WO2025190861A1 - Mutants de fucosyltransférase à faible formation de dfl - Google Patents

Mutants de fucosyltransférase à faible formation de dfl

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Publication number
WO2025190861A1
WO2025190861A1 PCT/EP2025/056456 EP2025056456W WO2025190861A1 WO 2025190861 A1 WO2025190861 A1 WO 2025190861A1 EP 2025056456 W EP2025056456 W EP 2025056456W WO 2025190861 A1 WO2025190861 A1 WO 2025190861A1
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fucosyltransferase
variant
seq
alpha
amino acid
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Margit Pedersen
Ferhat BÜKE
René Marcel de Jong
Marianne RUDOLPH HANSEN
Carlos RAMIREZ PALACIOS
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DSM IP Assets BV
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DSM IP Assets BV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01069—Galactoside 2-alpha-L-fucosyltransferase (2.4.1.69)

Definitions

  • the present disclosure relates to alpha-1 ,2-fucosyltransferease variants with decreased alpha- 1 ,3-fucosyl transferase activity, in particular variants of FutC from Helicobacter pylori.
  • HMO Human Milk Oligosaccharide
  • 2’FL 2’-fucosyllactose
  • DFL byproduct HMO difucosyllactose
  • the disclosure further relates to genetically engineered cells comprising the alpha-1 ,2-fucosyltransferease variants, wherein the cells are suitable for use in methods for biosynthetic production of 2’FL.
  • HMOs Human Milk Oligosaccharides
  • HMOs fucosylated Human Milk Oligosaccharides
  • the human milk oligosaccharide 2’FL (2’-fucosyllactose) is the most abundant HMO in breast milk and favors intestinal integrity and promotes a healthy intestinal bacterial flora while also providing cognition and memory benefits. Thus, it is in particular of importance to improve the production of 2’FL by reducing by-products such in particular DFL and 3FL without affecting the 2’FL yield or even improving 2’FL yields further, to obtain a robust 2’FL production platform that is scalable for industrial production of 2’FL.
  • the most commonly used enzyme when producing 2’FL is the a-1 ,2-fucosyltransferases FutC from Helicobacter pylori. FutC is also one of the enzyme used in the commercial production of 2’FL and is known to form the by-product difucosyllactose (DFL) (see Bych et al 2019, Current Opinion in Biotechnology 56:130-137). Efforts to improve the activity of FutC have been described in Park et al 2022 Biotechnol Bioeng.119:1264-1277, CN116676288, CN117402804, CN113528480 and CN116064455 without mentioning any desire to decrease DFL formation by the enzyme. CN117343888 also describes FutC variants and combine these with a-1 ,3- fucosyltransferases to produce high levels of DFL.
  • DFL by-product difucosyllactose
  • alpha-1 , 2-fucosyltransferase variant enzymes originating from a parent alpha-1 , 2-fucosyltransferase from Helicobacter pylori, their uses, genetically engineered cells comprising a variant of the present disclosure and methods for the production of 2’- fucosyllactose.
  • the variant enzymes may also be used to produce LNFP-I.
  • a first aspect relates to a functional alpha-1 , 2-fucosyltransferase variant with at least 85 % sequence identity to SEQ ID NO: 1 , wherein the variant comprises a substitution in one or more positions selected from the group consisting of C9F,N,G,W,A,K; G10A,S; S39A,W; V93E; I175D; C177H,Q,D,S,F; and L273R.Q, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.
  • the variant has reduced alpha-1 ,3- fucosyltransferase activity compared to the alpha-1 ,3-fucosyltransferase activity of the parent alpha-1 , 2-fucosyltransferase, such as the alpha-1 , 2-fucosyltransferase of SEQ ID NO: 1.
  • the variants of the present disclosure comprises at least one further substitution in one or more positions selected from the group consisting of position 40, 80, 110, 121 , 124, 125, 150, 15, 239 and 297, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.
  • a second aspect relates to genetically engineered cells which can produce 2’FL, wherein the genetically engineered cell comprises an alpha- 1 , 2-fucosyltransferase variant of the present disclosure.
  • 2’FL is the primary oligosaccharide produced by said cell.
  • a third aspect relates to a method for producing at least one fucosylated oligosaccharide, in particular 2’fucosyllactose (2’FL), wherein said method comprises the following steps: a. providing a genetically engineered cell as disclosed herein comprising an alpha-1 , 2- fucosyltransferase variant of the present disclosure; b. culturing the genetically engineered cell of a. in a suitable cell culture medium to produce said fucosylated oligosaccharide; and optionally c. purifying said fucosylated oligosaccharide.
  • a- 1 , 2-fucosyltransferase used is capable of fucosylating lactose at the galactose (Gal) moiety in an a-1 ,2 configuration. Accordingly, contacting the a- 1 , 2-fucosyltransferase disclosed herein with a substrate in the presence of a fucose donor, such as GDP-fucose, results in formation of 2’FL when lactose is the substrate.
  • a fucose donor such as GDP-fucose
  • a fourth aspect relates to the use of an alpha-1 , 2-fucosyltransferase variant of the present disclosure, in the production of a fucosylated oligosaccharide, in particular 2’FL.
  • Figure 1 Overview of the synthesis of the fucosylated HMOs 2’FL, 3FL and DFL.
  • FIG. 1 Overview of the synthesis of the fucosylated HMOs LNFP-I and LNDFH-I.
  • FIG. 3 Amino acid scan at indicated positions of the parent FutC enzyme of SEQ ID NO: 1 (wt).
  • Figure 4 % 2’FL and % DFL formation of genomically integrated variants.
  • Figure 6 % DFL produced by the individual strains relative to 2’FL produced by the same strain. The order of the variants is the same as in figure 5, having the highest 2’FL producer at the top.
  • Figure 7 Three-dimensional structure of FutC with the amino acid sequence of SEQ ID NO: 1 , showing the amino acids in positions 9, 10, 40, 80, 121 , 124, 125, 150, 151 , 175, 177, 273, 239, and 297 relative to the GDP-fucose and the DFL embedded in the active site.
  • Figure 8 Three-dimensional structure of FutC with the amino acid sequence of SEQ ID NO: 1 with the GDP-fucose and the DFL embedded in the active site.
  • a and B shows the structure from different angles identifying positions that are conserved across H. pylori alpha-1 , 2- fucosyltransferases.
  • Figure 9 Alignment of FutC homologues from Helicobacter pylori, the amino acids indicated in bold correspond to position F110, Q150, C151 , C177 and Q239 in the amino acids sequence of SEQ ID NO: 1 (FutC-wt).
  • the present disclosure investigates the opportunity to improve enzymes used in the production of fucosylated oligosaccharides that only contain one fucosyl monosaccharide in an alpha- 1 ,2- linkage, such as the fucosylated oligosaccharides 2’FL, LNFP-I, and LNFP-IV. Potentially an additional fucose in an alpha-1 ,4-linkage where the second fucose is added by an alpha-1 ,4- fucosyltransferase may also be of interest.
  • the enzyme variants of the present disclosure can be applied in in vitro processes where a fucosyl donor, such as GDP-fucose, is combined with an acceptor molecule, e.g., a mono-, di- or oligosaccharide or larger glycans, proteins and lipids and the enzyme facilitates the transfer of the fucose to the acceptor molecule, to produce for example fucosylated oligosaccharides (see for example scheme 3 of Petschacher and Nidetzky 2016 Journal of Biotechnology 235: 61-83).
  • a fucosyl donor such as GDP-fucose
  • the enzyme variants of the present disclosure can also be used in biosynthetic production, also termed in vivo production, using the production machinery of a suitable host cell as illustrated for example in Bych et al 2019, Current Opinion in Biotechnology 56:130-137 and Petschacher and Nidetzky 2016 Journal of Biotechnology 235: 61-83.
  • biosynthetic production also termed in vivo production, using the production machinery of a suitable host cell as illustrated for example in Bych et al 2019, Current Opinion in Biotechnology 56:130-137 and Petschacher and Nidetzky 2016 Journal of Biotechnology 235: 61-83.
  • the in vivo process is often the most effective.
  • the present disclosure therefore offers specific strain engineering solutions to produce specific fucosylated oligosaccharides, in particular HMOs, in particular 2’FL (as the primary HMO), by exploiting the substrate specificity of the alpha-1 , 2-fucosyltransferase variants described herein, which have decreased a-1 ,3-fucosyltransferase activity towards the glucose (Glc) moiety, preferably while maintaining the high a-1 , 2-fucosyltransferase activity towards the galactose (Gal) moiety on lactose, which improves the production of 2’FL while reducing byproduct formation, in particular reduced difucosyllactose (DFL) formation and potentially increased 2’FL product yields in the fermentation broth.
  • HMOs in particular 2’FL
  • 2’FL as the primary HMO
  • the fermented product such as e.g., 2’FL is very pure and essentially free from any HMO by-products, such as in particular 3FL and DFL, i.e., 2’FL is the primary HMO constituting more than 95 wt%, such as more than 96 wt%, 97 wt%, 98 wt%, 98.5 wt%, 99 wt%, 99.5 wt% of the total amount of oligosaccharide/HMO produced in the fermentation process/fermentation broth.
  • An increased ratio of product (2’FL) over by-product (DFL) can also increase the overall yield relative to the amount of carbon-source used, since less energy is spent on making by-products leaving more over to form the product.
  • the present disclosure describes newly identified a-1 , 2-fucosyltransferase enzyme variants with reduced or no a-1 ,3-fucosyltransferase activity while maintaining or potentially increasing a-1 , 2-fucosyltransferase activity as compared to a wildtype or parent a-1 ,2- fucosyltransferase enzyme, in particular compared to the enzyme with the amino acid sequence of SEQ ID NO: 1 , also termed the parent FutC enzyme.
  • Such FutC variant enzymes allow for production with lower, or no amount of the HMO by-product DFL compared to the parent FutC enzyme or other homologous FutC enzymes.
  • the variants also allow for production of 2’FL amounts which are close to (not below 10-15%) or better than the 2’FL amounts produced by the parent FutC enzyme (see example 2, 3 and 4).
  • the a-1 , 2-fucosyltransferase, FutC from Helicobacter pylori, has been reported to be the best producer of 2’FL in a strain with the conventional improvements used in strains to improve fucosyllactose formation (see for example in Huang et al 2017 Metabolic Engineering 41 (2017) 23-38 and Li et al 2022 Microbial Biotechnology, 15, 1561-1573).
  • coli and other microorganisms comprises overexpression of the de novo GDP-fucose pathway genes responsible for the formation of GDP-fucose, which is needed to provide sufficient donor substrate for the fucosyl lactose formation, along with deletion of wcaJ, to prevent GDP-fucose degradation, and deletion of lacZ to prevent degradation of lactose, which acts as the acceptor in the production of fucosyllactose (see for example figure 2b of Bych et al 2019, Current Opinion in Biotechnology 56:130-137).
  • the enzyme variants presented herein serve as favourable alternatives to the parent and homologous FutC enzymes for improved large-scale manufacturing of 2’FL, in particular with reduced DFL formation.
  • oligosaccharide means a sugar polymer containing at least three monosaccharide units, i.e., a tri-, tetra-, penta-, hexa- or higher oligosaccharide.
  • the oligosaccharide can have a linear or branched structure containing monosaccharide units that are linked to each other by interglycosidic linkages.
  • the oligosaccharide comprises a lactose residue at the reducing end and one or more naturally occurring monosaccharides of 5-9 carbon atoms selected from aldoses (e.g., glucose, galactose, ribose, arabinose, xylose, etc.), ketoses (e.g., fructose, sorbose, tagatose, etc.), deoxysugars (e.g. rhamnose, fucose, etc.), deoxy-aminosugars (e.g.
  • aldoses e.g., glucose, galactose, ribose, arabinose, xylose, etc.
  • ketoses e.g., fructose, sorbose, tagatose, etc.
  • deoxysugars e.g. rhamnose, fucose, etc.
  • deoxy-aminosugars e.g.
  • the oligosaccharide is an HMO.
  • HMO Human milk oligosaccharide
  • oligosaccharides of the disclosure are human milk oligosaccharides (HMOs).
  • human milk oligosaccharide in the present context means a complex carbohydrate found in human breast milk.
