Fucosyltransferase mutants

Abstract

The present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation. The present invention relates to a de novo designed enzyme having alpha-1, 4- fucosyltransferase activity on the N-acetylglucosamine (GIcNAc) residue of Gal-β1,3-GlcNAc (lacto-N- biose, LNB), wherein said LNB resides in free form and / or is part of a saccharide. The present invention also relates to a de novo designed enzyme having alpha-1, 3-fucosyltransferase activity on the glucose (Glc) residue of Gal-β1,4-Glc (lactose). The present invention further relates to methods for the production of a fucosylated compound comprising Gal-β1,3-[Fuc-al,4]-GlcNAc and / or for the production of 3-fucosyllactose (3-FL, Gal-β1,4-[Fuc-al,3]-Glc). The present invention also provides a cell for expression of a de novo designed enzyme having alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB, wherein said LNB resides in free form and / or is part of a saccharide. The present invention also provides a cell for expression of a de novo designed enzyme having alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose. The present invention further relates to a cell for production of a fucosylated compound comprising Gal-β1,3-[Fuc-al,4]-GlcNAc. The present invention further relates to a cell for production of 3-FL. The present invention also relates to the use of said cells in a cultivation or incubation.

Description

[0001] Fucosyltransferase mutants

[0002] Field of the invention

[0003] The present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation. The present invention relates to a de novo designed enzyme having alpha-1, 4- fucosyltransferase activity on the N-acetylglucosamine (GIcNAc) residue of Gal-pi,3-GlcNAc (lacto-N- biose, LNB), wherein said LNB resides in free form and / or is part of a saccharide. The present invention also relates to a de novo designed enzyme having alpha-1, 3-fucosyltransferase activity on the glucose (Glc) residue of Gal-pi,4-Glc (lactose). The present invention further relates to methods for the production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or for the production of 3-fucosyllactose (3-FL, Gal-pi,4-[Fuc-al,3]-Glc). The present invention also provides a cell for expression of a de novo designed enzyme having alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB, wherein said LNB resides in free form and / or is part of a saccharide. The present invention also provides a cell for expression of a de novo designed enzyme having alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose. The present invention further relates to a cell for production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc. The present invention further relates to a cell for production of 3-FL. The present invention also relates to the use of said cells in a cultivation or incubation.

[0004] Background

[0005] Saccharides like e.g., disaccharides and oligosaccharides, are widely distributed in living organisms and are involved in many vital phenomena such as differentiation, development and biological recognition processes related to the development and progress of fertilization, embryogenesis, inflammation, metastasis, and host pathogen adhesion. Saccharides are often present as glyco-conjugated forms to proteins and lipids but can also be present as unconjugated glycans. An important group of free saccharides comprises mammalian milk oligosaccharides (MMOs) and human milk oligosaccharides (HMOs) found in mammalian and human milk, respectively. The Lewis a epitope Gal-pi,3-[Fuc-al,4]- GIcNAc-R is formed by alpha-1, 4-fucosylation of the N-acetylglucosamine (GIcNAc) residue of the type 1 core structure Gal-pi,3-GlcNAc, also known as lacto-N-biose or LNB. Lewis a (Lea) is a precursor for the synthesis of the Lewis b epitope Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-R (Leb). Both Leaand Lebare antigens of the Lewis blood group system and structurally related to the sugar determinants of the human ABH(O) blood group antigen system. Lewis epitopes are found on the surface of many cells and secreted in various body fluids and are reported to be involved in tumour metastasis, the pathogenesis of stomach disorders and embryo development. The Leaand Lebepitopes are often present in oligosaccharides present in human milk, such as for example in lacto-N-fucopentaose II (LNFP-II, Gal-pi,3-[Fuc-al,4]- GlcNAc-pi,3-Gal-pi,4-Glc), lacto-N-difucohexaose II (LNDFH-II, Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal- pi,4-[Fuc-al,3]-Glc) or in lacto-N-difucohexaose I (LNDFH I, Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3- Gal-pi,4-Glc). Another oligosaccharide that is present in human milk is 3-fucosyllactose (3-FL, Gal-pi,4- [Fuc-al,3]-Glc), which is formed by alpha-1, 3-fucosylation of the glucose (Glc) residue of lactose (Gal- pi,4-Glc). Although HMOs represent only a minor amount of total human milk nutrients, their beneficial effects on the development of breast-fed infants became evident over the past decades (Bode, Early Hum. Dev. 1-4 (2015). There is large scientific and commercial interest in these structures or compounds, yet the availability is limited.

[0006] Description

[0007] Summary of the invention

[0008] It is an object of the present invention to provide for tools and methods by means of which a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, 3-fucosyllactose (3-FL), a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or a mixture comprising 3-FL can be produced, preferably in an efficient, time and cost-effective way and which yields high amounts of the desired fucosylated compound, 3-FL, mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or mixture comprising 3-FL. According to the invention, this and other objects are achieved by providing a de novo designed enzyme, methods and a cell for production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, 3-FL a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or a mixture comprising 3-FL as described herein. Furthermore, the present invention provides a cell which is genetically engineered as described herein. This invention also provides a purified fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, purified 3-FL, a purified mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or a purified mixture comprising 3-FL by the above-referenced process. Further benefits of the teachings of this invention will be apparent to one skilled in the art from reading this invention.

[0009] Definitions

[0010] The words used in this specification to describe the invention and its various embodiments are to be understood not only in the sense of their commonly defined meanings, but to include by special definition in this specification structure, material or acts beyond the scope of the commonly defined meanings. Thus, if an element can be understood in the context of this specification as including more than one meaning, then its use in a claim must be understood as being generic to all possible meanings supported by the specification and by the word itself.

[0011] The various aspects and embodiments of the invention disclosed herein are to be understood not only in the order and context specifically described in this specification, but to include any order and any combination thereof. Each embodiment as identified herein may be combined together unless otherwise indicated. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Whenever the context requires, unless specifically stated otherwise, all words used in the singular number shall be deemed to include the plural and vice versa. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described herein are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications.

[0012] In the drawings and specification, there have been disclosed embodiments of the invention, and although specific terms are employed, the terms are used in a descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the following claims. It must be understood that the illustrated embodiments have been set forth only for the purposes of example and that it should not be taken as limiting the invention. It will be apparent to those skilled in the art that alterations, other embodiments, improvements, details and uses can be made consistent with the letter and spirit of the disclosure herein and within the scope of this disclosure, which is limited only by the claims, construed in accordance with the patent law, including the doctrine of equivalents. In the claims that follow, reference characters used to designate claim steps are provided for convenience of description only, and are not intended to imply any particular order for performing the steps, unless specifically stated otherwise.

[0013] Throughout the application, unless explicitly stated otherwise, the features "synthesize", "synthesized" and "synthesis" are interchangeably used with the features "produce", "produced" and "production", respectively. Throughout the application, unless explicitly stated otherwise, the expressions "capable of...<verb>" and "capable to...<verb>" are preferably replaced with the active voice of said verb and vice versa. For example, the expression "capable of expressing" is preferably replaced with "expresses" and vice versa, i.e., "expresses" is preferably replaced with "capable of expressing". In this document and in its claims, the verb "to comprise", "to have" and "to contain" and their conjugations are used in their nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Throughout the application, the verb "to comprise" may be replaced by "to consist" or "to consist essentially of" and vice versa. In addition, the verb "to consist" may be replaced by "to consist essentially of" meaning that a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In this document and in its claims, unless specifically stated otherwise, the verbs "to comprise", "to have" and "to contain", and their conjugations, may be replaced by "to consist of" (and its conjugations) or "to consist essentially of" (and its conjugations) and vice versa. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". Throughout the application, unless explicitly stated otherwise, the articles "a" and "an" are preferably replaced by "at least two", more preferably by "at least three", even more preferably by "at least four", even more preferably by "at least five", even more preferably by "at least six", most preferably by "at least two". The word "about" or "approximately" when used in association with a numerical value (e.g., "about 10") or with a range (e.g., "about x to approximately y") preferably means that the value or range is interpreted as being as accurate as the method used to measure it. If no error margins are specified, the expression "about" or "approximately" when used in association with a numerical value is interpreted as having the same roundoff as the given value. Throughout this document and its claims, unless otherwise stated, the expression "from x to y", wherein x and y represent numerical values, refers to a range of numerical values wherein x is the lower value of the range and y is the upper value of the range. Herein, x and y are also included in the range. Throughout the application, the term "one or more" is interchangeably used with the term "at least one".

[0014] According to the present invention, the term "polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triplestranded regions, or a mixture of single- and double-stranded regions. In addition, "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term "polynucleotide(s)" also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" according to the present invention. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, are to be understood to be covered by the term "polynucleotides". It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. The term "polynucleotide(s)" also embraces short polynucleotides often referred to as oligonucleotide(s). "Polypeptide(s)" refers to any peptide or protein comprising two or more amino acid residues joined to each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acid residues other than the 20 gene encoded amino acids. "Polypeptide(s)" include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to the skilled person. The same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Furthermore, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid sidechains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, crosslinking, cyclization, disulphide bond formation, demethylation, formation of covalent cross-links, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post- translational natural processes and may be made by entirely synthetic methods, as well.

[0015] The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions that also may contain coding and / or non-coding sequences.

[0016] "Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Similarly, a "synthetic" sequence, as the term is used herein, means any sequence that has been generated synthetically and not directly isolated from a natural source. "Synthesized", as the term is used herein, means any synthetically generated sequence and not directly isolated from a natural source. "Recombinant" means genetically engineered DNA prepared by transplanting or splicing genes from one species into the cells of a host organism of a different species. Such DNA becomes part of the host's genetic makeup and is replicated.

[0017] The term "control sequences" refers to sequences recognized by the cells transcriptional and translational systems, allowing transcription and translation of a polynucleotide sequence to a polypeptide. Such DNA sequences are thus necessary for the expression of an operably linked coding sequence in a particular host cell, cell or organism. Such control sequences can be, but are not limited to, promoter sequences, ribosome binding sequences, Shine Dalgarno sequences, Kozak sequences, transcription terminator sequences. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. DNA for a presequence or secretory leader may be operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Said control sequences can furthermore be controlled with external chemicals, such as, but not limited to, IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose via an inducible promoter or via a genetic circuit that either induces or represses the transcription or translation of said polynucleotide to a polypeptide. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous.

[0018] The term "wildtype" refers to the commonly known genetic or phenotypical situation as it occurs in nature.

