WO2025190861A1

WO2025190861A1 - Fucosyltransferase mutants with low dfl formation

Info

Publication number: WO2025190861A1
Application number: PCT/EP2025/056456
Authority: WO
Inventors: Margit Pedersen; Ferhat BÜKE; René Marcel de Jong; Marianne RUDOLPH HANSEN; Carlos RAMIREZ PALACIOS
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2024-03-11
Filing date: 2025-03-10
Publication date: 2025-09-18
Anticipated expiration: 2026-09-11

Abstract

The present disclosure relates to alpha-1,2-fucosyltransferase variants with reduced alpha-1,3- fucosyltransferase activity and genetically engineered cells comprising an alpha-1,2- fucosyltransferase variant for the biosynthetic production of 2'FL. Use of the variants and the genetically engineered cells in the production of 2'-fucosyllactose (2'FL) with reduced amounts of the by-product difucosyllactose (DFL).

Description

FUCOSYLTRANSFERASE MUTANTS WITH LOW DFL FORMATION

FIELD

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. The use of the variants in the production of fucosylated oligosaccharides, such as Human Milk Oligosaccharide (HMO), in particular 2’-fucosyllactose (2’FL) with reduced amounts of the byproduct HMO difucosyllactose (DFL). 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.

BACKGROUND

The construction of bacterial cell factories to produce Human Milk Oligosaccharides (HMOs), especially fucosylated Human Milk Oligosaccharides (HMOs) started in the early 2000 and has continuously developed over the last two decades as reviewed by Petschacher and Nidetzky 2016 Journal of Biotechnology 235: 61-83 and Bych et al 2019, Current Opinion in Biotechnology 56: 130-137.

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.

In industrial scale manufacturing even an increase of production yields of a few %, which can for example be achieved by increasing product formation over by-product formation, is of high value. Therefore, there is a continuous interest in identifying enzymes which are even better than wildtype FutC from Helicobacter pylori in producing 2’FL and/or reducing by-product formation. SUMMARY

Provided herein are 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. In alternative embodiments 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. Preferably, 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.

Preferably, 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. Preferably, 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.

Production of 2’FL requires that the 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 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. BRIEF DESCRIPTION OF FIGURES

Figure 1. Overview of the synthesis of the fucosylated HMOs 2’FL, 3FL and DFL.

Figure 2. Overview of the synthesis of the fucosylated HMOs LNFP-I and LNDFH-I.

Figure 3. Amino acid scan at indicated positions of the parent FutC enzyme of SEQ ID NO: 1 (wt). A) 2’FL produced by the individual mutations relative to 2’FL produced by the parent FutC (set to 1). B) DFL produced by the individual mutations relative to DFL produced by the parent FutC (set to 1).

Figure 4. % 2’FL and % DFL formation of genomically integrated variants. A) % 2’FL produced by the individual variants relative to 2’FL produced by the parent FutC (set to 100%). B) % DFL produced by the individual variants relative to 2’FL produced by the same variant.

Figure 5. % 2’FL produced by individual strains each expressing a different FutC variant (see table 6) relative to 2’FL produced by the parent (wt) FutC (SEQ ID NO:1), wt FutC is the bold bar set to 100%.

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).

DETAILED DESCRIPTION

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).

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. In particular for smaller oligosaccharides such as molecules of 5 monosaccharides or less, 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.

To aid the subsequent purification of the 2’FL it is highly advantageous that 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.

In essence, 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. Preferably, 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). The conventional stain modification used to improve 2’FL formation in E. 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.

In the following sections, individual elements of the disclosure, and in particular of the variant enzymes and the genetically engineered cell are described. It is understood that these elements can be combined across the individual sections.

Oligosaccharides

In the present context, the term “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. Particularly, 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. N-acetyl-glucosamine, N-acetyl-mannosamine, N-acetyl- galactosamine, etc.), uronic acids and ketoaldonic acids (e.g. N-acetylneuraminic acid). Preferably, the oligosaccharide is an HMO.

Human milk oligosaccharide (HMO)

Preferred oligosaccharides of the disclosure are human milk oligosaccharides (HMOs).

The term “human milk oligosaccharide" or "HMO" 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.

The present disclosure focuses on fucosylated HMO’s. Examples of fucosylated HMOs 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- difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-para-lacto-N-hexaose I (FpLNH-l), fucosyl-para-lacto-N-neohexaose II (F-pLNnH II), fucosyl-lacto-N-neohexaose (FLNnH), 3-fucosyl-3’-fucosyllactose (FSL), fucosyl-LST-a (FLST-a), fucosyl-LST b (FLST b), fucosyl-LST-c (FLST-c), fucosyl-LST d (FLST-d) and fucosyl-lacto-N-hexaose (SLNH).

In particular, 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.

The 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.

Variant alpha-1 ,2-fucosyltransferases

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”.

The a-1 ,2-fucosyltransferase variants of the present disclosure are preferably non-natural variants, i.e., they have not been found in nature. In describing the various a-1 , 2- fucosyltransferase variants of the present invention, 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.

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. In some positions 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). Alternatively, "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.

Across different strains of Helicobacter pylori there is natural diversity in the a-1 ,2- fucosyltransferase enzymes, also termed FutC homologues or a-1 ,2-fucosyltransferase homologues. At the date of filing this application there were 190 a-1 ,2-fucosyltransferase homologues from Helicobacter pylori in the UNiProt database example of such homologous wildtype H. 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 . The term "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%.

In addition to the functional substitutions (affecting alpha-1 ,3- or alpha- 1 ,2-fucosyltransferase activity) of the alpha-1 ,2-fucosyltransferase variant described herein, there are also examples of conservative substitutions that can be allowed without any significant change in functionality of the enzyme . Such 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)), and small amino acids (glycine (gly, G), alanine (ala, A), serine (ser, S) and threonine thr, T)). 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.

Likewise, 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.

In particular, it is desired that 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. In relation to 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. In particular 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. Of the positions indicated in Figure 8A and B (13, 45, 114, 166, 168, 169, 171 , 203, 204, 205, 206, 234, 248, 250, 253, 254) the following 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.

To maintain activity for the alpha-1 ,2-fucoayltransferase variants of the present disclosure it is desired that 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. Preferably, at least R45 and D171 , relative to the positions of SEQ ID NO: 1 , are maintained in the variants of the present disclosure. More preferably, all the positions G13, R45, Q114, H166, R168, D171 , E205, D234 and/or T250, relative to the positions of SEQ ID NO: 1 , are maintained in the variants of the present disclosure.

In specific embodiments 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.

In specific embodiments 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. Specifically, variant PE16 in example 4 shows that substituting D171 with N results in an enzyme with almost no alphal , 2-fucosyltransferase activity.

In the examples disclosed herein 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. Although SEQ ID NO: 1 is technically not a wildtype sequence it is sometimes denoted WT, wtFutC or futC_wt in the examples and figures. In embodiments of the present disclosure, 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. In embodiments, the a-1 ,2-fucosyltransferase variants disclosed herein produces 2’FL in the presence of a fucosyl donor and lactose as acceptor. In addition, the 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). To produce LNDFH-I an additional fucosyltransferase activity is required, namely an a-1 ,4-fucosyltransferase (see figure 2). In preferred embodiments 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. Preferably, 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.

The term “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-

1 .2-configuration.

The term “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.

The functional mutations introduced into the parent a-1 ,2-fucosyltransferase to generate the a-

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. Preferably, 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.

In embodiments of the present disclosure, it is advantageous to combine at least one mutation from group i) and at least one mutation from group ii) to achieve an a-1 ,2-fucosyltransferase variant with reduced the a-1 ,3-fucosyltransferase activity without significantly affecting the a-1 ,2- fucosyltransferase activity of the variant enzyme. Preferably, 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. More preferably, 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. Furthermore, when introduced into the genetically engineered cell of the present disclosure the nucleic acid sequence encoding the a-1 ,2-fucosyltransferase variant is preferably of heterologous origin. Preferably, 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.

In the context of the present disclosure, 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. Preferably, 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. Preferably, 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. In the examples of the present disclosure the DFL production of the host strain expressing the parent enzyme (parent strain) 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. Alternatively, 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. In preferred embodiments 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.

Similarly, 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.

In the context of the present disclosure, 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). For the indicated single mutation group i) variants 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. In the present disclosure 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. In further embodiments 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.

In further embodiments 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.

In embodiments 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 . Preferably, the variant produces less than 0.3%, such as less than 0.2% DFL relative to the 2’FL produced by the variant. Preferably, 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. In preferred embodiments 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.

In embodiments 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. Preferably, the variant produces less than 0.2%, such as 0% of DFL relative to the 2’FL produced by the variant. Preferably, 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. In embodiments alpha-1 , 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. In preferred embodiments 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.

In embodiments 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. Preferably, the variant produces less than 0.2%, such as 0% of DFL relative to the 2’FL produced by the variant. Preferably, 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. In preferred embodiments alpha-1 , 2-fucosyltransferase variants with a C9Y,W,F,G 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.

In embodiments 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. Preferably, 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. Preferably, 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. In preferred embodiments 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;

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.

In embodiments 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. Preferably, the variant produces less than 50 %, such as less than 45% of DFL relative to the 2’FL produced by the variant. Preferably, 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. Preferably the 2’FL production is at least at the same level as 2’FL production by the parent alpha-1 , 2-fucosyltransferase. In embodiments 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 . Preferably, the variant produces less than 1 %, such as less than 0.8% of DFL relative to the 2’FL produced by the variant. Preferably, 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.

In embodiments 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 . Preferably, the variant produces less than 0.2 %, such as 0% of DFL relative to the 2’FL produced by the variant. Preferably, 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. In preferred embodiments 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.

In some embodiments the variant only comprises two of the group i) mutations.

In some embodiments it may be beneficial only to have two out of the three substitutions I175D, C177H,Q,D,S and L273R in the same variant. Preferably, the substitutions I175D and L273R are not combined. Preferably, if the substitution I175D is present in the variant then position C177 and L273 are unsubstituted. The substitutions C177H and L273R is preferred over the substitution C177Q and L273R, which are preferably not combined.

In embodiments 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.

In embodiments 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 .

In some embodiments the variant contains one of the single group i) substitutions or combinations of group i) substitutions in table 3.

Table 3 preferred alpha-1 ,3-fucosyltransferase activity (DFL) reducing substitutions (group i)

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 . Furthermore, the 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, P124A, L125R, Q150H; C151 R, Q239S and/or Q297Q.D, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.

Furthermore, 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 .

Furthermore, 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. In a further embodiment 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%, 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

F40D, V93E;

F40D, C177Q;

F40D, C177D;

F40D, C177S;

F40D, C177F;

F40D, C177H

C9Y, F40D;

C9Y, F40D, C177Q;

C9F, F40D;

C9F, F40D, C177Q;

C9W, F40D;

C9W, F40D, C177Q;

G10A, F40D;

G10A, F40D, C177Q;

G10S, F40D;

G10S, F40D, C177Q;

F40D, L273Q;

F40D, L273R;

F40D, C177H, L273R;

F40D L80C A121 D P124A L125R C151 R L273R;

F40D, I175D;

F40D, I175D, C177Q;

C9Y, F40D, C177H, L273R;

F40S, V93E;

F40S, C177Q;

F40S, C177D;

F40S, C177S;

F40S, C177F;

F40S, C177H;

C9Y, F40S;

C9Y, F40S, C177Q;

C9F, F40S;

C9F, F40S, C177Q;

C9W, F40S;

C9W F40S, C177Q;

G10A, F40S;

G10A, F40S, C177Q;

G10S, F40S;

G10S, F40S, C177Q;

G10A F40S F110H A121 D Q150H C151 R C177Q Q239S;

F40S, L273Q;

F40S, L273R; F40S, C177H, L273R;

F40S Q150H C151R C177H Q239S L273R;

F40S Q150H C151 R C177Q Q239S L273R;

F40S, I175D;

F40S, I175D, C177Q;

C9Y, F40S, C177H, L273R;

F40T, V93E;

F40T, C177Q;

F40T, C177D;

F40T, C177S;

F40T, C177F;

F40T, C177H;

C9Y, F40T;

C9Y, F40T, C177Q;

C9F, F40T;

C9F, F40T, C177Q;

C9W, F40T;

C9W F40T, C177Q;

G10A, F40T;

G10A, F40T, C177Q;

G10S, F40T;

G10S, F40T, C177Q;

F40T, L273Q;

F40T, L273R;

F40T, C177H, L273R;

F40T, I175D;

F40T, I175D, C177Q; and

C9Y, F40T, C177H, L273R; wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 . Preferably, 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;

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 F 110A or F110H or F110S, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .

Furthermore, 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. In a further embodiment 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 or consists of a combination of substitutions selected from the group consisting of

F110A, V93E;

F110A, C177H

F110A, C177H, H276Y;

F110A, C177H, Q239S

F110A, C177Q;

F110A, C177Q, K297D

F110A, C177Q, K297Q

F110A, C177Q, Q239S

F110A, C177D;

F110A, C177S;

F110A, C177F;

C9Y, F110A;

C9Y, F110A, C177Q;

C9Y, F110A, C177Q; Q239S

C9F, F110A;

C9F, F110A, C177Q;

C9F, F110A, C177Q; Q239S

C9W, F110A;

C9W, F110A, C177Q;

C9W, F110A, C177Q; Q239S

G10A, F110A;

G10A, F110A, C177Q;

G10A, F110A, C177Q; Q239S

G10S, F110A;

G10S, F110A, C177Q;

G10S, F110A, C177Q; Q239S

F110A, L273Q;

F110A, C177H, L273R;

F110A, I175D;

F110A, I175D, C177Q;

F40S, F110A, C177H, L273R;

C9Y, F110A, C177H, L273R;

G10A, F110A, C177H, L273R;

F110H, V93E;

F110H, C177Q;

F110H, C177D;

F110H, C177Q, K297D

F110H, C177Q, K297Q;

F110H, C177Q, Q239S;

F110H Q150H C151 R C177Q Q239S

F110H Q150H C151 R C177Q Q239S K297D

F110H Q150H C151 R C177Q Q239S K297Q F110H P124A C177Q

F110H P124A L125R C177Q

F110H A121 D P124A L125R C177Q

L80C F110H C177Q

L80C F110H A121 D P124A L125R C151 R C177Q

L80C F110H A121 D P124A L125R C151 R L273R

L80C F110H A121 D P124A L125R Q150H C151 R C177Q Q239S

F110H, C177S;

F110H, C177F, Q239S;

F110H, C177F;

F110H, C177F, Q239S;

F110H, C177H;

F110H, C177H, Q239S;

F110H Q150H C151 R C177H Q239S

C9Y, F110H;

C9Y, F110H, C177Q;

C9Y, F110H, C177Q; Q239S

C9Y F110H Q150H C151 R C177Q Q239S

C9F, F110H;

C9F, F110H, C177Q;

C9F, F110H, C177Q; Q239S

C9W, F110H;

C9W, F110H, C177Q;

C9W, F110H, C177Q; Q239S

G10A, F110H;

G10A, F110H, C177Q;

G10A, F110H, C177Q; Q239S

G10A S39W F110H A121 D Q150H C151 R C177Q Q239S

G10S, F110H;

G10S, F110H, C177Q;

G10S, F110H, C177Q; Q239S

F110H, L273Q;

F110H, C177H, L273R;

F110H, I175D;

F110H, I175D, C177Q;

F40S, F110H, C177H, L273R;

C9Y, F110H, C177H, L273R;

G10A, F110H, C177H, L273R;

S39W F110H Q150H C151 R C177H Q239S L273R

F110S, V93E;

F110S, C177Q;

F110S, C177Q, K297D

F110S, C177Q, K297Q;

F110S, C177Q, Q239S;

F110S, C177D;

F110S, C177S;

F110S, C177F;

F110S, C177F, Q239S;

F110S, C177H; F110S, C177F, Q239S;

C9Y, F110S;

C9Y, F110S, C177Q;

C9Y, F110S, C177Q, Q239S;

C9W, F110S;

C9W, F110S, C177Q;

C9W, F110S, C177Q, Q239S;

C9F, F110S;

C9F, F110S, C177Q;

C9F, F110S, C177Q, Q239S;

G10A, F110S;

G10A, F110S, C177Q;

G10A, F110S, C177Q; Q239S

G10S, F110S;

G10S, F110S, C177Q;

G10S, F110S, C177Q; Q239S

F110S, L273Q;

F110S, C177H, L273R;

F110S, I175D;

F110S, I175D, C177Q;

F40S, F110S, C177H, L273R;

C9Y, F110S, C177H, L273R; and

G10A, F110S, C177H, L273R wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 . Preferably, 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.

Furthermore, 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.

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 substitutions C151 R and Q239S, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .

Furthermore, 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;

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 Q150H and C151 R and Q239S, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.

In a further embodiment 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 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

C9Y Q150H, C151 R, Q239S;

C9W Q150H, C151 R, Q239S,;

C9F Q150H, C151 R, Q239S,

G10A Q150H, C151 R, Q239S;

G10S Q150H, C151 R, Q239S;

V93E Q150H, C151 R, Q239S,; Q150H, C151R, Q239S, I175D;

Q150H, C151R, Q239S, C177H;

Q150H, C151 R, Q239S, C177Q;

Q150H, C151 R, Q239S, C177D;

Q150H, C151 R, Q239S, C177S;

Q150H, C151 R, Q239S, L273R;

F110H, Q150H, C151 R, C177Q Q239S

L80C F110H A121 D P124A L125R Q150H C151 R C177Q Q239S

F110H, Q150H, C151 R, C177Q, Q239S, K297D;

F110H, Q150H, C151 R, C177Q, Q239S, K297Q;

Q150H, C151 R, I175D, C177Q, Q239S, K297Q;

C9Y, F110H, Q150H, C151 R, C177Q, Q239S;

G10A, F110H, Q150H, C151 R, C177Q, Q239S;

G10A S39W F110H A121 D Q150H C151 R C177Q Q239S

F110H, Q150H, C151 R, C177H Q239S

F110H, Q150H, C151 R, C177H, Q239S, K297D;

F110H, Q150H, C151 R, C177H, Q239S, K297Q;

Q150H, C151 R, I175D, C177H, Q239S, K297Q;

F40S, Q150H, C151 R, C177H, Q239S, L273R;

Q150H, C151 R, I175D, C177H, Q239S, K297Q;

F110H, Q150H, C151 R, C177H, Q239S L273R;

S39W F110H Q150H C151 R C177H Q239S L273R

C9Y, F110H, Q150H, C151 R, C177H, Q239S, L273R; and

G10A, F110H, Q150H, C151 R, C177H, Q239S, L273R wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 . Preferably, the variant has at least 90% sequence identity to SEQ ID NO:1 .

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 L80C, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .

C9Y L80C F110H;

C9Y L80C F110H C177Q;

C9Y L80C F110H C177Q Q239S;

C9F L80C F110H;

C9F L80C F110H C177Q;

C9F L80C F110H C177Q Q239S;

C9W L80C F110H;

C9W L80C F110H C177Q;

C9W L80C F110H C177Q Q239S;

G10A L80C F110H;

G10A L80C F110H C177Q;

G10A L80C F110H C177Q Q239S;

G10S L80C F110H;

G10S L80C F110H C177Q;

G10S L80C F110H C177Q Q239S;

L80C F110H A121 D P124A L125R C151R C177Q;

L80C A121 D P124A L125R C151 R C177Q;

F40S L80C A121 D P124A L125R Q150H C151 R C177Q Q239S;

L80C F110H A121 D P124A L125R Q150H C151 R C177Q Q239S;

L80C F110H C177Q;

L80C A121 D P124A L125R C151 R L273R;

L80C F110H A121 D P124A L125R C151 R L273R; and

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 . Furthermore, 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. In a further embodiment 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 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

F110H A121D P124A C151R C177Q;

F110H A121D P124A L125R C151R C177Q;

L80C F110H A121D P124A C151R C177Q;

L80C F110H A121D P124A L125R C151R C177Q;

A121D P124A C151R C177Q

A121D P124A L125R C151R C177Q

L80C A121D P124A L125R C151R C177Q;

G10A F40S F110H A121D C151R C177Q Q239S;

G10A F40S F110H A121D Q150H C151R C177Q Q239S;

A121D P124AC151R L273R;

A121D P124A L125R C151R L273R;

L80C A121D P124AC151R L273R;

L80C A121D P124A L125R C151R L273R;

A121D P124A C151R Q239S L273R;

L80C A121D P124A C151R Q239S L273R;

F110H A121D C151R C177Q Q239S

F110H P124A C151R C177Q Q239S

F110H A121D P124A C151R C177Q Q239S;

F110H A121D P124A L125R Q150H C151R C177Q Q239S;

L80C F110H A121D P124A C151R C177Q Q239S;

L80C F110H A121D P124A L125R Q150H C151R C177Q Q239S;

G10A S39W F110H A121D C151R C177Q Q239S;

G 10A S39W F110H A121 D Q150H C151 R C177Q Q239S;

A121D P124AC151R L273R;

F110H A121D P124AC151R L273R;

L80C F110H A121D P124A C151R L273R;

L80C F110H A121D P124A L125R C151R L273R;

F110H A121DC177Q;

F110H A121D P124AC177Q; and

F110H A121D P124A L125R C177Q; 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.

Preferably, 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. Preferably, 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.

In embodiments 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. Table 4: alpha-1 ,2-fucosyltransferase variants with increased 2’FL when compared to the parent and low DFL In an alternative aspect of the present disclosure 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.

Embodiments relating to the alternative aspect of only increasing 2’FL formation 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 .

In a further embodiment 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 or consists of a combination of substitutions selected from the group consisting of

F110A F110H F110S F110A Q239S; F110H Q239S; F110S Q239S;

F110A Q239S K297Q;

F110H Q239S K297Q;

F110S Q239S K297Q;

F110A Q239S K297D;

F110H Q239S K297D;

F110S Q239S K297D;

L80C F110A A121 D P124A C151 R

L80C F110H A121 D P124A C151 R

L80C F110S A121 D P124A C151 R

L80C F110A A121 D P124A C151 R Q239S;

L80C F110H A121 D P124A C151 R Q239S; and

L80C F110S A121 D P124A C151 R Q239S;

As all the alpha- 1 , 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.

In preferred embodiments the 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.

In preferred embodiments 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 .In a preferred embodiment 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.

In a preferred embodiment 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: 37.

In a preferred embodiment the 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.

In a preferred embodiment the 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.

In a preferred embodiment the 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.

In a preferred embodiment the 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.

In a preferred embodiment the 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.

In a preferred embodiment the 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.

For the in vivo production of 2’FL the present disclosure also relates to a genetically engineered cell expressing an a-1 , 2-fucosyltransferase variant as described herein. Preferably, 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. For production of 2’FL 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. As such, 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. In the present disclosure 2’FL is the intended or desired product and DFL and 3FL are unwanted byproducts.

In embodiments, 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 comparison between an a-1 ,2-fucosyltransferase variant described herein and the parent alpha-1 , 2-fucosyltransferase, such as the alpha- 1 ,2-fucosyltransferase of SEQ ID NO: 1 , should be done in a strain with the same genetic background and with the enzymes being under control of identical promoters. Preferably, 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.

Additional glycosyltransferases

In alternative aspects of the present disclosure the intended or desired product may be a larger fucosylated HMO than 2’FL. In this aspect 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. In embodiments, 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.

Where it is desired to produce more complex fucosylated HMOs using the fucosyltransferase described herein, 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. In embodiments, 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. In one embodiment, a a-1 ,2-fucosyltransferase variant as described herein is introduced into a genetically engineered cell wherein said cell further comprises a |3-1 ,4-galactosyltransferase, or a p-1 ,3-galactosyltransferase, and preferably also further comprises a |3-1 ,3-N-acetyl- glucosaminyl-transferase.

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. gonorrhoeae (e.g., GenBank protein Accession No’s ACF31229.1 , or AAK70338.1), or p-1 ,3-N-acetylglucosaminyltransferases genes from Haemophilus ducreyi (e.g., GenBank protein Accession AAN05638.1), or p-1 ,3-N- acetylglucosaminyltransferases genes from Pasteurella multocida (e.g., GenBank protein Accession AAK02595.1), or p-1 ,3-N-acetylglucosaminyltransferases genes from Neisseria cinerea (e.g., GenBank protein Accession EEZ72046.1).