  • the HMOs have a core structure comprising a lactose unit at the reducing end that can be elongated by one or more beta-N-acetyl- lactosaminyl and/or one or more beta-lacto-N-biosyl unit, and this core structure can be substituted by an alpha-L-fucopyranosyl and/or an alpha-N-acetyl-neuraminyl (fucosyl) moiety.
  • HMO structures are e.g., disclosed by X/ Chen in Chapter 4 of Advances in Carbohydrate Chemistry and Biochemistry 2015 vol 72.
  • fucosylated HMOs examples include, 2'-fucosyllactose (2’FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3- fucosyllactose (3FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N- fucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-111) , fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-fucopentaose VI (LNFP-VI), lacto-N- difucohex
  • fucosylated HMOs with a fucosyl moiety in an alpha-1 ,2 linkage such as for example 2’FL, DFL, LNFP-I and LNDFH-I, and sialylated derivatives thereof, are desired HMOs of the present disclosure, more preferably the fucosylated HMO does not contain a fucosyl moiety in an alpha-1 ,3 linkage.
  • a-1 ,2-fucosyltransferase variants disclosed herein facilitate the transfer of a fucose moiety from a glycosyl donor to an acceptor molecule to generate a fucosylated molecule, preferably a fucosylated oligosaccharide, such as an HMO.
  • the present disclosure relates to variants of a parent a-1 ,2-fucosyltransferase from Helicobacter pylori, comprising an alteration/mutation, preferably in the form of a substitution and/or an insertion and/or a deletion at one or more (several) positions, where the numbering of the positions correspond to the numbering of the positions of the reference sequence, such as SEQ ID NO:1 , when the variant is aligned to the reference sequence using the Needle program of the EMBOSS package with the settings described under the heading “sequence identity”.
  • a-1 ,2-fucosyltransferase variants of the present disclosure are preferably non-natural variants, i.e., they have not been found in nature.
  • the nomenclature described below is adapted for ease of reference. In all cases, the accepted IUPAC single letter or triple letter amino acid abbreviation is employed.
  • valine substitutions For amino acid substitutions, the following nomenclature is used: original amino acid/position/substituted amino acid. Accordingly, the substitution of valine with glutamic acid at position 93 is designated as "Val93Glu” or “V93E”. Multiple mutations in the same sequence are separated by commas (",”), e.g., " V93E, F110A” or a space e.g., “V93E F110A”, representing mutations at positions 93 and 110 substituting valine (V) with glutamic acid (E), and phenylalanine (F) with alanine (A), respectively.
  • the original amino acid may be substituted by an amino acid selected from a group of amino acids which is indicated as follows C9Y,N,G,A,K representing the substitution of a cysteine (C) at position 9 with an amino acid selected from the group consisting of: tyrosine (Y), asparagine (N), glycine (G), alanine (A), and lysine (K).
  • C9Y,N,G,A,K could be written as C9Y or C9N or C9F or C9A or C9K.
  • the original amino acid is the amino acid found at the indicated position of the reference sequence.
  • the reference sequence may also be considered as the parent sequence/enzyme from which the variant originates.
  • the relationship between the parent sequence and a specified variant is that the sequence of the parent and the variant are identical except for the indicated substitutions in the variant, i.e., the parent is the unmodified version of the variant.
  • the parent a-1 ,2-fucosyltransferase is generally used as the comparative a-1 ,2-fucosyltransferase when assessing the effect of a specific mutation in a variant a-1 ,2-fucosyltransferase.
  • the parent a-1 ,2-fucosyltransferase from which the variants of the present disclosure originate is preferably FutC from Helicobacter pylori ATCC 26695 with NCBI accession nr WP_080473865.1 (SEQ ID NO: 6) or a sequence with at least 98%, such as 99% identity to WP_080473865.1 such as SEQ ID NO: 1.
  • the FutC enzyme with the accession nr WP_080473865.1 is considered to be a wildtype FutC a-1 ,2-fucosyltransferase enzyme.
  • pylori a-1 ,2-fucosyltransferase are WP_104758319.1 (SEQ ID NO: 44), WP_324286887.1 (SEQ ID NO: 45), WP_130778061 .1 (SEQ ID NO: 46), WP_128060420.1 (SEQ ID NO: 47), WP_026938579.1 , AAD29869.1 , WP_000874761 .1 , WP_126474003.1 WP_101020196.1 , WP_187942380.1 WP_187832504.1 and WP_001970518.1 which have a sequence identity between 85% and 98% identity to WP_080473865.1 .
  • wildtype a- 1 ,2-fucosyltransferase denotes an a-1 ,2-fucosyltransferase expressed by a naturally occurring microorganism.
  • the existence of multiple wildtype alpha-1 , 2-fucosyltransferases in Helicobacter pylori is an indication that quite some variation is possible outside the positions selected in the variants of the present disclosure, in principle any of these alternative wildtype a-1 ,2- fucosyltransferases from H. pylori can act as the parent enzyme for the alterations disclosed herein, which supports functional alpha-1 , 2-fucosyltransferase variants with at least 85% sequence identity to SEQ ID NO: 1 or alternative reference sequences.
  • Example 5 of the present disclosure shows that four different homologous futC enzymes from H. pylori behave in similar manners as the parent FutC of SEQ ID NO: 1 , when subjected to the same substitutions as SEQ ID NO: 1 , indicating that the substitutions identified in the present disclosure work in different alpha-1 , 2-fucosyltransferases down to at least a sequence identity of 86%.
  • alpha-1 ,3- or alpha- 1 ,2-fucosyltransferase activity there are also examples of conservative substitutions that can be allowed without any significant change in functionality of the enzyme .
  • substitutions are for example within the group of basic (positively charged) amino acids (arginine (arg, R), lysine (lys, K) and histidine (his, H)), acidic (negatively charged) amino acids (glutamic acid (glu, E) and aspartic acid (asp, D)), amides of the negatively charged amino acids (glutamine (gin, Q) and asparagine (asn, N)), polar amino acids with uncharged side chains (serine (ser,S), Threonine (thr, T), glutamine (gin, Q) and asparagine (asn, N)), hydrophobic amino acids (alanine (ala , A), valine (val, V), leucine (leu, L), isoleucine (ile, I) and methionine (met, M)), aromatic amino acids (phenylalanine (phe, F), tryptophan (trp, W) and tyrosine (tyr, Y)
  • Proline (pro, P) and cysteine (cys ,C) are non-charged but does not fit into any of the respective groups.
  • Amino acid substitutions which do not generally alter specific activity are known in the art and are described, for example, by Neurath and Hill, 1979, In, The Proteins, Academic Press, New York.
  • the most commonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, LeuA/al, Ala/Glu, and Asp/Gly.
  • amino acids which are important to the functionality of the alpha-1 ,2- fucosyltransferase enzyme variants of the present disclosure can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-1085, 1989). Identification of the active site in an enzyme can also be found using bioinformatic tools such as those described in (Izidoro et al 2015 Bioinformatics 31 :864-870) or the artificial intelligence (Al) based alpha fold structures as for example described in Jumper et al 2021 Nature. 596:583-589.
  • a variant with at least 85 % sequence identity to SEQ ID NO: 1 maintain the functional activity, i.e., it is a functional alpha- 1 ,2-fucosyltransferase variant.
  • alpha-1 ,2-fucosyltransferase variants this means that the enzyme maintains the ability of transferring a fucose moiety from GDP-fucose to a substrate present in the active site, preferably the substrate is lactose and the fucose moity is transferred to the galactose moiety of lactose in an alpha-1 ,2 configuration, so essentially the variant maintains its alpha-1 , 2- fucosyltransferase activity and thereby it’s ability to produce 2’FL.
  • Figures 7 and 8A and 8B shows the three-dimensional structure of the fucosyltransferase enzyme of SEQ ID NO: 1 with the GDP-fucose and DFL docketed into the active site.
  • figures 8A and 8B identify amino acids which are conserved across all the homologous alpha- 1 ,2-fucosyltransferases from H. pylori. Conservation of amino acids across a large number of homologous enzymes can indicate that such a position is important for the functionality of the enzyme and it should be tested if a change can be made in such an amino acid without losing functionality of the enzyme.
  • positions indicated in Figure 8A and B 13, 45, 114, 166, 168, 169, 171 , 203, 204, 205, 206, 234, 248, 250, 253, 254
  • positions are predicted to be important for the functionality of the enzyme (position refer to SEQ ID NO: 1) for the reasons given in the below table.
  • the amino acids at the following positions G13, R45, Q114, H166, R168, D171 , E205, D234 and/or T250, relative to the positions of SEQ ID NO: 1 are maintained/not deleted or substituted.
  • at least R45 and D171 , relative to the positions of SEQ ID NO: 1 are maintained in the variants of the present disclosure.
  • the alpha-1 , 2-fucosyltransferase variant has an R at position 45, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .
  • This position is believed to be the catalytical residue of the enzyme the variants M10 and M11 in example 2 shows that substituting R45 with E results in an enzyme with almost no alphal ,2- fucosyltransferase activity.
  • the alpha-1 , 2-fucosyltransferase variant has a D at position 171 , wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO: 1 .
  • This position is believed to be relevant for position the GDP-fucose and the substrate in the catalytical pocket of the enzyme.
  • variant PE16 in example 4 shows that substituting D171 with N results in an enzyme with almost no alphal , 2-fucosyltransferase activity.
  • the a-1 ,2-fucosyltransferases used for comparative purposes (the parent sequence) is disclosed in SEQ ID NO: 1 and contains two additional amino acids, leucine (L) and glycine (G), in the C-terminal compared to the wildtype FutC with the NCBI accession nr WP_080473865.1 .
  • the FutC enzyme of SEQ ID NO: 1 is considered as the preferred parent a-1 , 2-fucosyltransferase for the variants of the present disclosure.
  • SEQ ID NO: 1 is technically not a wildtype sequence it is sometimes denoted WT, wtFutC or futC_wt in the examples and figures.
  • the a-1 ,2-fucosyltransferase variants disclosed herein predominantly fucosylate a galactose moiety, in particular a terminal galactose moiety, such as the galactose (Gal) moiety of lactose or the terminal galactose moiety in LNT or LNnT.
  • the a-1 ,2-fucosyltransferase variants disclosed herein produces 2’FL in the presence of a fucosyl donor and lactose as acceptor.
  • a-1 ,2-fucosyltransferase variants disclosed herein may also be capable of/can produce additional fucosylated HMOs such as, but not limited to fucosylated oligosaccharides with an LNT or LNnT backbone.
  • Fucosylated oligosaccharides with an LNnT backbone may in this regard be lacto-/V- fucopentaose IV (LNFP-IV).
  • Fucosylated oligosaccharides, such as HMOs, with an LNT backbone may in the regard be lacto-/V-fucopentaose I (LNFP-I), and Lacto-N-difucohexaose I (LNDFH-I).
  • an additional fucosyltransferase activity is required, namely an a-1 ,4-fucosyltransferase (see figure 2).
  • the a-1 ,2-fucosyltransferase variants as disclosed herein produces an HMO with only one fucosyl moiety, wherein the fucosyl moiety is an a-1 ,2-linked fucosyl moiety and the HMO is 2’FL or LNFP-I, most preferably 2’FL.
  • the a-1 ,2-fucosyltransferase variants as disclosed herein has very low or no a- 1 ,3-fucosyltransferase activity, which can for example be assessed by the levels of DFL being less than less than 2%, such as less than 1%, such as less than 0.5%, such as less than 0.2%, such as 0% of the total fucosylated oligosaccharide produced by the reaction.
  • a-1 ,2-fucosyltransferase or “a-1 ,2-fucosyltransferase activity” refers to a glycosyltransferase that catalyses the transfer of a fucosyl moiety from a donor substrate, such as GDP-fucose, to an acceptor molecule, such as lactose, LNT or LNnT, in an a-1 ,2-linkage/ a-
  • a-1 ,3-fucosyltransferase or “a-1 ,3-fucosyltransferase activity” refers to a glycosyltransferase that catalyses the transfer of fucosyl moiety from a donor substrate, such as GDP-fucose, to an acceptor molecule, such as lactose, in an a-1 ,3-linkage/ a-1 ,3-configuration.
  • 1 .2-fucosyltransferase variants of the present disclosure fall into two groups: i) mutations that reduce the a-1 ,3-fucosyltransferase activity of the parent enzyme; or ii) mutations which improve the a-1 ,2-fucosyltransferase activity of the parent enzyme or the variant enzyme with a group i) mutation.