[0019] The term "modified activity" of a protein relates to a non-native activity of the protein in any phase of the production process of the desired fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, 3-FL, mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or mixture comprising 3-FL as described herein. The term "non-native", as used herein with reference to the activity of a protein indicates that the protein has been modified to have an abolished, impaired, reduced, delayed, higher, accelerated or improved activity compared to the native activity of said protein. A modified activity of a protein is obtained by modified expression of said protein or is obtained by expression of a modified, i.e., mutant form of the protein. A mutant form of the protein can be obtained by expression of a mutant form of the gene encoding the protein, e.g., comprising a deletion, an insertion and / or a mutation of one or more nucleotides compared to the native gene sequence. A mutant form of a gene can be obtained by techniques well-known to a person skilled in the art, such as but not limited to site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis. The term "non-native", as used herein with reference to a cell producing a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, 3-FL, a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or a mixture comprising 3-FL as described herein, indicates that said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, said 3-FL, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or said mixture comprising 3-FL is i) not naturally produced or ii) when naturally produced not in the same amounts by the cell; and that the cell has been genetically engineered to be able to produce said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc,said 3-FL, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or said mixture comprising 3-FL or to have a higher production of the fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc, 3-FL, the mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc as described herein and / or the mixture comprising 3-FL.

[0020] Throughout the application, the term "differs in at least one" can be interchangeably used with "differs in one" or "only differs in".

[0021] Throughout the application, the terms "de novo designed enzyme", "enzyme of present invention", "mutant enzyme" and "mutant fucosyltransferase" are used interchangeably.

[0022] Throughout the application, the sequence of a polypeptide can be represented by a SEQ ID NO or alternatively by an UniProt ID. Therefore, the terms "polypeptide SEQ ID NO" and "polypeptide UniProt ID" can be interchangeably used, unless explicitly stated otherwise.

[0023] The term "positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196 and / or 244-254 of SEQ ID NO 01" as used herein refers to positions 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195,

[0024] 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01.

[0025] The term "positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01" as used herein refers to positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102,

[0026] 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193,

[0027] 194, 195 and / or 196 of SEQ ID NO 01.

[0028] The term "positions 19-31, 49-60, 84-90, 101-106, 131-140, 175-177, 178-183, and / or 231-241 of SEQ ID NO 02" as used herein refers to positions 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 84, 85, 86, 87, 88, 89, 90, 101, 102, 103, 104, 105, 106, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 175, 176, 177, 178, 179, 180, 181, 182, 183, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, and / or 241 of SEQ ID NO 02.

[0029] The term "positions 49-60, 84-90, 101-106, 175-177, and / or 178-183 of SEQ ID NO 02" as used herein refers to positions 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 84, 85, 86, 87, 88, 89, 90, 101, 102, 103,

[0030] 104, 105, 106, 175, 176, 177, 178, 179, 180, 181, 182 and / or 183 of SEQ ID NO 02.

[0031] The term "positions 10-21, 37-46, 65-72, 84-89, 93-97, 124-129, 171-173, 174-179, and / or 227-237 of SEQ ID NO 03" as used herein refers to positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 65, 66, 67, 68, 69, 70, 71, 72, 84, 85, 86, 87, 88, 89, 93, 94, 95, 96, 97, 124, 125, 126, 127, 128, 129, 171, 172, 173, 174, 175, 176, 177, 178, 179, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, and / or 237 of SEQ ID NO 03.

[0032] The term "positions 37-46, 65-72, 84-97, 171-173, and / or 174-179 of SEQ ID NO 03" as used herein refers to positions 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 65, 66, 67, 68, 69, 70, 71, 72, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 171, 172, 173, 174, 175, 176, 177, 178 and / or 179 of SEQ ID NO 03.

[0033] The term "positions 10-21, 44-50, 61-66, 92-98, 124-126, 128-133, and / or 177-187 of SEQ ID NO 04" as used herein refers to positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 44, 45, 46, 47, 48, 49, 50, 61, 62, 63, 64, 65, 66, 92, 93, 94, 95, 96, 97, 98, 124, 125, 126, 128, 129, 130, 131, 132, 133, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, and / or 187 of SEQ ID NO 04.

[0034] The term "positions 10-21, 44-50, 61-66, 124-126, and / or 128-133 of SEQ ID NO 04" as used herein refers to positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 44, 45, 46, 47, 48, 49, 50, 61, 62, 63, 64, 65, 66, 124, 125, 126, 128, 129, 130, 131, 132 and / or 133 of SEQ ID NO 04.

[0035] The term "positions 10-21, 44-50, 61-66, 92-98, 124-126, 128-133, and / or 177-187 of SEQ ID NO 05" as used herein refers to positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 44, 45, 46, 47, 48, 49, 50, 61, 62, 63, 64, 65, 66, 92, 93, 94, 95, 96, 97, 98, 124, 125, 126, 128, 129, 130, 131, 132, 133, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, and / or 187 of SEQ ID NO 05.

[0036] The term "positions 10-21, 44-50, 61-66, 124-126, and / or 128-133 of SEQ ID NO 05" as used herein refers to positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 44, 45, 46, 47, 48, 49, 50, 61, 62, 63, 64, 65, 66, 124, 125, 126, 128, 129, 130, 131, 132 and / or 133 of SEQ ID NO 05.

[0037] The term "positions 10-19, 43-56, 68-74, 100-106, 134-136, 138-143, and / or 194-204 of SEQ ID NO 06" as used herein refers to positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 68, 69, 70, 71, 72, 73, 74, 100, 101, 102, 103, 104, 105, 106, 134, 135, 136, 138, 139, 140, 141, 142, 143, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203 and / or 204 of SEQ ID NO 06.

[0038] The term "positions 10-19, 43-56, 68-74, 134-136, and / or 138-143 of SEQ ID NO 06" as used herein refers to positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 68, 69, 70, 71, 72, 73, 74, 134, 135, 136, 138, 139, 140, 141, 142 and / or 143 of SEQ ID NO 06.

[0039] The term "positions 2-13, 30-41, 62-70, 81-86, 89-92, 118-123, 161-163, 164-169 and / or 217-227 of SEQ ID NO 07" as used herein refers to positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 62, 63, 64, 65, 66, 67, 68, 69, 70, 81, 82, 83, 84, 85, 86, 89, 90, 91, 92, 118, 119, 120, 121, 122, 123, 161, 162, 163, 164, 165, 166, 167, 168, 169, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, and / or 227 of SEQ ID NO 07.

[0040] The term "positions 30-41, 62-70, 81-92, 161-163 and / or 164-169 of SEQ ID NO 07" as used herein refers to positions 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 62, 63, 64, 65, 66, 67, 68, 69, 70, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 161, 162, 163, 164, 165, 166, 167, 168, and / or 169 of SEQ ID NO 07.

[0041] The term "positions 4-15, 32-42, 61-68, 79-84, 89-94, 120-125, 159-161, 162-167 and / or 215-225 of SEQ ID NO 08" as used herein refers to positions 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 61, 62, 63, 64, 65, 66, 67, 68, 79, 80, 81, 82, 83, 84, 89, 90, 91, 92, 93, 94, 120, 121, 122, 123, 124, 125, 159, 160, 161, 162, 163, 164, 165, 166, 167, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, and / or 225 of SEQ ID NO 08.

[0042] The term "positions 32-42, 61-68, 79-94, 159-161, and / or 162-167 of SEQ ID NO 08" as used herein refers to positions 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 61, 62, 63, 64, 65, 66, 67, 68, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 159, 160, 161, 162, 163, 164, 165, 166, and / or 167 of SEQ ID NO 08. The term "positions 7-17, 37-48, 71-78, 90-95, 98-101, 127-132, 170-172, 173-178, and / or 226-236 of SEQ ID NO 09" as used herein refers to positions 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 37, 38, 39, 40, 41, 42,

[0043] 43, 44, 45, 46, 47, 48, 71, 72, 73, 74, 75, 76, 77, 78, 90, 91, 92, 93, 94, 95, 98, 99, 100, 101, 127, 128, 129, 130, 131, 132, 170, 171, 172, 173, 174, 175, 176, 177, 178, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, and / or 236 of SEQ ID NO 09.

[0044] The term "positions 37-48, 71-78, 90-101, 170-172, and / or 173-178 of SEQ ID NO 09" as used herein refers to positions 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 71, 72, 73, 74, 75, 76, 77, 78, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 170, 171, 172, 173, 174, 175, 176, 177, and / or 178 of SEQ ID NO 09.

[0045] The term "position 19-31, 49-60, 84-90, 101-106, 131-140, 175-177, 178-183, and / or 231-241 of SEQ ID NO 10" as used herein refers to positions 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 84, 85, 86, 87, 88, 89, 90, 101, 102, 103, 104, 105, 106, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 175, 176, 177, 178, 179, 180, 181, 182, 183, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, and / or 241 of SEQ ID NO 10.

[0046] The term "positions 49-60, 84-90, 101-106, 175-177, and / or 178-183 of SEQ ID NO 10" as used herein refers to positions 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 84, 85, 86, 87, 88, 89, 90, 101, 102, 103, 104, 105, 106, 175, 176, 177, 178, 179, 180, 181, 182 and / or 183 of SEQ ID NO 10.

[0047] The term "position 19-31, 49-60, 84-90, 101-106, 131-140, 175-177, 178-183, and / or 231-241 of SEQ ID NO 11" as used herein refers to positions 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 84, 85, 86, 87, 88, 89, 90, 101, 102, 103, 104, 105, 106, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 175, 176, 177, 178, 179, 180, 181, 182, 183, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, and / or 241 of SEQ ID NO 11.

[0048] The term "positions 49-60, 84-90, 101-106, 175-177, and / or 178-183 of SEQ ID NO 11" as used herein refers to positions 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 84, 85, 86, 87, 88, 89, 90, 101, 102, 103, 104, 105, 106, 175, 176, 177, 178, 179, 180, 181, 182 and / or 183 of SEQ ID NO 11.

[0049] The term "positions 11-22, 41-51, 71-81, 91-96, 101-107, 151-156, 194-196, 197-202, and / or 250-260 of SEQ ID NO 12" as used herein refers to positions 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 41, 42, 43,

[0050] 44, 45, 46, 47, 48, 49, 50, 51, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 91, 92, 93, 94, 95, 96, 101, 102, 103, 104, 105, 106, 107, 151, 152, 153, 154, 155, 156, 194, 195, 196, 197, 198, 199, 200, 201, 202, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, and / or 260 of SEQ ID NO 12. The term "positions 41-51, 71-81, 91-107, 194-196, and / or 197-202 of SEQ ID NO 12" as used herein refers to positions 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 194, 195, 196, 197, 198, 199, 200, 201, and / or 202 of SEQ ID NO 12.

