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, GlcNAc-beta-l,6-(Gal-beta-

[0115] 1.3-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, monofucosyllacto-N-hexaose III (MFLNH III), difucosyllacto-N-hexaose (a) (DFLNH (a)), difucosyllacto-N-hexaose (DFLNH) and trifucosyllacto-N- hexaose (TFLNH); preferably selected from the list consisting of Lacto-N-triose II (LN3, LNT-II), GIcNAc- 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-l,3-)Gal-beta-l,4-Glc, GlcNAc-beta-1,6- (Gal-beta-l,3-GlcNAc-beta-l,3-)Gal-beta-l,4-Glc, Lacto-N-pentaose, lacto-N-neopentaose, para-Lacto-N- neopentaose, 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-beta-l,3-LNT, Lacto-N-hexaose (LNH), para-lacto-N-hexaose (pLNH), lacto-N-neohexaose (LNnH), para-Lacto-N-neohexaose (pLNnH), para-lacto-N-neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N- neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose, 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,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-Glc, lacto-N-neodifucohexaose (LNnDFH), Fuc- alphal,2-Gal-beta-l,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N- fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose V (LNFP V), Gal-LNFP I, GalNAc-LNFP I, Fuc-alphal,2-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N- difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), lewis b-lewis x, GlcNAc-beta-l,3-Gal-beta-

[0116] 1.4-(Fuc-alpha-l,3-)Glc, GlcNAc-beta-l,6-(GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, GIcNAc- beta-1, 6-(Gal-beta-l,4-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, GlcNAc-beta-l,6-(Gal-beta-

[0117] 1.3-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, monofucosyllacto-N-hexaose III (MFLNH III), difucosyllacto-N-hexaose (a) (DFLNH (a)) and difucosyllacto-N-hexaose (DFLNH); more preferably 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-l,3-)Gal-beta-l,4-Glc, GlcNAc-beta-l,6-(Gal-beta-l,3-GlcNAc-beta-l,3-)Gal-beta-

[0118] 1.4-Glc, Lacto-N-pentaose, lacto-N-neopentaose, para-Lacto-N-neopentaose, 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-beta-l,3-LNT, Lacto-N-hexaose (LNH), para-lacto-N-hexaose (pLNH), lacto-N-neohexaose (LNnH), para-Lacto-N-neohexaose (pLNnH), para-lacto-N-neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), 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,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-Glc, lacto-N-neodifucohexaose (LNnDFH), Fuc-alphal,2-Gal-beta-l,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose V (LNFP V), Gal- LNFP I, GalNAc-LNFP I, Fuc-alphal,2-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N-difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), lewis b-lewis x, GlcNAc-beta-1,3- Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, GlcNAc-beta-l,6-(GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, GlcNAc-beta-l,6-(Gal-beta-l,4-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc and GlcNAc-beta-1,6- (Gal-beta-l,3-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc; even more preferably 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,3-Gal-beta-

[0119] 1.3-GlcNAc-beta-l,3-Gal-beta-l,4-Glc, GalNAc-beta-l,3-LNT, Gal-beta-1, 3-GalNAc-beta-l,3-LNT, Lacto-N- hexaose (LNH), para-lacto-N-hexaose (pLNH), lacto-N-neohexaose (LNnH), para-Lacto-N-neohexaose (pLNnH), para-lacto-N-neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), 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,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-Glc, lacto-N-neodifucohexaose (LNnDFH), Fuc- alphal,2-Gal-beta-l,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N- fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose V (LNFP V), Gal-LNFP I, GalNAc-LNFP I, Fuc-alphal,2-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N- difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), lewis b-lewis x, GlcNAc-beta-l,3-Gal-beta-

[0120] 1.4-(Fuc-alpha-l,3-)Glc, GlcNAc-beta-l,6-(GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, GIcNAc- beta-l,6-(Gal-beta-l,4-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc and GlcNAc-beta-l,6-(Gal- beta-1, 3-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc; even more preferably 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), para-Lacto-N-neohexaose (pLNnH), para-lacto-N-neohexaose II (pLNnH II), para- lacto-N-hexaose II (pLNH II), 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,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-Glc, lacto-N- neodifucohexaose (LNnDFH), Fuc-alphal,2-Gal-beta-l,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4- (Fuc-alpha-l,3-)Glc, lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N- fucopentaose V (LNFP V), Gal-LNFP I, GalNAc-LNFP I, Fuc-alphal,2-Gal-beta-l,3-GlcNAc-beta-l,3-Gal- beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N-difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), lewis b-lewis x, GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc and GlcNAc-beta-l,6-(GlcNAc-beta-l,3-)Gal- beta-l,4-(Fuc-alpha-l,3-)Glc; even more preferably 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), 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), 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 x, GlcNAc-beta-l,3-Gal-beta- l,4-(Fuc-alpha-l,3-)Glc and GlcNAc-beta-l,6-(GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc; most preferably 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 x and GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc; optionally wherein said 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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6- linkage.

[0121] Throughout the application and claims, 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). More than 50 different examples of a sialic acid having a nine-carbon backbone are known (Essentials of Glycobiology, 2ndedition, 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, 2ndedition, 2009, Chapter 14, Varki and Schauer). Hence, the term "sialic acid having an eight-carbon backbone" is preferably replaced with "eight-carbon 2-keto-3-deoxyoctonic acid".

[0122] In another preferred embodiment, and in fact particularly preferred in the context of the present invention, 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.

[0123] In the context of the present invention, throughout the application and claims, the expression "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. Preferably, throughout the application and claims, said LNnT-containing oligosaccharide is on oligosaccharide comprising LNnT at its reducing end.

[0124] In a preferred embodiment, 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-neoheptaose, para lacto-N-neoheptaose, lacto-N- neooctaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose and lacto-N- neodecaose; more preferably 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-neoheptaose, para lacto-N- neoheptaose, lacto-N-neooctaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose and para lacto-N- neooctaose; even more preferably selected from the list consisting of Lacto-N-neotetraose (LNnT), Gal- alpha-l,3-Gal-beta-l,4-GlcNAc-beta-l,3-Gal-beta-l,4-Glc, GlcNAc-beta-l,6-(Gal-beta-l,4-GlcNAc-beta-

[0125] 1.3-)Gal-beta-l,4-Glc, lacto-N-neopentaose, para-Lacto-N-neopentaose, para-lacto-N-hexaose (pLNH), lacto-N-neohexaose (LNnH) and para-Lacto-N-neohexaose (pLNnH); even more preferably selected from the list consisting of Lacto-N-neotetraose (LNnT), Gal-alpha-1, 3-Gal-beta-l,4-GlcNAc-beta-l,3-Gal-beta-

[0126] 1.4-Glc, para-lacto-N-hexaose (pLNH), lacto-N-neohexaose (LNnH) and para-Lacto-N-neohexaose (pLNnH); even more preferably selected from the list consisting of Lacto-N-neotetraose (LNnT), para- lacto-N-hexaose (pLNH), lacto-N-neohexaose (LNnH) and para-Lacto-N-neohexaose (pLNnH); even more preferably Lacto-N-neotetraose (LNnT) or para-Lacto-N-neohexaose (pLNnH); most preferably LNnT; 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-linkage, preferably an alpha-1,2- or an alpha-1, 3-linkage, more preferably an alpha- 1,3-linkage; and / or 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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage.

[0127] In an alternative preferred embodiment, 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-neoheptaose, para lacto-N- neoheptaose, lacto-N-neooctaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N- neooctaose, lacto-N-neodecaose, 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-

[0128] 1.4-(Fuc-alphal,3-)Glc, Fuc-alphal,2-Gal-beta-l,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-Glc, lacto-N-neodifucohexaose (LNnDFH), Fuc-alphal,2-Gal-beta-l,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal- beta-l,4-(Fuc-alpha-l,3-)Glc and GlcNAc-beta-l,6-(Gal-beta-l,4-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc- alpha-l,3-)Glc; more preferably 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-neoheptaose, para lacto-N- neoheptaose, lacto-N-neooctaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N- neooctaose, 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,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-Glc, lacto-N- neodifucohexaose (LNnDFH), Fuc-alphal,2-Gal-beta-l,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4- (Fuc-alpha-l,3-)Glc and GlcNAc-beta-l,6-(Gal-beta-l,4-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3- )Glc; even more preferably 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-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,4-(Fuc-alpha-l,3-)GlcNAc- beta-l,3-Gal-beta-l,4-Glc, lacto-N-neodifucohexaose (LNnDFH), Fuc-alphal,2-Gal-beta-l,4-(Fuc-alpha-

[0129] 1.3-)GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc and GlcNAc-beta-l,6-(Gal-beta-l,4-GlcNAc-beta-

[0130] 1.3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc; even more preferably 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, 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,4-(Fuc-alpha-l,3- )GlcNAc-beta-l,3-Gal-beta-l,4-Glc, lacto-N-neodifucohexaose (LNnDFH), Fuc-alphal,2-Gal-beta-l,4-(Fuc- alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc and GlcNAc-beta-l,6-(Gal-beta-l,4-GlcNAc- beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc; even more preferably 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,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-Glc, lacto-N- neodifucohexaose (LNnDFH) and Fuc-alphal,2-Gal-beta-l,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-

[0131] 1.4-(Fuc-alpha-l,3-)Glc; even more preferably 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) and lacto-N-neodifucohexaose (LNnDFH); even more preferably selected from the list consisting of LNnT, Lacto-N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose III (LNFP III), lacto-N- neofucopentaose V (LNnFP V, LNFP VI) and lacto-N-neodifucohexaose (LNnDFH); most preferably selected from the list consisting of LNnT, Lacto-N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose III (LNFP III) and lacto-N-neofucopentaose V (LNnFP V, LNFP VI); optionally wherein said LNnT-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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage. In the context of the present invention, throughout the application and claims, the expression "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. Preferably, throughout the application and claims, said LNT-containing oligosaccharide is on oligosaccharide comprising LNT at its reducing end.