  • the variant enzymes with the mutations of group i) once introduced into a suitable cell only produce very low or no amounts of DFL and no 3FL.
  • the DFL level produced by these variants is less than 2%, such as less than 1 .5%, such as less than 1%, such as less than 0.5%, such as less than 0.2%, such as 0% of the total fucosylated oligosaccharide products produced by the cell.
  • the group i) mutant variants may display reduced 2’FL production compared to the parent FutC. This may in itself not be an issue if the primary goal is to reduce HMO by-products.
  • the 2’FL production from a-1 ,2- fucosyltransferase variants with at least one mutation from both group i) and ii) has a 2’FL formation that is not reduced more than 30% compared to the parent a-1 ,2-fucosyltransferase, such as not reduced by more than 20% compared to the parent a-1 ,2-fucosyltransferase such as not reduced by more than 15% compared to the parent a-1 ,2-fucosyltransferase, such as not reduced more than 10%, 8%, 5% or 3% compared to the parent a-1 ,2-fucosyltransferase.
  • the 2’FL production from the variants with at least one mutation from both group i) and ii) is at least at the same level as the 2’FL production from the parent a-1 ,2- fucosyltransferase and even more preferably an increased 2’FL production of at least 2%, such as at least 5%, such as at least 8%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50% is achieved with the variants described herein compared to the parent a-1 ,2-fucosyltransferase enzyme.
  • the a-1 ,2-fucosyltransferase variants described herein are non-natural variants which do not occur in nature.
  • the variant enzymes disclosed herein are recombinant enzymes in that they are encoded by recombinant nucleic acid sequences encoding an a-1 ,2-fucosyltransferase variant with at least 85% sequence identity to FutC of SEQ ID NO: 1 or SEQ ID NO: 6.
  • the nucleic acid sequence encoding the a-1 ,2-fucosyltransferase variant is preferably of heterologous origin.
  • the host cell does not naturally encode an a-1 ,2- fucosyltransferase.
  • the group i) mutations of the present disclosure are alterations, such as a substitution, at position 9, 10, 39, 93, 175, 177 and/or 273 wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 and wherein the alteration leads to reduced a-1 ,3- fucosyltransferase activity when compared to the parent a-1 ,2-fucosyltransferase.
  • the group ii) mutations of the present disclosure are alterations, such as a substitution, at position 40, 80, 110, 121 , 124, 125, 150, 151 , 239 and/or 297 wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 and wherein the alteration either alone or in combination with other group ii) alterations leads to a similar or increased a-1 ,2- fucosyltransferase activity compared to the parent a-1 ,2-fucosyltransferase.
  • An aspect the present disclosure relates to an alpha-1 ,2-fucosyltransferase variant with at least 85%, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 6, wherein the variant comprises an alteration in one or more positions selected from the group consisting of position 9, 10, 39, 93, 175, 177 and 273, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.
  • the alteration(s) can be selected from i) an insertion of one or more amino acid downstream of the amino acid which occupies the position, and/or ii) a deletion of the amino acid which occupies the position, and/or iii) a substitution of the amino acid which occupies the position with a different amino acid.
  • the alpha-1 , 2-fucosyltransferase variant obtained by the alteration is a functional variant of a parent a-1 ,2-fucosyltransferases from H. pylori, such as the a-1 ,2-fucosyltransferases with NCBI accession nr. WP_080473865.1 (SEQ ID NO: 6) and in particular the FutC enzyme of SEQ ID NO: 1 , in that it is capable of producing 2’FL.
  • Embodiment of the present disclosure relates to an alpha-1 , 2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, wherein the variant comprises a substitution in one or more positions selected from the group consisting of position 9, 10, 39, 93, 175, 177 and 273, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .
  • Said alpha-1 ,2- fucosyltransferase variant is a functional variant of a parent a-1 ,2-fucosyltransferases from H.
  • pylori such as the a-1 ,2-fucosyltransferases with NCBI accession nr. WP_080473865.1 (SEQ ID NO: 6) and preferably the FutC enzyme of SEQ ID NO: 1 , in that it is capable of producing 2’FL.
  • the primary effect of the alteration, such as substitution, at any of the positions 9, 10, 39, 93, 175, 177 and 273 is that the variant has reduced alpha-1 ,3-fucosyltransferase activity compared to the parent alpha-1 , 2-fucosyltransferase, such as the alpha- 1 , 2-fucosyltransferase of SEQ ID NO: 1 , while maintaining the ability to produce 2’FL.
  • the term “reduced alpha-1 , 3-fucosyltransferase activity” is a comparative measure to the parent alpha-1 , 2-fucosyltransferase, such as the alpha-1 ,2- fucosyltransferase of SEQ ID NO: 1 , where the only difference between the comparative parent and the selected variant is the indicated alterations, such as substitutions.
  • the alpha-1 ,3- fucosyltransferase activity of an enzyme can be assessed in multiple ways known by the skilled person in the art.
  • the activity can be assessed by cell free systems such as the once described in for example Nguyen et al 1998 the Journal of Biological Chemistry 273, 25244-25249 or Hood et al 1998 Analytical Biochemistry 255, 8 - 12.
  • the activity is assessed in vivo using a production host with the machinery for producing 2’FL production e.g., as described in Bych et al 2019, Current Opinion in Biotechnology 56:130-137, where the alpha-1 , 3- fucosyltransferase activity is assessed by the ability to produce the by-product DFL or 3FL.
  • the a-1 ,3-fucosyltransferase activity of the a-1 ,2-fucosyltransferase variants disclosed herein is assessed as the DFL production by the strain expressing the variant enzyme relative to the DFL production of the same strain with the parent enzyme, such as the a- 1 ,2- fucosyltransferase of SEQ ID NO: 1 instead of the variant enzymes.
  • the DFL production of the host strain expressing the parent enzyme is calculated as the percentage relative to the 2’FL production in the same strain.
  • the DFL production of the strain with the variant enzyme is calculated as the as the percentage relative to the 2’FL production in the parent strain, thereby clearly indicating the reduction of DFL relative to the desired product of the parent strain.
  • the DFL formation can be calculated in relation to the 2’FL produced by the variant strain and compared amount of DFL relative to the amount of 2’FL produced by the parent strain. With the alternative calculation variant strains that produce less 2’FL than the parent strain will have higher DFL percentages than if they are calculated relative to the 2’FL production of the parent strain.
  • the a-1 ,2-fucosyltransferase variants disclosed herein has very low or no a-1 ,3- fucosyltransferase activity, which can for example be assessed by the levels of DFL being less than 2%, such as less than 1.5%, such as less than 1%, such as less than 0.5%, such as less than 0.2%, such as 0% of the 2’FL produced by the parent strain or alternatively by the variant strain.
  • the a-1 ,2-fucosyltransferase activity can be assessed using similar cell free systems as described above or it can be assessed in vivo as described for the a-1 ,3-fucosyltransferase activity above measuring 2’FL formation of the parent strain and calculating the 2’FL produced by the variant strain relative to the 2’FL produced by the parent strain.
  • the term “maintaining the ability to produce 2’FL” indicates that 2’FL can be detected in a fermentation broth where a genetically engineered with an enzyme variant of the present disclosure has been fermented under conditions allowing for 2’FL production (see for example Bych et al 2019, Current Opinion in Biotechnology 56: ISO- 137).
  • the level of 2’FL does not need to be at the same level as what can be produced by the parent alpha-1 , 2-fucosyltransferase, such as FutC of SEQ ID NO: 1.
  • 2’FL levels produced by a variant with a substitution in one or more positions selected from the group consisting of position 9, 10, 39, 93, 175, 177 and 273 can be at the same level as that produced by the parent alpha-1 ,2- fucosyltransferase, such as FutC of SEQ ID NO: 1 , it can however also be significantly less, preferably the 2’FL production in a functional variant is not reduced by more than 70%, such as not reduced by more than 60%, such as not reduced by more than 50%, such as not reduced by more than 40%, such as not reduced by more than 30%, such as not reduced by more than 20%, such not reduced by more than 15%, such not reduced by more than 10% of the 2’FL produced by the parent alpha-1 , 2-fucosyltransferase, such as FutC of SEQ ID NO: 1.
  • the alpha-1 ,2-fucosyltransferase variant with at least 85 % sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises a substitution in one or more positions selected from the group consisting C9Y, F, W, N,Q,T,S,G,A,V,K;R; G10A,S; S39A,W; V93E,D, I175D; C177H,Q,N,D,E,S,F and L273R,K,Q,N wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.
  • the alpha-1 ,2-fucosyltransferase variant with at least 85 % sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises a substitution in one or more positions selected from the group consisting C9Y,F,W,N,G,A,K,F,W; G10A,S; S39A,W; V93E; I175D; C177H,Q,D,S and L273R.Q wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 , and the variant has reduced alpha-1 ,3-fucosyltransferase activity compared to the alpha-1 ,2- fucosyltransferase of SEQ ID NO: 1.
  • the alpha-1 ,2-fucosyltransferase variant with at least 85 % sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises one of the following substitutions C177Q or C177S or C177H or C177F, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .
  • the variant produces less than 0.3%, such as less than 0.2% DFL relative to the 2’FL produced by the variant.
  • the 2’FL production by the variant is not reduced by more than 30% relative to the 2’FL production by the parent alpha-1 ,2-fucosyltransferase.
  • alpha-1 , 2-fucosyltransferase variants with a C177Q,H,S,F substitution also have at least one of the following group ii) substitutions F40D; L80C; F110A,H,S; A121 D; P124A; L125R; Q150H; C151 R; Q239S; and /or K297Q,D such as at least one of the substitutions F40D, L80C, F110A,H,S; P124A; Q239S and /or K297Q,D.
  • the alpha-1 ,2-fucosyltransferase variant with at least 85 % sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises the following substitution L273R, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.
  • the variant produces less than 0.2%, such as 0% of DFL relative to the 2’FL produced by the variant.
  • the 2’FL production by the variant is not reduced by more than 30% relative to the 2’FL production by the parent alpha-1 ,2- fucosyltransferase.
  • 2-fucosyltransferase variants with the substitution L273R preferably do not contain any of the following group i) substitutions C177Q, I175D and/or C9Y, in particular the double substitution L273R and I175D should be avoided.
  • alpha-1 , 2-fucosyltransferase variants with a L273R substitution also have at least one of the following group ii) substitutions F40D; L80C; F110A,H,S; A121 D; P124A; L125R; Q150H; C151R; Q239S; and /or K297Q.D such as at least one of the substitutions F40D, L80C, F110A,H,S; P124A; Q239S and /or K297Q.D, such as at least one of the substitutions F40D, L80C, F110A.H and/or Q239S.
  • the alpha-1 , 2-fucosyltransferase variant with at least 85 % sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises one of the following substitution C9Y or C9N or C9G or C9A or C9K or C9F or C9W, preferably C9Y or C9W or C9F or C9G, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.
  • the variant produces less than 0.2%, such as 0% of DFL relative to the 2’FL produced by the variant.
  • the 2’FL production by the variant is not reduced by more than 55% relative to the 2’FL production by the parent alpha-1 ,2- fucosyltransferase.
  • alpha-1 , 2-fucosyltransferase variants with a C9Y,W,F,G substitution also have at least one of the following group ii) substitutions F40D;
  • the alpha-1 , 2-fucosyltransferase variant with at least 85 % sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises the following substitution G10A or G10S, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.
  • the variant produces less than 0.8 %, such as less than 0.5%, such as less than 0.2% of DFL relative to the 2’FL produced by the variant.
  • the 2’FL production by the variant is not reduced by more than 30% relative to the 2’FL production by the parent alpha-1 , 2-fucosyltransferase.
  • alpha- 1 , 2-fucosyltransferase variants with a G10A,S substitution also have at least one of the following group ii) substitutions F40D; L80C; F110A,H,S; A121 D; P124A; L125R; Q150H;
  • the alpha-1 , 2-fucosyltransferase variant with at least 85 % sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises the following substitution S39A or S39W, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.
  • the variant produces less than 50 %, such as less than 45% of DFL relative to the 2’FL produced by the variant.
  • the 2’FL production by the variant is not reduced by more than 10% relative to the 2’FL production by the parent alpha-1 ,2-fucosyltransferase.