[0051] The term "positions 13-23, 32-46, 73-81, 92-97, 123-131, 188-190, 191-196, and / or 240-250 of SEQ ID NO 13" as used herein refers to positions 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 73, 74, 75, 76, 77, 78, 79, 80, 81, 92, 93, 94, 95, 96, 97, 123, 124, 125, 126, 127, 128, 129, 130, 131, 188, 189, 190, 191, 192, 193, 194, 195, 196, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, and / or 250 of SEQ ID NO 13.

[0052] The term "positions 32-46, 73-81, 92-97, 188-190, and / or 191-196 of SEQ ID NO 13" as used herein refers to positions 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 73, 74, 75, 76, 77, 78, 79, 80, 81, 92,

[0053] 93, 94, 95, 96, 97, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 13.

[0054] The term "positions 13-24, 33-47, 74-82, 93-98, 124-132, 189-191, 192-197, and / or 241-251 of SEQ ID NO 14" as used herein refers to positions 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 93, 94, 95, 96, 97, 98, 124, 125, 126, 127, 128, 129, 130, 131, 132, 189, 190, 191, 192, 193, 194, 195, 196, 197, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 and / or 251 of SEQ ID NO 14.

[0055] The term "positions 33-47, 74-82, 93-98, 189-191, and / or 192-197 of SEQ ID NO 14" as used herein refers to positions 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 93,

[0056] 94, 95, 96, 97, 98, 189, 190, 191, 192, 193, 194, 195, 196 and / or 197 of SEQ ID NO 14.

[0057] The term "positions 10-21, 43-49, 59-64, 90-96, 123-125, 128-133 and / or 177-187 of SEQ ID NO 15" as used herein refers to positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 43, 44, 45, 46, 47, 48, 49, 59, 60, 61, 62, 63, 64, 90, 91, 92, 93, 94, 95, 96, 123, 124, 125, 128, 129, 130, 131, 132, 133, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, and / or 187 of SEQ ID NO 15.

[0058] The term "positions 10-21, 43-49, 59-64, 123-125, and / or 128-133 of SEQ ID NO 15" as used herein refers to positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 43, 44, 45, 46, 47, 48, 49, 59, 60, 61, 62, 63, 64,

[0059] 123, 124, 125, 128, 129, 130, 131, 132 and / or 133 of SEQ ID NO 15.

[0060] The term "positions 19-31, 49-60, 84-90, 101-106, 131-140, 175-177, 178-183, and / or 231-241 of SEQ ID

[0061] NO 16" as used herein refers to positions 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 49, 50, 51, 52,

[0062] 53, 54, 55, 56, 57, 58, 59, 60, 84, 85, 86, 87, 88, 89, 90, 101, 102, 103, 104, 105, 106, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 175, 176, 177, 178, 179, 180, 181, 182, 183, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240 and / or 241 of SEQ ID NO 16.

[0063] The term "positions 49-60, 84-90, 101-106, 175-177, and / or 178-183 of SEQ ID NO 16" as used herein refers to positions 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 84, 85, 86, 87, 88, 89, 90, 101, 102, 103, 104, 105, 106, 175, 176, 177, 178, 179, 180, 181, 182 and / or 183 of SEQ ID NO 16. The term "positions 3-17, 29-40, 58-67, 78-83, 88-95, 123-130, 167-169, 170-175, and / or 223-233 of SEQ ID NO 17" as used herein refers to positions 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 78, 79, 80, 81, 82, 83, 88, 89, 90, 91, 92, 93, 94, 95, 123, 124, 125, 126, 127, 128, 129, 130, 167, 168, 169, 170, 171, 172, 173, 174, 175, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, and / or 233 of SEQ ID NO 17.

[0064] The term "positions 29-40, 58-67, 78-95, 167-169, and / or 170-175 of SEQ ID NO 17" as used herein refers to positions 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 167, 168, 169, 170, 171, 172, 173, 174, and / or 175 of SEQ ID NO 17.

[0065] The term "positions 9-19, 33-43, 63-70, 82-101, 106-113, 150-155, 189-191, 192-197, and / or 245-255 of SEQ ID NO 44" as used herein refers to positions 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 63, 64, 65, 66, 67, 68, 69, 70, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 106, 107, 108, 109, 110, 111, 112, 113, 150, 151, 152, 153, 154, 155, 189, 190, 191, 192, 193, 194, 195, 196, 197, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 and / or 255 of SEQ ID NO 44.

[0066] The term "positions 33-43, 63-70, 82-113, 189-191, and / or 192-197 of SEQ ID NO 44" as used herein refers to positions 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 63, 64, 65, 66, 67, 68, 69, 70, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 189, 190, 191, 192, 193, 194, 195, 196 and / or 197 of SEQ ID NO 44.

[0067] The term "AA 22-254 of SEQ ID NO 01" as used herein refers to SEQ ID NO 46, which is a polypeptide sequence consisting of amino acid residues 22 to 254 of SEQ ID NO 01, including amino acid residues 22 and 254. The term "AA 10-237 of SEQ ID NO 03" as used herein refers to SEQ ID NO 47, which is a polypeptide sequence consisting of amino acid residues 10 to 237 of SEQ ID NO 03, including amino acid residues 10 and 237. The term "AA 11-260 of SEQ ID NO 12" as used herein refers to SEQ ID NO 48, which is a polypeptide sequence consisting of amino acid residues 11 to 260 of SEQ ID NO 12, including amino acid residues 11 and 260. The term "AA 3-233 of SEQ ID NO 17" as used herein refers to SEQ ID NO 49, which is a polypeptide sequence consisting of amino acid residues 3 to 233 of SEQ ID NO 17, including amino acid residues 3 and 233. The term "AA 7-236 of SEQ ID NO 09" as used herein refers to SEQ ID NO

[0068] 50, which is a polypeptide sequence consisting of amino acid residues 7 to 236 of SEQ ID NO 09, including amino acid residues 7 and 236. The term "AA 1-1 1 of SEQ ID NO 07" as used herein refers to SEQ ID NO

[0069] 51, which is a polypeptide sequence consisting of amino acid residues 2 to 227 of SEQ ID NO 07, including amino acid residues 2 and 227. The term "AA 4-225 of SEQ ID NO 08" as used herein refers to SEQ ID NO

[0070] 52, which is a polypeptide sequence consisting of amino acid residues 4 to 225 of SEQ ID NO 08, including amino acid residues 4 and 225. The term "AA 13-250 of SEQ ID NO 13" as used herein refers to SEQ ID NO

[0071] 53, which is a polypeptide sequence consisting of amino acid residues 13 to 250 of SEQ ID NO 13, including amino acid residues 13 and 250. The term "AA 1-187 of SEQ ID NO 15" as used herein refers to SEQ ID NO 54, which is a polypeptide sequence consisting of amino acid residues 1 to 187 of SEQ ID NO 15, including amino acid residues 1 and 187. The term "AA 1-187 of SEQ ID NO 04" as used herein refers to SEQ ID NO

[0072] 55, which is a polypeptide sequence consisting of amino acid residues 1 to 187 of SEQ ID NO 04, including amino acid residues 1 and 187. The term "AA 19-241 of SEQ ID NO 10" as used herein refers to SEQ ID NO

[0073] 56, which is a polypeptide sequence consisting of amino acid residues 19 to 241 of SEQ ID NO 10, including amino acid residues 19 and 241. The term "AA 1-204 of SEQ ID NO 06" as used herein refers to SEQ ID NO

[0074] 57, which is a polypeptide sequence consisting of amino acid residues 1 to 204 of SEQ ID NO 06, including amino acid residues 1 and 204.

[0075] The term "one or more amino acid residue insertion(s) at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01" as used herein refers to one or more consecutive and / or non-consecutive amino acid residue insertion(s) before and / or after one or more amino acid residue(s) corresponding to positions 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01.

[0076] The term "one or more amino acid residue insertion(s) at amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01" as used herein refers to one or more consecutive and / or non-consecutive amino acid residue insertion(s) before and / or after one or more amino acid residue(s) corresponding to positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01.

[0077] A domain can be characterized, for example, by a Pfam (El-Gebali et al., Nucleic Acids Res. 47 (2019) D427- D432) and / or an IPR (InterPro domain) (http: / / ebi.ac.uk / interpro) (Mitchell et al., Nucleic Acids Res. 47 (2019) D351-D360). InterPro provides functional analysis of proteins by classifying them into families and predicting domains and important sites. To classify proteins in this way, InterPro uses predictive models, known as signatures, provided by several different databases (referred to as member databases) that make up the InterPro consortium. Protein signatures from these member databases are combined into a single searchable resource, capitalizing on their individual strengths to produce a powerful integrated database and diagnostic tool. It should be understood for those skilled in the art that for the databases used herein, comprising Pfam 32.0 (released Sept 2018) and InterPro 99.0 (released 28thMarch 2024), the content of each database is fixed at each release and is not to be changed. When the content of a specific database is changed, this specific database receives a new release version with a new release date. All release versions for each database with their corresponding release dates and specific content as annotated at these specific release dates are available and known to those skilled in the art. Protein or polypeptide sequence information and functional information can be provided by a comprehensive resource for protein sequence and annotation data like e.g., the Universal Protein Resource (UniProt) (www.uniprot.org) (Nucleic Acids Res. 2021, 49(D1), D480-D489). UniProt comprises the expertly and richly curated protein database called the UniProt Knowledgebase (UniProtKB), together with the UniProt Reference Clusters (UniRef) and the UniProt Archive (UniParc). The UniProt identifiers (UniProt ID) are unique for each protein present in the database. Throughout the application, the sequence of a polypeptide is represented by a SEQ ID NO or an UniProt ID. Unless stated otherwise, the UniProt IDs of the proteins described correspond to their sequence version 01 as present in the UniProt Database (www.uniprot.org) version release 2021_03 and consulted on 09 June 2021.

[0078] The terms "alanine", "Ala" and "A" are used interchangeably. The terms "arginine", "Arg" and "R" are used interchangeably. The terms "asparagine", "Asn" and "N" are used interchangeably. The terms "aspartic acid", "Asp" and "D" are used interchangeably. The terms "cysteine", "Cys" and "C" are used interchangeably. The terms "glutamine", "Gin" and "Q" are used interchangeably. The terms "glutamic acid", "Glu" and "E" are used interchangeably. The terms "glycine", "Gly" and "G" are used interchangeably. The terms "histidine", "His" and "H" are used interchangeably. The terms "isoleucine", "He" and "I" are used interchangeably. The terms "leucine", "Leu" and "L" are used interchangeably. The terms "lysine", "Lys" and "K" are used interchangeably. The terms "methionine", "Met" and "M" are used interchangeably. The terms "phenylalanine", "Phe" and "F" are used interchangeably. The terms "Proline", "Pro" and "P" are used interchangeably. The terms "Serine", "Ser" and "S" are used interchangeably. The terms "Threonine", "Thr" and "T" are used interchangeably. The terms "tryptophan", "Trp" and "W" are used interchangeably. The terms "tyrosine", "Tyr" and "Y" are used interchangeably. The terms "valine", "Vai" and "V" are used interchangeably.