For production of e.g. LNDH-I an 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 molecule

In the context of the present disclosure, 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. In the present description the term 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”. Preferably, 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. In the present context, said acceptor saccharide is preferably lactose for the production of 2’FL. The production of more complex HMOs or oligosaccharides, such as LNFP-I, LNFP-IV, and/or LNDFH-I may also include more complex acceptor molecules such as lacto-N-tetraose (LNT) or (lacto-N-neotetraose) LNnT, which are produced from the precursor molecules lactose and/or lacto-N-triose II (LNT-II). In both cases, the precursor molecule can be fed to the genetically modified cell which is capable of producing, or which produces the fucosylated HMO from the precursor or the cell can be modified to produce the more complex acceptor molecule from lactose.

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.

As an alternative to adding the initial substrate for the production of the first oligosaccharide to the fermentation medium, the genetically modified cell may be further engineered to produce the initial substrate, such as lactose, inside the cell (see for example WO2015/150328).

Glycosyl-donor - nucleotide-activated sugar pathways

When carrying out the method of this disclosure, preferably 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. Thus, preferably the following 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. In one embodiment of the current disclosure, the genetically engineered cell is capable of producing one or more activated sugar nucleotides mentioned above by a de novo pathway. In this regard, 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.

In another embodiment, the genetically engineered cell can utilize salvaged monosaccharides for sugar nucleotide. In the salvage pathway, 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.

Colanic acid gene cluster

For the production of fucosylated HMOs, the de novo GDP-fucose pathway is important to ensure presence of sufficient GDP-fucose. 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. An alternative pathway for producing GDP-fucose is known as the salvage pathway. Both GDP-fucose producing pathways are shown in figure 2 of the review article by Petschacher and Nidetzky (Journal of Biotechnology 235 (2016) 61-83).

To secure sufficient amounts of GDP-fucose 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. Furthermore, 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.

In embodiments, 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.

In relation to the present disclosure, 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;

Hi) 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; iv) 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-fucose.

Accordingly, it is preferred that 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

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. In oligosaccharide- production, especially in the production of human milk oligosaccharides (HMOs), lactose is often the initial substrate being decorated to produce any HMO of interest in a bioconversion that happens in the cell interior. Thus, in the production of HMOs, there is a desire to be able to import lactose into the cell, e.g., by expression and/or overexpression of a lactose permease such as lacY from E. coll. In embodiments, 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.

In embodiments, the expression of the lactose permease is regulated by a promoter according to the present disclosure.

P-galactosidase

A host cell suitable for HMO production, e.g., E. coll, may comprise an endogenous |3- galactosidase gene or an exogenous p-galactosidase gene, e.g., E. coli comprises an endogenous lacZ gene (e.g., GenBank Accession Number V00296 (GI:41901)). For the purposes of the disclosure, when producing an HMO, it is preferred that, 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. In embodiments 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. In this way the HMO-producing bacterium accumulates an increased intracellular lactose pool which is beneficial for the production of HMOs.

Importer proteins

Most commonly HMO producing cells are genetically engineered to use lactose as the initial substrate since this is easily taken up by e.g., a lactose permease. Lactose permeases are described above.

However, in the production of for example LNFP-I, it may be desired to use an initial substrate that will require the presence of fewer glycosyltransferases in the cell, since this will reduce the strain on the cell in terms of producing multiple enzymes. In addition, it can reduce the byproduct profile, e.g. if lactose is not used as initial substrate a cell comprising a fucosyltransferase will not produce 2’FL as by-product allowing the fucose to be used to produce e.g. more LNFP-I.

Examples of suitable 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).

Accordingly, in embodiments, 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.

Exporter proteins

The oligosaccharide product, such as the HMO produced by the cell, can be accumulated both in the intra- and the extracellular matrix. 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.

In one embodiment the genetically engineered cell of the present disclosure preferably expresses a heterologous Major Facilitator Superfamily (MFS) transporter protein.

The term “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.

In one embodiment 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 In one embodiment 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.

The genetically engineered cell

In the present context, the terms “a genetically engineered cell” and "a genetically modified cell” are used interchangeably. As used herein “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. In one embodiment 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.

The genetically engineered cell may comprise further modifications that facilitate the production of the desired fucosylated oligosaccharide. Such 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. In a preferred embodiment the 2’FL is the primary product produced by the genetically engineered cell.

In embodiments, the genetically engineered cell capable of producing a fucosylated oligosaccharide, such as at least one 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. Preferably, the a-1 ,2-fucosyltransferase variant disclosed herein is capable of fucosylating lactose at the Gal moiety with an a-1 , 2 linkage. Preferably, the a-1 , 2- fucosyltransferase variant disclosed herein is fucosylates lactose at the Gal moiety with an a-1 ,2 linkage. In preferred embodiments 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). In preferred embodiments the 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. In some embodiments, 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. Coll, through introduction of a nucleic acid construct encoding the CA as shown in SEQ ID NO: 5, allowing for formation of GDP-fucose, which enables the cell to produce a higher level of fucosylated oligosaccharides from lactose. Alternatively, the native promoter regulating the endogenous colanic acid gene cluster (CA) may be exchanged or supplemented with a stronger promoter. Depending on the intended use of substrate and desirable final product, 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.

Host cells

In embodiments, 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 (host cell) may be e.g., a bacterial or yeast cell.

In one embodiment the genetically engineered cell may be a prokaryotic cell e.g., a bacterial cell.

Regarding 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. Preferably, the host cell has the property to allow cultivation to high cell densities. Non-limiting examples of 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. Similarly, 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. Also included as part of this invention are 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., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa).

Examples of 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. (such as Pectobacterium carotovorum), Pseudomonas sp., Stenotrophomonas sp., Xanthomonas sp. (such as Xanthomonas campestris).

In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of Escherichia sp., Bacillus sp., Lactobacillus sp and Corynebacterium sp. Campylobacter sp.

In one embodiment, the genetically engineered cell is selected from the group consisting of E. coll, C. glutamicum, L. lactis, B. subtilis, S. lividans.

In one embodiment, the genetically engineered cell is Bacillus subtilis.

In one embodiment, the genetically engineered cell is Corynebacterium glutamicum.

Preferably, the genetically engineered cell is gram-negative bacteria.

In a preferred embodiment, the genetically engineered cell is Escherichia coli.

In one or more exemplary embodiments, the disclosure relates to a genetically engineered cell, wherein the cell is derived from the E. coli K-12 strain or DE3.

In other embodiments the microbial cell is an eucaryotic cell such as a yeast or a filamentous fungus. In one embodiment the genetically engineered cell may be a yeast cell such as Komagataella phaffii, Kluyveromyces lactis, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

In one embodiment 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.

In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

In one embodiment, the genetically engineered cell is Pichia pastoris.

In one embodiment, the genetically engineered cell is Yarrowia lipolytica.

A recombinant nucleic acid sequence

The present disclosure also relates a recombinant nucleic acid sequence encoding an a-1 ,2- fucosyltransferase variant as disclosed herein.

In the present context, the term “recombinant 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.

The term "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. As used herein "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.

The term “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. E.g., 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.

Generally, promoter sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.

In one exemplified embodiment, 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. In another embodiment, the nucleic acid construct it is an expression cassette/cartridge that is integrated in the genome of a host cell.

Accordingly, the term “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. Preferably 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.

Accordingly, in embodiments, 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.

In embodiments, said recombinant nucleic acid sequence is encoded on a plasmid. In additional embodiments, the plasmid is a high copy number plasmid, preferably, a pUC57 or pBB-B9 plasmid.

Preferably, 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.

Table 2 - Selected promoter sequences *The promoter activity is assessed in the LacZ assay described below with the PglpF promoter run as positive reference in the same assay. To compare across assays the activity is calculated relative to the PglpF promoter, a range indicates results from multiple assays.

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 |3- galactosidase activity is assayed as described previously (see e.g., Miller J. H. Experiments in molecular genetics, Cold spring Harbor Laboratory Press, NY, 1972). Briefly the cells are diluted in Z-buffer and permeabilized with sodium dodecyl sulfate (0.1%) and chloroform. The LacZ assay is performed at 30°C. Samples are preheated, the assay initiated by addition of 200 pl ortho-nitro-phenyl-p-galactosidase (4 mg/ml) and stopped by addition of 500 pl of 1 M Na2COs when the sample had turned slightly yellow. The release of ortho-nitrophenol is subsequently determined as the change in optical density at 420 nm. The specific activities are reported in Miller Units (MU) [A420/(min*ml*A600)]. A regulatory element with an activity above 10,000 MU is considered strong and a regulatory element with an activity below 3,000 MU is considered weak, what is in between has intermediate strength. An example of 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. In preferred embodiments, 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.

In embodiments 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. Further suitable variants of PglpF, PglpA_70UTR, PglpT_70UTR and PmglB_70UTR promoter sequences are described in or WO2019/123324 and W02020/255054 respectively (hereby incorporated by reference).

In preferred embodiments, 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.

Integration 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. (1998);180(8):2063-7; Zhang et al., Nature Genetics (1998) 20: 123-128 Muyrers et al., EMBO Rep. (2000) 1 (3): 239-243); methods based on Red/ET recombination (Wenzel et al., Chem Biol. (2005), 12(3):349-56.; Vetcher et al., Appl Environ Microbiol. (2005) ;71 (4): 1829-35); or positive clones, i.e., clones that carry the expression cassette, can be selected e.g., by means of a marker gene, or loss or gain of gene function.

Sequence identity

The term "sequence identity" as used herein 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. For purposes disclosed herein, the 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. 16: 276-277), preferably version 5.0.0 or later (available at https://www.ebi.ac.uk/Tools/psa/emboss_needle/). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, -endopen 10.0, -endextend 0.5 and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. The output of Needle labelled " identity" (obtained using the -nobrief option) is used as the percent identity. Generally sequence identity may be calculated as follows: (Identical Residues x 100)/(aligned region).

For purposes disclosed herein, the 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. Generally sequence identity may be calculated as follows: (Identical nucleotide residues x 100)/(aligned region).

Functional variant

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). Preferably, 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. Preferably, 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.

Use of a genetically engineered cell or an alpha-1 ,2-fucosyltransferase variant enzyme

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.

Accordingly, 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. In preferred embodiments, the fucosylated product is 2’FL.

In embodiments, 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.

In an exemplified embodiment, the genetically engineered cell and/or the nucleic acid construct described herein is used in the manufacturing of HMOs. Preferably, in the manufacturing of 2’FL. Preferably, 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). In embodiments, 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.

A method for producing fucosylated human milk oligosaccharides (HMOs)

The present disclosure also relates to a method for producing one or more fucosylated human milk oligosaccharide (HMO), preferably 2’FL.

In one embodiment 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. The a-1 ,2- fucosyltransferase variant can be expressed and purified from known cellular production hosts, including those described in the section “host cells”.

In another embodiment 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.

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. Preferably 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. Even more preferably, 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. It is further preferred, that no other 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.

Even more preferably, 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 . Preferably, the molar ratio of 2’FL:DFL is more than 900: 1 .

In embodiments 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. In further embodiments 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.

As mentioned herein, it is preferable that 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.

In embodiments 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. In preferred embodiments essentially no DFL is produced by said method. In further embodiments essentially no 3FL is produced by said method.

In additional embodiments, 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 . Preferably, the molar ratio of 2’FL:DFL is more than 900:1.

In addition to the a-1 ,2-fucosyltransferase variant it is understood that 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. Preferably, 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.

In one embodiment, 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. In a preferred embodiment 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 (HMO) is retrieved from the culture, either from the culture medium and/or the genetically engineered cell.

Culturing/ferm enting

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. By 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. The term culture broth is a common term describing the mixture of culture medium, carbon source and the cells being fermented.

The terms “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. Usually, 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. In addition to the large volume, a manufacturing scale method, as opposed to simple lab scale methods like shake flask cultivation, 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.). To a large extent, the behaviour of an expression system in a lab scale method, such as shake flasks, benchtop bioreactors or the deep well format described in the examples of the disclosure, does allow to predict the behaviour of that system in the complex environment of a bioreactor.

With regards to the suitable cell medium used in the fermentation process, there are no limitations. 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. In one or more exemplary embodiments, the culturing media is supplemented with one or more energy and carbon sources selected form the group containing glycerol, sucrose and glucose. In additional embodiments, lactose is added during the cultivation of the genetically engineered cells as a substrate for the HMO formation.