  • the 2’FL production is at least at the same level as 2’FL production by the parent alpha-1 , 2-fucosyltransferase.
  • the alpha-1 , 2- fucosyltransferase variant with at least 85 % sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises the following substitution V93E, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .
  • the variant produces less than 1 %, such as less than 0.8% of DFL relative to the 2’FL produced by the variant.
  • the 2’FL production by the variant is not reduced by more than 30% relative to the 2’FL production by the parent alpha-1 ,2-fucosyltransferase.
  • the alpha-1 ,2-fucosyltransferase variant with at least 85 % sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises the following substitution I175D, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .
  • the variant produces less than 0.2 %, such as 0% of DFL relative to the 2’FL produced by the variant.
  • the 2’FL production by the variant is not reduced by more than 70% relative to the 2’FL production by the parent alpha-1 ,2- fucosyltransferase.
  • alpha-1 , 2-fucosyltransferase variants with a I175D substitution also have at least one of the following group ii) substitutions F40D; L80C; F110A,H,S; A121 D; P124A; L125R; Q150H; C151R; Q239S; and/or K297Q.D such as at least one of the substitutions F40D, L80C, F110A,H,S; P124A; Q239S and/or K297Q.D.
  • the variant only comprises two of the group i) mutations.
  • substitutions I175D, C177H,Q,D,S and L273R may be beneficial only to have two out of the three substitutions I175D, C177H,Q,D,S and L273R in the same variant.
  • the substitutions I175D and L273R are not combined.
  • position C177 and L273 are unsubstituted.
  • the substitutions C177H and L273R is preferred over the substitution C177Q and L273R, which are preferably not combined.
  • the alpha-1 ,2-fucosyltransferase variant with at least 85 % sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises the substitution L273R and one of the following substitutions C177S or C177H or C177F, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.
  • the alpha-1 ,2-fucosyltransferase variant with at least 85 % sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises the substitution I175D and one of the following substitutions C177Q or C177S or C177H or C177F, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .
  • the variant contains one of the single group i) substitutions or combinations of group i) substitutions in table 3.
  • the individual variants above which belong to group i) all produce low amounts, such as less than 2%, 1 .5%, 1%, 0.5%, 0.3% or such as less than 0.2%, such as 0% of by-product HMDs, such as DFL and/or 3FL relative to the amount of 2’FL produced by said variant.
  • a second aspect of the present disclosure is to improve these group i) variants by combining one or more of the mutations from group i), particularly those in table 3 above, with one or more mutations from group ii).
  • Embodiments of the present disclosure relates to an alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, wherein the variant comprises i) at least one first alteration in one or more positions selected from the group consisting of position 9, 10, 39, 93, 175, 177 and 273, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 ; and ii) at least one second alteration in one or more positions selected from the group consisting of position 40, 80, 110, 121 , 124, 125, 150, 151 , 239 and 297, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .
  • alpha-1 , 2-fucosyltransferase variant described herein is a functional variant of a parent a-1 ,2-fucosyltransferases from H. pylori, such as the a-1 ,2-fucosyltransferases with NCBI accession nr. WP_080473865.1 (SEQ ID NO: 6), NCBI accession WP_128060420.1 (SEQ ID NO: 47) or WP_130778061 .1 (SEQ ID NO: 46) or preferably the FutC enzyme of SEQ ID NO: 1 , in that it is capable of producing 2’FL.
  • Embodiments of the present disclosure relates to an alpha-1 , 2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, wherein the variant comprises i) at least one first alteration selected from one or more of the substitutions C9Y,F,W,N,Q,T,S,G,A,V,K;R,F,W; G10A,S; V93E,D, I175D; C177H,Q,N,D,E,S,F and/or L273R,K,Q,N, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 ; and ii) at least one second alteration selected from selected from one or more of the substitutions F40S,D,T; L80C, F110A,H,S; A121 D,
  • said alpha-1 , 2-fucosyltransferase variant is a functional variant of a parent a-1 ,2- fucosyltransferases from H. pylori, such as the a-1 ,2-fucosyltransferases with NCBI accession nr. WP_080473865.1 (SEQ ID NO: 6) or NCBI accession WP_128060420.1 (SEQ ID NO: 47) or WP_130778061.1 (SEQ ID NO: 46) or preferably the FutC enzyme of SEQ ID NO: 1 , in that it is capable of producing 2’FL.
  • Embodiments of the present disclosure relates to an alpha-1 , 2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, wherein the variant comprises i) at least one first alteration selected from one or more of the substitutions C9F,W,Y,G,; G10A,S; V93E; I175D; C177H,Q,D,S,F and/or L273R, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 ; and ii) a second alteration comprising the substitution F40S or F40D or F40T, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .
  • said alpha-1 , 2-fucosyltransferase variant is a functional variant of a parent a-1 ,2- fucosyltransferases from H. pylori, such as the a-1 ,2-fucosyltransferases with NCBI accession nr. WP_080473865.1 (SEQ ID NO: 6) or NCBI accession WP_128060420.1 (SEQ ID NO: 47) or WP_130778061.1 (SEQ ID NO: 46) or preferably the FutC enzyme of SEQ ID NO: 1 , in that it is capable of producing 2’FL.
  • alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98%, at least 98.5% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises or consists of a combination of substitutions selected from the group consisting of
  • the variant has at least 90% sequence identity to SEQ ID NO:1 or 6.
  • Embodiments of the present disclosure relates to an alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, wherein the variant comprises i) at least one first alteration selected from one or more of the substitutions C9F,W,Y,G;
  • said alpha-1 ,2-fucosyltransferase variant is a functional variant of a parent a-1 ,2- fucosyltransferases from H. pylori, such as the a-1 ,2-fucosyltransferases with NCBI accession nr. WP_080473865.1 (SEQ ID NO: 6) or NCBI accession WP_128060420.1 (SEQ ID NO: 47) or WP_130778061.1 (SEQ ID NO: 46) or preferably the FutC enzyme of SEQ ID NO: 1 , in that it is capable of producing 2’FL.
  • alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98%, or at least 98.5% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises or consists of a combination of substitutions selected from the group consisting of
  • the variant has at least 90% sequence identity to SEQ ID NO:1 .
  • Embodiments of the present disclosure relates to an alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, wherein the variant comprises i) at least one first alteration selected from one or more of the substitutions C9F,W,Y,G; G10A,S; V93E; I175D; C177H,Q,D,S,F and/or L273R, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 ; and ii) at least one second alteration comprising the following substitutions Q150H and C151R, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.
  • alpha-1 ,2-fucosyltransferase variant is a functional variant of a parent a-1 ,2- fucosyltransferases from H. pylori, such as the a-1 ,2-fucosyltransferases with NCBI accession nr. WP_080473865.1 (SEQ ID NO: 6) or NCBI accession WP_128060420.1 (SEQ ID NO: 47) or
  • WP_130778061.1 (SEQ ID NO: 46) or preferably the FutC enzyme of SEQ ID NO: 1 , in that it is capable of producing 2’FL.
  • Embodiments of the present disclosure relates to an alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, wherein the variant comprises i) at least one first alteration selected from one or more of the substitutions C9F,W,Y,G;
  • said alpha-1 ,2-fucosyltransferase variant is a functional variant of a parent a-1 ,2- fucosyltransferases from H. pylori, such as the a-1 ,2-fucosyltransferases with NCBI accession nr. WP_080473865.1 (SEQ ID NO: 6) or NCBI accession WP_128060420.1 (SEQ ID NO: 47) or WP_130778061.1 (SEQ ID NO: 46) or preferably the FutC enzyme of SEQ ID NO: 1 , in that it is capable of producing 2’FL.
  • Embodiments of the present disclosure relates to an alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 98.5% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, wherein the variant comprises i) at least one first alteration selected from one or more of the substitutions C9F,W,Y,G;
  • said alpha-1 ,2-fucosyltransferase variant is a functional variant of a parent a-1 ,2- fucosyltransferases from H. pylori, such as the a-1 ,2-fucosyltransferases with NCBI accession nr. WP_080473865.1 (SEQ ID NO: 6) or NCBI accession WP_128060420.1 (SEQ ID NO: 47) or WP_130778061.1 (SEQ ID NO: 46) or preferably the FutC enzyme of SEQ ID NO: 1 , in that it is capable of producing 2’FL.
  • alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least 97%, at least 97.5%, at least 98% or at least 98.5% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises or consists of a combination of substitutions selected from the group consisting of
  • the variant has at least 90% sequence identity to SEQ ID NO:1 .
  • Embodiments of the present disclosure relates to an alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 98.5% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, wherein the variant comprises i) at least one first alteration selected from one or more of the substitutions C9F,W,Y,G;
  • said alpha-1 ,2-fucosyltransferase variant is a functional variant of a parent a-1 ,2- fucosyltransferases from H. pylori, such as the a-1 ,2-fucosyltransferases with NCBI accession nr. WP_080473865.1 (SEQ ID NO: 6) or NCBI accession WP_128060420.1 (SEQ ID NO: 47) or WP_130778061.1 (SEQ ID NO: 46) or preferably the FutC enzyme of SEQ ID NO: 1 , in that it is capable of producing 2’FL.
  • alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least 97%, at least 97.5%, at least 98% or at least 98.5% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises or consists of a combination of substitutions selected from the group consisting of
  • F40S L80C A121 D P124A L125R Q150H C151 R Q239S L273R; wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 and the variant has at least 90% sequence identity to SEQ ID NO:1 or 6.
  • Embodiments of the present disclosure relates to an alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 98.5% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, wherein the variant comprises i) at least one first alteration selected from one or more of the substitutions C9F,W,Y,G; G10A,S; V93E; I175D; C177H,Q,D,S,F and/or L273R, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 ; and ii) a second alteration comprising the following substations A121 D and/or P124A, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .
  • said alpha-1 ,2-fucosyltransferase variant is a functional variant of a parent a-1 ,2- fucosyltransferases from H. pylori, such as the a-1 ,2-fucosyltransferases with NCBI accession nr. WP_080473865.1 (SEQ ID NO: 6) or NCBI accession WP_128060420.1 (SEQ ID NO: 47) or WP_130778061.1 (SEQ ID NO: 46) or preferably the FutC enzyme of SEQ ID NO: 1 , in that it is capable of producing 2’FL.
  • alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least 97%, at least 97.5%, at least 98% or at least 98.5% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises or consists of a combination of substitutions selected from the group consisting of
  • an alpha-1, 2-fucosyltransferase variant comprising an alteration from group i) and an alteration from group ii) as disclosed above has similar or increased alpha-1, 2- fucosyltransferase activity on lactose compared to the parent alpha-1, 2-fucosyltransferase, such as the alpha-1 ,2-fucosyltransferase of SEQ ID NO: 1.
  • this can be translated into an increased production of 2’FL of at least 2%, such as at least 5%, such as at least 8%, such as at least 10% such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50% more 2’FL compared to the parent alpha-1 ,2-fucosyltransferase, such as the alpha-1 ,2- fucosyltransferase of SEQ ID NO: 1.
  • the alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98%, or at least 98.5% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises a combination of substitutions selected from the group presented in table 4, wherein the variant produces at least 5% more 2’FL relative to the 2’FL produced by the parent alpha-1 , 2-fucosyltransferase, such as relative to SEQ ID NO: 1 , and wherein the DFL is less than 0.8% relative to the 2’FL produced by the variant sequence.
  • alpha-1 ,2-fucosyltransferase variants with increased 2’FL when compared to the parent and low DFL the alpha-1 , 2-fucosyltransferase variant only comprise group ii) mutations with the purpose of increasing the 2’FL formation compared to the parent alpha-1 , 2-fucosyltransferase.
  • the alpha-1 ,2- fucosyltransferase variant has at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, wherein the variant comprises an alteration comprising the substitution F110A or F110H or F110S, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .
  • said alpha-1 , 2-fucosyltransferase variant is a functional variant of a parent a-1 ,2- fucosyltransferases from H. pylori, such as the a-1 ,2-fucosyltransferases with NCBI accession nr. WP_080473865.1 (SEQ ID NO: 6) or NCBI accession WP_128060420.1 (SEQ ID NO: 47) or WP_130778061.1 (SEQ ID NO: 46) or preferably the FutC enzyme of SEQ ID NO: 1 , in that it is capable of producing 2’FL.