[0079] The terms "identical" or "percent identity" or "% identity" in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using sequence comparison algorithms or by visual inspection. For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are inputted into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the % sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. The percentage of sequence identity can be, preferably is, determined by alignment of the two sequences and identification of the number of positions with identical residues divided by the number of residues in the shorter of the sequences x 100. Percent identity may be calculated globally over the full-length sequence of a given SEQ ID NO, i.e. the reference sequence, resulting in a global % identity score. Alternatively, % identity may be calculated over a partial sequence of the reference sequence, resulting in a local percent identity score. A partial sequence preferably means at least about 50 %, 60 %, 70 %, 80 %, 90 % or 95 % of the full-length reference sequence. In another preferred embodiment, a partial sequence of a reference polypeptide sequence means a stretch of at least 150 amino acid residues up to the total number of amino acid residues of a reference polypeptide sequence. In another more preferred embodiment, a partial sequence of a reference polypeptide sequence means a stretch of at least 200 amino acid residues up to the total number of amino acid residues of a reference polypeptide sequence. Using the full-length of the reference sequence in a local sequence alignment results in a global percent identity score between the test and the reference sequence. Percent identity can be determined using different algorithms like for example BLAST and PSI-BLAST (Altschul et al., 1997, Nucleic Acids Res 25: 17, 3389-402), the Clustal Omega method (Sievers et al., 2011, Mol. Syst. Biol. 7:539), the MatGAT method (Campanella et al., 2003, BMC Bioinformatics, 4:29) or EMBOSS Needle.

[0080] As used herein, a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence having 80 % or more sequence identity to the full-length sequence of a reference polypeptide sequence is to be understood as that the amino acid sequence has 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 90.5 %, 91 %, 91.5 %, 92 %, 92.5 %, 93 %, 93.5 %, 94 %, 94.5 %, 95 %, 95.5 %, 96 %, 96.5 %, 97 %, 97.5 %, 98 %, 98.5 %, 99 %, 99.5 %, 99.6 %, 99.7 %, 99.8 %, 99.9 %, 100 % sequence identity to the full-length of the amino acid sequence of the reference polypeptide sequence.

[0081] Throughout the application, unless explicitly specified otherwise, a polypeptide comprising, consisting of or having an amino acid sequence having 80 % or more sequence identity to the full-length amino acid sequence of a reference polypeptide, usually indicated with a SEQ ID NO or UniProt ID, preferably has 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 %, more preferably has at least 80 %, even more preferably has at least 85 %, even more preferably has at least 90 %, even more preferably has at least 95 %, even more preferably has at least 97.5 % sequence identity to the full length reference sequence.

[0082] For the purposes of this invention, percent identity is determined using MatGAT2.01 (Campanella et al., 2003, BMC Bioinformatics 4:29). The following default parameters for protein are employed: (1) Gap cost Existence: 12 and Extension: 2; (2) The Matrix employed was BLOSUM50. In a preferred embodiment, sequence identity is calculated based on the full-length sequence of a given SEQ ID NO, i.e. the reference sequence, or a part thereof. Part thereof preferably means at least 50 %, 60 %, 70 %, 80 %, 90 % or 95 % of the complete reference sequence.

[0083] The term "glycosyltransferase" as used herein refers to an enzyme capable of catalysing the transfer of a sugar moiety of a donor to a specific acceptor, forming glycosidic bonds. Said donor can be a precursor as defined herein. A classification of glycosyltransferases using nucleotide diphospho-sugar, nucleotide monophospho-sugar and sugar phosphates and related proteins into distinct sequence-based families has been described (Campbell et al., Biochem. J. 326, 929-939 (1997)) and is available on the CAZy (CArbohydrate-Active EnZymes) website (www.cazy.org). As used herein the glycosyltransferase can be selected from the list comprising but not limited to: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N- acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N- glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino- 4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases.

[0084] The term "monosaccharide" as used herein refers to a sugar that is not decomposable into simpler sugars by hydrolysis, is classed either an aldose or ketose, and contains one or more hydroxyl groups per molecule. Monosaccharides are saccharides containing only one simple sugar. The term "Gal" refers to galactose, "GIcNAc" to N-acetylglucosamine, "Neu5Ac" to N-acetylneuraminic acid, "Glc" to glucose, "ManNAc" to N-acetylmannosamine, "GalNAc" to N-acetylgalactosamine, "Fuc" to fucose, and "Fruc" to fructose. The term "phosphorylated monosaccharide" as used herein refers to a monosaccharide that is phosphorylated. Examples of phosphorylated monosaccharides include but are not limited to glucose-1- phosphate, glucose-6-phosphate, glucose-l,6-bisphosphate, galactose-l-phosphate, fructose-6- phosphate, fructose-l,6-bisphosphate, fructose-l-phosphate, glucosamine-l-phosphate, glucosamine-6- phosphate, N-acetylglucosamine-l-phosphate, mannose-l-phosphate, mannose-6-phosphate or fucose- 1-phosphate. Some, but not all, of these phosphorylated monosaccharides are precursors or intermediates for the production of activated monosaccharide. The terms "activated monosaccharide", "nucleotide-activated sugar", "nucleotide-sugar", "activated sugar", "nucleoside" or "nucleotide donor" are used herein interchangeably and refer to activated forms of monosaccharides. Nucleotide-sugars act as glycosyl donors in glycosylation reactions. Glycosylation reactions are reactions that are catalysed by glycosyltransferases. The term "CMP-sialic acid" as used herein refers to a nucleotide-activated form of sialic acid comprising but not limited to CMP-Neu5Ac, CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac?, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) and CMP-KDO. The term "disaccharide" as used herein refers to a saccharide polymer containing two simple sugars, i.e. monosaccharides. "Oligosaccharide" as the term is used herein and as generally understood in the state of the art, refers to a saccharide polymer containing a small number, typically three to twenty, preferably three to ten, of simple sugars, i.e., monosaccharides. The oligosaccharide as used in the present invention can be a linear structure or can include branches. The linkage (e.g., glycosidic linkage, galactosidic linkage, glucosidic linkage, etc.) between two sugar units can be expressed, for example, as 1,4, l->4, or (1-4), used interchangeably herein. For example, the terms "Gal-bl,4-Glc", "Gal- pi,4-Glc", "b-Gal-(l->4)-Glc", "P-Gal-(l->4)-Glc", "Galbetal-4-Glc", "Gal-b(l-4)-Glc" and "Gal-P(l-4)-Glc" have the same meaning, i.e. a beta-glycosidic bond links carbon-1 of galactose (Gal) with the carbon-4 of glucose (Glc). Each monosaccharide can be in the cyclic form (e.g., pyranose or furanose form). Linkages between the individual monosaccharide units may include alpha l->2, alpha l->3, alpha l->4, alpha l->6, alpha 2->l, alpha 2->3, alpha 2->4, alpha 2->6, beta l->2, beta l->3, beta l->4, beta l->6, beta 2->l, beta 2->3, beta 2->4, and beta 2->6. An oligosaccharide can contain both alpha- and beta-glycosidic bonds or can contain only alpha-glycosidic or only beta-glycosidic bonds. The term "polysaccharide" refers to a compound consisting of a large number, typically more than twenty, of monosaccharides linked glycosidically. Examples of oligosaccharides include but are not limited to Lewis-type antigen oligosaccharides, mammalian (including human) milk oligosaccharides, O-antigen, enterobacterial common antigen (ECA), the glycan chain present in lipopolysaccharides (LPS), the oligosaccharide repeats present in capsular polysaccharides, peptidoglycan (PG), amino-sugars, antigens of the human ABO blood group system, non-charged (neutral) oligosaccharides, negatively charged oligosaccharides, fucosylated oligosaccharides, sialylated oligosaccharides, N-acetylglucosamine containing oligosaccharides, N- acetyllactosamine containing oligosaccharides, lacto-N-biose containing oligosaccharides, lactose containing oligosaccharides, non-fucosylated non-charged (neutral) oligosaccharides, N- acetyllactosamine containing fucosylated oligosaccharides, N-acetyllactosamine non-fucosylated oligosaccharides, lacto-N-biose containing fucosylated oligosaccharides, lacto-N-biose containing non- fucosylated oligosaccharides, N-acetyllactosamine containing negatively charged oligosaccharides, lacto- N-biose containing negatively charged oligosaccharides, animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans, and plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans.

[0085] "Charged oligosaccharides" are oligosaccharide structures that contain one or more negatively charged monosaccharide subunits including N-acetylneuraminic acid (Neu5Ac), commonly known as sialic acid, N- glycolylneuraminic acid (Neu5Gc), glucuronate and galacturonate. Charged oligosaccharides are also referred to as acidic oligosaccharides. Sialic acid belongs to the family of derivatives of neuraminic acid (5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid). Neu5Gc is a derivative of sialic acid, which is formed by hydroxylation of the N-acetyl group at C5 of Neu5Ac. The terms "sialic acid", "N- acetylneuraminate", "N-acylneuraminate", "N-acetylneuraminic acid" and "Neu(n)Ac molecule" are used interchangeably and refer to an acidic sugar with a nine-carbon backbone comprising but not limited to Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and KDO (2-keto-3-deoxymanno- octulonic acid). The terms "negatively charged oligosaccharide" or "acidic oligosaccharide" are used interchangeably and refer to an oligosaccharide with a negative charge. In a preferred embodiment, the negatively charged oligosaccharide is a sialylated oligosaccharide. As used herein, a 'sialylated oligosaccharide' is to be understood as a negatively charged sialic acid containing oligosaccharide, i.e., an oligosaccharide having one or more sialic acid residue(s). It has an acidic nature. Some examples are 3'SL (3'-sialyllactose, Neu5Ac-a2,3-Gal-pi,4-Glc), 3'-sialyllactosamine, 6'SL (6'sialyllactose, Neu5Ac-a2,6-Gal- pi,4-Glc), 8'SL (8'sialyllactose, Neu5Ac-a2,8-Gal-pi,4-Glc), 3,6-disialyllactose (Neu5Ac-a2,3-(Neu5Ac- a2,6)-Gal-pi,4-Glc), 6,6'-disialyllactose (Neu5Ac-a2,6-Gal-pi,4-(Neu5Ac-a2,6)-Glc), 8,3-disialyllactose (Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-pi,4-Glc), 6'-sialyllactosamine, oligosaccharides comprising 6'sialyllactose, SGG hexasaccharide (Neu5Aca-2,3Gaip -l,3GalNacP-l,3Gala-l,4Gaip-l,4Gal), sialylated tetrasaccharide, sialylated pentasaccharide, sialylated lacto-N-triose, sialylated lacto-N-tetraose, sialyllacto-N-neotetraose, LSTc (Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), LSTd (Neu5Ac-a2,3- Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a (LSTa, Neu5Ac-a2,3-Gal- i,3- GlcNAc-pi,3-Gal-pi,4-Glc), disialyllacto-N-hexaose I, sialyllacto-N-tetraose b (LSTb, Gal-pi,3-(Neu5Ac- a2,6)-GlcNAc-pi,3-Gal-pi,4-Glc), 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyl lacto-N- fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose and oligosaccharides bearing one or several sialic acid residue(s).