In one or more exemplary embodiments, the culturing media contains sucrose as the sole carbon and energy source. In one or more exemplary embodiments, 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.

In one or more exemplary embodiments, 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. Preferably, the expression of the scrYA and/or scrBR operons is/are under control of a Pscr promoter (SEQ ID NO: 6)

After carrying out the method of this disclosure, the fucosylated HMO produced can be collected from the cell culture or fermentation broth in a conventional manner.

Retrieving/Harvesting

The fucosylated human milk oligosaccharide (HMO) is retrieved from the culture medium and/or the genetically engineered cell. In the present context, 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).

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.

Manufactured product

The term “manufactured 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). Preferably, the product HMO or composition is produced by a method described herein using a genetically engineered cell described herein.

The data presented in example 2 and 4, shows that selected a-1 ,2-fucosyltransferase variants disclosed herein produces a higher relative amount of 2’FL, with much lower DFL levels produced than FutC, thus underlining its suitability in large scale manufacturing.

Advantageously, 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. ITEMS

1 . A functional alpha-1 ,2-fucosyltransferase variant derived from a parent alpha-1 ,2- fucosyltransferase originating from Helicobacter pylori, 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 or SEQ ID NO: 6.

2. A functional alpha-1 ,2-fucosyltransferase variant with at least 85% sequence identity to 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.

3. A functional alpha-1 ,2-fucosyltransferase variant with at least 85 % sequence identity to SEQ ID NO: 1 or 6, wherein the variant comprises a substitution in one or more positions selected from the group consisting C177 H,Q,S, F,N,D,E; L273 R,K,Q,N;

C9F,Y,W,N,Q,T,S,G,A,V,K;R; G10A,S; S39A,W; V93E,D; I175D; wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 ,

4. The functional alpha-1 ,2-fucosyltransferase variant according to any one of items 1 to 3, wherein the variant has 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.

5. The functional alpha- 1 ,2-fucosyltransferase variant according to item 1 to 4, wherein the variant comprises a substitution in one or more positions selected from the group consisting C177H,Q,D,S,F; L273R.Q; C9F,W,N,G,A,K; G10A,S; S39A,W; V93E; I175D; wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO: 1.

6. The functional alpha- 1 ,2-fucosyltransferase variant according to item 1 to 5, wherein the variant comprises a substitution in one or more positions selected from the group consisting C177H,Q,D,S,F; L273R; C9W,G,F; G10A,S; wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .

7. The alpha-1 ,2-fucosyltransferase variant according to any one of items 1 to 6, where the variant only is mutated in two of the indicated positions.

8. The 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. 9. The alpha-1 ,2-fucosyltransferase variant according to any one of items 1 to 8, wherein 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.

10. The 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.

11 . The alpha-1 , 2-fucosyltransferase variant according to items 1 to 5, wherein the variant comprises one of the following combinations of substitutions:

I175D and C9Y,F,W,G;

I175D and G10A,S

I175D and S39A,W;

I175D and V93E;

C177H,Q,D,S,F and C9Y,F,W,G;

C177H,Q,D,S,F and G10A,S;

C177H,S,F and L273R;

C177H,Q,D,S,F and S39A,W;

C177H,Q,D,S,F and 93E;

L273R and S39A,W;

L273R and C9Y,F,W,G;

L273R and C177H and C9Y; or

L273R and C177H and S39W.

12. The 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.

13. The 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.

14. The alpha-1 , 2-fucosyltransferase variant according to item 13, wherein the parent alpha- 1 ,2-fucosyltransferase is SEQ ID NO: 1 , 6, 45, 46 or 47 or a sequence with at least 98%, such as 99% identity to SEQ ID NO: 1 , 6, 45, 46 or 47.

15. The 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 .

16. The alpha-1 , 2-fucosyltransferase variant according to any one of the preceding items, wherein the variant further comprises at least one of the following substitutions F40S,D,T; L80C, F110A,H,S; A121 D, P124A, L125R, Q150H; C151 R, Q239S and/or Q297Q.D, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1. 7. The alpha-1 , 2-fucosyltransferase variant according to any one of the proceeding items, wherein the variant comprises one of the following combinations of substitutions:

F40D, V93E;

F40D, C177Q;

F40D, C177D;

F40D, C177S;

F40D, C177F;

C9Y, F40D;

C9Y, F40D, C177Q;

F40D, L273Q;

F40D, L273R;

F40D_L273R;

F40D, C177Q, L273R;

F40D, I175D;

F40D, I175D, C177Q;

F40D, I175D, C177Q, L273R;

C9Y, F40D, C177Q, L273R;

C9Y, F40D, I175D, C177Q, L273R

F40S, V93E;

F40S, C177Q;

F40S, C177D;

F40S, C177S;

F40S, C177F;

C9Y, F40S;

C9Y, F40S, C177Q;

F40S, L273Q;

F40S, L273R;

F40S_L273R;

F40S, C177Q, L273R;

F40S, I175D;

F40S, I175D, C177Q;

F40S, I175D, C177Q, L273R;

C9Y, F40S, C177Q, L273R;

C9Y, F40S, I175D, C177Q, L273R

F110A, V93E;

F110A, C177H

F110A, C177H, H276Y;

F110A, C177Q;

F110A, C177Q, K297D

F110A, C177D;

F110A, C177S;

F110A, C177F;

C9Y, F110A;

C9Y, F110A, C177Q;

F110A, L273Q; F110A, L273R;

F110A, C177Q, L273R;

F110A, I175D;

F110A, I175D, C177Q;

F110A, I175D, C177Q, L273R;

F40S, F110A, C177Q, L273R;

C9Y, F110A, C177Q, L273R;

C9Y, F110A, I175D, C177Q, L273R

F110H, V93E;

F110H, C177Q;

F110H, C177D;

F110H, C177Q, K297D

F110H, C177S;

F110H, C177F;

F110H, C177H;

C9Y, F110H;

C9Y, F110H, C177Q;

F110H, L273Q;

F110H, L273R;

F110H, C177Q, L273R;

F110H, I175D;

F110H, I175D, C177Q;

F110H, I175D, C177Q, L273R;

F40S, F110H, C177Q, L273R;

C9Y, F110H, C177Q, L273R;

C9Y, F110H, I175D, C177Q, L273R

Q150H, C151 R, Q239S, C9Y;

Q150H, C151 R, Q239S, C9G;

Q150H, C151 R, Q239S, V93E;

Q150H, C151 R, Q239S, I175D;

Q150H, C151 R, Q239S, C177H;

Q150H, C151 R, Q239S, C177Q;

Q150H, C151 R, Q239S, C177D;

Q150H, C151 R, Q239S, C177S;

Q150H, 0151 R, Q239S, L273R;

F110H, Q150H, C151 R, C177Q, Q239S

F110H, Q150H, C151 R, C177Q, Q239S, K297D;

F110H, Q150H, C151 R, C177Q, Q239S, K297Q;

Q150H, C151 R, I175D, C177H, Q239S, K297Q;

C9Y, F110H, Q150H, C151 R, C177Q, Q239S;

F40S, Q150H, C151 R, C177H, Q239S, L273R;

Q150H, C151 R, I175D, C177H, Q239S, K297Q;

Q150H, C151 R, I175D, Q239S, L273R;

F110H, Q150H, C151 R, C177Q, Q239S L273R;

F110H, Q150H, C151 R, I175D, C177Q, Q239S;

F110H, Q150H, C151 R, I175D, C177Q, Q239S, L273R;

C9Y, F110H, Q150H, C151 R, C177Q, Q239S, L273R; or

C9Y, F110H, Q150H, C151 R, I175D, C177Q, 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 The alpha-1 , 2-fucosyltransferase variant according to any one of the preceding items, wherein the variant further comprises the following substitution F110A or F110H, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 . The alpha-1 , 2-fucosyltransferase variant according to item 18, wherein the variant comprises one of the following combinations of substitutions:

F110A, V93E;

F110A, C177H

F110A, C177H, H276Y;

F110A, C177H, Q239S

F110A, C177Q;

F110A, C177Q, K297D

F110A, C177Q, K297Q

F110A, C177Q, Q239S

F110A, C177D;

F110A, C177S;

F110A, C177F;

C9Y, F110A;

C9Y, F110A, C177Q;

C9Y, F110A, C177Q; Q239S

C9F, F110A;

C9F, F110A, C177Q;

C9F, F110A, C177Q; Q239S

C9W, F110A;

C9W, F110A, C177Q;

C9W, F110A, C177Q; Q239S

G10A, F110A;

G10A, F110A, C177Q;

G10A, F110A, C177Q; Q239S

G10S, F110A;

G10S, F110A, C177Q;

G10S, F110A, C177Q; Q239S

F110A, L273Q;

F110A, C177H, L273R;

F110A, I175D;

F110A, I175D, C177Q;

F40S, F110A, C177H, L273R;

C9Y, F110A, C177H, L273R;

G10A, F110A, C177H, L273R;

F110H, V93E;

F110H, C177Q;

F110H, C177D;

F110H, C177Q, K297D

F110H, C177Q, K297Q;

F110H, C177Q, Q239S; F110H Q150H C151RC177Q Q239S

F110H Q150H 0151 R C177Q Q239S K297D

F110H Q150H C151R C177Q Q239S K297Q

F110H P124AC177Q

F110H P124A L125R C177Q

F110H A121D P124A L125R C177Q

L80C F110H C177Q

L80C F110H A121D P124A L125R C151R C177Q

L80C F110H A121D P124A L125R C151R L273R

L80C F110H A121D P124A L125R Q150H C151RC177Q Q239S

F110H, C177S;

F110H, C177F, Q239S;

F110H, C177F;

F110H, C177F, Q239S;

F110H, C177H;

F110H, C177H, Q239S;

F110H Q150H C151RC177H Q239S

C9Y, F110H;

C9Y, F110H, C177Q;

C9Y, F110H, C177Q; Q239S

C9Y F110H Q150H C151R C177Q Q239S

C9F, F110H;

C9F, F110H, C177Q;

C9F, F110H, C177Q; Q239S

C9W, F110H;

C9W, F110H, C177Q;

C9W, F110H, C177Q; Q239S

G10A, F110H;

G10A, F110H, C177Q;

G10A, F110H, C177Q; Q239S

G10AS39WF110H A121DQ150H C151RC177Q Q239S

G10S, F110H;

G10S, F110H, C177Q;

G10S, F110H, C177Q; Q239S

F110H, L273Q;

F110H, C177H, L273R;

F110H, I175D;

F110H, I175D, C177Q;

F40S, F110H, C177H, L273R;

C9Y, F110H, C177H, L273R;

G10A, F110H, C177H, L273R;

S39WF110H Q150H C151RC177H Q239S L273R

F110S, V93E;

F110S, C177Q;

F110S, C177Q, K297D

F110S, C177Q, K297Q;

F110S, C177Q, Q239S;

F110S, C177D;

F110S, C177S; F110S, C177F;

F110S, C177F, Q239S;

F110S, C177H;

F110S, C177F, Q239S;

C9Y, F110S;

C9Y, F110S, C177Q;

C9Y, F110S, C177Q, Q239S;

C9W, F110S;

C9W, F110S, C177Q;

C9W, F110S, C177Q, Q239S;

C9F, F110S;

C9F, F110S, C177Q;

C9F, F110S, C177Q, Q239S;

G10A, F110S;

G10A, F110S, C177Q;

G10A, F110S, C177Q; Q239S

G10S, F110S;

G10S, F110S, C177Q;

G10S, F110S, C177Q; Q239S

F110S, L273Q;

F110S, C177H, L273R;

F110S, I175D;

F110S, I175D, C177Q;

F40S, F110S, C177H, L273R;

C9Y, F110S, C177H, L273R; or

G10A, F110S, C177H, 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. The alpha-1 , 2-fucosyltransferase variant according to any one of the preceding items, wherein the variant further comprises the following substitution F40D or F40S, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 . The alpha-1 , 2-fucosyltransferase variant according to item 20, wherein the variant comprises a combination of substitutions selected from the group consisting of

F40D, V93E;

F40D, C177Q;

F40D, C177D;

F40D, C177S;

F40D, C177F;

F40D, C177H

C9Y, F40D;

C9Y, F40D, C177Q;

C9F, F40D;

C9F, F40D, C177Q;

C9W, F40D;

C9W, F40D, C177Q;

G10A, F40D; G10A, F40D, C177Q;

G10S, F40D;

G10S, F40D, C177Q;

F40D, L273Q;

F40D, L273R;

F40D, C177H, L273R;

F40D L80C A121 D P124A L125R

C151 R L273R;

F40D, I175D;

F40D, I175D, C177Q;

C9Y, F40D, C177H, L273R;

F40S, V93E;

F40S, C177Q;

F40S, C177D;

F40S, C177S;

F40S, C177F;

F40S, C177H;

C9Y, F40S;

C9Y, F40S, C177Q;

C9F, F40S;

C9F, F40S, C177Q;

C9W, F40S;

C9W F40S, C177Q;

G10A, F40S;

G10A, F40S, C177Q;

G10S, F40S;

G10S, F40S, C177Q;

G10A F40S F110H A121 D Q150H

C151 R C177Q Q239S;

F40S, L273Q;

F40S, L273R;

F40S, C177H, L273R;

F40S Q150H C151 R C177H Q239S

L273R;

F40S Q150H C151 R C177Q Q239S

L273R;

F40S, I175D;

F40S, I175D, C177Q;

C9Y, F40S, C177H, L273R;

F40T, V93E;

F40T, C177Q;

F40T, C177D;

F40T, C177S;

F40T, C177F;

F40T, C177H;

C9Y, F40T;

C9Y, F40T, C177Q;

C9F, F40T;

C9F, F40T, C177Q;

C9W, F40T; C9W F40T, C177Q;

G10A, F40T;

G10A, F40T, C177Q;

G10S, F40T;

G10S, F40T, C177Q;

F40T, L273Q;

F40T, L273R;

F40T, C177H, L273R;

F40T, I175D;

F40T, I175D, C177Q; and

C9Y, F40T, C177H, 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.