[0132] In a preferred embodiment, 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-beta-l,3-LNT, Lacto-N-hexaose (LNH), para-lacto-N- neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), lacto-N-heptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), iso 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; more preferably selected from the list consisting of Lacto-N-tetraose (LNT), Gal-alpha-1, 3-Gal-beta-l, 3-GlcNAc- beta-1, 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-beta-l,3-LNT, Lacto-N-hexaose (LNH), para-lacto-N- neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), lacto-N-heptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), iso lacto-N-octaose and para lacto-N-octaose; even more preferably 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-beta-l,3-LNT, Lacto-N-hexaose (LNH), para-lacto-N-neohexaose II (pLNnH II) and para- lacto-N-hexaose II (pLNH II); even more preferably 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-1, 3-Gal-beta-l, 3- GlcNAc-beta-1, 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); even more preferably Lacto-N-tetraose (LNT) or Lacto-N-hexaose (LNH); most preferably LNT; 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-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; and / or 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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage.

[0133] In an alternative preferred embodiment, 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-beta-l,3-LNT, Lacto-N-hexaose (LNH), para- lacto-N-neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), lacto-N-heptaose, para lacto-N- heptaose, lacto-N-octaose (LNO), iso 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, novo lacto-N-decaose, lacto-N- fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose V (LNFP V), Gal-LNFP I, GalNAc-LNFP I, Fuc-alphal,2-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N- difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), lewis b-lewis x, GlcNAc-beta-l,6-(Gal-beta-

[0134] 1.3-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, monofucosyllacto-N-hexaose III (MFLNH III), difucosyllacto-N-hexaose (a) (DFLNH (a)), difucosyllacto-N-hexaose (DFLNH) and trifucosyllacto-N- hexaose (TFLNH); more preferably selected from the list consisting of Lacto-N-tetraose (LNT), Gal-alpha-

[0135] 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-l,3-LNT, Gal-beta-1, 3-GalNAc-beta-l,3-LNT, Lacto-N-hexaose (LNH), para- lacto-N-neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), lacto-N-heptaose, para lacto-N- heptaose, lacto-N-octaose (LNO), iso lacto-N-octaose, para lacto-N-octaose, lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose V (LNFP V), Gal-LNFP I, GalNAc-LNFP I, Fuc- alphal,2-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N-difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), lewis b-lewis x, GlcNAc-beta-l,6-(Gal-beta-l,3-GlcNAc-beta-l,3- )Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, monofucosyllacto-N-hexaose III (MFLNH III), difucosyllacto-N-hexaose (a) (DFLNH (a)) and difucosyllacto-N-hexaose (DFLNH); even more preferably 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-l,3-LNT, Gal- beta-1, 3-GalNAc-beta-l,3-LNT, Lacto-N-hexaose (LNH), para-lacto-N-neohexaose II (pLNnH II), para-lacto- N-hexaose II (pLNH II), lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N- fucopentaose V (LNFP V), Gal-LNFP I, GalNAc-LNFP I, Fuc-alphal,2-Gal-beta-l,3-GlcNAc-beta-l,3-Gal- beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N-difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), lewis b-lewis x, GlcNAc-beta-l,6-(GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc and GlcNAc-beta-l,6-(Gal- beta-l,3-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc; even more preferably 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, Lacto-N-hexaose (LNH), para-lacto-N-neohexaose II (pLNnH II), para-lacto-N- hexaose II (pLNH II), lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N- fucopentaose V (LNFP V), Gal-LNFP I, GalNAc-LNFP I, Fuc-alphal,2-Gal-beta-l,3-GlcNAc-beta-l,3-Gal- beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N-difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), lewis b-lewis x, and GlcNAc-beta-l,6-(Gal-beta-l,3-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc; even more preferably selected from the list consisting of Lacto-N-tetraose (LNT), Lacto-N-hexaose (LNH), para- lacto-N-neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), lacto-N-fucopentaose I (LNFP I), lacto- N-fucopentaose II (LNFP II), lacto-N-fucopentaose V (LNFP V), Gal-LNFP I, GalNAc-LNFP I, Fuc-alphal,2- Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N-difucohexaose I (LNDFH I), lacto- N-difucohexaose II (LNDFH II) and lewis b-lewis x; even more preferably selected from the list consisting of Lacto-N-tetraose (LNT), Lacto-N-hexaose (LNH), 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) and lewis b-lewis x; even more preferably selected from the list consisting of LNT, 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) and lewis b-lewis x; even more preferably selected from the list consisting of LNT, 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 x; most preferably selected from the list consisting of LNT, lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II) and lacto-N-fucopentaose V (LNFP V); optionally wherein said LNT- 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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage.

[0136] In another preferred embodiment, 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.

[0137] In an additional and / or alternative preferred embodiment, said LN3-containing oligosaccharide according to the invention does not comprise a fucose.

[0138] In the context of the present invention, 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 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; as described herein is particularly useful in a cell which is genetically engineered for the production of LN3, a LNT-containing oligosaccharide or a LNnT-containing oligosaccharide; or an oligosaccharide mixture comprising LN3, a LNT-containing oligosaccharide and / or a LNnT-containing oligosaccharide.

[0139] In the context of the present invention, 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 the activity of the full-length sequence represented by SEQ ID NO 13; as described herein is particularly useful in a cell which is genetically engineered for the production of a LNT-containing oligosaccharide (preferably LNT); or an oligosaccharide mixture comprising a LNT- containing oligosaccharide (preferably LNT).

[0140] In the context of the present invention, 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 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; as described herein is particularly useful in a cell which is genetically engineered for the production of a LNnT-containing oligosaccharide (preferably LNnT); or an oligosaccharide mixture comprising a LNnT- containing oligosaccharide (preferably LNnT).

[0141] In the context of the present invention, a transporter protein comprising an amino acid:

[0142] - selected from SEQ ID NO 02, 03, 04, 05 or 06 (preferably SEQ ID NO 03, 04, 05 or 06); or having at least 80.0 % sequence identity to the full-length amino acid sequence of SEQ ID NO 02, 03, 04, 05 or 06 (preferably SEQ ID NO 03, 04, 05 or 06); or that is a functional fragment of SEQ ID NO 02, 03, 04, 05 or 06 (preferably SEQ ID NO 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 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 amino acid sequence of SEQ ID NO 02, 03, 04, 05 or 06 (preferably SEQ ID NO 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 02, 03, 04, 05 or 06 (preferably SEQ ID NO 03, 04, 05 or 06), respectively; as described herein is particularly useful in a cell which is genetically engineered for the production of LN3 or an oligosaccharide mixture comprising LN3 (especially when one wants to enrich the relative abundance of LN3 in the oligosaccharide mixture). Method for the production of an oligosaccharide

[0143] In a second aspect, 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:

[0144] (a) cultivating a cell (preferably a single cell) according to the first aspect of the invention, 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;

[0145] (b) optionally separating said LN3-containing oligosaccharide from the cultivation broth or separating 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.

[0146] 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).

[0147] Throughout the application and claims, unless specified otherwise, the verbs "cultivate" (and its conjugations) and "culture" are interchangeably used in the context of the present invention. Said "cultivate" also comprises fermentation as understood by the skilled person.

[0148] Throughout the application and claims, unless explicitly stated otherwise, the terms "synthesize", "synthesized" and "synthesis" are interchangeably used with the features "produce", "produced" and "production", respectively.

[0149] In the context of the present invention, the term "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. Preferably, the permissive conditions include a temperature-range of 30 + / - 20 degrees centigrade and / or a pH-range of 7 + / - 3.

[0150] In a preferred embodiment, 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. In this context, it is preferred to cultivate said cell of the invention in a fermentation process.

[0151] Step (a)

[0152] In an embodiment of the second aspect of the invention, 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. In a preferred embodiment, said cell according to the invention is cultivated in a minimal salt medium with a carbon source on which said cell grows. Preferably, 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.