  • alpha-1 , 2-fucosyltransferase variant with at least 85% sequence identity to SEQ ID NO: 1 or 6, such as at least 90%, at least 95%, at least about 97%, at least 98%, or at least 98.5% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 6, comprises or consists of a combination of substitutions selected from the group consisting of
  • F110A F110H F110S F110A Q239S; F110H Q239S; F110S Q239S;
  • 2-fucosyltransferase variants described herein may have additional variation compared to the parent alpha-1 , 2-fucosyltransferase since they may for example originate from another natural H. pylori alpha- 1 , 2-fucosyltransferase homologue figure 8A and 8B identify amino acids which are conserved across most of the H. pylori alpha-1 , 2-fucosyltransferase homologues and therefore may have some common function relevant for the activity of the enzyme. Relative to SEQ ID NO: 1 the conserved positions are 13, 45, 114, 166, 168, 169, 171 , 203, 204, 205, 206, 234, 248, 250, 253, 254.
  • alpha-1 , 2-fucosyltransferase variant of the present disclosure is not substituted in one or more of the following positions G13, R45, Q114, H166, R168, D171 , E205, D234 and/orT250, wherein the position and amino acid corresponds to the position and the amino acid in SEQ ID NO:1 or SEQ ID NO: 6.
  • the alpha-1 , 2-fucosyltransferase variants described herein have an R at position 45 and preferably a D at position 171 , wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .
  • the a-1 ,2- fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 36.
  • a-1 , 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 37.
  • alpha-1 , 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 40.
  • alpha-1 , 2-fucosyltransferase variant at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 41.
  • alpha-1 , 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 42.
  • alpha-1 , 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 43.
  • alpha-1 , 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 48.
  • alpha-1 , 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 49.
  • the present disclosure also relates to a genetically engineered cell expressing an a-1 , 2-fucosyltransferase variant as described herein.
  • the genetically engineered cell comprises at least one, such as too or three genomically integrated copies of a nucleic acid sequence encoding an a-1 , 2-fucosyltransferase variant as described herein.
  • the genetically engineered cell preferably only expresses one recombinant glycosyltransferase activity, namely a recombinant glycosyltransferase with the activity of an a-1 , 2-fucosyltransferase variant as described herein.
  • a genetically engineered cell which is capable of producing high titres of the intended product, while low titers of by-product or even no by-product at all is highly advantageous as it simplifies the downstream processing and purification of the product substantially.
  • 2’FL is the intended or desired product and DFL and 3FL are unwanted byproducts.
  • a genetically engineered cell expressing an alpha- 1 ,2-fucosyltransferase variant disclosed herein produces at least 2%, such as at least 5%, such as at least 8%, such as at least 10% such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50% more 2’FL than a genetically engineered cell expressing the parent alpha-1 ,2- fucosyltransferase.
  • the expression of an a- 1 ,2-fucosyltransferase variant described herein and the and the parent alpha-1 ,2-fucosyltransferase, FutC is regulated via a glp promoter, such as a PglpF promoter (see for example table 1 of WO 2019/123324 for glp promoter examples), alternative other strong promoters such as the modified PmglB promoters described in WO 2020/255054 can be used. Suitable promoters are also disclosed herein in table 2.
  • the intended or desired product may be a larger fucosylated HMO than 2’FL.
  • the a-1 ,2-fucosyltransferase variant disclosed herein may be capable of transferring a fucose unit onto the Gal, moiety of LNT, LNFP-I, or LST-b, to generate LNFP-I, LNDFH-I, or FLST-b respectively.
  • the a-1 , 2- fucosyltransferase variant disclosed herein is capable of producing LNFP-I.
  • the additional glycosyltransferase is preferably selected from the group consisting of, galactosyltransferases, glucosaminyltransferases, fucosyltransferases, N-acetylglucosaminyl transferases and sialyltransferases.
  • additional glycosyltransferases may be needed to produce the desired acceptor molecule for the a-1 , 2-fucosyltransferase, to enable the production of more complex fucosylated HMOs.
  • such glycosyltransferases may be introduced into the cell and are preferably a p-1 ,3-galactosyltransferase, or a p-1 ,4-galactosyltransferase, or a a-1 , 4- fucosyltransferase or a combination of a p-1 ,3-galactosyltransferase and a p-1 ,3-N-acetyl- glucosaminyl-transferases, or a combination of a p-1 ,3-galactosyltransferase and a p-1 ,3-N- acetyl-glucosaminyl-transferases and an a-1 ,4-fucosyltransferase.
  • a a-1 ,2-fucosyltransferase variant as described herein is introduced into a genetically engineered cell wherein said cell further comprises a
  • the p-1 ,3-galactosyltransferases can be obtained from a number of sources, e.g., the galTK gene from H. pylori (homologous to GenBank protein Accession BD182026.1), or the WbgO gene from E. coli 055:H7 (GenBank Accession WP_000582563.1), or the jhp0563 gene from H. pylori (GenBank Accession AEZ55696.1).
  • the p-1 ,4-glycosyltransferases can be obtained from a number of sources, e.g., GalT from Helicobacter pylori (homologous to GenBank protein Accession WP_001262061 .1), or LgtB from Neisseria meningitidis MC58 (homologous to GenBank protein Accession AAF42257.1).
  • the p-1 ,3-N-acetylglucosaminyltransferases can be obtained from a number of sources, e.g., the IgtA genes from N. meningitidis (e.g., GenBank protein Accession ID’s AAF42258.1 , WP_002248149.1 , or WP_033911473.1 or ELK60643.1), or -1 ,3-N- acetylglucosaminyltransferases genes from N.
  • N. meningitidis e.g., GenBank protein Accession ID’s AAF42258.1 , WP_002248149.1 , or WP_033911473.1 or ELK60643.1
  • -1 ,3-N- acetylglucosaminyltransferases genes from N e.g., the IgtA genes from N. meningitidis (e.g., GenBank protein Accession ID’s
  • gonorrhoeae e.g., GenBank protein Accession No’s ACF31229.1 , or AAK70338.1
  • p-1 ,3-N-acetylglucosaminyltransferases genes from Haemophilus ducreyi e.g., GenBank protein Accession AAN05638.1
  • p-1 ,3-N- acetylglucosaminyltransferases genes from Pasteurella multocida e.g., GenBank protein Accession AAK02595.1
  • p-1 ,3-N-acetylglucosaminyltransferases genes from Neisseria cinerea e.g., GenBank protein Accession EEZ72046.1.
  • a-1 ,4-fucosyltransferase can be incorporated into the genetically modified cell.
  • a-1 ,4-fucosyltransferase can for example be obtained from Helicobacter pylori (e.g. GenBank protein Accession AY450598.1 or WP_000487428.1) or from Mediterranea sp. An20 (e.g. GenBank protein Accession WP_087337236.1) or from Bacteroides gallinaceum (e.g. GenBank protein Accession WP_204430034.1).
  • an acceptor saccharide is a disaccharide or an oligosaccharide that can act as a substrate for a glycosyltransferase capable of transferring a glycosyl moiety from a glycosyl donor to the acceptor saccharide.
  • acceptor saccharide and acceptor oligosaccharide may be used interchangeably.
  • the glycosyl donor is preferably a nucleotide-activated sugar as described in the section on “Glycosyl-donor-nucleotide-activated sugar pathways”.
  • the acceptor saccharide is a precursor for making an HMO and can also be termed the precursor molecule or substrate.
  • the acceptor saccharide can be either an intermediate product of the present fermentation process, an end-product of a separate fermentation process employing a separate genetically engineered cell, or an enzymatically or chemically produced molecule.
  • said acceptor saccharide is preferably lactose for the production of 2’FL.
  • LNFP-I lacto-N-tetraose
  • LNT-II lacto-N-neotetraose
  • LNT-II lacto-N-triose II
  • the acceptor saccharide can be either an intermediate product of the present fermentation process, an end-product of a separate fermentation process employing a separate genetically modified cell, or an enzymatically or chemically produced molecule.
  • a genetically engineered cell according to the present disclosure comprises a recombinant nucleic acid sequence encoding an a-1 ,2-fucosyltransferase variant of the present disclosure capable of transferring fucose from an activated sugar to the Gal moiety of an acceptor molecule or acceptor saccharide, such as a disaccharide or oligosaccharide, such as lactose, in an a-1 ,2 linkage while possessing very limited or no a-1 ,3-fucosyltransferase activity.
  • the limited a-1 ,3-fucosyltransferase activity result in very limited fucosylation of the glucose moiety in the acceptor molecule.
  • the a-1 ,2-fucosyltransferase variant of the present disclosure may also be capable of transferring a fucosyl moiety to a terminal Gal moiety, of more complex acceptor oligosaccharides, such as LNT or LNnT.
  • the genetically modified cell may be further engineered to produce the initial substrate, such as lactose, inside the cell (see for example WO2015/150328).
  • a glycosyltransferase mediated glycosylation reaction takes place in which an activated sugar nucleotide serves as glycosyl- donor.
  • An activated sugar nucleotide generally has a phosphorylated glycosyl residue attached to a nucleoside.
  • a specific glycosyl transferase enzyme accepts only a specific sugar nucleotide.
  • activated sugar nucleotides are involved in the glycosyl transfer: glucose-UDP-GIcNAc, UDP-galactose, UDP-glucose, UDP-N- acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc) and CMP-N- acetylneuraminic acid (CMP-Neu5Ac).
  • the genetically engineered cell according to the present disclosure can comprise one or more pathways to produce a nucleotide-activated sugar selected from the group consisting of glucose-UDP-GIcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and CMP-N-acetylneuraminic acid.
  • the genetically engineered cell is capable of producing one or more activated sugar nucleotides mentioned above by a de novo pathway.
  • an activated sugar nucleotide is made by the cell under the action of enzymes involved in the de novo biosynthetic pathway of that respective sugar nucleotide in a stepwise reaction sequence starting from a simple carbon source like glycerol, sucrose, fructose or glucose (for a review for monosaccharide metabolism see e.g. H. H. Freeze and A. D. Elbein: Chapter 4: Glycosylation precursors, in: Essentials of Glycobiology, 2nd edition (Eds. A. Varki et al.), Cold Spring Harbour Laboratory Press (2009)).
  • the enzymes involved in the de novo biosynthetic pathway of an activated sugar nucleotide can be naturally present in the cell or introduced into the cell by means of gene technology or recombinant DNA techniques, all of them are parts of the general knowledge of the skilled person.
  • the genetically engineered cell can utilize salvaged monosaccharides for sugar nucleotide.
  • monosaccharides derived from degraded oligosaccharides are phosphorylated by kinases and converted to nucleotide sugars by pyrophosphorylases.
  • the enzymes involved in the procedure can be heterologous ones, or native ones of the host cell.
  • the colanic acid gene cluster of Escherichia coll encodes selected enzymes involved in the de novo synthesis of GDP-fucose gmd, wcaG, wcaH, weal, manB, manC), whereas one or several of the genes downstream of GDP-L-fucose such as wcaJ, which are responsible for the production of the extracellular polysaccharide colanic acid, a major oligosaccharide of the bacterial cell wall, can be deleted to prevent conversion of GDP-fucose to colanic acid.
  • the promoter of the native colanic acid gene cluster may be exchanged with a stronger promoter, generating a recombinant colanic acid gene cluster, to drive additional production of GDP-fucose.
  • an extra copy of the colanic acid gene cluster or selected genes thereof can be introduced in the genetically engineered cells as described in the examples.
  • the colanic acid gene cluster may be expressed from its native genomic locus.
  • the expression may be actively modulated.
  • the expression can be modulated by swapping the native promoter with a promoter of interest, and/or increasing the copy number of the colanic acid genes coding said protein(s) by expressing the gene cluster from another genomic locus than the native, or episomally expressing the colanic acid gene cluster or specific genes thereof.
  • the term “native genomic locus”, in relation to the colanic acid gene cluster, relates to the original and natural position of the gene cluster in the genome of the genetically engineered cell.