22. The 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

NO:1.

23. The alpha-1 ,2-fucosyltransferase variant according to according to item 22, wherein the variant further comprises the following substitution Q150H, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 24. The alpha-1 ,2-fucosyltransferase variant according to item 20 or 23, wherein the variant comprises one of the following combinations of substitutions:

C9Y Q150H, C151 R, Q239S;

C9W Q150H, C151 R, Q239S,;

C9F Q150H, C151 R, Q239S,

G10A Q150H, C151 R, Q239S;

G10S Q150H, C151 R, Q239S;

V93E Q150H, C151 R, Q239S,;

Q150H, C151 R, Q239S, I175D;

Q150H, C151 R, Q239S, C177H;

Q150H, C151 R, Q239S, C177Q;

Q150H, C151 R, Q239S, C177D;

Q150H, C151 R, Q239S, C177S;

Q150H, C151 R, Q239S, L273R;

F110H, Q150H, C151 R, C177Q Q239S

L80C F110H A121 D P124A L125R Q150H C151 R C177Q Q239S

F110H, Q150H, C151 R, C177Q, Q239S, K297D;

F110H, Q150H, C151 R, C177Q, Q239S, K297Q;

Q150H, C151 R, I175D, C177Q, Q239S, K297Q;

C9Y, F110H, Q150H, C151 R, C177Q, Q239S;

G10A, F110H, Q150H, C151 R, C177Q, Q239S;

G10A S39W F110H A121 D Q150H C151 R C177Q Q239S

F110H, Q150H, C151 R, C177H Q239S

F110H, Q150H, C151 R, C177H, Q239S, K297D; F110H, Q150H, C151R, C177H, Q239S, K297Q;

Q150H, C151R, I175D, C177H, Q239S, K297Q;

F40S, Q150H, C151 R, C177H, Q239S, L273R;

Q150H, C151 R, I175D, C177H, Q239S, K297Q;

F110H, Q150H, C151 R, C177H, Q239S L273R;

S39W F110H Q150H C151 R C177H Q239S L273R

C9Y, F110H, Q150H, C151 R, C177H, Q239S, L273R; or

G10A, F110H, Q150H, C151 R, C177H, 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

25. The 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 .

26. The alpha-1 , 2-fucosyltransferase variant according to item 25, wherein the variant comprises one of the following combinations of substitutions:

L80C F110H A121D P124A L125R C151 R C177Q;

L80C A121 D P124A L125R C151 R C177Q;

L80C F110H C177Q;

L80C A121 D P124A L125R C151 R L273R;

L80C F110H A121 D P124A L125R Q150H C151R C177Q Q239S;

L80C F110H A121 D P124A L125R C151 R L273R; or

L80C A121 D P124A L125R Q150H C151 R Q239S; 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. 27. The alpha-1 , 2-fucosyltransferase variant according to any one of the preceding items, wherein the variant further comprises one of the substitutions A121 D and/or P124A, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.

28. The alpha-1 , 2-fucosyltransferase variant according to item 25, wherein the variant comprises one of the following combinations of substitutions:

L80C F110H A121 D P124A L125R C151 R C177Q;

L80C A121 D P124A L125R C151 R C177Q;

G10A F40S F110H A121 D Q150H C151 R C177Q Q239S;

L80C A121 D P124A L125R C151 R L273R;

L80C F110H A121 D P124A L125R Q150H C151R C177Q Q239S;

G 10A S39W F110H A121 D Q150H C151 R C177Q Q239S;

L80C F110H A121 D P124A L125R C151 R L273R;

F110H A121 D C177Q;

L80C A121 D P124A L125R Q150H C151 R Q239S; or

F110H A121 D P124A L125R C177Q; 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. The alpha-1, 2-fucosyltransferase variant according to items 15 to 24, wherein the variant has increased alpha- 1, 2-fucosyltransferase activity on lactose compared to the alpha-1, 2- fucosyltransferase of SEQ ID NO: 1. The alpha-1, 2-fucosyltransferase variant according to item 29, wherein the variant comprises one of the following combinations of substitutions:

F110S C177Q Q239S K297Q;

L80C F110H A121D P124A L125R C151R C177Q;

F110H C177Q Q239S;

L80C A121D P124A L125R C151R C177Q;

C9F F110H C177Q Q239S;

F110H C177H Q239S;

G10A F40S F110H A121D Q150H C151R C177Q Q239S;

F110H C177S Q239S;

L80C F110H C177Q;

F110H Q150H C151RC177Q Q239S;

F110H Q150H C151RC177Q Q239S K297D;

F110H Q150H C151R C177Q Q239S K297Q;

F110S C177Q Q239S;

G10AF110H C177Q Q239S;

L80C A121D P124A L125R C151R L273R;

L80C F110H A121D P124A L125R Q150H C151R C177Q Q239S;

F110H P124AC177Q;

G10AS39WF110H A121DQ150H C151RC177Q Q239S;

L80C F110H A121D P124A L125R C151R L273R;

C9Y F110H C177Q Q239S;

F110H C177Q;

S39WF110H Q150H C151RC177H Q239S L273R;

G10S F110H C177Q Q239S;

F110H C177Q K297D;

F110H A121DC177Q;

F40S Q150H C151R C177H Q239S L273R;

F110H A121D P124A L125R C177Q;

F110H P124A L125R C177Q;

F110S C177Q;

C9Y F110H Q150H C151R C177Q Q239S;

F110AC177H H276Y

G10AF110AC177Q;

V93E F110H;

C9Y F110H C177Q;

V93E Q150H C151R Q239S;

F110H L273R;

F110H C177F; or

S39WF110H 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. The alpha-1, 2-fucosyltransferase variant according to any of items 1 to 18, wherein 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. The alpha-1 , 2-fucosyltransferase variant according to any of items 1 to 18, wherein 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: 37. The alpha-1 , 2-fucosyltransferase variant according to any of items 1 to 18, wherein 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: 38. The alpha-1 , 2-fucosyltransferase variant according to any of items 1 to 18, wherein 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: 39. The alpha-1 , 2-fucosyltransferase variant according to any of items 1 to 18, wherein 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: 40. The alpha-1 , 2-fucosyltransferase variant according to any of items 1 to 18, wherein 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: 41. The alpha-1 , 2-fucosyltransferase variant according to any of items 1 to 18, wherein 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: 42. The alpha-1 , 2-fucosyltransferase variant according to any of items 1 to 18, wherein 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: 43. The alpha-1 , 2-fucosyltransferase variant according to any of items 1 to 18, wherein 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: 48. The alpha-1 , 2-fucosyltransferase variant according to any of items 1 to 18, wherein 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: 49. The alpha-1 , 2-fucosyltransferase variant according to any of the preceding items, wherein the variant 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 . The alpha-1 , 2-fucosyltransferase variant according to any of the preceding items, wherein the variant has an R/Arg at position 45, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1. 43. The alpha-1 , 2-fucosyltransferase variant according to any of the preceding items, wherein the variant has a D/Asp at position 171 , wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.

44. 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.

45. The genetically engineered cell according to item 44, wherein the fucosylated oligosaccharide produced by said cell is 2’FL.

46. The genetically engineered cell according to item 44 or 45, wherein the cell produces at least 5% more 2’FL, such as at least 10% more 2’FL, relative to the 2’FL produced by a similar strain expressing the parent alpha-1 , 2-fucosyltransferase, such as the alpha-1 ,2- fucosyltransferase of SEQ ID NO: 1.

47. The genetically engineered cell according to item 44 to 46, wherein the cell produces less than 2%, such as less than 1.5%, such as less than 1%, such as less than 0.8%, such as less than 0.5% of DFL relative to the content of 2’FL produced by said cell.

48. The genetically engineered cell according to any of the items 44 to 47, wherein the alpha- 1 , 2-fucosyltransferase variant is expressed from a recombinant nucleic acid sequence.

49. The genetically engineered cell according to item 48, wherein the recombinant nucleic acid sequence is 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) and variants thereof.

50. The genetically engineered cell according to items 48 or 49, wherein the recombinant nucleic acid sequence is expressed from a plasmid or is genomically integrated.

51 . The genetically engineered cell according to any of the items 48 to 50, wherein the cell comprises two or three or more genomic copies of the recombinant nucleic acid sequence encoding the a-1 , 2-fucosyltransferase variant according to any one of items 1 to 30.

52. The genetically engineered cell according to any of items 44 to 51 , wherein the cell further comprises a recombinant nucleic acid sequence encoding a recombinant colanic acid (CA) gene cluster.

53. The genetically engineered cell according to any of items 44 to 52, wherein the cell further comprises a lactose importer, such as a lactose permease.

54. The genetically engineered cell according to any of items 44 to 53, wherein the cell further comprises a nucleic acid sequence encoding an MFS transporter protein capable of exporting the fucosylated HMO out of the cell. 55. The genetically engineered cell according to item 54, wherein the MFS transporter comprises or consists of SEQ ID NO: 4 or a functional homologue with at least 80% identity thereto.

56. The genetically engineered cell according to any of the preceding items, wherein the genetically engineered cell is capable of producing or importing LNT and wherein the fucosylated oligosaccharide is LNFP-I.

57. The genetically engineered cell according to item 56, wherein the cell further comprises one or more recombinant nucleic acid sequence(s) encoding a |3-1 ,4-galactosyltransferase.

58. The genetically engineered cell according to item 56 or 57, wherein the cell further comprises one or more recombinant nucleic acid sequence(s) encoding a [3-1 ,3-N- acetylglucosaminyltransferase.

59. The genetically engineered cell according to any of items 56 or 57, wherein the cell comprises a substrate importer selected from a lacto-N-triose-l I (LNT-II) importer or a LNT importer.

60. The genetically engineered cell according to any one of items 44 to 59, wherein said engineered cell is a microorganism, preferably a bacterium or a fungi.

61 . The genetically engineered cell according to item 60, wherein the microorganism is selected from the group consisting of Escherichia Coll, Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

62. The genetically engineered cell according to any of one of items 44 to 61 , wherein said engineered cell is an E. coll strain.

63. A nucleic acid construct comprising a recombinant nucleic acid sequence encoding an a-

1 ,2-fucosyltransferase variant, wherein said recombinant nucleic acid sequence consists or comprises of a nucleic acid sequence encoding an 1 ,2-fucosyltransferase variant according to any one of items 1 to 43.

64. The nucleic acid construct according to item 63, wherein the nucleic acid construct is a plasmid, preferably a high copy number plasmid capable of encoding the recombinant nucleic acid sequence.