[0153] In an additional and / or alternative preferred embodiment, 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. With the term "main" is meant the most important carbon source for the bioproducts of interest, biomass formation, carbon dioxide and / or by-products formation (such as acids and / or alcohols, such as acetate, lactate, and / or ethanol), i.e. 20.0%, 25.0%, 30.0%, 35.0%, 40.0%, 45.0%, 50.0 %, 55.0%, 60.0 %, 65.0%, 70.0 %, 75.0%, 80.0 %, 81.0 %, 82.0 %, 83.0 %, 84.0 %, 85.0 %, 86.0 %, 87.0 %, 88.0 %, 89.0 %, 90.0 %, 91.0 %, 92.0 %, 93.0 %, 94.0 %, 95.0 %, 95.5%, 96.0 %, 96.5 %, 97.0 %, 97.5 %, 98.0 %, 98.5 %, 99.0 %, 99.5 %, 100 % of all the required carbon is derived from the aboveindicated carbon source. In a more preferred embodiment of the invention, 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. With the term "complex medium" is meant a medium for which the exact constitution is not determined. Examples are molasses, corn steep liquor, peptone, tryptone or yeast extract. 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.

[0154] In an additional and / or alternative preferred embodiment, 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.

[0155] In a more preferred embodiment of the invention, 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 m3(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 of 1 day, 2 days, 3 days, 4 days or 5 days, by means of a feeding solution and wherein the concentration of said lactose feeding solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, even more preferably 125 g / L, even more preferably 150 g / L, even more preferably 175 g / L, even more preferably 200 g / L, even more preferably 225 g / L, even more preferably 250 g / L, even more preferably 275 g / L, even more preferably 300 g / L, even more preferably 325 g / L, even more preferably 350 g / L, even more preferably 375 g / L, even more preferably 400 g / L, even more preferably 450 g / L, even more preferably 500 g / L, even more preferably 550 g / L, most preferably 600 g / L; wherein preferably the pH of said solution is set between 3 and 7 and wherein preferably the temperature of said feed solution is kept between 20°C and 80°C; said method resulting in a concentration of said saccharide or said mixture of saccharides of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, even more preferably at least 175 g / L, most preferably at least 200 g / L in the final volume of said culture medium.

[0156] In an additional and / or alternative more preferred embodiment of the invention, 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.

[0157] In an additional and / or alternative more preferred embodiment of the invention, 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.

[0158] In another preferred embodiment, 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.

[0159] In an additional and / or alternative preferred embodiment of the invention, 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.

[0160] In a more preferred embodiment of the invention, 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.

[0161] In an alternative more preferred embodiment, the lactose is added already in the first phase of exponential growth together with the carbon-based substrate.

[0162] Step (b)

[0163] In an optional embodiment of the second aspect of the invention, 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.

[0164] 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.

[0165] In the context of the present invention, 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.

[0166] Throughout the application and claims, 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.

[0167] In a preferred embodiment, 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.

[0168] In an additional and / or alternative preferred embodiment, said separation step, preferably purification 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.

[0169] In an additional and / or alternative preferred embodiment, 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. Preferably 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.

[0170] In a more preferred embodiment, 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. Alcohols, such as ethanol, and aqueous alcohol mixtures can also be used.

[0171] It is particularly preferred that said separation step, preferably purification step, of a method according to the invention comprises:

[0172] (i) clarifying the cultivation broth, and / or

[0173] (ii) removing salts and / or medium components from said clarified cultivation broth, and / or

[0174] (iii) concentrating said LN3-containing oligosaccharide or said oligosaccharide mixture in said clarified cultivation broth.

[0175] In an embodiment, the clarification is combined with the removal of salts and / or medium components.

[0176] In an embodiment, the clarification is combined with the step of concentrating the LN3-containing oligosaccharide or oligosaccharide mixture in the clarified cultivation. In an embodiment, 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. In an embodiment, 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. Advantageously said LN3-containing oligosaccharide or said mixture of oligosaccharides are obtained in large quantities and at high purity.

[0177] In the context of the present invention, step (iii) preferably comes before step (ii).

[0178] 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. In this step, the cultivation broth containing the produced LN3-containing oligosaccharide or oligosaccharide mixture can be clarified in a conventional manner. Preferably, clarification is done by centrifugation, flocculation, decantation, ultrafiltration and / or filtration. In another embodiment, 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. In an additional and / or alternative preferred embodiment, step i) comprises subjecting the cultivation broth to two membrane filtration steps using different membranes (i.e. different cut-offs). In an additional and / or alternative preferred embodiment, step i) of clarifying the cultivation broth further comprises use of a filtration aid, preferably an adsorbing agent, more preferably active carbon.

[0179] In an additional and / or alternative embodiment, step (i) comprises a first step of clarification by microfiltration. Alternatively, step i) comprises a first step of clarification by centrifugation. Alternatively, step i) comprises a first step of clarification by flocculation. Alternatively, step i) comprises a first step of clarification by ultrafiltration.

[0180] In a preferred embodiment, step (i) comprises ultrafiltration. Preferably, 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. Alternatively or preferably, 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.

[0181] In another preferred embodiment, step i) is preceded by an enzymatic treatment. Preferably, 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. Preferably or alternatively, 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. In this step, 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. With the exception of size exclusion chromatography, proteins and related impurities are retained by a chromatography medium or a selected membrane, while the LN3-containing oligosaccharide or oligosaccharide mixture remains in the (clarified) cultivation broth.

[0182] In an embodiment, 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. Preferably, 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.

[0183] In an embodiment, 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. Such 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. As such, 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.

[0184] In an embodiment, 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. In an embodiment, 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. In other words, this involves an anion exchange resin which has selectivity for negatively charged minerals, but not for sialyllactose. As described in the art, see e.g. W02009 / 113861, to this end, it is necessary that the moisture content, that is, the water content, is not greater than 48%, and preferably not greater than 45 %. At moisture contents lower than 35%, and more so at moisture contents lower than 30 %, the desalting capacity starts to become too low to yield an effective process. The moisture content in the anion exchanger is determined in the following manner: prior to measurement of the moisture content of the resin, adhering water is removed, for instance by wrapping the resin in a cloth and then subjecting it to centrifugation (centrifuge: 30 cm diameter; 3,000 rpm); the resin is then weighed, for instance in a weighing bottle; after which the resin is dried for 4 hours at a constant temperature of 105°C; the resin is then cooled down in an exicator for 30 minutes; after which in turn the weight of the dry resin is determined; the moisture percentage (weight percent) = [(weight loss after drying (g)) / (weight of the wet resin)] * 100 percent. Through this desalting, an important part of the negatively charged ions is removed without substantial amounts of sialyllactose (despite the negative charge) being thereby removed.

[0185] The 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.

[0186] In an embodiment, step ii) comprises a treatment with a mixed bed ion exchange resin. In an embodiment, such 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. In an embodiment, 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.

[0187] In an additional and / or alternative embodiment, step ii) comprises nanofiltration and / or electrodialysis. Preferably, said nanofiltration and / or electrodialysis is performed twice. More preferably, said nanofiltration and / or electrodialysis steps are performed consecutively. In some embodiments, the ultrafiltration permeate of step i) is nanofiltered and / or electrodialysed in step ii).

[0188] In an embodiment, 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.

[0189] In some embodiments, step (i) is ultrafiltration, and step (ii) is nanofiltration and / or electrodialysis treatment combined with treatment with an ion exchange resin and / or chromatography. Preferably, 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.

[0190] In still another preferred embodiment of the method of the invention, 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. Preferably, 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. In a preferred embodiment of the method of the invention, the method does not comprise electrodialysis. In an embodiment of the invention wherein said step (i) is ultrafiltration, said 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.

[0191] The separation, preferably purification, preferably involves concentrating the cultivation broth containing the LN3-containing oligosaccharide or oligosaccharide mixture [i.e. step (iii)]. In an embodiment, step (iii) precedes the second step. In an embodiment, the step of concentrating [i.e. step (iii)] precedes the second step and is once more applied after the second step as described above.

[0192] In an embodiment, step (iii) of concentrating comprises one or more of nanofiltration, diafiltration, reverse osmosis, evaporation, wiped film evaporation and falling film evaporation.

[0193] In another embodiment, the separated, preferably purified, LN3-containing oligosaccharide or oligosaccharide mixture is concentrated to a syrup of at least 40% dry matter.

[0194] In an additional and / or alternative preferred embodiment, the method further comprises decolorization.

[0195] In an additional and / or alternative preferred embodiment, the method comprises a step of sterile filtration and / or endotoxin removal, preferably by filtration of the separated, preferably purified, saccharide mixture or saccharide mixture through a 3 kDa filter.

[0196] Drying

[0197] In an optional embodiment of the second aspect of the invention, 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.

[0198] Several drying techniques are known to the skilled person which can be used to obtain a slurry, preferably a solid, more preferably a powder, from a solution containing said LN3-containing oligosaccharide or said mixture of different oligosaccharides according to the invention. 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.

[0199] In the context of the present invention, 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.

[0200] Use

[0201] In a third aspect, 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. 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).

[0202] In a fourth aspect, 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. 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).