  • the de novo GDP-fucose pathway genes responsible for the formation of GDP-fucose comprises or consists of the following genes: i) manA which encodes the protein mannose-6 phosphate isomerase (EC 5.3.1 .8, UniProt accession nr. P00946), which facilitates the interconversion of fructose 6- phosphate (F6P) and mannose-6-phosphate; ii) manB which encodes the protein phosphomannomutase (EC 5.4.2.8, UniProt accession nr P24175), which is involved in the biosynthesis of GDP-mannose by catalyzing conversion mannose-6-phosphate into mannose-1 -phosphate;
  • ManC which encodes the protein mannose-1 -phosphate guanylyltransferase guanylyltransferase (EC:2.7.7.13, UniProt accession nr P24174), which is involved in the biosynthesis of GDP-mannose through synthesis of GDP-mannose from GTP and a-D-mannose-1-phosphate;
  • gmd which encodes the protein GDP-mannose-4,6-dehydratase (UniProt accession nr P0AC88), which catalyzes the conversion of GDP-mannose to GDP-4-dehydro-6- deoxy-D-mannose;
  • v) wcaG (fcl) which encodes the protein GDP-L-fucose synthase (EC 1 .1 .1 .271 , UniProt accession nr P32055) which catalyses the two-step NADP-dependent conversion of GDP-4-dehydro-6-deoxy-D-mannose to GDP-fuco
  • the genetically engineered cell when producing one or more fucosylated heterologous products, overexpresses either the entire colonic acid gene cluster and/or one or more individual genes of the de novo GDP-fucose pathway selected from the group consisting of manA, manB, manC, gmd and wcaG.
  • Lactose permease is a membrane protein which is a member of the major facilitator superfamily and can be classified as a symporter, which uses the proton gradient towards the cell to transport p-galactosides such as lactose in the same direction into the cell.
  • lactose is often the initial substrate being decorated to produce any HMO of interest in a bioconversion that happens in the cell interior.
  • HMOs human milk oligosaccharides
  • the lactose permease is as shown in SEQ ID NO: 3, or a functional homologue thereof having an amino acid sequence which is at least 80 % identical, such as at least 85 %, such as at least 90%, or such as at least 95% identical to SEQ ID NO: 3.
  • the expression of the lactose permease is regulated by a promoter according to the present disclosure.
  • a host cell suitable for HMO production may comprise an endogenous
  • the genetically engineered cell does not express a functional p-galactosidase to avoid the degradation of lactose, if lactose is used as the initial substrate for producing the complex fucosylated HMO.
  • the gene may be inactivated by a complete or partial deletion of the corresponding nucleic acid sequence from the bacterial genome, or the gene sequence is mutated in the way that it is not transcribed, or, if transcribed, the transcript is not translated or if translated to a protein (i.e., p-galactosidase), the protein does not have the corresponding enzymatic activity.
  • a protein i.e., p-galactosidase
  • lactose permeases are described above.
  • LNT-II and LNnT importers are described in WO 2023/099680 and includes for example,
  • Lactose permease (LacY) mutants such as LacY mutant Y236H or LacY mutant A177V+S306T, wherein the mutations are equivalent with the corresponding position in the sequence of SEQ ID NO: 3,
  • ABC transporter protein complexes such as ABC transporter from B. pseudocatenulatum JCM 1200 BBPC_1775, 1776, 1777, (NCBI accession Nrs BAR04453.1 , BAR04454.1 and BAR04455.1 , respectively) or ABC transporter from B. breve UCC2003 BBR_0527/lntP1 , BBR_0528/lntP2, BBR_0530/lntS and BBR_0531 (NCBI accession Nrs ABE95224.1 , ABE95225.1 , ABE95226.1 and ABE95228.1), and/or MFS transporters, such as but not limited to Blon_0962 (NCBI accession Nr ACJ52061.1).
  • a nucleic acid, or a cluster of nucleic acids encoding one, or more of these transporters may be introduced into a genetically modified cell as described herein.
  • the expression of such transporters enables the production of complex fucosylated oligosaccharide with LNT-II as the initial substrate.
  • the oligosaccharide product such as the HMO produced by the cell
  • the product can be transported to the supernatant in a passive way, i.e., it diffuses outside across the cell membrane.
  • the more complex HMO products may remain in the cell, which is likely to eventually impair cellular growth, thereby affecting the possible total yield of the product from a single fermentation.
  • the HMO transport can be facilitated by major facilitator superfamily transporter proteins that promote the effluence of sugar derivatives from the cell to the supernatant.
  • the transporter can be present exogenously or endogenously and can be overexpressed under the conditions of the fermentation to enhance the export of the oligosaccharide produced by the genetically modified cell.
  • the genetically engineered cell according to the present disclosure can further comprise a nucleic acid sequence encoding a transporter protein capable of exporting the fucosylated human milk oligosaccharide product, in particular 2’FL, such a transporter protein can for example be a member of the major facilitator superfamily transport proteins.
  • the genetically engineered cell of the present disclosure preferably expresses a heterologous Major Facilitator Superfamily (MFS) transporter protein.
  • MFS Facilitator Superfamily
  • MFS transporter in the present context means a protein that facilitates transport of an oligosaccharide, preferably an HMO, through or across a cell membrane, from the cell cytosol to the cell periplasm and/or medium.
  • the genetically engineered cell comprises an MFS transporter protein from Rosenbergiella nectarea with an amino acid sequence corresponding the GenBank accession WP_092672081 .1 , or a functional homologue thereof, having an amino acid sequence which is at least 80 %, such as at least 90 %, such as at least 95 %, such as at least 99 %, such as 100% identical to the amino acid sequence of GenBank accession WP_092672081.1 or SEQ ID NO: 4.
  • the MFS transporter is also disclosed in WO2021/148615
  • the genetically engineered cell comprises an sugar efflux transporter with an amino acid sequence corresponding the GenBank accession WP_000637847, or a functional homologue thereof, having an amino acid sequence which is at least 80 %, such as at least 90 %, such as at least 95 %, such as at least 99 %, such as 100% identical to the amino acid sequence of GenBank accession WP_000637847.
  • the sugar efflux transporter is also disclosed in WO2010/142305.
  • a genetically engineered cell and "a genetically modified cell” are used interchangeably.
  • a genetically engineered cell is a host cell whose genetic material has been altered by human intervention using a genetic engineering technique, such a technique is e.g., but not limited to transformation or transfection e.g., with a heterologous and/or recombinant polynucleotide sequence, Crisper/Cas editing and/or random mutagenesis.
  • the genetically engineered cell has been transformed or transfected with a recombinant nucleic acid sequence.
  • the genetically engineered cell according to the present disclosure comprises at least one recombinant nucleic acid sequence encoding a fucosyltransferase variant of the present disclosure, capable of transferring a fucosyl residue from a fucosyl donor to an acceptor saccharide to synthesize one or more fucosylated oligosaccharide products, such as human milk oligosaccharide (HMO) products.
  • HMO human milk oligosaccharide
  • the genetically engineered cell may comprise further modifications that facilitate the production of the desired fucosylated oligosaccharide.
  • modifications can be selected from inclusion of glycosyltransferases, and/or metabolic pathway engineering, deletion of repressors or undesired enzymes and inclusion of transporters as described in the above sections, which the skilled person will know how to combine into a genetically engineered cell that is capable of producing a fucosylated oligosaccharide, such as 2’FL or LNFP-I or LNFP-IV.
  • the 2’FL is the primary product produced by the genetically engineered cell.
  • the genetically engineered cell capable of producing a fucosylated oligosaccharide comprises a a-1 , 2- fucosyltransferase variant as described herein, with an amino acid sequence that is at least 80 %, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 98.5%, or such as at least 99% identical to SEQ ID NO: 1.
  • the a-1 ,2-fucosyltransferase variant disclosed herein is capable of fucosylating lactose at the Gal moiety with an a-1 , 2 linkage.
  • the a-1 , 2- fucosyltransferase variant disclosed herein is fucosylates lactose at the Gal moiety with an a-1 ,2 linkage.
  • the a-1 ,2-fucosyltransferase variant is selected from the group of variants disclosed in table 1.
  • Table 1 Preferred a-1 ,2-fucosyltransferase variants of the present disclosure
  • the position indicated in Table 1 corresponds to the position in the amino acid sequence of SEQ ID NO:1 and the variant in table 1 has at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 98.5% or such as at least 99% sequence identity to SEQ ID NO:1 or FutC from Helicobacter pylori ATCC 26695 with NCBI accession nr WP_080473865.1 (SEQ ID NO: 6) or NCBI accession WP_128060420.1 (SEQ ID NO: 47) or WP_130778061.1 (SEQ ID NO: 46).
  • sequence of a specific variant in table 1 corresponds to the sequence of SEQ ID NO: 1 or FutC from Helicobacter pylori ATCC 26695 with NCBI accession nr WPJ380473865.1 with the only difference being the amino acid substitutions indicated for the specific variant.
  • the genetically engineered cell described herein preferably expresses genes encoding key enzymes for the biosynthesis of fucosylated HMOs.
  • a genetically engineered cell disclosed herein further expresses the de novo GDP-fucose pathway genes responsible for the formation of GDP-fucose manA, manB, manC, gmd and wcaG. It may be advantageous to overexpress one or more of these genes and/or to upregulate the colanic acid gene cluster (CA), including the genes gmd, wcaG, wcaH, weal, manC and manB from E.
  • CA colanic acid gene cluster
  • CA colanic acid gene cluster
  • one or more additional glycosyltransferases and pathways for producing nucleotide-activated sugars such as glucose-UDP-GIcNAc, CMP-N-acetylneuraminic acid, UDP-galactose, UDP-glucose, UDP-N- acetylglucosamine, UDP-N-acetylgalactosamine and/or CMP-N-acetylneuraminic acid can also be present in the genetically engineered cell.
  • the engineered cell is a microorganism.
  • the genetically engineered cell is preferably a microbial cell, such as a cell of eucaryotic or prokaryotic origin.
  • Appropriate microbial cells that may function as a host cell include yeast cells, bacterial cells, archaebacterial cells, algae cells, and fungal cells.
  • the genetically engineered cell may be e.g., a bacterial or yeast cell.
  • the genetically engineered cell may be a prokaryotic cell e.g., a bacterial cell.
  • the bacterial host cells there are, in principle, no limitations; they may be eubacteria (gram-positive or gram-negative) or archaebacteria, as long as they allow genetic manipulation for insertion of a gene of interest and can be cultivated on a manufacturing scale.
  • the host cell has the property to allow cultivation to high cell densities.
  • suitable bacterial host cells are Escherichia coll, Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Campylobacter sp, Corynebacterium sp., Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris.
  • Bacteria of the genus Bacillus may also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans.
  • bacteria of the genera Lactobacillus and Lactococcus may be used, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus easel, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis.
  • Streptococcus thermophiles Streptomyces lividans, and Proprionibacterium freudenreichii are also suitable bacterial species for the present invention.
  • strains from the genera Enterococcus e.g., Enterococcus faecium and Enterococcus thermophiles
  • Bifidobacterium e.g., Bifidobacterium long urn, Bifidobacterium infantis, and Bifidobacterium bifidum
  • Sporolactobacillus spp. Micromomospora spp., Micrococcus spp., Rhodococcus spp.
  • Pseudomonas e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa.
  • suitable gram-negative host cells are host cell are Bdellovibrio sp., Campylobacter sp., Citrobacter sp. (such as Citrobacter freundii), Erwinia sp. (such as Erwinia herbicola) Haemophilus sp, Helicobacter sp., Legionella sp, Moraxella sp, Neisseria sp, Pantoea sp. (such as Pantoea agglomerans), Pantoea sp. (such as Pantoea citrea), Pectobacterium sp.
  • the genetically engineered cell is selected from the group consisting of Escherichia sp., Bacillus sp., Lactobacillus sp and Corynebacterium sp. Campylobacter sp.
  • the genetically engineered cell is selected from the group consisting of E. coll, C. glutamicum, L. lactis, B. subtilis, S. lividans.
  • the genetically engineered cell is Bacillus subtilis.
  • the genetically engineered cell is Corynebacterium glutamicum.
  • the genetically engineered cell is gram-negative bacteria.
  • the genetically engineered cell is Escherichia coli.
  • the disclosure relates to a genetically engineered cell, wherein the cell is derived from the E. coli K-12 strain or DE3.
  • the microbial cell is an eucaryotic cell such as a yeast or a filamentous fungus.
  • the genetically engineered cell may be a yeast cell such as Komagataella phaffii, Kluyveromyces lactis, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.
  • the genetically engineered cell may be a filamentous fungus such as Aspargillus sp, Fusarium sp or Thricoderma sp, exemplary species are A. niger, A. nidulans, A. oryzae, F. solani, F. graminearum and T. reesei.