[0086] The terms 'neutral oligosaccharide' and 'non-charged oligosaccharide' as used herein are used interchangeably and refer, as generally understood in the state of the art, to an oligosaccharide that has no negative charge originating from a carboxylic acid group. Neutral oligosaccharides are non-sialylated oligosaccharides, and thus do not contain an acidic monosaccharide subunit. Neutral oligosaccharides comprise non-charged fucosylated oligosaccharides that contain one or more fucose subunits in their glycan structure as well as non-charged non-fucosylated oligosaccharides that lack any fucose subunit. Examples of such neutral oligosaccharides comprise 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4- fucosyllactose (4FL), 6-fucosyllactose (6FL), 2', 3-difucosyllactose (diFL), lacto-N-triose II (LN3), lacto-N- tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP I), lacto-N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-fucopentaose VI, lacto-N-neofucopentaose V (LNnFP V), lacto-N-difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto- N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, fucosyl-lacto-N-hexaose, difucosyl- lacto-N-hexaose, difucosyl-lacto-N-neohexaose (LNnDFH II), difucosyl-para-lacto-N-neohexaose, trifucosyllacto-N-hexaose, para-lacto-N-fucohexaose and lacto-N-trifucoheptaose.

[0087] A 'fucosylated oligosaccharide' as used herein and as generally understood in the state of the art is an oligosaccharide that is carrying a fucose-residue. Such fucosylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of said monosaccharide subunit is a fucose. A fucosylated oligosaccharide can contain more than one fucose residue, e.g., two, three or more. A fucosylated oligosaccharide can be a neutral oligosaccharide or a charged oligosaccharide e.g., also comprising sialic acid structures. Fucose can be linked to other monosaccharide subunits comprising glucose, galactose, GIcNAc via alpha-glycosidic bonds comprising alpha-1,2 alpha-1,3, alpha-1,4, alpha-1,6 linkages. Examples comprise 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL), Lacto-N- fucopentaose I (LNFP I), Lacto-N-fucopentaose II (LNFP II), Lacto-N-fucopentaose III (LNFP III), lacto-N- fucopentaose V (LNFP V), lacto-N-fucopentaose VI (LNFP VI), lacto-N-neofucopentaose I, lacto-N- difucohexaose I (LDFH I), lacto-N-difucohexaose II (LDFH II), Monofucosyllacto-N-hexaose III (MFLNH III), Difucosyllacto-N-hexaose (DFLNHa), difucosyl-lacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, disialomonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyl lacto-N- fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose.

[0088] Mammalian milk oligosaccharides or MMOs comprise oligosaccharides present in milk found in any phase during lactation including colostrum milk from humans (i.e. human milk oligosaccharides or HMOs) and mammals including but not limited to cows (Bos Taurus), sheep (Ovis aries), goats (Capra aegagrus hircus), bactrian camels (Camelus bactrianus), horses (Eguusferus caballus), pigs (Sus scropha), dogs (Canis lupus familiaris), ezo brown bears (Ursus arctos yesoensis), polar bear (Ursus maritimus), Japanese black bears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asian elephants (Elephas maximus), African elephant (Loxodonta africana), giant anteater (Myrmecophaga tridactyla), common bottlenose dolphins (Tursiops truncates), northern minke whales (Balaenoptera acutorostrata), tammar wallabies (Macropus eugenii), red kangaroos (Macropus rufus), common brushtail possum (Trichosurus Vulpecula), koalas (Phascolarctos cinereus), eastern quolls (Dasyurus viverrinus), platypus (Ornithorhynchus anatinus). As used herein, "mammalian milk oligosaccharide" or MMO refers to oligosaccharides such as but not limited to 3-fucosyllactose, 2'-fucosyllactose, 6-fucosyllactose, 2', 3- difucosyllactose, 2',2-difucosyllactose, 3,4-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, 3,6- disialyllactose, 6,6'-disialyllactose, 8,3-disialyllactose, 3,6-disialyllacto-N-tetraose, lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N- fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N-tetraose c, sialyl lacto-N- tetraose b, sialyllacto-N-tetraose a, lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, monofucosylmonosialyllacto-N-tetraose c, monofucosyl para-lacto-N-hexaose, monofucosyllacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose I, sialyllacto-N-hexaose, sialyllacto-N-neohexaose II, difucosyl-para-lacto-N- hexaose, difucosyllacto-N-hexaose, difucosyllacto-N-hexaose a, difucosyllacto-N-hexaose c, galactosylated chitosan, fucosylated oligosaccharides, non-charged (neutral) oligosaccharide and / or sialylated oligosaccharides.