[0203] Specific embodiments

[0204] The present invention preferably relates to the following specific embodiments:

[0205] 1. 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:

[0206] - selected from 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 that is a functional fragment of a polypeptide 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, 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. Cell according to embodiment 1, wherein said transporter protein consists of at least 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. 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. Cell according to any one of embodiments 1 to 3, wherein said LN3-containing oligosaccharide is a milk oligosaccharide, preferably a mammalian milk oligosaccharide, most preferably a human milk oligosaccharide. Cell according to any one of embodiments 1 to 4, wherein said LN3-containing oligosaccharide comprises a lactose at its reducing end. Cell according to any one of embodiments 1 to 5, wherein 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,

[0207] GlcNAc-beta-l,6-(Gal-beta-l,4-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, lacto-N-neopentaose, para-Lacto-N- neopentaose, 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-beta-l,3-LNT, Lacto-N-hexaose (LNH), para-lacto-N- hexaose (pLNH), lacto-N-neohexaose (LNnH), para-Lacto-N-neohexaose (pLNnH), para-lacto-N- neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto- N-neooctaose, iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose, lacto-N-decaose, iso lacto-N-decaose, novo lacto-N-decaose and lacto-N-neodecaose; optionally wherein said LN3- 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-linkage, preferably an alpha-1,2- or an alpha-1, 3-linkage, more preferably an alpha-1, 3-linkage; and / or 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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage. Cell according to any one of embodiments 1 to 6, wherein 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), 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 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; and / or 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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage. Cell according to any one of embodiments 1 to 5, wherein 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-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, lacto-N-neopentaose, para-Lacto-N- neopentaose, 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-beta-l,3-LNT, Lacto-N-hexaose (LNH), para-lacto-N- hexaose (pLNH), lacto-N-neohexaose (LNnH), para-Lacto-N-neohexaose (pLNnH), para-lacto-N- neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto- N-neooctaose, iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose, lacto-N-decaose, iso lacto-N-decaose, novo lacto-N-decaose, lacto-N-neodecaose, 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,4-(Fuc-alpha-

[0208] 1.3-)GlcNAc-beta-l,3-Gal-beta-l,4-Glc, lacto-N-neodifucohexaose (LNnDFH), Fuc-alphal,2-Gal-beta-

[0209] 1.4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose V (LNFP V), Gal-LNFP I, GalNAc-LNFP I, Fuc- alphal,2-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N-difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), lewis b-lewis x, GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc- alpha-l,3-)Glc, GlcNAc-beta-l,6-(GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, GIcNAc-beta- l,6-(Gal-beta-l,4-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, GlcNAc-beta-l,6-(Gal-beta-

[0210] 1.3-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, monofucosyllacto-N-hexaose III (MFLNH III), difucosyllacto-N-hexaose (a) (DFLNH (a)), difucosyllacto-N-hexaose (DFLNH) and trifucosyllacto-N- hexaose (TFLNH); optionally wherein said 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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage. Cell according to any one of embodiments 1 to 5, wherein said 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 x and GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc; optionally wherein said 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-

[0211] 2.3-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage. Cell according to any one of embodiments 1 to 5, wherein said LN3-containing oligosaccharide is LN3, a LNT-containing oligosaccharide or a LNnT-containing oligosaccharide. Cell according to embodiment 10, wherein said 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-

[0212] 1.3-LNT, Gal-beta-1, 3-GalNAc-beta-l,3-LNT, Lacto-N-hexaose (LNH), para-lacto-N-neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), lacto-N-heptaose, para lacto-N-heptaose, lacto-N- octaose (LNO), iso 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; 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-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; and / or 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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage. Cell according to embodiment 10, wherein said 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-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; and / or 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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage. Cell according to embodiment 10, wherein said 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-l,3-LNT, Lacto-N-hexaose (LNH), para-lacto-N-neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), lacto-N-heptaose, para lacto-N-heptaose, lacto-N- octaose (LNO), iso 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, novo lacto-N-decaose, lacto-N- fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose V (LNFP V), Gal-LNFP I, GalNAc-LNFP I, Fuc-alphal,2-Gal-beta-l,3-GlcNAc-beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto- N-difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), lewis b-lewis x, GlcNAc-beta-1,6- (Gal-beta-l,3-GlcNAc-beta-l,3-)Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, monofucosyllacto-N-hexaose III (MFLNH III), difucosyllacto-N-hexaose (a) (DFLNH (a)), difucosyllacto-N-hexaose (DFLNH) and trifucosyllacto-N-hexaose (TFLNH); optionally wherein said LNT-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-, alpha-2,4- or alpha- 2,8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage.

[0213] 14. Cell according to embodiment 10, wherein said LNT-containing oligosaccharide is selected from the list consisting of Lacto-N-tetraose (LNT), Lacto-N-hexaose (LNH), para-lacto-N-neohexaose II (pLNnH II), para-lacto-N-hexaose II (pLNH II), lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose V (LNFP V), Gal-LNFP I, GalNAc-LNFP I, Fuc-alphal,2-Gal-beta-l,3-GlcNAc- beta-l,3-Gal-beta-l,4-(Fuc-alpha-l,3-)Glc, lacto-N-difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II) and lewis b-lewis x; optionally wherein said LNT-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-, alpha-2,4- or alpha- 2,8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage.

[0214] 15. Cell according to any one of embodiments 10 to 14, wherein said 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-neoheptaose, para lacto-N-neoheptaose, lacto- N-neooctaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose and lacto- N-neodecaose; 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-linkage, preferably an alpha-1,2- or an alpha-1, 3-linkage, more preferably an alpha-1, 3-linkage; and / or 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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage.

[0215] 16. Cell according to any one of embodiments 10 to 14, wherein said 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-linkage, preferably an alpha-1,2- or an alpha-1, 3-linkage, more preferably an alpha-l,3-linkage; and / or 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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage. Cell according to any one of embodiments 10 to 14, wherein said 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-neoheptaose, para lacto-N-neoheptaose, lacto- N-neooctaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose, lacto-N- neodecaose, 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,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3-Gal-beta-l,4-Glc, lacto-N-neodifucohexaose (LNnDFH), Fuc-alphal,2-Gal-beta-l,4-(Fuc-alpha-l,3-)GlcNAc-beta-l,3- Gal-beta-l,4-(Fuc-alpha-l,3-)Glc and GlcNAc-beta-l,6-(Gal-beta-l,4-GlcNAc-beta-l,3-)Gal-beta-l,4- (Fuc-alpha-l,3-)Glc; optionally wherein said LNnT-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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage. Cell according to any one of embodiments 10 to 14, wherein said 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,4-(Fuc-alpha- l,3-)GlcNAc-beta-l, 3-Gal-beta-l, 4-Glc, lacto-N-neodifucohexaose (LNnDFH) and Fuc-alphal,2-Gal- beta-l,4-(Fuc-alpha-l,3-)GlcNAc-beta-l, 3-Gal-beta-l, 4-(Fuc-alpha-l,3-)Glc; optionally wherein said LNnT-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-, alpha-2,4- or alpha-2, 8-linkage, preferably an alpha-2,3- or an alpha-2, 6-linkage. Cell according to any one of embodiments 1 to 18, wherein said LN3-containing oligosaccharide is a neutral oligosaccharide. Cell according to any one of embodiments 1 to 19, wherein said LN3-containing oligosaccharide does not comprise a fucose.

[0216] 21. Cell according to any one of embodiments 1 to 20, wherein said LN3-containing oligosaccharide consists of at least 3, preferably at least 4, monosaccharides.

[0217] 22. Cell according to any one of embodiments 1 to 21, wherein said LN3-containing oligosaccharide consists of < 10, preferably < 9, more preferably < 8, even more preferably < 7, even more preferably < 6, most preferably < 5, monosaccharides.

[0218] 23. Cell according to any one of embodiments 1 to 5, wherein said LN3-containing oligosaccharide is LN3, LNT or LNnT.

[0219] 24. Cell according to any one of embodiments 1 to 23, wherein said cell 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.

[0220] 25. Cell according to any one of embodiments 1 to 24, wherein said cell 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 galactosyltransferase, most preferably a galactosyltransferase; preferably wherein 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, preferably a beta-1, 3-galactosyltransferase or a beta-1, 4- galactosyltransferase, more preferably a beta-1, 3-galactosyltransferase; 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, 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;

[0221] N-acetylglucosaminyltransferase is a beta-1, 6-N-acetylglucosaminyltransferase;

[0222] 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.

[0223] 26. Cell according to embodiment 24 or 25, wherein said cell is modified in the expression or activity of at least one of said glycosyltransferases. 27. Cell according to any one of embodiments 1 to 26, wherein said cell 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 precursor saccharide 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-linkage, preferably an alpha-1,2- or an alpha-1, 3-linkage, more preferably an alpha-1, 3-linkage.

[0224] 28. Cell according to any one of embodiments 1 to 27, wherein said cell is selected from a list consisting of a microorganism, a plant cell, an animal cell, an insect cell or a protozoan cell, preferably a microorganism, more preferably a bacterium, fungus or yeast, even more preferably a bacterium.