  • the genetically engineered cell is selected from the group consisting of Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.
  • the genetically engineered cell is Pichia pastoris.
  • the genetically engineered cell is Yarrowia lipolytica.
  • the present disclosure also relates a recombinant nucleic acid sequence encoding an a-1 ,2- fucosyltransferase variant as disclosed herein.
  • nucleic acid sequence “recombinant gene/nucleic acid/nucleotide sequence/DNA encoding” or “coding nucleic acid sequence” is used interchangeably and intended to mean an artificial nucleic acid sequence (i.e. produced in vitro using standard laboratory methods for making nucleic acid sequences) that comprises a set of consecutive, non-overlapping triplets (codons) which is transcribed into mRNA and translated into a protein when under the control of the appropriate control sequences, i.e., a promoter sequence.
  • the boundaries of the coding sequence are generally determined by a ribosome binding site located just upstream of the open reading frame at the 5’end of the mRNA, a transcriptional start codon (AUG, GUG or UUG), and a translational stop codon (UAA, UGA or UAG).
  • a coding sequence can include, but is not limited to, genomic DNA, cDNA, synthetic, and recombinant nucleic acid sequences.
  • nucleic acid includes RNA, DNA and cDNA molecules. It is understood that, as a result of the degeneracy of the genetic code, a multitude of nucleic acid sequences encoding a given protein may be produced.
  • the recombinant nucleic acid sequence encoding a variant alpha-1 ,2-fucosyltrantsferase of the present disclosure is a non-natural sequence that does not occur in nature.
  • Non-natural sequences are also considered to be heterologous to the host cell they are introduced into.
  • heterologous refers to a polypeptide, amino acid sequence, nucleic acid sequence or nucleotide sequence that is foreign to a cell or organism, i.e., to a polypeptide, amino acid sequence, nucleic acid molecule or nucleotide sequence that does not naturally occurs in said cell or organism.
  • the disclosure also relates to a nucleic acid construct comprising a coding nucleic sequence, i.e. recombinant DNA sequence of an a-1 ,2-fucosyltransferase variant disclosed herein.
  • the nucleic acid construct may furthermore comprise a non-coding regulatory DNA sequence, e.g., a promoter DNA sequence, e.g., a recombinant promoter sequence derived from the promoter sequence of the lac operon or the glp operon, or a promoter sequence derived from another genomic promoter DNA sequence, or a synthetic promoter sequence, wherein the coding and promoter sequences are operably linked.
  • operably linked refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. It refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
  • a promoter sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
  • the nucleic acid construct of the disclosure may be a part of the vector DNA, such as a plasmid for insertion into a host cell.
  • the nucleic acid construct it is an expression cassette/cartridge that is integrated in the genome of a host cell.
  • nucleic acid construct means an artificially constructed segment of nucleic acids, in particular a DNA segment, which is intended to be inserted into a target cell, e.g., a bacterial cell, to modify expression of a gene of the genome or expression of a gene/coding DNA sequence which may be included in the construct.
  • An embodiment of the present disclosure is a nucleic acid construct comprising a recombinant nucleic acid sequence encoding an a-1 ,2-fucosyltransferase variant of the present disclosure.
  • the nucleic acid construct is an expression cassette/cartridge that is integrated in the genome of a host cell allowing expression of the recombinant nucleic acid sequence, preferably regulated by a promoter sequence which is also present in the nucleic acid construct.
  • the genetically engineered cell according to the present disclosure may also comprise multiple copies of the recombinant nucleic acid sequence encoding an a-1 ,2-fucosyltransferase variant of the present disclosure. Enhancing the copy number of the a-1 ,2-fucosyltransferase variant may be used to further enhance the 2’FL production.
  • the genetically engineered cell disclosed herein comprises one, two, three or more genomic copies of the recombinant nucleic acid sequence encoding an a- 1 ,2-fucosyltransferase variant disclosed herein, comprising or consisting of a variant of table 1 , more preferably a variant of table 4.
  • said recombinant nucleic acid sequence is encoded on a plasmid.
  • the plasmid is a high copy number plasmid, preferably, a pUC57 or pBB-B9 plasmid.
  • the a-1 ,2-fucosyltransferase variant encoding sequence is under the control of a promoter sequence selected from promotor sequences with a nucleic acid sequence as identified in Table 2.
  • the promoter may be of heterologous origin, native to the genetically engineered cell or it may be a recombinant promoter, combining heterologous and/or native elements.
  • One way to increase the production of a product may be to regulate the production of the desired enzyme activity used to produce the product, such as the glycosyltransferases or enzymes involved in the biosynthetic pathway of the glycosyl donor.
  • Increasing the promoter strength driving the expression of the desired enzyme may be one way of doing this.
  • the strength of a promoter can be assessed using a lacZ enzyme assay where
  • a strong regulatory element is the PglpF promoter with an activity of approximately 14.000 MU and an example of a weak promoter is Plac which when induced with IPTG has an activity of approximately 2300 MU.
  • the expression of said nucleic acid sequences are under control of a strong promoter selected from the group consisting of SEQ ID NOs 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18.
  • the expression of said nucleic acid sequences disclosed herein is under control of a PglpF (SEQ ID NO: 19) or Plac (SEQ ID NO: 28) promoter or PmglB_UTR70 (SEQ ID NO: 16) or PglpA_70UTR (SEQ ID NO: 17) or PglpT_70UTR (SEQ ID NO: 18) or variants thereof such as promoters identified in Table 3, in particular the PglpF_SD4 variant of SEQ ID NO: 14 or Plac_70UTR variant of SEQ ID NO: 10, or PmglB_70UTR variants of SEQ ID NO: 7, 8, 11 , 12, 13, 15 and 16.
  • PglpF, PglpA_70UTR, PglpT_70UTR and PmglB_70UTR promoter sequences are described in or WO2019/123324 and W02020/255054 respectively (hereby incorporated by reference).
  • the recombinant nucleic acid sequences individually are under the control of one or more promoters selected from the group consisting of PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 19, 28, 16, 17 and 18, respectively) and variants thereof.
  • nucleic acid construct of interest comprised in the construct (expression cassette) into the bacterial genome
  • introduction of the nucleic acid construct of interest comprised in the construct (expression cassette) into the bacterial genome can be achieved by conventional methods, e.g. by using linear cartridges that contain flanking sequences homologous to a specific site on the chromosome, as described for the attTn7-site (Waddell C.S. and Craig N.L, Genes Dev. (1988) Feb;2(2):137-49.
  • methods for genomic integration of nucleic acid sequences in which recombination is mediated by the Red recombinase function of the phage A or the RecE/RecT recombinase function of the Rac prophage (Murphy, J Bacteriol.
  • sequence identity describes the relatedness between two amino acid sequences or between two nucleotide sequences, i.e., a candidate sequence (e.g., a sequence of the disclosure) and a reference sequence (such as a prior art sequence) based on their pairwise alignment.
  • sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.
  • sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1 970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), 10 preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, -endopen 10.0, -endextend 0.5 and the DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labelled " identity" (obtained using the -nobrief option) is used as the percent identity.
  • sequence identity may be calculated as follows: (Identical nucleotide residues x 100)/(aligned region).
  • a functional alpha- 1 ,2-fucosyltransferase variant or functional homologue of a protein/nucleic acid sequence as described herein is a protein/nucleic acid sequence with alterations in the genetic code, which retain its ability to fucosylate an acceptor molecule with the fucose in an alpha-1 ,2 configuration (functionality).
  • a functional variant is a non-natural variant, for example obtained by mutagenesis.
  • the functional variant should have a remaining functionality in terms of 2’FL formation of at least 30%, such as at least 40%, 50%, 50% 70%, 80 %, 90% or 100% compared to the functionality of the protein/nucleic acid sequence of SEQ ID NO: 1.
  • a functional alpha- 1 ,2-fucosyltransferase variant of any one of the disclosed variants can also have a higher functionality.
  • a functional variant as shown in table 1 with an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 , should be able to produce 2’FL to the same level as the parent alpha-1 ,2-fucosyltransferase, such as the alpha-1 ,2-fucosyltransferase of SEQ ID NO: 1 .
  • a functional variant of the present disclosure may in particular contribute to a decreased by-product formation, such as DFL and 3FL formation compared to the alpha-1 ,2- fucosyltransferase of SEQ ID NO: 1.
  • a functional alpha- 1 ,2-fucosyltransferase variant also contribute with an increased 2’FL yield, reduction in biomass formation, viability of the genetically engineered cell, robustness of the genetically engineered cell according to the disclosure, and/or reduction in consumables needed for the production.
  • the disclosure also relates to any commercial use of the enzyme variants, genetically engineered cells or the nucleic acid constructs disclosed herein, such as, but not limited to, in a method for producing one or more fucosylated human milk oligosaccharide (HMO), preferably, 2’FL.
  • HMO fucosylated human milk oligosaccharide
  • the present disclosure also relates to the use of an a-1 ,2-fucosyltransferase variant in production of a fucosylated product, wherein the a-1 ,2-fucosyltransferase variant is selected from table 1 , comprising or consisting of an amino acid sequence that is at least 85% identical, such as at least 90% identical, such as at least 95% identical, such as at least 97% identical to SEQ ID NO: 1.
  • the fucosylated product is 2’FL.
  • the a-1 ,2-fucosyltransferase variant disclosed herein is also used in the manufacturing of a fucosylated product, wherein the fucosylated product is one or more fucosylated oligosaccharides, such as one or more HMOs, preferably, 2’FL, most preferred 2’FL constitute at least 98%, such as at least 99%, such as at least 99.5% of the fucosylated oligosaccharides produced using the a-1 ,2-fucosyltransferase variant disclosed herein.
  • the fucosylated product is one or more fucosylated oligosaccharides, such as one or more HMOs, preferably, 2’FL, most preferred 2’FL constitute at least 98%, such as at least 99%, such as at least 99.5% of the fucosylated oligosaccharides produced using the a-1 ,2-fucosyltransferase variant disclosed herein.
  • the genetically engineered cell and/or the nucleic acid construct described herein is used in the manufacturing of HMOs.
  • the manufacturing of 2’FL in the manufacturing of 2’FL.
  • 2’FL in the manufacturing of 2’FL where 2’FL constitute at least 98%, such as at least 99%, such as at least 99.5% of the fucosylated oligosaccharides produced at the end of fermentation (before any subsequent purification).
  • the a-1 ,2-fucosyltransferase variants disclosed herein are also used in the manufacturing of a fucosylated product, wherein the fucosylated product is a fucosylated oligosaccharide of 5, 6 or 7 monosaccharide units, such as LNFP-I, LNFP-IV, LNDFH-I or FLSTb. Production of these HMO’s may require the presence of two or more glycosyltransferase activities.
  • HMOs fucosylated human milk oligosaccharides
  • the present disclosure also relates to a method for producing one or more fucosylated human milk oligosaccharide (HMO), preferably 2’FL.
  • HMO fucosylated human milk oligosaccharide
  • said method is an in vitro production of at least one fucosylated HMO, such as 2’FL, encompassing addition of a purified a-1 ,2-fucosyltransferase variant to an acceptor molecule (e.g., lactose) and a fucose donor molecule in a suitable reaction medium.
  • a-1 ,2- fucosyltransferase variant can be expressed and purified from known cellular production hosts, including those described in the section “host cells”.
  • said method comprises an in vivo production of at least one fucosylated HMO, such as 2’FL, encompassing culturing a genetically engineered cell encoding/expressing an a-1 ,2-fucosyltransferase variant of the present disclosure under conditions that allows the genetically modified cell to produce 2’FL.
  • at least one fucosylated HMO such as 2’FL
  • An aspect of the present disclosure is a method for producing at least one fucosylated HMO, such as 2’FL, wherein said method comprises the following steps: a. providing a genetically engineered cell a disclosed herein; b. culturing the genetically engineered cell of a. in a suitable cell culture medium to produce said fucosylated HMO; and optionally c. purifying said fucosylated HMO.
  • the method provided herein involves cultivating a genetically modified cell, wherein said method significantly optimizes the production of 2’FL, while concurrently minimizing the generation of the by-product DFL.
  • This shift towards a preference for producing 2’FL instead of by-product can increase the yield of the desired 2’FL, and most interesting ensures a purer form of the product, as the occurrence of DFL is markedly decreased.