[0089] The terms "lacto-N-biose" and "LNB" are used interchangeably and refer to Gal-pi,3-GlcNAc. The terms "LNT II", "LNT-II", "LN3", "lacto-N-triose II", "lacto- / V-triose II", "lacto-N-triose", "lacto- / V-triose" or "GlcNAcpi-3Gaipi-4Glc" as used in the present invention, are used interchangeably. The terms "LNT", "lacto-N-tetraose", "lacto- / V-tetraose" or "Gaipi-3GlcNAc i-3Gaipi-4Glc" as used in the present invention, are used interchangeably. The terms "LNnT", "lacto-N-neotetraose", "lacto- / V-neotetraose", "neo-LNT" or "Gaipi-4GlcNAc i-3Gaipi-4Glc" as used in the present invention, are used interchangeably. The terms "lacto-N-pentaose" and "LN5" are used interchangeably and refer to GlcNAc-pi,3-Gal-pi,4- GlcNAc-pi,3-Gal-pi,4-Glc. The terms "lacto-N-neohexaose" and "LNnH" are used interchangeably and refer to Gal-pi,4-GlcNAc-pi,6-(Gal-pi,4-GlcNAc-pi,3)-Gal-pi,4Glc. The term "pLNnH" refers to Gal-pi,4- GlcNAc-pi,3-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc. The terms "para-lacto-N-neohexaose II" and "pLNnH-ll" are used interchangeably and refer to Gal-pi,4-GlcNAc-pi,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc. The term "pLNH" refers to Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc. The terms "para-lacto- N-hexaose II" and "pLNH-ll" are used interchangeably and refer to Gal-pi,3-GlcNAc-pi,3-Gal-pi,3- GlcNAc-pi,3-Gal-pi,4-Glc. The term "pLNnO" refers to Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-GlcNAc-pi,3-Gal- pi,4-GlcNAc-pi,3-Gal-pi,4-Glc. The term "pLNnD" refers to Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-GlcNAc-pi,3- Gal-pi GIcNAc-pi,3-Gal-pi,4-GlcNAC-pi,3-Gal-pi,4-Glc. The term "LNH" refers to Gal-pi,4-GlcNAc- pi,6-(Gal-pi,3-GlcNAc-pi,3)-Gal-pi,4-Glc. The terms "N-acetyllactosamine" and "LacNAc" are used interchangeably and refer to Gal-pi,4-GlcNAc. The terms "iso-LNO" and "iso-lacto-N-octaose" are used interchangeably and refer to Gal-pi,3-GlcNAc-pi,3-(Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-GlcNAc-pi,6-)Gal- pi,4-Glc. The terms "LND" and "lacto-N-decaose" are used interchangeably. The terms "LNnD" and "lacto- N-neodecaose" are used interchangeably. The terms "2' fucosyllactose", "2'-fucosyllactose", "alpha-1, 2- fucosyllactose", "alpha 1,2 fucosyllactose", "a-l,2-fucosyllactose", "a 1,2 fucosyllactose", "Fuc-al,2-Gal- pi,4-Glc", "2FL" and "2'FL" are used interchangeably. The terms "3-fucosyllactose", "alpha-1, 3- fucosyllactose", "alpha 1,3 fucosyllactose", "a-1, 3-fucosyllactose", "a 1,3 fucosyllactose", "Gal- i,4-[Fuc- al,3]-Glc", "Gal-pi,4-(Fuc-al,3)-Glc", "Gal-pi,4-(Fuc-al,3-)Glc", "3FL" and "3-FL" are used interchangeably. The terms "difucosyllactose", "di-fucosyllactose", "lactodifucotetraose", "2',3- difucosyllactose", "2', 3 difucosyllactose", "a-2', 3-fucosyllactose", "a 2', 3 fucosyllactose", "Fuc-al,2-Gal- pi,4-(Fuc-al,3-)Glc", "DFLac", "2', 3 diFL", "DFL", "DiFL" and "diFL" are used interchangeably. The terms "LNFP-I", "lacto-N-fucopentaose I", "LNFP I", "LNF I OH type I determinant", "LNF I", "LNF1", "LNF 1", "Blood group H antigen pentaose type 1" and "Fuc-al,2-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc" are used interchangeably. The terms "GalNAc-LNFP-l", "blood group A antigen hexaose type I", and "GalNAc-al,3- (Fuc-al,2)-Gal- pi,3-GlcNAc- pi,3-Gal-pi,4-Glc" are used interchangeably. The terms "Gal-LNFP-I", "blood group B antigen hexaose type I" and "Gal-al,3-(Fuc-al,2)-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc" are used interchangeably. The terms "LNFP-II", "lacto-N-fucopentaose II" and "Gal-pi,3-(Fuc-al,4)-GlcNAc- pi,3-Gal-pi,4-Glc" are used interchangeably. The terms "LNFP-III", "lacto-N-fucopentaose III" and "Gal- pi,4-(Fuc-al,3)-GlcNAc-pi,3-Gal-pi,4-Glc" are used interchangeably. The terms "LNFP-V", "lacto-N- fucopentaose V" and "Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-(Fuc-al,3)-Glc" are used interchangeably. The terms "LNDFH I", "Lacto-N-difucohexaose I", "LNDFH-I", "LDFH I", "Leb-lactose", "Lewis-b hexasaccharide" and "Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-Glc" are used interchangeably. The terms "LNDFH II", "Lacto-N-difucohexaose II", "Lewis a-Lewis x", "LDFH II" and "Fuc-al,4-(Gal-pi,3)-GlcNAc- pi,3-Gal-pi,4-(Fuc-al,3)-Glc" are used interchangeably. The terms "lewis b-lewis x" and "Fucal,4-[Fuc- al,2-Gaipi,3]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-Glc" are used interchangeably. The terms "MFLNH III", "monofucosyllacto-N-hexaose-lll" and "Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6-[Gal-pi,3-GlcNAc-pi,3]-Gal- pi,4-Glc" are used interchangeably. The terms "DFLNH (a)", "difucosyllacto-N-hexaose (a)" and "Gal-pi,4- [Fuc-al,3]-GlcNAc-pi,6-[Fuc-al,2-Gal-pi,3-GlcNAc-pi,3]-Gal-pi,4-Glc" are used interchangeably. The terms "DFLNH", "difucosyllacto-N-hexaose" and "Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6-[Fuc-al,4-[Gal-pi,3]- GlcNAc-pi,3]-Gal-pi,4-Glc" are used interchangeably. The terms "TFLNH", "trifucosyllacto-N-hexaose" and "Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6-[Fuc-al,4-[Fuc-al,2-Gal-pi,3]-GlcNAc-pi,3]-Gal-pi,4-Glc" are used interchangeably. The terms "LNnFP I", "Lacto-N-neofucopentaose I" and "Fuc-al,2-Gal-pi,4-GlcNAc- pi,3-Gal-pi,4-Glc" are used interchangeably. The terms "LNFP-VI", "LNnFP V", "lacto-N-neofucopentaose V" and "Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-(Fuc-al,3)-Glc" are used interchangeably. The terms "LNnDFH", "Lacto-N-neoDiFucohexaose", "Lewis x hexaose" "Gal-pi,4-(Fuc-al,3)-GlcNAc-pi,3-Gal-pi,4-(Fuc-al,3)- Glc" are used interchangeably. The terms "2'-fucosyllacto-N-biose", "2'FLNB" and "Fuc-al,2-Gal-pi,3- GIcNAc" are used interchangeably. The terms "4-fucosyllacto-N-biose", "4FLNB" and "Fuc-al,4-[Gal-pi,3- ]GlcNAc" are used interchangeably. The terms "difucosyllacto-N-biose", "diFLNB" and "Fuc-al,4-[Fuc- al,2-Gal-pi,3-]GlcNAc" are used interchangeably. The terms "2'-fucosyl-N-acetyllactosamine", "2'FlacNAc" and "Fuc-al,2-Gal-pi,4-GlcNAc" are used interchangeably. The terms "3-fucosyl-N- acetyllactosamine", "3FlacNAc", "3-FLacNAc" and "Gal-pi,4-(Fuc-al,3-)GlcNAc" are used interchangeably. The terms "difucosyl-N-acetyllactosamine", "diFlacNAc" and "Fuc-al,2-Gal-pi,4-[Fuc- al,3-]GlcNAc" are used interchangeably. The terms "3' sialyllactose", "3'-sialyllactose", "alpha-2, 3- sialyllactose", "alpha 2,3 sialyllactose", "a-2,3-sialyllactose", "a 2,3 sialyllactose", "3SL", "Sia-a2,3-Gal- pi,4-Glc", "Neu5Ac-a2,3-Gal-pi,4-Glc" and "3'SL" are used interchangeably. The terms "6' sialyllactose", "6' -sialyllactose", "alpha-2, 6-sialyllactose", "alpha 2,6 sialyllactose", "a-2,6-sialyllactose", "a 2,6 sialyllactose", "6SL", "Sia-a2,6-Gal-pi,4-Glc", "Neu5Ac-a2,6-Gal- i,4-Glc" and "6'SL" are used interchangeably. The terms "3,6-disialyllactose" and "Neu5Ac-a2,3-Neu5Ac-a2,6- Gal-pi,4-Glc" are used interchangeably. The terms "6,6'-disialyllactose" and "Neu5Ac-a2,6-Neu5Ac-a2,6- Gal-pi,4-Glc" are used interchangeably. The terms "8,3-disialyllactose" and "Neu5Ac-a2,8-Neu5Ac-a2,3- Gal-pi,4-Glc" are used interchangeably. The terms "3'S-2'FL", "3' -sialyl-2' -fucosyllactose" and "Neu5Ac-a2,3-[Fuc-al,2-]Gal- pi,4-Glc" are used interchangeably. The terms "6'S-2'FL", "6' -sialyl-2' -fucosyllactose" and "Neu5Ac-a2,6- [Fuc-al,2-]Gal-pi,4-Glc" are used interchangeably. The terms "3'S-3-FL", "3'-sialyl-3-fucosyllactose" and "Neu5Ac-a2,3-Gal-pi,4-[Fuc-al,3]Glc" are used interchangeably. The terms "6'S-3-FL", "6'-sialyl-3- fucosyllactose" and "Neu5Ac-a2,6-Gal-pi,4-[Fuc-al,3]Glc" are used interchangeably. The terms "LSTa", "LS-Tetrasaccharide a", "Sialyl-lacto-N-tetraose a", "sialyllacto-N-tetraose a" and "Neu5Ac-a2,3-Gal-pi,3- GlcNAc-pi,3-Gal- i,4-Glc" are used interchangeably. The terms "LSTb", "LS-Tetrasaccharide b", "Sialyl- lacto-N-tetraose b", "sialyllacto-N-tetraose b" and "Gal- i,3-(Neu5Ac-a2,6)-GlcNAc- i,3-Gal- i,4-Glc" are used interchangeably. The terms "LSTc", "LS-Tetrasaccharide c", "Sialyl-lacto-N-tetraose c", "sialyllacto-N-tetraose c", "sialyllacto-N-neotetraose c" and "Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal- pi,4-Glc" are used interchangeably. The terms "LSTd", "LS-Tetrasaccharide d", "Sialyl-lacto-N-tetraose d", "sialyllacto-N-tetraose d", "sialyllacto-N-neotetraose d" and "Neu5Ac-a2,3-Gal-pi,4-GlcNAc-pi,3-Gal- pi,4-Glc" are used interchangeably. The terms "3'-sialyllacto-N-biose", "3'SLNB" and "Neu5Ac-a2,3-Gal- pi,3-GlcNAc" are used interchangeably. The terms "6'-sialyllacto-N-biose", "6'SLNB" and "Neu5Ac-a2,6-

[0090] Gal-pi,3-GlcNAc" are used interchangeably. The terms "monofucosylmonosialyllacto-N-octaose", "sialyl

[0091] Lewis a", "sialyl Lea", "5-acetylneuraminyl-(2-3)-galactosyl-(l-3)-(fucopyranosyl-(l-4))-N- acetylglucosamine" and "Neu5Ac-a2,3-Gal-pi,3-[Fuc-al,4]-GlcNAc" are used interchangeably. The terms "3'-sialyllactosamine", "3'SLacNAc" and "Neu5Ac-a2,3-Gal-pi,4-GlcNAc" are used interchangeably. The terms "6'-sialyllactosamine", "6'SLacNAc" and "Neu5Ac-a2,6-Gal-pi,4-GlcNAc" are used interchangeably. The terms "sialyl Lewis x" , "sialyl Lex", "5-acetylneuraminyl-(2-3)-galactosyl-(l-4)-(fucopyranosyl-(l-3))- N-acetylglucosamine" and "Neu5Ac-a2,3-Gal-pi,4-[Fuc-al,3-]GlcNAc" are used interchangeably. The terms "DSLNnT" and "Disialyllacto-N-neotetraose" are used interchangeably and refer to Neu5Ac-a2,6- [Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3]-Gal-pi,4-Glc. The terms "DSLNT" and "Disialyllacto-N-tetraose" are used interchangeably and refer to Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,3-)GlcNAc-pi,3-Gal-pi,4-Glc. The terms "DS'LNT" and "disialyllacto-N-tetraose analog" are used interchangeably and refer to Neu5Ac-a2,6- (Neu5Ac-a2,6-Gal-pi,3-GlcNAc-pi,3-)Gal-pi,4-Glc. The terms "DS'LNnT" and "disialyllacto-N- neotetraose analog" are used interchangeably and refer to Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,4-GlcNAc- pi,3-)Gal-pi,4-Glc.

[0092] The term "fucosylated compound produced according to the present invention" as used herein refers to a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein. The term "fucosylated compound produced according to the present invention" as used herein also refers to 3-FL. The term "fucosylated compound produced according to the present invention" as used herein also refers to both a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and 3-FL.

[0093] A "genetic pathway" or "biosynthetic pathway" as used herein preferably refers to a set of at least two different coding sequences, where the coding sequences encode enzymes that catalyze different parts of a synthetic pathway to form a desired product (e.g., a fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc, 3-FL, a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or a mixture comprising 3-FL). In a genetic pathway, a first encoded enzyme uses a substrate to make a first product which in turn is used as a substrate for a second encoded enzyme to make a second product. In some embodiments, the genetic pathway includes 3 or more members (e.g., 3, 4, 5, 6, 7, 8, 9, etc.), wherein the product of one encoded enzyme is the substrate for the next enzyme in the synthetic pathway.

[0094] The term "transporter protein" as used herein refers to a protein that is part of or interacts with the cell membrane and controls the flow of molecules and information across the cell. The transporter protein is thus involved in transport, be it import into or export out of the cell. Such transporter protein can be but is not limited to porters, P-P-bond-hydrolysis-driven transporters, -Barrel Porins, auxiliary transport proteins and phosphotransfer-driven group translocators. The term "enabled efflux" means to introduce the activity of transport of a solute over the cytoplasm membrane and / or the cell wall. Said transport may be enabled by introducing and / or increasing the expression of a transporter protein and / or by expressing and / or overexpressing a membrane protein as described in the present invention. The term "enhanced efflux" means to improve the activity of transport of a solute over the cytoplasm membrane and / or the cell wall. Transport of a solute over the cytoplasm membrane and / or cell wall may be enhanced by introducing and / or increasing the expression of a transporter protein and / or by expressing and / or overexpressing a membrane protein as described in the present invention. "Expression" of a transporter protein is defined as "overexpression" of the gene encoding said transporter protein in the case said gene is an endogenous gene or "expression" in the case the gene encoding said transporter protein is a heterologous gene that is not present in the wild-type strain or cell.

[0095] The term "purified" refers to material that is substantially or essentially free from components that interfere with the activity of the biological molecule. For cells, saccharides, nucleic acids, and polypeptides, the term "purified" refers to material that is substantially or essentially free from components that normally accompany the material as found in its native state. Typically, purified saccharides, oligosaccharides, proteins or nucleic acids of the invention are at least about 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 % or 85 % pure, usually at least about 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99.0 % pure as measured by band intensity on a silver-stained gel or other method for determining purity. Purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized. For di- and oligosaccharides, purity can be determined using methods such as but not limited to thin layer chromatography, gas chromatography, NMR, HPLC, capillary electrophoresis or mass spectroscopy. Further herein, the terms "contaminants" and "impurities" preferably mean particulates, cells, cell components, metabolites, cell debris, proteins, peptides, amino acids, nucleic acids, glycolipids and / or endotoxins which can be present in an aqueous medium like e.g., a cultivation or an incubation.