[0225] 29. Cell according to any one of embodiments 1 to 28, wherein said cell is a bacterium, preferably Escherichia coli, more preferably Escherichia coli (K-12 strain), even more preferably Escherichia coli MG1655.

[0226] 30. Cell according to any one of embodiments 1 to 29, wherein said cell has an improved production of said LN3-containing oligosaccharide compared to said cell with an identical genetic background but that lacks said transporter protein.

[0227] 31. Cell according to embodiment 30, 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.

[0228] 32. Cell according to embodiment 30, 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). 3. Cell according to any one of embodiments 1 to 32, wherein said transporter protein 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-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 26, 30, 33 and / or 34 of TMl of the transporter protein represented by SEQ ID NO 16, o 22, 26, 29 and / or 30 of TMl of the transporter protein represented by SEQ ID NO 02, o 22 of TMl of the transporter protein represented by SEQ ID NO 03, o 27 , 31, 34 and / or 35 of TMl of the transporter protein represented by SEQ ID NO 04, o 28, 32, 35 and / or 36 of TMl of the transporter protein represented by SEQ ID NO 05, o 26, 30, 33 and / or 34 of TMl of the transporter protein represented by SEQ ID NO 06; optionally wherein said transporter protein 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.

[0229] 34. Cell according to embodiment 33, wherein TMl of SEQ ID NO 01 is represented by SEQ ID NO 07.

[0230] 35. Cell according to embodiment 33, wherein TMl of SEQ ID NO 13 is represented by SEQ ID NO 14.

[0231] 36. Cell according to embodiment 33, wherein TMl of SEQ ID NO 02 is represented by SEQ ID NO 08.

[0232] 37. Cell according to embodiment 33, wherein TMl of SEQ ID NO 03 is represented by SEQ ID NO 09.

[0233] 38. Cell according to embodiment 33, wherein TMl of SEQ ID NO 04 is represented by SEQ ID NO 10.

[0234] 39. Cell according to embodiment 33, wherein TMl of SEQ ID NO 05 is represented by SEQ ID NO 11.

[0235] 40. Cell according to embodiment 33, wherein TMl of SEQ ID NO 06 is represented by SEQ ID NO 12.

[0236] 41. Cell according to embodiment 33, wherein TMl of SEQ ID NO 15 is represented by SEQ ID NO 20.

[0237] 42. Cell according to embodiment 33, wherein TMl of SEQ ID NO 16 is represented by SEQ ID NO 213. 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:

[0238] (a) cultivating a cell (preferably a single cell) according to any one of embodiments 1 to 42, 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;

[0239] (b) optionally separating said LN3-containing oligosaccharide from the cultivation broth or separating 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. 44. Method according to embodiment 43, 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 (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.

[0240] 45. 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.

[0241] 46. 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.

[0242] 47. Use of a cell according to any one of embodiments 1 to 42 for the production of a LN3-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide.

[0243] 48. Use of a 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.

[0244] The present invention more preferably relates to the following specific embodiments:

[0245] 1. 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 LN3-containing oligosaccharide is LN3, a lacto-N-tetraose (LNT)-containing oligosaccharide or a lacto-N-neotetraose (LNnT)-containing oligosaccharide, characterized in that said cell expresses a transporter protein comprising an amino acid sequence:

[0246] (i) wherein said 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 full-length of said polypeptide;

[0247] (ii) wherein said 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;

[0248] (iii) wherein said 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;

[0249] (iv) wherein said 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.

[0250] 2. A cell according to embodiment 1, wherein said transporter is able to transport said LN3-containing oligosaccharide.

[0251] 3. A cell according to embodiment 1 or 2, wherein said transporter protein is heterologous.

[0252] 4. A cell according to any one of embodiments 1 to 3, wherein said LN3-containing oligosaccharide is a neutral oligosaccharide.

[0253] 5. A cell according to any one of embodiments 1 to 4, wherein said cell is selected from the list consisting of a microorganism, a plant cell, an animal cell, an insect cell and a protozoan cell.

[0254] 6. A cell according to any one of embodiments 1 to 5, wherein said cell is a bacterium.

[0255] 7. A cell according to any one of embodiments 1 to 6, wherein said cell expresses a galactoside beta- 1,3-N-acetylglucosaminyltransferase that is involved in the synthesis of said LN3-containing oligosaccharide.

[0256] 8. A cell according to any one of embodiments 1 to 7, wherein said cell 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.

[0257] 9. A cell according to any one of embodiments 1 to 8, wherein said cell 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.

[0258] 10. A cell according to embodiment 9, wherein said precursor saccharide is lactose, optionally wherein said lactose further comprises a fucose.

[0259] 11. A cell according to any one of embodiments 1 to 10, wherein said cell has an improved production of said LN3-containing oligosaccharide compared to said cell with an identical genetic background but that lacks said transporter protein.

[0260] 12. 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.

[0261] 13. 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).

[0262] 14. Cell according to any one of embodiments 1 to 13, wherein said transporter protein 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; 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 26, 30, 33 and / or 34 of TMl of the transporter protein represented by SEQ ID NO 16, o 22, 26, 29 and / or 30 of TMl of the transporter protein represented by SEQ ID NO 02, o 22 of TMl of the transporter protein represented by SEQ ID NO 03, o 27 , 31, 34 and / or 35 of TMl of the transporter protein represented by SEQ ID NO 04, o 28, 32, 35 and / or 36 of TMl of the transporter protein represented by SEQ ID NO 05, o 26, 30, 33 and / or 34 of TMl of the transporter protein represented by SEQ ID NO 06; optionally wherein said transporter protein lacks all amino acids N-terminally from its TMl domain compared to the transporter protein represented by SEQ ID NO 01, 13, 02, 03, 04, 05 or 06, respectively.

[0263] 15. 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:

[0264] (a) cultivating a cell according to any one of embodiments 1 to 14, 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;

[0265] (b) optionally separating said LN3-containing oligosaccharide from the cultivation broth or separating any one, preferably all, of the oligosaccharides in said mixture from the cultivation broth.

[0266] 16. 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.

[0267] 17. 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.

[0268] 18. 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.

[0269] 19. Use of a cell according to any one of embodiments 1 to 14 for the production of a LN3-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide.

[0270] 20. Use of a transporter protein as defined in any one of embodiments 1, 2, 3 and 14 in the production of a LN3-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide.

[0271] Definitions

[0272] The words used in this specification to describe the invention and its various embodiments are to be understood not only in the sense of their commonly defined meanings, but to include by special definition in this specification structure, material or acts beyond the scope of the commonly defined meanings. Thus, if an element can be understood in the context of this specification as including more than one meaning, then its use in a claim must be understood as being generic to all possible meanings supported by the specification and by the word itself.

[0273] The various aspects and embodiments of the invention disclosed herein are to be understood not only in the order and context specifically described in this specification, but to include any order and any combination thereof. Each embodiment as identified herein may be combined together unless otherwise indicated. All publications, patents and patent applications cited in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Unless specifically stated otherwise, all words used in the singular number shall be deemed to include the plural and vice versa. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described herein are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications.

[0274] In the drawings and specification, there have been disclosed embodiments of the invention, and although specific terms are employed, the terms are used in a descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the following claims. It must be understood that the illustrated embodiments have been set forth only for the purposes of example and that it should not be taken as limiting the invention. It will be apparent to those skilled in the art that alterations, other embodiments, improvements, details and uses can be made consistent with the letter and spirit of the invention herein and within the scope of this invention, which is limited only by the claims, construed in accordance with the patent law, including the doctrine of equivalents. In the claims which follow, reference characters used to designate claim steps are provided for convenience of description only, and are not intended to imply any particular order for performing the steps (unless specifically stated otherwise).

[0275] In this document and in its claims, 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. Throughout the document and claims, unless specifically stated otherwise, 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). In addition, reference to an element by the 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. Preferably, the word "about" or "approximately" or "around" when used in association with a numerical value (e.g. about 10) 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.

[0276] Throughout the description and claims, unless specifically stated otherwise, the expression "from x to y", wherein x and y represent numerical values, refers to a range of numerical values wherein both x and y are included and wherein x represents the lowest value and y represents the highest value. Hence, both x and y are also included in the range in addition to any value in-between.

[0277] Throughout the application and claims, unless explicitly stated otherwise, the expressions "capable of...<verb>" and "capable to...<verb>" are preferably replaced with the active voice of said verb and vice versa. For example, the expression "capable of expressing" is preferably replaced with "expresses" and vice versa, i.e. "expresses" is preferably replaced with "capable of expressing".

[0278] The term "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. For example, 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.

[0279] The term "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,...) 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.

[0280] As used herein, the term "cell productivity index (CPI)" 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.

[0281] The terms "LNT", "lacto-N-tetraose", "lacto- / V-tetraose" and "Gal-pi,3-GlcNAc-pi,3-Gal-pi,4Glc" are used interchangeably.