65. The nucleic acid construct according to item 63, wherein the nucleic acid construct is an expression cassette/cartridge suitable for integration into the genome of a host cell.

66. The nucleic acid construct according to item 63 to 65, wherein the recombinant nucleic acid sequence is regulated by a promoter sequence, wherein said promoter is also present in the nucleic acid construct. The nucleic acid construct according to item 66, wherein said promoters is selected from the group consisting of PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 19, 28, 16, 17 and 18) and variants thereof, preferably the variants of table 2. A method for producing at least one fucosylated oligosaccharide, preferably 2’FL, wherein said method comprises the steps of a. Providing a genetically engineered cell according to any one of items 44 to 62; b. Culturing/fermenting the genetically engineered cell of a. in a suitable cell culture medium to produce said fucosylated oligosaccharide, preferably 2’FL; and optionally c. purifying said fucosylated oligosaccharide, preferably 2’FL. The method according to item 68, wherein the fucosylated oligosaccharide is a human milk oligosaccharide (HMO). The method according to item 68 or 69, wherein said method primarily produces 2’FL. The method according to any one of items 68 to 70, wherein prior to purification less than 2% DFL is produced, such as less than 1.5%, such as less than 1%, such as less than 0.8%, such as less than 0.5% DFL is produced, relative to the amount of 2’FL produced by said method. The method according to any one of items 68 to 71 , wherein said method produces at least 5% more 2’FL, such as at least 10% more 2’FL, relative to the 2’FL produced in the same method using a similar strain expressing the parent alpha-1 , 2-fucosyltransferase, such as the alpha-1 , 2-fucosyltransferase of SEQ ID NO: 1. The method according to any of items 68 to 71 , wherein 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 method according to any of items 68 to 73, wherein the genetically modified cell is cultivated in in the presence of lactose as the substrate for the a-1 , 2-fucosyltransferase variant. The method according to item 68, wherein said method produces LNFP-I. The method according to any of items 75, wherein the genetically modified cell is cultivated in in the presence of lactose to produce LNT as the substrate for the a-1 ,2- fucosyltransferase variant. The method according to item 68 or 75, wherein the genetically modified cell is cultivated in in the presence of LNT-II or LNT. 78. The method according to any one of items 68 to 77, wherein the genetically engineered cell is cultivated in the presence of a carbon source selected from sucrose, glucose, glycerol, fructose and xylose.

79. Use of a a-1 ,2-fucosyltransferase variant according to any one of items 1 to 43 in the production of 2’FL or LNFP-I or a mixture thereof.

80. Use of a genetically engineered cell according to any one of items 44 to 62, or a nucleic acid construct according to any one of items 63 to 66, in the production of 2’FL or LNFP-I or a mixture thereof.

SEQUENCES The current application contains a sequence listing in text format and electronical format which is hereby incorporated by reference.

An overview of the SEQ ID NOs used in the present application can be found in table 5.

Table 5 - sequences in the application

EXAMPLES

Methods

Unless stated otherwise, standard techniques, vectors, control sequence elements, and other expression system elements known in the field of molecular biology are used for nucleic acid manipulation, transformation, and expression. Such standard techniques, vectors, and elements can be found, e.g., in: Ausubel et al. (eds.), Current Protocols in Molecular Biology (1995) (John Wiley & Sons); Sambrook, Fritsch, & Maniatis (eds.), Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, NY); Berger & Kimmel, Methods in Enzymology 152: Guide to Molecular Cloning Techniques (1987) (Academic Press); Bukhari et al. (eds.), DNA Insertion Elements, Plasmids and Episomes (1977) (Cold Spring Harbor Laboratory Press, NY); Miller, J.H. Experiments in molecular genetics (1972.) (Cold spring Harbor Laboratory Press, NY)

The examples described below are selected to illustrate the invention and are not limiting the invention in any way.

Enzymes

The alpha-1 , 2-fucosyltransferase variants tested in the present examples were generated using the alpha-1 , 2-fucosyltransferase with SEQ ID NO: 1 as the parent sequence. The nucleic acid sequences encoding the variants were ordered at Twist Bioscience or ThermoFisher Scientific. The sequences were either introduced into a host strain (see below) on a plasmid or integrated into the genome as a single copy.

Table 6. List of the variant enzymes tested in the framework disclosed herein

*The sequence of the variants corresponds to SEQ ID NO: 1 except for the indicated substitutions

Strains

The E. coll strains (genetically engineered cells) constructed in the present application were based on Escherichia coli K-12 DH1 with the genotype: F", A~, gyrA96, recA1, relA1, endA1, thi- 1, hsdR17, supE44. Additional modifications were made to the E. coli K-12 DH1 strain to generate the MDO strain with the following modifications: lacZ: deletion of 1 .5 kbp, /acA: deletion of 0.5 kbp, nanKETA'. deletion of 3.3 kbp, melA'. deletion of 0.9 kbp, wcaJ deletion of 0.5 kbp, mdoH’. deletion of 0.5 kbp, and insertion of Plac promoter upstream of the gmd gene.

The MDO strain was further engineered by inserting an additional colonic acid cluster (gmd- wcaG-wcaH-wcal-manC-manB, SEQ ID NO: 5) under the control of a PglpF promoter (SEQ ID NO: 19) at a locus that is different than the native locus and lacl was deleted (MDO-CA strain).

For initial screening the parent alpha-1 ,2-fucosyltransferase, FutC with SEQ ID NO: 1 or an alpha-1 , 2-fucosyltransferase variant was expressed under the control of a PglpF promoter (SEQ ID NO: 19) from a low copy number plasmid in the MDO-CA strain.

For the second round of screening the parent alpha-1 , 2-fucosyltransferase, FutC with SEQ ID NO: 1 or an alpha-1 ,2-fucosyltransferase variant was expressed from a single gene copy inserted into the genome under the control of a PglpF promoter (SEQ ID NO: 19). The MDO-CA strain was further modified expressing the MFS transporter nec (SEQ ID NO: 4) and the scrYA, scrBR operons encoding the proteins facilitating growth on sucrose ( SEQ ID NO: 32 (ScrY) and 33 (ScrA) and SEQ ID NO: 34 (ScrB) and 35 (ScrR)) under control of the Pscr promoter (SEQ ID NO: 35). Methods of inserting or deleting gene(s) of interest into the genome of E. coli are well known to the person skilled in the art. Insertion of genetic cassettes into the E. coli chromosome can be done using gene gorging (see e.g., Herring and Blattner 2004 J. Bacteriol. 186: 2673-81 and Warming et al 2005 Nucleic Acids Res. 33(4): e36) with specific selection marker genes and screening methods.

Deep well assay for plasmid strains

Deep Well Assays in the current examples were performed as originally described by Lv et al (Bioprocess Biosyst Eng 20 (2016) 39:1737 — 1747) and optimized for the purposes of the current invention. More specifically, the strains disclosed in the present example were screened in 96 deep well plates (DWPs) using a 5 or 6 day protocol. During the first 24 hours, precultures were inoculated by picking colonies from agar plates into multi well plates (MTP’s) and grown to high densities (OD600 up to 5). Optionally, the precultures were frozen in 50% Glycerol until use. Biomass from either fresh or thawed pre-cultures was transferred to fresh growth medium for overnight growth to form a Starter culture. The starter culture was transferred to a medium in DWP’s that allowed induction of gene expression and product formation in main cultures for 3 days. On day 5 or 6 after initiation of the preculture, samples were collected.

More specifically, during day 1 , precultures were prepared by picking colonies from agar plates into multi well plates (MTPs) with 150uL LB media composed of: Tryptone 10g/L, NaCL 10g/L, Yeast extract 5g/L and pH set to 7.0 using 5M NaOH. Precultures were inoculated overnight (~20hrs) at 37°C, 1000 RPM shaking and 80% humidity after which optionally 50% glycerol was added, allowing plates to be stored at -80 °C. To start the screening on day 1 or 2, 15uL from fresh precultures or thawed frozen plates was used to inoculate 350uL starter cultures in half well deep plates (HDWP’s) in a basal minimal medium (BMM) (pH 7,0) supplemented with magnesium sulphate (0.12 g/L), thiamine (0.004 g/L) and glucose (5.5 g/L). Basal Minimal medium had the following composition: NaOH (1 g/L), KOH (2.5 g/L), KHzPO4 (7 g/L), NH&HzPO4 (7 g/L), Citric acid (0.5 g/l), trace mineral solution (5 mL/L). The trace mineral stock solution contained; ZnSO~*7H~O 0.82 g/L, Citric acid 20 g/L, MnSO4*H&O 0.98 g/L, FeS04*7H&0 3.925 g/L, CuSO4*5H~O 0.2 g/L. The pH of the Basal Minimal Medium was adjusted to 7.0 with 5 N NaOH and autoclaved. After 20hrs of growth at 34°C, 1000 RPM shaking and 80% humidity, on day 2 or 3, 15uL Starter culture was then transferred into 0.75 mL of a new BMM (pH 7,5) in DWP’s to start the main culture. The new BMM was supplemented with magnesium sulphate (0.12 g/L), thiamine (0.02 g/L), a bolus of glucose solution (0.1-0.15 g/L) and a bolus of either lactose solution (20 g/L) or 2’FL solution (20 g/L) leading to 2 parallel screens. Moreover, a 20 % stock solution of maltodextrin (19-20 g/L) was provided as carbon source, accompanied by the addition of a hydrolytic enzyme with glycoamylase activity, such that glucose was released at a rate suitable for carbon-limited growth and similar to that of a typical fed-batch fermentation process. The main cultures were incubated for 72 hours at 28 °C and 1000 rpm shaking. On day 5 or 6 for the analysis of total broth, the 96 DWP’s were boiled at 100°C in a water bath, subsequently centrifuged, and finally the supernatants were analysed by HPLC to assess 2FL and DFL productivities of FutC variants.

Deep well assay for genomic integrated strains

The strains were screened in 96 deep well plates using a 5 day protocol for assessment of HMO production. During the first 24 hours, fresh precultures were grown to high densities. More specifically, fresh precultures were prepared using a basal minimal medium supplemented with magnesium sulphate, thiamine and glucose. The pH was adjusted to pH 7.0 with NaOH. The precultures were incubated for 24 hours at 34 °C and 1000 rpm shaking.

For HMO production an aliquot of the pre-culture was transferred to a new deep well plate with basal minimal medium (BMM7, pH 7,5) to start the main culture. The new BMM was supplemented with thiamine and magnesium sulphate, 0.01% of glucose, 2% lactose, 1.75 % of Maltodextrin, thiamine, and Glucoamylase for optimized hydrolysis of maltodextrin. The HMO main cultures were incubated for 72 hours at 28°C and 1000 rpm shaking. The assay was generally performed in triplicates.

To be able to compare results across plates all plates contained the FutC parent strain and the variants were normalized to the FutC parent strain on the same plate.

After incubation of the main cultures were boiled and centrifuged. Samples were analysed by HPLC for 2’FL, DFL and 3FL.

Fermentation

The E. coli strains were cultivated in 250 mL fermenters (Ambr 250 Bioreactor system, Sartorius) starting with 100 mL of mineral culture medium consisting of 8.5 g/kg sucrose, lactose monohydrate, H₃PO₄, MgSO₄ x 7H₂O, KOH, citric acid, trace element solution, antifoam and thiamine. The dissolved oxygen level was kept at 20% by a cascade of first agitation and then airflow starting at 1200 rpm (up to max 4500 rpm) and 0.1 WM (up to max 3 WM). The pH was set and kept at 6.8 by titration with 8.5% NH₄OH solution. The cultivations were started with 2% (v/v) inoculums from pre-cultures grown in a similar sucrose containing medium. After depletion of the sucrose contained in the batch medium, a feed solution containing sucrose, lactose (supplied in sufficient quantity to not become limiting for the 2’FL yield) and the basal medium components was continuously added to the fermenter at a rate that maintained carbon-limiting conditions. The temperature was set to 33°C. The growth and metabolic activity and state of the cells were followed by on-line measurements of reflectance and CO2 evolution rate. Throughout the fermentations, samples were taken to determine the concentration of HMO products, lactose and other minor by-products using HPLC. Example 1 - Amino acid scan at selected single positions

The following example aimed to identify positions and substitutions in the parent alpha-1 , 2- fucosyltransferase of SEQ ID NO: 1 , FutC, with an impact on the enzymes ability to form DFL, e.g., with an impact on the alpha- 1 ,3-fucosyltransferase side-activity.