[0096] The term "clarifying" as used herein refers to the act of treating an aqueous medium like e.g., a cultivation or an incubation, to remove suspended particulates and contaminants from the production process, like e.g., cells, cell components, insoluble metabolites and debris, that could interfere with the eventual purification of the saccharide. Such treatment can be carried out in a conventional manner by centrifugation, flocculation, flocculation with optional ultrasonic treatment, gravity filtration, microfiltration, foam separation or vacuum filtration (e.g., through a ceramic filter which can include a Celite™ filter aid).

[0097] Throughout present specification, the term "fucosylated compound" refers to a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein, 3-FL, a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or a mixture comprising 3-FL. The term "cultivation" refers to the culture medium wherein the cell is cultivated, or fermented, the cell itself, and a fucosylated compound produced according to the present invention by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracellularly) of the cell. The terms "culture medium" and "cultivation medium" as used herein are used interchangeably and refer to the medium wherein the cell is cultivated. The term "incubation" refers to a mixture wherein a fucosylated compound is produced according to the present invention. Said mixture can comprise one or more enzyme(s), one or more precursor(s) as defined herein present in a buffered solution and incubated for a certain time at a certain temperature enabling production of a fucosylated compound according to the present invention, catalysed by said one or more enzyme(s) using said one or more precursor(s) in said mixture. Said mixture can also comprise i) the cell obtained after cultivation or incubation, optionally said cell is subjected to cell lysis, ii) a buffered solution or the cultivation or incubation medium wherein the cell was cultivated or fermented, and iii) a fucosylated compound produced according to the present invention by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracellularly) of the cell. Said incubation can also be the cultivation as defined herein. The terms "reactor" and "incubator" refer to the recipient filled with the cultivation or incubation. Examples of reactors and incubators comprise but are not limited to microfluidic devices, well plates, tubes, shake flasks, fermenters, bioreactors, process vessels, cell culture incubators, CO2 incubators. As used herein, the term "cell productivity index (CPI)" refers to the mass of the desired fucosylated compound produced according to the present invention by the cells divided by the mass of the cells produced in the culture.

[0098] The term "precursor" as used herein refers to substances which are taken up or synthetized by the cell for the specific production of a fucosylated compound produced according to the present invention. In this sense a precursor can be an acceptor as defined herein, but can also be another substance, metabolite, which is first modified within the cell as part of the biochemical synthesis route of a fucosylated compound produced according to the present invention. The term "precursor" as used herein is also to be understood as a chemical compound that participates in a chemical or enzymatic reaction to produce another compound like e.g. an intermediate or an acceptor as defined herein, as part in the metabolic pathway of a fucosylated compound produced according to the present invention. The term "precursor" as used herein is also to be understood as a donor that is used by a glycosyltransferase to modify an acceptor as defined herein with a sugar moiety in a glycosidic bond, as part in the metabolic pathway of a fucosylated compound produced according to the present invention. Examples of such precursors used in the production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or in the production of a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc as described herein comprise the acceptors as defined herein, and / or fucose, dihydroxyacetone, glucosamine, sialic acid, N-acetylglucosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, galactosyllactose, phosphorylated sugars or sugar phosphates like e.g. but not limited to glucose-l-phosphate, galactose-l-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-l,6-bisphosphate, mannose-6-phosphate, mannose-l-phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N- acetylglucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N-acetylglucosamine-1- phosphate, N-acetylneuraminic acid-9-phosphate and nucleotide-activated sugars like nucleotide diphospho-sugars and nucleotide monophospho-sugars as defined herein like e.g. UDP-glucose, UDP- galactose, UDP-N-acetylglucosamine, GDP-mannose, GDP-4-dehydro-6-deoxy-a-D-mannose, GDP- fucose, CMP-sialic acid, CMP-KDO. Examples of such precursors used in the production of 3-FL and / or in the production of a mixture comprising 3-FL comprise the acceptors as defined herein, and / or phosphorylated sugars or sugar phosphates like e.g. but not limited to glucose-l-phosphate, galactose-1- phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-l,6-bisphosphate, and nucleotide- activated sugars like nucleotide diphospho-sugars and nucleotide monophospho-sugars as defined herein like e.g. UDP-glucose, UDP-galactose and GDP-fucose.

[0099] Optionally, the cell is transformed to comprise and to express at least one nucleic acid sequence encoding a protein selected from the group consisting of lactose transporter, N-acetylneuraminic acid transporter, fucose transporter, glucose transporter, galactose transporter, transporter for a nucleotide-activated sugar wherein said transporter internalizes a to the medium added precursor for the synthesis of the fucosylated compound produced according to the present invention.

[0100] The term "acceptor" as used herein refers to a mono-, di- or oligosaccharide, which can be modified by a glycosyltransferase and that is used in the production of a fucosylated compound produced according to the present invention. Examples of such acceptors used in the production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or in the production of a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprise glucose, galactose, fructose, glycerol, fucose, mannose, maltose, sucrose, lactose, sialic acid, LNB, a saccharide comprising LNB, sialylated oligosaccharide, fucosylated oligosaccharide, lacto-N-triose, lacto-N-tetraose (LNT), lacto- N-neotetraose (LNnT), lacto-N-pentaose (LNP), lacto-N-neopentaose, para lacto-N-pentaose, para lacto- N-neopentaose, lacto-N-novopentaose I, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para lacto- N-neohexaose (pLNnH), para lacto-N-hexaose (pLNH), lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, iso lacto-N- octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N- neooctaose, iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose, lacto-N-decaose, iso lacto-N- decaose, novo lacto-N-decaose, lacto-N-neodecaose, and oligosaccharide containing 1 or more N- acetyllactosamine units and / or 1 or more lacto-N-biose units or an intermediate into oligosaccharide and fucosylated versions thereof, ceramide, N-acylated sphingoid, glucosylceramide, lactosylceramide, sphingosine, phytosphingosine, sphingosine synthons, peptide backbones with beta-GIcNAc-Asn residues, glycoproteins with terminal GIcNAc and Gal residues, immunoglobulins. Examples of such acceptors used in the production of 3-FL and / or in the production of a mixture comprising 3-FL comprise glucose, galactose, fructose, glycerol, fucose, mannose, maltose, sucrose and lactose. Detailed description of the invention

[0101] According to a first aspect, the present invention provides a de novo designed enzyme having alpha-1, 4- fucosyltransferase activity on the N-acetylglucosamine (GIcNAc) residue of Gal-pi,3-GlcNAc (lacto-N- biose, LNB) and comprising, consisting of or consisting essentially of when compared to SEQ ID NO 01 one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01. Specifically, said de novo designed enzyme is obtained by modification of a progenitor fucosyltransferase wherein said modification comprises one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196 and / or 244-254 of SEQ ID NO 01. Herein, said progenitor fucosyltransferase (1) comprises the IPR001503 domain as defined by InterPro 99.0 as released on 28th March 2024, (2) has a GDP-fucose binding domain, (3) has alpha-1,3 fucosyltransferase activity and / or alpha-1,4 fucosyltransferase activity and (4) comprises, consists of or consists essentially of a polypeptide as represented by any one of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 or 44.

[0102] In other words and as understood herein, the enzyme of present specification is a mutant fucosyltransferase derived from a progenitor fucosyltransferase. In a preferred embodiment of the enzyme of present invention, the enzyme is a mutant fucosyltransferase derived from a progenitor fucosyltransferase, wherein the mutant fucosyltransferase is a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to the full-length amino acid sequence of said progenitor fucosyltransferase.

[0103] The person skilled in the art will understand that the mutant fucosyltransferase as described herein is not identical to its progenitor fucosyltransferase. The terms "derived from", "mutated from" and "modified from" are used herein interchangeably and are to be understood as that the polypeptide sequence of the mutant fucosyltransferase differs in at least one amino acid residue substitution, insertion and / or deletion compared to the polypeptide sequence of a progenitor fucosyltransferase at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01. A mutant form of a progenitor fucosyltransferase can be obtained by expression of a mutant form of the gene encoding the progenitor fucosyltransferase, e.g., comprising a deletion, an insertion and / or a mutation of one or more nucleotides compared to the native gene sequence. A mutant form of a gene can be obtained by techniques well-known to a person skilled in the art, such as but not limited to site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis. An enzyme having alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB is to be understood as that said enzyme is capable of transferring, preferably transfers, a fucose (Fuc) residue from a suitable fucose donor to the GIcNAc residue of LNB in an alpha-1, 4-glycosidic linkage resulting in the production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein. Preferably, the enzyme of present invention is capable of using and / or uses GDP-fucose as suitable fucose donor.

[0104] In a preferred embodiment of the enzyme of present invention, LNB resides in free form. The term "LNB in free form" means that LNB is present as a disaccharide consisting of only a galactose (Gal) residue and a GIcNAc residue that are beta-1,3 glycosidically linked to each other, i.e. Gal-pi,3-GlcNAc. In this free form, LNB is not linked to one or more monosaccharides as described herein or to other molecules.

[0105] In another and / or additional preferred embodiment of the enzyme of present invention, LNB is part of a saccharide. Within the context of present invention, the terms "LNB is part of a saccharide", "LNB as part of a saccharide", "LNB as part of a saccharide comprising LNB", "saccharide comprising LNB" and "saccharide comprising an LNB epitope" are used interchangeably and mean that LNB is linked to one or more monosaccharide subunits forming a saccharide structure having a degree of polymerization (DP) of three or more, i.e. a saccharide structure having three or more monosaccharide subunits. Said saccharide can be an oligosaccharide, a polysaccharide or a glycan. Herein, the one or more monosaccharide subunits can be linked to the Gal residue of LNB and / or to the GIcNAc residue of LNB excluding a fucose that is linked to the GIcNAc residue of LNB in an alpha-1,4 glycosidic linkage. Said saccharide comprising LNB can have one LNB molecule but can also have more than one LNB molecule, i.e. two LNB molecules, three LNB molecules or more than three LNB molecules. An enzyme having alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB is thus also to be understood as that said enzyme is capable of transferring, preferably transfers, a fucose (Fuc) residue from a suitable fucose donor to the GIcNAc residue of LNB, wherein said LNB is part of a saccharide, in an alpha-1, 4-glycosidic linkage resulting in the production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein.