[0282] The terms "LNnT", "lacto-N-neotetraose", "lacto- / V-neotetraose", "neo-LNT" and "Gaipi-4GlcNAcpi- 3Gaipi-4Glc" are used interchangeably.

[0283] The terms "lacto-N-pentaose" and "LN5" are used interchangeably and refer to GlcNAC-bl,3-Gal-bl,4- GlcNAC-bl,3-Gal-bl,4-Glc.

[0284] The terms "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.

[0285] The term "pLNnH" refers to Gal-bl,4-GlcNAC-bl,3-Gal-bl,4-GlcNAC-bl,3-Gal-bl,4-Glc.

[0286] The terms "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.

[0287] The term "pLNH" refers to Gal-bl,3-GlcNAC-bl,3-Gal-bl,4-GlcNAC-bl,3-Gal-bl,4-Glc.

[0288] The terms "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.

[0289] The term "pLNnO" refers to Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4- Glc.

[0290] The term "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.

[0291] The term "LNH" refers to Gal-bl,4-GlcNAC-bl,6-(Gal-bl,3-GlcNAc-bl,3)-Gal-bl,4-Glc.

[0292] The terms "lacto-N-biose" and "LNB" are used interchangeably and refer to Gal-bl,3-GlcNAc.

[0293] The terms "N-acetyllactosamine" and "LacNAc" are used interchangeably and refer to Gal-bl,4-GlcNAc.

[0294] The terms "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.

[0295] The terms "LND" and "lacto-N-decaose" are used interchangeably.

[0296] The terms "LNnD" and "lacto-N-neodecaose" are used interchangeably.

[0297] The terms "2' fucosyllactose", "2'-fucosyllactose", "alpha-1, 2-fucosyllactose", "alpha 1,2 fucosyllactose", "a-l,2-fucosyllactose", "a 1,2 fucosyllactose", "Fuc-al,2-Gal-pi,4-Glc", 2FL" and "2'FL" are used interchangeably.

[0298] The terms "3-fucosyllactose", "alpha-1, 3-fucosyllactose", "alpha 1,3 fucosyllactose", "a-1,3- fucosyllactose", "a 1,3 fucosyllactose", "Gal-pi,4-(Fuc-al,3-)Glc", 3FL" and "3-FL" are used interchangeably.

[0299] The terms "difucosyllactose", "di-fucosyllactose", "lactodifucotetraose", "2',3-difucosyllactose", "2', 3 difucosyllactose", "a-2', 3-fucosyllactose", "a 2', 3 fucosyllactose, "Fuc-al,2-Gal-pi,4-(Fuc-al,3-)Glc", "DFLac", 2', 3 diFL", "DFL", "DiFL" and "diFL" are used interchangeably.

[0300] The terms "LNFP-I", "lacto-N-fucopentaose I", "LNFP I", "LNF I OH type I determinant", "LNF I", "LNF1", "LNF 1" , "Blood group H antigen pentaose type 1" and "Fuc-al,2-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc" are used interchangeably.

[0301] The terms "GalNAc-LNFP-l", "blood group A antigen hexaose type I", and "GalNAc-al,3-(Fuc-al,2)-Gal- pi,3-GlcNAc- pi,3-Gal-pi,4-Glc" are used interchangeably.

[0302] The terms "Gal-LNFP-I", "blood group B antigen hexaose type I" and "Gal-al,3-(Fuc-al,2)-Gal-pi,3- GlcNAc-pi,3-Gal-pi,4-Glc" are used interchangeably.

[0303] The terms "LNFP-II", "lacto-N-fucopentaose II" and "Gal-pi,3-(Fuc-al,4)-GlcNAc-pi,3-Gal-pi,4-Glc" are used interchangeably.

[0304] The terms "LNFP-III", "lacto-N-fucopentaose III" and "Gal-pi,4-(Fuc-al,3)-GlcNAc-pi,3-Gal-pi,4-Glc" are used interchangeably.

[0305] The terms "LNFP-V", "lacto-N-fucopentaose V" and "Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-(Fuc-al,3)-Glc" are used interchangeably.

[0306] The terms "LNDFH I", "Lacto-N-difucohexaose I", "LNDFH-I", "LDFH I", "Leb-lactose", "Lewis-b hexasaccharide" and "Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-Glc" are used interchangeably. The terms "LNDFH II", "Lacto-N-difucohexaose II", "Lewis a-Lewis x", "LDFH II" and "Fuc-al,4-(Gal-pi,3)- GlcNAc-pi,3-Gal-pi,4-(Fuc-al,3)-Glc" are used interchangeably.

[0307] The terms "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.

[0308] The terms "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.

[0309] The terms "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.

[0310] The terms "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.

[0311] The terms "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.

[0312] The terms "LNnFP I", "Lacto-N-neofucopentaose I" and "Fuc-al,2-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc" are used interchangeably.

[0313] The terms "LNFP-VI", "LNnFP V", "lacto-N-neofucopentaose V" and "Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-(Fuc- al,3)-Glc" are used interchangeably.

[0314] The terms "LNnDFH", "Lacto-N-neoDiFucohexaose", "Lewis x hexaose" "Gal-pi,4-(Fuc-al,3)-GlcNAc-pi,3- Gal-pi,4-(Fuc-al,3)-Glc" are used interchangeably.

[0315] The terms "2'-fucosyllacto-N-biose", "2'FLNB" and "Fuc-al,2-Gal-pi,3-GlcNAc" are used interchangeably. The terms "4-fucosyllacto-N-biose", "4FLNB" and "Fuc-al,4-[Gal-pi,3-]GlcNAc" are used interchangeably. The terms "difucosyllacto-N-biose", "diFLNB" and "Fuc-al,4-[Fuc-al,2-Gal-pi,3-]GlcNAc" are used interchangeably.

[0316] The terms "2'-fucosyl-N-acetyllactosamine", "2'FlacNAc" and "Fuc-al,2-Gal-pi,4-GlcNAc" are used interchangeably.

[0317] The terms "3-fucosyl-N-acetyllactosamine", "3FlacNAc" and "Gal-pi,4-(Fuc-al,3-)GlcNAc" are used interchangeably.

[0318] The terms "difucosyl-N-acetyllactosamine", "diFlacNAc" and "Fuc-al,2-Gal-pi,4-[Fuc-al,3-]GlcNAc" are used interchangeably.

[0319] The terms "3' sialyllactose", "3'-sialyllactose", "alpha-2, 3-sialyllactose", "alpha 2,3 sialyllactose", "a-2,3- sialyllactose", "a 2,3 sialyllactose", "3SL", "Sia-a2,3-Gal-pi,4-Glc" and "3'SL" are used interchangeably.

[0320] The terms "6' sialyllactose", "6'-sialyllactose", "alpha-2, 6-sialyllactose", "alpha 2,6 sialyllactose", "a-2,6- sialyllactose", "a 2,6 sialyllactose", "6SL", "Sia-a2,6-Gal-pi,4-Glc" and "6'SL" are used interchangeably.

[0321] The terms "3,6-disialyllactose" and "Neu5Ac-a2,3-Neu5Ac-a2,6- Gal-pi,4-Glc" are used interchangeably.

[0322] The terms "6,6'-disialyllactose" and "Neu5Ac-a2,6-Neu5Ac-a2,6- Gal-pi,4-Glc" are used interchangeably.

[0323] The terms "8,3-disialyllactose" and "Neu5Ac-a2,8-Neu5Ac-a2,3- Gal-pi,4-Glc" are used interchangeably.

[0324] The terms "3'S-2'FL", "3' -sialyl-2' -fucosyllactose" and "Neu5Ac-a2,3-[Fuc-al,2-]Gal-pi,4-Glc" are used interchangeably.

[0325] The terms "6'S-2'FL", "6' -sialyl-2' -fucosyllactose" and "Neu5Ac-a2,6-[Fuc-al,2-]Gal-pi,4-Glc" are used interchangeably.

[0326] The terms "3'S-3-FL", "3'-sialyl-3-fucosyllactose" and "Neu5Ac-a2,3-Gal-pi,4-[Fuc-al,3]Glc" are used interchangeably.

[0327] The terms "6'S-3-FL", "6'-sialyl-3-fucosyllactose" and "Neu5Ac-a2,6-Gal-pi,4-[Fuc-al,3]Glc" are used interchangeably.

[0328] The terms "LSTa", "LS-Tetrasaccharide a", "Sialyl-lacto-N-tetraose a", "sialyllacto-N-tetraose a" and "Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc" are used interchangeably.

[0329] The terms "LSTb", "LS-Tetrasaccharide b", "Sialyl-lacto-N-tetraose b", "sialyllacto-N-tetraose b" and "Gal- bl,3-(Neu5Ac-a2,6)-GlcNAc-bl,3-Gal-bl,4-Glc" are used interchangeably.

[0330] The terms "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.

[0331] The terms "LSTd", "LS-Tetrasaccharide d", "Sialyl-lacto-N-tetraose d", "sialyllacto-N-tetraose d", "sialyllacto-N-neotetraose d" and "Neu5Ac-a2,3-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc" are used interchangeably.