The following positions C9, G10, S39, S43, R45, E91 , V93, F110, Y112, 1175, C177, L273, K297 and Y298 in the alpha-1 ,2-fucosyltransferase of SEQ ID NO: 1 were selected and substituted with all 19 amino acids.

A library of nucleic acid sequences encoding individual variants with the mutations under control of the PglpF promoter was generated. A library of plasmids was build using golden gate cloning technology. The plasmid used was a low copy number plasmid with P15A origin. The plasmid library was transformed into competent E. coli host cells, with the genotype described under “strains” in the method section, using heat shock.

The transformed cells were screened for their ability to produce 2’FL and DFL using the deep well assay for plasmid strains described in the method section above.

The formation of 2’FL and DFL by the variant strains was assessed relative to the 2’FL and DFL, respectively, formation of the parent FutC strain.

The results are shown in Figure 3A (2’FL) and 3B (DFL). To select individual mutations for future variants it was desired to pick variant strains which were not significantly impaired in the 2’FL formation (did not have less than 0.8 relative to the parent FutC (wt)) while they show low DFL formation relative to the parent FutC (wt) (below 0.75 relative to the parent FutC).

Based on this the following individual mutants C9Y,N,S,G,A,K; S39A,W; V93E,D, I175D; C177H,Q,D,S and L273R.Q were identified as potentially relevant for reduction of DFL, and F110A,H,S and K297D,Q,Y were identified as potential 2’FL increasers without increasing DFL significantly.

Example 2 - Deep well screening of selected variants

In the present example the most promising individual mutants identified in example 1 , were screened as single genomic inserts in the host cell described above and compared to the same strain with the parent alpha- 1 ,2-fucosyltransferase of SEQ ID NO: 1.

In addition, variants with multiple mutations were made. These contained various combinations of the single mutations identified in example 1 potentially in combination with mutations at one or more of the additional positions F40, Q150, C151 and Q239 in SEQ ID NO: 1 which have previously been described to increase the 2’FL (and DFL) formation in a genetically modified cell (Park et al 2022 Biotechnol Bioeng. 119:1264-1277). The tested variants are disclosed in table 6 above and their ability to produce 2’FL and DFL was assessed using the Deep well assay for genomically integrated strains described above. The samples were at least tested as replicates of 3. The results of the assay is shown in table 7 and figure 4A and B. 2’FL is calculated relative to the 2’FL produced by the strain containing the reference enzyme, FutC, which is set to 100%. The percentage of DFL is calculated relative to the 2’FL produced by the same strain, e.g., % DFL in M7 variant strain is relative to the amount of 2’FL produced by the M7 strain.

Table 7 Content of 2’FL as % of 2’FL content produced by the parent FutC strain and DFL as % of 2’FL produced by the same strain No 3FL was identified in any of the samples.

From the data it can be seen that all the single mutations at position C9, 1175, C177 and L273 reduce DFL levels to 0.5% or less relative to the 2’FL produced by the strain (group i). Of these variants C9Y,G, C177S,H, I175D and L273R maintain 2’FL levels that are not reduced by more than 70% of the parent strain C177D is slightly lower in 2’FL production, but it may still be beneficial if combined with 2’FL increasing mutations (group ii)). It cannot be concluded if the combinations of multiple mutations from group i) in the absence of 2’FL increasing mutations (e.g., M15 and M14) have an additional negative impact on 2’FL formation or whether the mutation K297Q has a negative impact on these combination variants.

It can also be seen that the known mutations Q239S (P1_Q239S) and Q150H, C151 R (P2) and F40S, Q150H, C151 R, Q239S (P4) indeed do increase both 2’FL and DFL when compared to the parent strain, FutC, so these are considered group ii) mutations. It also appears that substitution at position F40 to a D has a positive effect on 2’FL activity in that it increases 2’FL formation in the L273R variant (M1 vs S_L273R).

Mutations in position F110, which was identified in example 1 as a possible 2’FL increaser, seem to have a positive impact on group i) mutants, see for example M7 and M8. F110 mutations can therefore be considered as group ii) mutations. The mutation H276Y in M8 was unintentional and unlikely to have any significant impact since it is not in the active site of the enzyme and it is not an uncommon variation in other wild type H. pylori alpha-1 , 2- fucosyltransferases. Mutations in position R45E in combination with C177Q or F110A seem to severely affect the ability of the variant to produce 2’FL.

From these data it can be seen that the combination of group i) and group ii) mutations can lead to variants that have significantly decreased DFL compared to the parent strain, FutC, and which are able to maintain 2’FL levels above 85% of the parent strain. Specifically, variants M7, M9, M8, M5, M4 and M1 show this ability and variants M7, M9, M8 and M5 all show improved 2’FL over the parent strain.

Example 3 - 2’FL synthesis by fermentation of selected variants

The two most promising variant, M7 (F110H Q150H C151R C177Q Q239S K297D), identified in Example 2 was fermented as described in the “Method” section to confirm that the increased 2’FL formation and reduced DFL formation was reproducible in larger scale.

The results are shown in table 8, where the 2’FL and DFL are calculated relative to the 2’FL produced by the parent FutC strain and in table 9, where the DFL is calculated relative to the 2’FL produced by the strain. Furthermore the molar ratio of 2’FL:DFL is given in table 9.

Table 8: Content of individual HMO’s as % of 2’FL content produced by the parent FutC strain at the end offermentation Table 9: Relative content of individual HMO’s as % of 2’ FL content produced by the specific strain at the end of fermentation

Table 9a: Relative content of individual HMO’s as % of total oligosaccharide content produced by the specific strain at the end of fermentation.

From the data presented table 8, it can be seen that the strain expressing the M7 alpha-1 ,2 fucosyltransferase variant produced about 25% more 2’FL, compared to the parent FutC strain. From table 9 and 9a it can be seen that the amount of DFL produced by the M7 strain is well below 0.5% of the total amount of 2’FL produced by the M7 strain and that the 2’FL can be increased to 99.71% of the total amount of oligosaccharide in the fermentation, which is a very high purity of 2’FL in the fermentation broth.

Accordingly, the fermentation shows that the M7 variant containing the following mutations F110H, Q150H, C151R, C177Q, Q239S, K297D in SEQ ID NO: 1 is especially suited for production of 2’FL, due to its enhanced production levels of 2’FL compared to FutC, but also due to the almost 7-fold reduction in DFL levels, which enables production of very pure 2’FL, directly from fermentation.

Example 4 - Designing further variants with increased 2’FL and decreased DFL formation Based on the results from Example 2, 81 additional mutants were designed (See PE mutants in table 6) to assess the effect of substitutions in specific positions.

All the variants in table 6 were tested for their ability to produce 2’FL and DFL in the deep well assay for genomically integrated strains described above, meaning that the strains from example 2 were tested again to make the data more comparative with the new variants. The strains were at least tested as replicates of 3. The result of the assay is shown in table 10. 2’FL is calculated relative to the 2’FL produced by the strain containing the reference enzyme, FutC of SEQ ID NO: 1 , which is set to 100%. The percentage of DFL is calculated relative to the 2’FL produced by the same strain, e.g., % DFL in PE125 variant strain is relative to the amount of 2’FL produced by the PE125 variant strain.

In addition, the 2’FL data from table 10 is presented in Figure 5 ranked with the highest 2’FL producer at the top. Figure 6 shows the production of DFL relative to the 2’FL produced within the same strain while maintaining the ranking according to 2’FL production in Figure 5. Table 10: Content of 2’ FL as % of 2’ FL content produced by the parent FutC strain and DFL as % of 2’FL produced by the same strain

Of the 107 different variant strains generated, 52 show an increase in 2’FL relative to the parent FutC (SEQ ID NO: 1) of these 52 variant strains 40 produced less DFL relative to the 2’FL produced by the same strain, when compared to the 2.14% DFL produce by the parent FutC. 32 of the variant strains produced less than 0.5% DFL of the total amount of 2’FL produced by the same strain. Specifically, the 32 strains are PE125, PE128, PE116, PE126, PE122, PE119, PE133, PE135, PE58, M7, PE60, PE117, PE120, PE129, PE114, PE137, PE132, PE131 , PE124, M9, PE148, PE121 , PE136, M5/PE11 , PE139, PE138, PE39, PE61 , PE75, PE8, PE44 and PE46, of these 29 variant strains contain a substitution in C177, five variant strains contain a substitution in position L273, four variant strain contain a substitution in position C9, five variant strains contain a substitution in position G10, two variant strains contain a substitution in position W39.

Variants with the I175D substitution all have reduced DFL formation. The I175D substitution does however also impact the formation of 2’FL. For example, PE63, PE50, PE49 all reduce DFL to below 0.5 % of the 2’FL produced by the cell while 2’FL is not reduced by more than 30% compared to the parent FutC. The substitution, I175D, should preferably not be combined with a substitution in L273R, since this may result in the formation of a salt bridge between the carboxylate of 175D and the guanidine group of Arginine in 273R.

It can also be seen that mutating the conservative position R45 (M10 and M11) and D171 (PE16) seem to have a negative impact on the fucosyltransferase activity of the enzyme. Below, the data set from table 10 is broken down to assess the contribution of some of the individual substitutions in more detail.

C177 substitutions as single DFL reducing substitution

The data set in table 10 contain 59 variants where position C177 has been mutated. In table 11 variants where C177 substitutions is the only DFL reducing (group i) substitution is show. The variants are sorted in groups with similar substitutions for comparative purposes. In addition, the %DFL relative to the total HMO produced by the cell DFL/HMO is calculated relative to the %DFL relative to total HMO of the parent FutC strain (DFL/HMO rel. to FutC_wt DFL/HMO), to illustrate the DFL is also reduced when compared to the parent FutC.

Table 11: Content of2’FL as % of 2’FL content produced by the parent FutC strain and DFL relative to DFL in the parent strain and DFL as % of 2’FL produced by the same strain

All C177Q,H,S,F,D substitutions decrease DFL significantly without resulting in total loss of 2’FL. C177D however has quite a negative impact on 2’FL production, which in part can be compensated by combining C177D with a group ii) substitution such as F110H (PE40).

Most strains with a C177Q,H,S,F variants in combination with one or more 2’FL increasing (group ii) substitutions has an at least 50% reduction in DFL while maintaining or increasing 2’FL.

Generally, C177Q appear to be better in reducing DFL while maintaining 2’FL formation.

Although, mutants with C177H also can be favourable, in particular if combined with the L273R substitution. In addition to table 11 above, table 12 below compares variant pairs with and without a C177 substitution.

Table 12: comparative variant pairs with and without a C177 substitution

These data illustrate the ability of most of the tested C177 substitutions to decrease DFL (bold). In addition, eight C177 variant strains are capable of increasing 2’FL by more than 5% (bold) when compared to a variant with the same substitutions except the C177 substitution. This is quite surprising since single C177 substitutions generally appear to decrease 2’FL compared to the parent FutC.

L273 substitutions for reducing DFL

The data set in table 10 contain 23 variants where position L273 has been substituted. They are all listed in table 13 below. The variant strains are sorted in groups with similar substitutions for comparative purposes. In addition, the %DFL relative to the total HMO produced by the cell DFL/HMO is calculated relative to the %DFL relative to total HMO of the parent FutC strain

(DFL/HMO rel. to FutC_wt DFL/HMO), to illustrate the DFL is also reduced when compared to the parent FutC.

Table 13: Content of 2 ’FL as % of 2 ’FL content produced by the parent FutC strain and DFL relative to DFL in the parent strain and DFL as % of 2’FL produced by the same strain

The L273R substitution is a highly efficient DFL reducing substitution and can bring the DFL level produced by the strain down to zero. The L273Q substitution appears to have a negative effect on 2’FL formation and is less efficient in reducing DFL than L273R.

The L273R substitution appear to have the lowest impact on 2’FL formation when it is not combined with other DFL reducing substitutions (e.g., PE129, PE131 , PE46, M1 and PE130).

Other DFL reducing substitutions that appear to have good compatibility with L273R substitutions are S39W and C177H (PE148 and M5). From figure 7 it can be seen that position C177 and R273 are in front of each other in the three-dimensional structure of the wt FutC, this leaves little space for two big amino acids. With an arginine in position 273 the structure therefore better accommodates the histidine (H) in position 177 than the larger glutamine (Q). In particular L273R combinations with I175D has a negative impact on 2’FL formation, most likely due to the formation of a salt bridge between the carboxylate of 175D and the guanidine group of arginine in 273R.