  • the present disclosure thus relates to a method for producing one or more fucosylated human milk oligosaccharide (HMO), said method comprising culturing a genetically engineered cell, said cell comprising a recombinant nucleic acid sequence encoding an a-1 ,2-fucosyltransferase variant as disclosed herein, in particular a variant as disclosed in table 1 comprising or consisting of an amino acid sequence that is at least 85% identical, such as at least 90% identical, such as at least 95% identical, such as at least 97% identical to SEQ ID NO: 1 , wherein said method produces a fucosylated HMO, such as at least one fucosylated HMO, preferably 2’FL.
  • HMO fucosylated human milk oligosaccharide
  • At least 95 wt%, such as at least 96 wt%, such as at least 97 wt%, such as at least 98 wt%, such as at least 99 wt%, such as at least 99.5 wt% of the total amount of oligosaccharide produced in the cultivation step of said method is 2’FL.
  • less than 2%, such as less than 1 .5%, such as less than 1%, such as less than 0.5%, such as less than 0.2%, such as 0% of the total oligosaccharide products produced by the cell is DFL.
  • HMOs such as 3FL and/ or tri-fucosylated lactose (trifucosyllactose) are produced, i.e. 2’FL and DFL are the sole HMOs produced by the method according to the present invention.
  • the molar ratio of 2’FL to DFL produced by the genetically engineered cell comprising a recombinant nucleic acid sequence encoding an a-1 ,2-fucosyltransferase variant as disclosed herein is more than 65:1 , preferably more than 100:1 , even more preferably more than 150:1 , such as most preferably more than 200:1. Further preferred ratios encompass more than 300:1 , 400:1 , 500:1 , 600:1 , 700:1 , 800:1 or such as more than 900:1 .
  • the molar ratio of 2’FL:DFL is more than 900: 1 .
  • the genetically engineered cell is cultured in a suitable medium providing a suitable carbon source, and in the presence of lactose as the initial substrate.
  • one or more fucosylated HMOs selected form the groups consisting of 2’FL, LNFP-I and LNDFH-I, and potentially with LNT, DFL, LNT-II and pLNH2 as by-products are produced by the method of the disclosure.
  • the HMOs produced are free of by-products, in particular fucosylated by-products, such as for the production of 2’FL it is highly beneficial that no 3FL or DFL is produced. In addition, it is also beneficial that no tri-fucosylated lactose (trifucosyllactose) it produced.
  • less than 2% such as less than 1%, such as less the 0.5%, such as less than 0.2% DFL and/or 3FL of the total amount of HMO produced, is produced by said method.
  • essentially no DFL is produced by said method.
  • essentially no 3FL is produced by said method.
  • the molar ratio of 2’FL:DFL is more than 65:1 , such as more than 100:1 , 200:1 , 300:1 , 400:1 , 500:1 , 600:1 , 700:1 , 800:1 or such as more than 900:1 .
  • the molar ratio of 2’FL:DFL is more than 900:1.
  • the genetically engineered cell used in the method for producing a fucosylated HMO may contain additional modifications as described herein.
  • the method disclosed herein comprises providing a glycosyl donor, which is synthesized separately by one or more genetically engineered cells and/or is exogenously added to the culture medium from an alternative source.
  • the glucosyl donor is produced by an endogenous or recombinant de novo pathway in the genetically engineered cell.
  • One embodiment disclosed herein further comprises providing an acceptor saccharide as substrate for the HMO formation, the acceptor saccharide comprising at least two monosaccharide units, which is exogenously added to the culture medium and/or has been produced by a separate microbial fermentation or is produced by the genetically modified cell.
  • the method disclosed herein comprises providing an acceptor saccharide comprising at least two monosaccharide units, which is exogenously added to the culture medium and/or has been produced by a separate microbial fermentation and which is selected form lactose, LNT-II and LNnT.
  • the substrate for HMO formation is lactose which is fed to the culture during the fermentation of the genetically engineered cell.
  • the fucosylated human milk oligosaccharide is retrieved from the culture, either from the culture medium and/or the genetically engineered cell.
  • Culturing, cultivation, fermenting or fermentation (used interchangeably herein) in a controlled bioreactor typically comprises (a) a first phase of exponential cell growth in a culture medium ensured by a carbon-source, and (b) a second phase of cell growth in a culture medium run under carbon limitation, where the carbon-source is added continuously together with the acceptor oligosaccharide, such as lactose, allowing formation of the HMO product in this phase.
  • carbon (sugar) limitation is meant the stage in the fermentation where the growth rate is kinetically controlled by the concentration of the carbon source (sugar) in the culture broth, which in turn is determined by the rate of carbon addition (sugar feed-rate) to the fermenter.
  • culture broth is a common term describing the mixture of culture medium, carbon source and the cells being fermented.
  • a “manufacturing” or “manufacturing scale” or “large-scale production” or “large-scale fermentation”, are used interchangeably and in the meaning of the disclosure defines a fermentation with a minimum volume of 100 L, such as WOOL, such as 10.000L, such as 100.000L, such as 200.000L culture broth.
  • a “manufacturing scale” process is defined by being capable of processing large volumes yielding amounts of the HMO product of interest that meet, e.g., in the case of a therapeutic compound or composition, the demands for toxicity tests, clinical trials as well as for market supply.
  • a manufacturing scale method is characterized by the use of the technical system of a bioreactor (fermenter) which is equipped with devices for agitation, aeration, nutrient feeding, monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.).
  • a bioreactor which is equipped with devices for agitation, aeration, nutrient feeding, monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.).
  • process parameters pH, temperature, dissolved oxygen tension, back pressure, etc.
  • the culture medium may be semi-defined, i.e., containing complex media compounds (e.g., yeast extract, soy peptone, casamino acids, etc.), or it may be chemically defined, without any complex compounds.
  • the carbon-source can for example be selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.
  • the culturing media is supplemented with one or more energy and carbon sources selected form the group containing glycerol, sucrose and glucose.
  • lactose is added during the cultivation of the genetically engineered cells as a substrate for the HMO formation.
  • the culturing media contains sucrose as the sole carbon and energy source.
  • the genetically engineered cell comprises one or more heterologous nucleic acid sequence encoding one or more heterologous polypeptide(s) which enables utilization of sucrose as sole carbon and energy source of said genetically engineered cell.
  • the genetically engineered cell comprises a PTS- dependent sucrose utilization system, further comprising the scrYA and scrBR operons as described in WO2015/197082 (hereby incorporated by reference).
  • the proteins encoded by the two operons are the sucrose-specific porin (scrY) represented by SEQ ID NO: 31 or a functional homologue thereof, sucrose transport pretein enzyme II (ScrA) represented by SEQ ID NO: 32 or a functional homologue thereof, scrB invertase enzyme (ScrB) represented by SEQ ID NO: 33 or a functional homologue thereof and Scr repressor protein (ScrR) represented by SEQ ID NO: 34 or a functional homologue thereof.
  • the expression of the scrYA and/or scrBR operons is/are under control of a Pscr promoter (SEQ ID NO: 6)
  • the fucosylated HMO produced can be collected from the cell culture or fermentation broth in a conventional manner.
  • the fucosylated human milk oligosaccharide is retrieved from the culture medium and/or the genetically engineered cell.
  • the term “retrieving” is used interchangeably with the term “harvesting”. Both “retrieving” and “harvesting” in the context relate to collecting the produced HMO(s) from the culture/ fermentation broth following the termination of fermentation. In one or more exemplary embodiments it may include collecting the HMO(s) included in both the biomass (i.e., the host cells) and cultivation media, i.e., before/without separation of the liquid in the fermentation broth from the biomass. In other embodiments, the produced HMOs may be collected separately from the biomass and the liquid part of the fermentation broth, i.e., after/following the separation of biomass from cultivation medium (supernatant).
  • the separation of cells from the medium can be carried out with any of the methods well known to the skilled person in the art, such as any suitable type of centrifugation or filtration.
  • the separation of cells from the medium can follow immediately after harvesting the fermentation broth or be carried out at a later stage after storing the fermentation broth at appropriate conditions.
  • Recovery of the produced HMO(s) from the remaining biomass (or total fermentation broth) include extraction thereof from the biomass (i.e., the production cells).
  • HMO(s) After recovery from fermentation, HMO(s) are available for further processing and purification.
  • the HMOs can be purified according to the procedures known in the art, e.g., such as described in WO2017/152918, WO2017/182965 or WO2015/188834, wherein the latter describes purification of fucosylated HMOs.
  • the purified HMOs can be used as nutraceuticals, pharmaceuticals, or for any other purpose, e.g., for research.
  • product refers to the one or more fucosylated HMOs, in particular 2’FL or LNFP-I, intended as the one or more product HMO(s).
  • product HMO or composition is produced by a method described herein using a genetically engineered cell described herein.
  • the methods disclosed herein utilizing selected a-1 ,2-fucosyltransferase variants disclosed herein, provide an increased overall yield of the product, with only limited byproduct formation.
  • This, reduced by-product formation in relation to product formation facilitates an elevated product production and increases efficiency of both the production and product recovery process, providing superior manufacturing procedure of HMOs.
  • the manufactured product may be a powder, a composition, a suspension, or a gel comprising one or more HMOs.
  • An aspect of the present disclosure relates to the use of the 2’FL and/or LNFP-I disclosed herein in infant nutrition.
  • the present disclosure also relates to the use of the 2’FL and/or LNFP-I disclosed herein as a dietary supplement or medical nutrition or a pharmaceutical composition.
  • alpha-1 ,2-fucosyltransferase variant according to any one of items 1 to 5, wherein the variant comprises or consists of a substitution of the amino acid C in position 177 to the amino acid Q,H, S or F, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 , preferably this is the only substitution of substitutions listed in items 1 to 3.
  • the variant comprises or consists of the following substitution L273R, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 , preferably this is the only substitution of substitutions listed in items 1 to 3.
  • alpha-1 , 2-fucosyltransferase variant according to any one of the preceding items, wherein the variant does not comprise the following two substitutions C177Q and L273R together.
  • alpha-1 , 2-fucosyltransferase variant according to any one of the preceding items, wherein the variant has reduced alpha-1 ,3-fucosyltransferase activity compared to the alpha-1 ,3-fucosyltransferase activity of the parent alpha-1 ,2-fucosyltransferase.
  • alpha-1 , 2-fucosyltransferase variant according to any one of the preceding items, wherein the variant originates from a parent alpha- 1 , 2-fucosyltransferase from a Helicobacter pylori strain.
  • alpha-1 , 2-fucosyltransferase variant according to any one of the preceding items, wherein the variant further comprises a substitution in one or more positions selected from the group consisting of position 40, 80, 110, 121 , 124, 125, 150, 151 , 239 and 297, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .
  • G10A, F40D G10A, F40D, C177Q;
  • alpha-1 ,2-fucosyltransferase variant according to any one of items 1 to 21 , wherein the variant further comprises at least the following substitutions C151 R and Q239S, wherein the position corresponds to the position in the amino acid sequence of SEQ ID
  • alpha-1 , 2-fucosyltransferase variant according to any one of the preceding items, wherein the variant further comprises the substitutions L80, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .
  • 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 36.
  • 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 37.
  • 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 38.
  • 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 39.
  • 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 40.
  • 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 41.
  • 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 42.
  • 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 43.
  • 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 48.
  • 2-fucosyltransferase variant is at least 90% identical, such as at least 92%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 49.
  • a genetically engineered cell capable of producing at least one fucosylated oligosaccharide, wherein said genetically engineered cell comprises an alpha-1 , 2-fucosyltransferase variant according to any one of claims 1 to 43.

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Abstract

La présente invention concerne des variants d'alpha-1,2-fucosyltransférase ayant une activité alpha-1,3-fucosyltransférase réduite et des cellules génétiquement modifiées comprenant un variant d'alpha-1,2-fucosyltransférase pour la production biosynthétique de 2'FL. Elle concerne également l'utilisation des variants et des cellules génétiquement modifiées dans la production de 2'-fucosyllactose (2'FL) avec des quantités réduites du difucosyllactose (DFL) sous-produit.
PCT/EP2025/056456 2024-03-11 2025-03-10 Mutants de fucosyltransférase à faible formation de dfl Pending WO2025190861A1 (fr)

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