[0332] The terms "3'-sialyllacto-N-biose", "3'SLNB" and "Neu5Ac-a2,3-Gal-bl,3-GlcNAc" are used interchangeably.

[0333] The terms "6'-sialyllacto-N-biose", "6'SLNB" and "Neu5Ac-a2,6-Gal-bl,3-GlcNAc" are used interchangeably.

[0334] The terms "monofucosylmonosialyllacto-N-octaose", "sialyl Lewis a", "sialyl Lea", "5-acetylneuraminyl-(2- 3)-galactosyl-(l-3)-(fucopyranosyl-(l-4))-N-acetylglucosamine" and "Neu5Ac-a2,3-Gal-pi,3-[Fuc-al,4]- GIcNAc" are used interchangeably.

[0335] The terms "3'-sialyllactosamine", "3'SLacNAc" and "Neu5Ac-a2,3-Gal-bl,4-GlcNAc" are used interchangeably.

[0336] The terms "6'-sialyllactosamine", "6'SLacNAc" and "Neu5Ac-a2,6-Gal-bl,4-GlcNAc" are used interchangeably.

[0337] The terms "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.

[0338] The terms "Neu4Ac", "4-O-acetyl-5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid" and "4-O-acetyl neuraminic acid" are used interchangeably and have C11H19NO9 as molecular formula. The terms "Neu5Ac", "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-non-2-ulopyranosonic acid" are used interchangeably and have C11H19NO9 as molecular formula.

[0339] The terms "Neu4,5Ac2", "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.

[0340] The terms "Neu5,7Ac2", "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.

[0341] The terms "Neu5,8Ac2" and "5-n-acetyl-8-o-acetyl neuraminic acid" are used interchangeably herein and have C13H21NO10 as molecular formula.

[0342] The terms "Neu5,9Ac2", "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" are used interchangeably herein and have C13H21NO10 as molecular formula. The terms "Neu4,5,9Ac3" and "5-N-acetyl-4,9-di-O-acetylneuraminic acid" are used interchangeably herein.

[0343] The terms "Neu5,7,9Ac3" and "5-N-acetyl-7,9-di-O-acetylneuraminic acid" are used interchangeably herein.

[0344] The terms "Neu5,8,9Ac3" and "5-N-acetyl-8,9-di-O-acetylneuraminic acid" are used interchangeably herein.

[0345] The terms "Neu4,5,7,9Ac4" and "5-N-acetyl-4,7,9-tri-O-acetylneuraminic acid" are used interchangeably herein.

[0346] The terms "Neu5,7,8,9Ac4" and "5-N-acetyl-7,8,9-tri-O-acetylneuraminic acid" are used interchangeably herein.

[0347] The terms "Neu4,5,7,8,9Ac5" and "5-N-acetyl-4,7,8,9-tetra-O-acetylneuraminic acid" are used interchangeably herein.

[0348] The terms "Neu5Gc", "N-glycolyl-neuraminic acid", "N-glycolylneuraminic acid", "N- glycolylneuraminate", "N-glycoloyl-neuraminate", "N-glycoloyl-neuraminic 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-glycero-D- galacto-non-2-ulopyranosonic acid", "D-glycero-5-glycolylamido-3,5-dideoxy-D-galacto-non-2-ulo- pyranosonic acid" are used interchangeably and have C11H19NO10 as molecular formula.

[0349] The terms "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.

[0350] The terms "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.

[0351] The terms "DS'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.

[0352] The terms "DS'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.

[0353] The term "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

[0354] The invention will be described in more detail in the examples. The following examples will serve as further illustration and clarification of the present invention and are not intended to be limiting in any way.

[0355] Example 1. Materials and Methods Escherichia coli

[0356] Media

[0357] 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 NH4CI, 5.00 g / L (NH4)2SO4, 2.993 g / L KH2PO4, 7.315 g / L K2HPO4, 8.372 g / L MOPS, 0.5 g / L NaCI, 0.5 g / L MgSO4.7H2O, 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. As specified in the respective examples, 20 g / L lactose was additionally added to the medium as precursor. The minimal medium was set to a pH of 7 with IM KOH. Vitamin solution consisted of 3.6 g / L FeCI2.4H2O, 5 g / L CaCI2.2H2O, 1.3 g / L MnCI2.2H2O, 0.38 g / L CUCI2.2H2O, 0.5 g / L COCI2.6H2O, 0.94 g / L ZnCI2, 0.0311 g / L H3BO4, 0.4 g / L Na2EDTA.2H2O and 1.01 g / L thiamine. HCI. The molybdate solution contained 0.967 g / L NaMoO4.2H2O. The selenium solution contained 42 g / L SeO2.

[0358] The minimal medium for fermentations contained 6.75 g / L NH4CI, 1.25 g / L (NH4)2SO4, 2.93 g / L KH2PO4and 7.31 g / L KH2PO4, 0.5 g / L NaCI, 0.5 g / L MgSO4.7H2O, 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. As specified in the respective examples, 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)).

[0359] 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.

[0360] Strains and mutations

[0361] 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).

[0362] In an example to produce LN3, 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. In an example for production of LN3 derived oligosaccharides like lacto- / V-tetraose (LNT, Gal- bl,3-GlcNAc-bl,3-Gal-bl,4-Glc), 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. In an example for production of lacto- / V-neotetraose (LNnT, Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc), 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. Alternatively, LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis can be used.

[0363] Optionally, 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).

[0364] 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.

[0365] 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).

[0366] 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).

[0367] 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).

[0368] In an example for sialic acid and CMP-sialic acid production, the mutant strain was derived from E. coli K12 MG1655 as described e.g. in W02022 / 034067, W02022 / 034068 or W02022 / 034070. To allow sialylated oligosaccharide production, 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). 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 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).

[0369] In an example for GDP-fucose production, 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. To allow fucosylated oligosaccharide production, 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).

[0370] In an example to produce one or more fucosylated non-charged oligosaccharide(s), 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. In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTa and LSTb, 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). In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTc and LSTd, 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).

[0371] Preferably but not necessarily, 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. 2016, 6: 160196). Optionally, 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 Biology™, vol 205. Humana Press).

[0372] All constitutive promoters, UTRs and terminator sequences originated from the libraries described by Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148), Dunn et al. (Nucleic Acids Res. 1980, 8, 2119- 2132), Edens et al. (Nucleic Acids Res. 1975, 2, 1811-1820), Kim and Lee (FEBS Letters 1997, 407, 353-356) and Mutalik et al. (Nat. Methods 2013, No. 10, 354-360). Genes were ordered synthetically at Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com) and the codon usage was adapted using the tools of the supplier. Proteins described in present disclosure are summarized in Table 1. All strains were stored in cryovials at -80°C (overnight LB culture mixed in a 1:1 ratio with 70% glycerol).

[0373] Cultivation conditions

[0374] A preculture of 96-well microtiter plate experiments was started from a cryovial, in 150 pL LB and was incubated overnight at 37 °C on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96well square microtiter plate, with 400 pL minimal medium by diluting 400x. These final 96-well culture plates were then incubated at 37°C on an orbital shaker at 800 rpm for 72h, or shorter, or longer. To measure sugar concentrations at the end of the cultivation experiment whole broth samples were taken from each well by boiling the culture broth for 1 hour at 60°C before spinning down the cells (= average of intra- and extracellular sugar concentrations).

[0375] 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, Melsungen, 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% NH4OH. The exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.

[0376] Optical density

[0377] Cell density of the cultures was frequently monitored by measuring optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or with a Spark 10M microplate reader, Tecan, Switzerland). The maximum growth speed (mumax) was calculated based on the observed optical densities at 600nm using the R package grofit. Heterologous and homologous expression

[0378] Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: IDT or Twist Bioscience. Proteins described in present application are summarized in Table 1. Unless stated otherwise, the UniProt IDs of the proteins described in the application and claims correspond to their sequence version 01 as present in the UniProt Database version release 2021_03 of 09 June 2021. Genes were optimized using the tools of the supplier.

[0379] Table 1. Overview of proteins with corresponding SEQ ID NOs or UniProt IDs (sequence version 01, UniProt Database 2021_03 of 09 June 2021) as described in the present application

[0380]

[0381] *Sequence version 03 (23 Jan 2007) as present in the UniProt Database 2021_03 of 09 June 2021

[0382] **Sequence version 04 (23 Jan 2007) as present in the UniProt Database 2021_03 of 09 June 2021

[0383] ***Sequence version 02 (23 Jan 2007) as present in the UniProt Database 2021_03 of 09 June 2021

[0384] ****Sequence version 02 (01 Dec 2000) as present in the UniProt Database 2021_03 of 09 June 2021 Analytical analysis

[0385] Standards such as but not limited to sucrose, lactose, LN3, LNT, LNnT, pLNnH were purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analyzed with in-house made standards.

[0386] Neutral oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Evaporative Light Scattering Detector (ELSD) or a Refractive Index (Rl) detection. 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 and the data rate 10 pps. The temperature of the Rl detector was set at 35 °C.