The L273R substitution should preferably be combined with substitutions capable of improving 2’FL formation significantly (e.g., PE129, PE131 , M5/PE11 , PE46, M1/PE19). In addition to table 13 above table 14 below, compares variant pairs with and without a L273 substitution.

Table 14: Comparative variant pairs with and without a L273 substitution

From this it can be seen that the L273R substitution is a very strong reducer of DFL, as it often is capable of reducing DFL to zero (bold). It can also be seen that the L273R substitution generally has some impact on 2’FL formation, but that this can be counter acted by combining with 2’FL increasing substitutions, in particular F110H and F40D (bold).

C9 substitutions for reducing DFL

The data set in table 10 contain 14 variants where position C9 has been mutated. In table 15 all the C9 substitutions are listed and compared with a similar variant without the C9 substitution. Table 15: Comparative variant pairs with and without a C9 substitution

C9F,Y,W all are good reducers of DFL. In particular C9Y and C9F does not seem to have any significant negative effect on 2’FL formation when combined with F110H or F110H and Q239S (PE43, PE122, PE124). G10 substitutions for reducing DFL

The data set in table 10 contain 9 variants where position G10 has been mutated, they are all listed in table 16 below. In addition, the %DFL relative to the total HMO produced by the cell DFL/HMO is calculated relative to the %DFL relative to total HMO of the parent FutC strain (DFL/HMO rel. to FutC_wt DFL/HMO), to illustrate the DFL is also reduced when compared to the parent FutC. Table 16: Content of 2’FL as % of 2’FL content produced by the parent FutC strain and DFL relative to DFL in the parent strain and DFL as % of 2’FL produced by the same strain

Table 17 below, in addition compares variant pairs with and without a G10 substitution.

Table 17: Comparative variant pairs with and without a G10 substitution From table 16 and 17 it can be seen that the substitution G10A and G10S are very efficient DFL reducers without resulting in too significant a 2’FL reduction when combined with at least an F110A or F110H substitution.

V93E substitutions for reducing DFL

The data set in table 10 include 3 variants with a substitution in position V93. In table 18 all the V93E substitutions are listed and compared with a similar variant without the V93Esubstitution. In addition, the %DFL relative to the total HMO produced by the cell DFL/HMO is calculated relative to the %DFL relative to total HMO of the parent FutC strain (DFL/HMO rel. to FutC_wt DFL/HMO), to illustrate the DFL is also reduced when compared to the parent FutC. Table 18: Content of 2’ FL as % of 2’ FL content produced by the parent FutC strain and DFL relative to DFL in the parent strain and DFL as % of 2’FL produced by the same strain

Variant strains with the substitution V93E appear to reduce DFL by at least 30% when compared the same mutant without the V39E substitution. V93E, as the only DFL reducing mutant, also seems to increase 2’FL slightly both when it is alone or when combined with a 2’FL increasing mutant, in particular when V39E combined with F110H (PE69).

S39 substitutions for reducing DFL

The data set in table 10 include 8 variants with a substitution in position S39. Table 19 below compare with mutants that do not have the S39 substitution. Table 19: Content of 2’FL as % of 2’FL content produced by the parent FutC strain and DFL as

% of 2’FL produced by the same strain

S39A,W mutants do have a reducing effect on DFL without impacting the 2’FL formation significantly. Of the S39A and S39W substitution, S39W seem to be preferred in that it has the highest DFL decrease and no impact or a slightly increase on 2’FL formation (PE24, PE68 vs PE23 and PE67 respectively). 2’FL increasing substitutions

Generally, it is desired to generate fucosyltransferase variants which do not have more than 15% reduction in the 2’FL formation compared to the parent FutC used to generate the variant.

Positions 50, 150, 151 and 239 have been published by Park et al (Biotechnol Bioeng. 2022119:1264-1277) and Positions 80, 121 , 124, 125 have been published in Lee et al (J

Biotechnol 2024 20:387:49-57) as having a benefit in increasing 2’FL titre. These positions have been tested without combining them with a DFL reducing substitution, see the following variants PE31 , PE32, P2, P1_Q239S, PE55, PE56, PE26, P4, PE28, PE46, PE113 in table 10. Except for Q150H alone and F40S alone all these substitutions have the ability to increase 2’FL and with that DFL is also increased.

In addition, the present disclosure has identified position F110 as a suitable position for increasing 2’FL when combined with at least one substitution that reduces DFL. Table 20 below compare varinats with and without a F110H or F110A substitution.

Table 20: Comparative variant pairs with and without a FHOsubstitution From this table it can be seen that the substituion F110H on its own does not have a large impact on 2’FL, and both the F110H and F 110A substitution have the ability to prevent a decrease in 2’FL when they are combined with a DFL reducing substitution, such as Q177H,Q,F; L273R; C9Y and I175D.

In particular the combination of two 2’FL increasing substitutions containing H110H or F110S with Q239S appear to provide a significant increase in 2’FL (see table 21 below). Table 21: Comparative variant pairs with a F11 OH or F110S substitution with or without Q239S substitution

Based on this is suggested that alpha-1 ,2-fucosyltransferase variants with one of the double substitutions H110H and Q239S or F110S and Q239S may function as 2’FL increasing substitutions that can be used independently of the DFL decreasing substitutions. Example 5 - Effect of substitutions on natural diverse of the a-1 ,2-fucosyltransferases The present example is made to illustrate that a similar effect on 2’FL and DFL formation can be achieved in other natural variants/FutC homologues from other Helicobacter pylori species. For this purpose, the substitutions in the alpha- 1 ,2-fucosyltransferase variant PE58 (F110H Q150H C151 R C177Q Q239S) with SEQ ID NO: 1 as the parent were introduced into four different alpha-1 ,2-fucosyltransferase Helicobacter pylori homologues with between 86% and 93% identity to the FutC of SEQ ID NO: 1 . The FutC homolouges and the PE58 homologue variants are shown in table 22. An alignment of SEQ ID NO: 1 (FutC-wt) and the four wildtype homologues are shown in figure 9, the positions that are substituted are shown in bold.

Table 22 Homologous Helicobacter pylori alpha-1 ,2-fucosyltransferases assessed using Emboss-Needle Matrix: EBLOSUM62 Gap_penalty: 10.0 Extend_penalty: 0.5

The parent sequences in Table 22 (except SEQ ID NO: 6) were compared to their corresponding variants using the deep well assay for genomic integrated strains described in the method section. The results are shown in in table 23.

Table 23: Content of 2’FL of the variant as % of 2’FL content produced by the parent FutC strain and DFL as % of 2’FL produced by the same strain.

From table 23 it can be seen that it is possible to use alpha-1 ,2-fucosyltransferase homologues from other Helicobacter pylori strains as the parent amino acid sequence to generate variants with substitutions in the positions equivalent to the substitutions in PE58 of SEQ ID NO: 1 and obtain an increase in 2’FL formation and a decrease in DFL. The alpha- 1 ,2-fucosyltransferase Helicobacter pylori homologue, WP_104758319.1 , did not produce any DFL, so here a further decrease of DFL was not possible, the 2’FL formation was however increased by the introduction of the substitutions in the WP_104758319.1 parent enzyme.

Claims

1 . 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 C177Q,H,S,F,D; L273R; C9F,N,G,W,A,K; G10A,S V93E; S39A,W and I175D, 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 , 3-fucosyltransferase activity of the alpha-1 ,2- fucosyltransferase of SEQ ID NO: 1.

2. The alpha-1 ,2-fucosyltransferase variant according to claim 1 , wherein the variant comprises 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.

3. The alpha-1 ,2-fucosyltransferase variant according to claim 1 or 2, wherein the variant comprises the following substitution L273R, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .

4. The alpha-1 ,2-fucosyltransferase variant according to claims 1 to 3, wherein the variant comprises one of the following substitutions:

C177H,Q,D,S,F;

C177H,Q,D,S,F and C9Y,F,W,G;

C177H,Q,D,S,F and G10A,S;

C177H,S,F and L273R;

C177H,Q,D,S,F and S39A,W;

C177H,Q,D,S,F and V93E;

L273R;

L273R and S39A,W;

L273R and C9Y,F,W,G;

L273R and C177H and C9Y; or L273R and C177H and S39W.

5. The alpha-1 ,2-fucosyltransferase variant according to any one of the preceding claims, 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 .

6. The alpha-1 ,2-fucosyltransferase variant according to any one of the proceeding claims, wherein the variant further comprises at least one of the following substitutions F40S,D; L80C; A121 D; P124A; L125R; F110A,H,S; Q150H; C151 R; Q239S and Q297Q.D, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1 .

7. The alpha-1 ,2-fucosyltransferase variant according to claim 5, wherein the variant comprises the following substitution F110S or F110H, wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.

8. The alpha-1 ,2-fucosyltransferase variant according to claim 5 or 7, wherein the variant further comprises the following substitution Q239S wherein the position corresponds to the position in the amino acid sequence of SEQ ID NO:1.

9. The alpha-1 ,2-fucosyltransferase variant according to any one of the proceeding claims, wherein the variant comprises one of the following combinations of substitutions:

F110S C177Q Q239S K297Q;

L80C F110H A121D P124A L125R C151R C177Q;

F110H C177Q Q239S;

L80C A121D P124A L125R C151R C177Q;

C9F F110H C177Q Q239S;

F110H C177H Q239S;

G10A F40S F110H A121D Q150H C151R C177Q Q239S;

F110H C177S Q239S;

L80C F110H C177Q;

F110H Q150H C151RC177Q Q239S;

F110H Q150H C151R C177Q Q239S K297D;

F110H Q150H C151R C177Q Q239S K297Q;

F110S C177Q Q239S;

G10AF110H C177Q Q239S;

L80C A121D P124A L125R C151R L273R;

L80C F110H A121D P124A L125R Q150H C151R C177Q Q239S;

F110H P124AC177Q;

G10AS39WF110H A121DQ150H C151RC177Q Q239S;

L80C F110H A121D P124A L125R C151R L273R;

C9Y F110H C177Q Q239S;

F110H C177Q;

S39WF110H Q150H C151RC177H Q239S L273R;

G10S F110H C177Q Q239S;

F110H C177Q K297D;

F110H A121DC177Q;

F40S Q150H C151R C177H Q239S L273R;

F110H A121D P124A L125R C177Q;

F110H P124A L125R C177Q;

F110S C177Q;

C9Y F110H Q150H C151R C177Q Q239S;

F110AC177H H276Y

G10AF110AC177Q;

S39W;

V93E F110H;

C9Y F110H C177Q;

V93E Q150H C151R Q239S;

V93E; F110H L273R;

F110H C177F; or

S39W F110H 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.

10. A genetically engineered cell capable of producing 2’FL, wherein the genetically engineered cell comprises an alpha-1 , 2-fucosyltransferase variant according to any one of claims 1 to 9.

11 . The genetically engineered cell according to claim 10, wherein the cell produces at least 5% more 2’FL relative to the 2’FL produced by a similar strain expressing the unmodified (parent) alpha-1 , 2-fucosyltransferase.

12. The genetically engineered cell according to claim 10 to 11 , wherein the cell produces less than 1% DFL relative to the content of 2’FL produced by said cell.

13. The genetically engineered cell according to any of claims 10 to 12, wherein said engineered cell is selected from the group consisting of Escherichia Coll, Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

14. A method for producing 2’FL, wherein the method comprises the following steps: a. providing a genetically engineered cell according to any one of claims 10 to 13; b. culturing the genetically engineered cell of a. in a suitable cell culture medium to produce 2’FL; and optionally c. purifying the 2’FL from the culture.

15. The method according to claim 14, wherein prior to purification less than 1 % DFL is produced relative to the amount of 2’FL produced by the method and the method produces at least 5% more 2’FL relative to the 2’FL produced in the same method using a similar strain expressing the parent alpha-1 , 2-fucosyltransferase, such as the alpha-1 , 2- fucosyltransferase of SEQ ID NO: 1.

16. Use of an alpha-1 , 2-fucosyltransferase variant according to any one of claims 1 to 9, in the production of a fucosylated oligosaccharide, such as 2’FL.