[0387] Sialylated oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Refractive Index (Rl) detection. 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.

[0388] Both neutral and sialylated sugars were analyzed on a Waters Acquity H-class UPLC with Refractive Index (Rl) detection. 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 72% acetonitrile and 28% ammonium acetate buffer (100 mM) to which 0.1% triethylamine was added. The method was isocratic with a flow of 0.260 mL / min. The temperature of the Rl detector was set at 35°C.

[0389] Normalization of the data

[0390] For all types of cultivation conditions, data obtained from the mutant strains was normalized against data obtained in identical cultivation conditions with reference strains having an identical genetic background as the mutant strains but lacking the transporter protein expression cassettes. All data is given in relative percentages to that setpoint.

[0391] Example 2. Lacto-N-triose (LN3) and 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

[0392] An 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. 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. In a next step, 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. The experiment demonstrated that expression of a transporter protein with SEQ ID NO 01 or 02 enhanced the production of LN3 and 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 wbgO (Uniprot ID D3QY14) from E. coli 055:1-17 (Tables 2 and 3). The expression of 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).

[0393] Table 2. The percentage of LN3 and LNT cell performance index (CPI) measured in the whole broth compared to the reference strain.

[0394] "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.

[0395] "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.

[0396] 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

[0397] An 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. 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. In a next step, 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. The experiment demonstrated that expression of a transporter protein with SEQ ID NO 01 enhanced the production of LN3 and LNnT that is being produced in a LNnT production host expressing 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

[0398] 2000) from N. meningitidis (Tables 4 and 5).

[0399] Table 4. The percentage of LN3 and LNnT cell performance index (CPI) measured in the whole broth compared to the reference strain.

[0400] "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.

[0401] Table 5. The percentage of LN3 and LNnT (g / L) measured in the whole broth compared to the reference strain.

[0402] "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.

[0403] 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

[0404] An 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. 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). In a next step, 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. 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).

[0405] Table 6. The percentage of LNFPI cell performance index (CPI) measured in the whole broth compared to the reference strain.

[0406] "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.

[0407] Table 7. The percentage of LNFPI (g / L) measured in the whole broth compared to the reference strain.

[0408] "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.

[0409] 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

[0410] An 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. 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. asymbioticus (UniProt ID A4SVF8). In a next step, 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. The experiment demonstrated that expression of a transporter protein with SEQ ID NO 01 enhanced the production of LNFPIII that is being produced in a LNFPIII production host expressing 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 and the alpha-1, 3- fucosyltransferase from P. asymbioticus (UniProt ID A4SVF8) (Table 8).

[0411] Table 8. The percentage of LNFPIII (g / L) measured in the whole broth compared to the reference strain.

[0412] "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.

[0413] 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

[0414] An 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. 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. catarrhinii (UniProt ID A0A4V6F2S0) and a constitutive transcriptional unit for the alpha-1, 2-fucosyltransferase from D. alaskensis (UniProt ID Q316B5). In a next step, 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. 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).

[0415] Table 9. The percentage of LNDFHI cell performance index (CPI) measured in the whole broth compared to the reference strain.

[0416] "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.

[0417] Table 10. The percentage of LNDFHI (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) is not expressed in the reference strain.

[0418] 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

[0419] An 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. 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. ligni (UniProt ID A0A3D9Z454). In a next step, 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. The experiment demonstrated that expression of a transporter protein with SEQ ID NO 01 enhanced the production of LNnDFH that is being produced in a LNnDFH production host expressing 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 and the alpha-1, 3- fucosyltransferase from M. ligni (UniProt ID A0A3D9Z454) (Tables 11 and 12).

[0420] Table 11. The percentage of LNnDFH cell performance index (CPI) measured in the whole broth compared to the reference strain.

[0421] "SD" represents the standard deviation (4 replicates of the same strain tested). The "reference strain

[0422] (REF)" is identical to the tested strains, except that the indicated transporter protein (SEQ ID NO 01) is not expressed in the reference strain.

[0423] Table 12. The percentage of LNnDFH (g / L) measured in the whole broth compared to the reference strain.

[0424] "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.

[0425] 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

[0426] An 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. 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.

[0427] In a next step, 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. 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.

[0428] 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.

[0429] "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.

[0430] 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

[0431] An 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. 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). In a next step, 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. The experiment demonstrated that expression of a transporter protein with SEQ ID NO 13 enhanced the production of LNnT that is being produced in a LNnT production host expressing 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) (Table 15 & 16).

[0432] Table 15. The percentage of LNnT cell performance index (CPI) measured in the whole broth compared to the reference strain.

[0433] "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.

[0434] Table 16. The percentage of LNnT (g / L) measured in the whole broth compared to the reference strain.

[0435] "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

[0436] An 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. 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). In a next step, 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. The experiment demonstrated that expression of a transporter protein with SEQ ID NO 15, 17, 18 or 19 enhanced the production of LNnT that is being produced in a LNnT production host expressing the galactoside beta-1, 3- N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6), 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) (Table 17 & 18).

[0437] Table 17. The percentage of LNnT cell performance index (CPI) measured in the whole broth compared to the reference strain.

[0438] "SD" represents the standard deviation (4 replicates of the same strain tested). The "reference strain

[0439] (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. Table 18. The percentage of LNnT (g / L) measured in the whole broth compared to the reference strain.

[0440] "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.

Claims

Claims1. 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 LN3-containing oligosaccharide is LN3, a lacto-N-tetraose (LNT)-containing oligosaccharide or a lacto-N-neotetraose (LNnT)-containing oligosaccharide, characterized in that said cell expresses a transporter protein comprising an amino acid sequence:(i) wherein said 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 full-length of said polypeptide;(ii) wherein said 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;(iii) wherein said 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 SEQID 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;(iv) wherein said 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.

2. A cell according to claim 1, wherein said transporter is able to transport said LN3-containing oligosaccharide.

3. A cell according to claim 1 or 2, wherein said transporter protein is heterologous.

4. A cell according to any one of claims 1 to 3, wherein said LN3-containing oligosaccharide is a neutral oligosaccharide.

5. A cell according to any one of claims 1 to 4, wherein said cell is selected from the list consisting of a microorganism, a plant cell, an animal cell, an insect cell and a protozoan cell.

6. A cell according to any one of claims 1 to 5, wherein said cell is a bacterium.

7. A cell according to any one of claims 1 to 6, wherein said cell expresses a galactoside beta-1, 3-N- acetylglucosaminyltransferase that is involved in the synthesis of said LN3-containing oligosaccharide.

8. A cell according to any one of claims 1 to 7, wherein said cell further expresses one or more glycosyltransferases involved in the synthesis of said LN3-containing oligosaccharide, wherein saidone or more glycosyltransferases is / are selected from the list consisting of a galactosyltransferase, a fucosyltransferare, a N-acetylglucosaminyltransferase, a N-acetylgalactosaminyltransferase and a sialyltransferase.

9. A cell according to any one of claims 1 to 8, wherein said cell 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.

10. A cell according to claim 9, wherein said precursor saccharide is lactose, optionally wherein said lactose further comprises a fucose.

11. A cell according to any one of claims 1 to 10, wherein said cell has an improved production of said LN3-containing oligosaccharide compared to said cell with an identical genetic background but that lacks said transporter protein.

12. A cell according to claim 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.

13. Cell according to claim 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).

14. Cell according to any one of claims 1 to 13, wherein said transporter protein 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; 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 26, 30, 33 and / or 34 of TMl of the transporter protein represented by SEQ ID NO 16,o 22, 26, 29 and / or 30 of TMl of the transporter protein represented by SEQ ID NO 02, o 22 of TMl of the transporter protein represented by SEQ ID NO 03, o 27 , 31, 34 and / or 35 of TMl of the transporter protein represented by SEQ ID NO 04, o 28, 32, 35 and / or 36 of TMl of the transporter protein represented by SEQ ID NO 05, o 26, 30, 33 and / or 34 of TMl of the transporter protein represented by SEQ ID NO 06; optionally wherein said transporter protein lacks all amino acids N-terminally from its TMl domain compared to the transporter protein represented by SEQ ID NO 01, 13, 02, 03, 04, 05 or 06, respectively.

15. 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:(a) cultivating a cell according to any one of claims 1 to 14, 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;(b) optionally separating said LN3-containing oligosaccharide from the cultivation broth or separating any one, preferably all, of the oligosaccharides in said mixture from the cultivation broth.

16. Method according to claim 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.

17. Method according to claim 15 or 16, further comprising the step of purifying said LN3-containing oligosaccharide or any one, preferably all, of the oligosaccharides in said mixture.

18. Method according to claim 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.

19. Use of a cell according to any one of claims 1 to 14 for the production of a LN3-containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide.

20. Use of a transporter protein as defined in any one of claims 1, 2, 3 and 14 in the production of a LN3- containing oligosaccharide or an oligosaccharide mixture comprising a LN3-containing oligosaccharide.

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