[0106] Optionally, LNB and / or said saccharide comprising LNB is / are linked to a peptide, a protein and / or a lipid.

[0107] Surprisingly, the de novo designed enzyme of present invention has an improved alpha-1,4 fucosyltransferase activity and / or improved alpha-1,4 fucosyltransferase specificity on the GIcNAc residue of LNB compared to an alpha-1, 3 / alpha-l, 4 fucosyltransferase that does not comprise one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01.

[0108] The expression "improved fucosyltransferase activity of an enzyme" is to be understood as a higher, adjusted, tweaked and / or faster catalytic fucosyltransferase activity of said enzyme on a specific compound which results in a higher production of a specific fucosylated compound and / or in the production of a specific fucosylated compound with a higher purity when using said enzyme compared to a reference fucosyltransferase. Specifically for the enzyme of present invention, the expression "enzyme having an improved alpha-1,4 fucosyltransferase activity on the GIcNAc residue of LNB" is to be understood as an enzyme of present invention having a higher, adjusted, tweaked and / or faster catalytic alpha-1,4 fucosyltransferase activity on the GIcNAc residue of LNB which results in a higher production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or in a production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein with a higher purity when using said enzyme of present invention compared to an alpha-1, 3 / alpha-l, 4 fucosyltransferase that does not comprise one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110- 117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01.

[0109] The expression "improved fucosyltransferase specificity of an enzyme" is to be understood as an increased or preferred specific substrate specificity of said enzyme towards a specific compound to be used in a fucosylation reaction catalyzed by said enzyme which results in a higher production of a specific fucosylated compound and / or in the production of a specific fucosylated compound with a higher purity when using said enzyme compared to a reference fucosyltransferase. Specifically for the enzyme of present invention, the expression "enzyme having an improved alpha-1,4 fucosyltransferase specificity on the GIcNAc residue of LNB" is to be understood as an enzyme of present invention having a higher, better or increased specific substrate specificity or preference towards the GIcNAc residue of LNB which results in a higher production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or in a production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein with a higher purity when using said enzyme of present invention compared to an alpha- 1, 3 / alpha-l, 4 fucosyltransferase that does not comprise one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01.

[0110] Additionally, and surprisingly, the de novo designed enzyme may have a declined alpha-1,3 fucosyltransferase activity and / or declined alpha-1,3 fucosyltransferase specificity on the glucose (Glc) residue of lactose compared to an alpha-1, 3 / alpha-l, 4 fucosyltransferase that does not comprise one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01. Herein, said lactose can reside in free form or can be present in an oligosaccharide like e.g. LNT, LNnT.

[0111] The expression "declined fucosyltransferase activity of an enzyme" is to be understood as a lower and / or slower catalytic fucosyltransferase activity of said enzyme on a specific compound which results in a lower production of a specific fucosylated compound and / or in the production of a specific fucosylated compound with a lower purity when using said enzyme compared to a reference fucosyltransferase. Specifically for the enzyme of present invention, the expression "enzyme having a declined alpha-1,3 fucosyltransferase activity on the Glc residue of lactose" is to be understood as an enzyme of present invention having a lower and / or slower catalytic alpha-1,3 fucosyltransferase activity on the Glc residue of lactose which results in a lower production of 3-FL and / or a fucosylated compound comprising Gal- pi,4-[Fuc-al,3]-Glc and / or in a production of 3-FL and / or a fucosylated compound comprising Gal-pi,4- [Fuc-al,3]-Glc with a lower purity when using said enzyme of present invention compared to an alpha- l,3 / alpha-l,4 fucosyltransferase that does not comprise one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01.

[0112] The expression "declined fucosyltransferase specificity of an enzyme" is to be understood as a decreased or less preferred specific substrate specificity of said enzyme towards a specific compound to be used in a fucosylation reaction catalyzed by said enzyme which results in a lower production of a specific fucosylated compound and / or in the production of a specific fucosylated compound with a lower purity when using said enzyme compared to a reference fucosyltransferase. Specifically for the enzyme of present invention, the expression "enzyme having a declined alpha-1,3 fucosyltransferase specificity on the Glc residue of lactose" is to be understood as an enzyme of present invention having a decreased specific substrate specificity or preference towards the Glc residue of lactose which results in a lower production of 3-FL and / or a fucosylated compound comprising Gal-pi,4-[Fuc-al,3]-Glc and / or in a production of 3-FL and / or a fucosylated compound comprising Gal-pi,4-[Fuc-al,3]-Glc with a lower purity when using said enzyme of present invention compared to an alpha-1, 3 / alpha-l, 4 fucosyltransferase that does not comprise one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188- 190, 191-196, and / or 244-254 of SEQ ID NO 01.

[0113] In a specific embodiment, the de novo designed enzyme of present invention is a biologically active enzyme. The skilled person can readily assess whether an enzyme having alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein is active or not (it is for example referred to Blixt et al, 1999, Glycobiology 9(10): p. 1061-1071; which is incorporated by reference), by bringing the enzyme into contact with (1) a suitable fucose donor, like e.g. GDP-fucose, and (2) LNB under conditions suitable for the transfer of fucose from said donor to the GIcNAc residue of said LNB in an alpha-1,4 glycosidic linkage. Herein, a cellular method or a cell-free method (e.g. enzymatic method) are suitable. Suitable conditions comprise a pH of 5 to 10, i.e. a pH of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, with an optimal pH between 7 to 8, i.e. a pH of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 and a temperature of 20°C to 50°C, i.e. a temperature of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C with an optimal temperature range of 20°C to 30°C, i.e. a temperature of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C. In the context of the invention, an enzyme of present invention is active when at least 10.0 %, preferably at least 20.0%, more preferably at least 25.0%, even more preferably at least 50.0% of LNB is modified by the enzyme, i.e., is modified by the addition of a fucose residue in an alpha- 1,4-linkage on the GIcNAc residue of LNB. LNB can herein be provided in free form or as part of a saccharide comprising LNB as described herein. Optionally, said LNB and / or saccharide comprising LNB is / are linked to a peptide, a protein and / or a lipid.

[0114] In an additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188- 190, 191-196, and / or 244-254 of SEQ ID NO 01. More specifically, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01. Preferably, said amino acid residue substitution(s), insertion(s) and / or deletion(s) are scattered along the polypeptide sequence of said enzyme. Additionally, and / or alternatively, said amino acid residue substitution(s), insertion(s) and / or deletion(s) can be integrated consecutively in the polypeptide sequence of said enzyme.

[0115] SEQ ID NO 01 is an alpha-1, 3 / 1, 4-fucosyltransferase originating from Porphyromonas catoniae that has alpha-1, 3 / alpha-l,4-fucosyltransferase activity on structures like e.g. lactose, LNnT, LNT and LNB as described e.g. in WQ2023 / 110994 and WQ2023 / 110995, which are to be incorporated by reference in their entirety herein. It is to be understood by the skilled person that the enzyme of present invention is not SEQ ID NO 01, i.e. the de novo designed enzyme differs from SEQ ID NO 01 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 01 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143- 150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0116] In a preferred embodiment, the enzyme of present invention is a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to the full-length amino acid sequence of SEQ ID NO 01 and having alpha-1, 4- fucosyltransferase activity on the GIcNAc residue of LNB as described herein. More specifically, the enzyme is a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence that is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to the full-length amino acid sequences of SEQ ID NO 01 and having alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0117] In a specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue substitution(s) at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01 and has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein. In a preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 only one amino acid residue substitution at an amino acid residue corresponding to position 22, 23, 24, 25, 26, Tl, 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 or 254 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 two amino acid residue substitutions at two consecutive or non-consecutive amino acid residues corresponding to positions 22, 23, 24, 25, 26, Tl, 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 three amino acid residue substitutions at three consecutive or non-consecutive amino acid residues corresponding to positions 22, 23, 24, 25, 26, Tl , 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 four or more amino acid residue substitutions at four or more consecutive or non-consecutive amino acid residues corresponding to positions 22, 23, 24, 25, 26, Tl, 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01.

[0118] In another and / or additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue insertion(s) at one or more amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188- 190, 191-196, and / or 244-254 of SEQ ID NO 01 and has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein. In a preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 only one amino acid residue insertion before or after an amino acid residue corresponding to position 22, 23, 24, 25, 26, Tl, 28, 29, 30, 31, 32, 33, 52, 53, 54, 55,

[0119] 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112,

[0120] 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 or 254 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 two consecutive or non-consecutive amino acid residue insertions before and / or after one or two amino acid residue(s) corresponding to positions 22, 23, 24, 25, 26, Tl , 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 three consecutive or non-consecutive amino acid residue insertions before and / or after one, two or three amino acid residue(s) corresponding to positions 22, 23, 24, 25, 26, Tl , 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56,

[0121] 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113,

[0122] 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196,

[0123] 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 four or more consecutive or non-consecutive amino acid residue insertions before and / or after one or more amino acid residue(s) corresponding to positions 22, 23, 24, 25, 26, Tl , 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57,

[0124] 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114,

[0125] 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244,

[0126] 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01.

[0127] In another and / or additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue deletion(s) at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01 and has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein. In a preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 only one amino acid residue deletion at an amino acid residue corresponding to position 22, 23, 24, 25, 26, Tl , 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82,

[0128] 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144,

[0129] 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249,

[0130] 250, 251, 252, 253 or 254 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 two amino acid residue deletions at two consecutive or non-consecutive amino acid residues corresponding to positions 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 three amino acid residue deletions at three consecutive or non-consecutive amino acid residues corresponding to positions 22, 23, 24, 25, 26, Tl , 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 four or more amino acid residue deletions at four or more consecutive or non-consecutive amino acid residues corresponding to positions 22, 23, 24, 25, 26, Tl , 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83,

[0131] 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145,

[0132] 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250,

[0133] 251, 252, 253 and / or 254 of SEQ ID NO 01.

[0134] In another and / or additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue substitution(s) and one or more amino acid residue insertion(s) at amino acid residue(s) corresponding to positions 22, 23, 24, 25, 26, Tl, 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01 and has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0135] In another and / or additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue substitution(s) and one or more amino acid residue deletion(s) at amino acid residue(s) corresponding to positions 22, 23, 24, 25, 26, Tl, 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01 and has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0136] In another and / or additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue insertions(s) and one or more amino acid residue deletion(s) at amino acid residue(s) corresponding to positions 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01 and has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein. In another and / or additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue substitution(s), one or more amino acid residue insertion(s) and one or more amino acid residue deletion(s) at amino acid residue(s) corresponding to positions 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117, 143, 144, 145, 146, 147, 148, 149, 150, 188, 189, 190, 191, 192, 193, 194, 195, 196, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 and / or 254 of SEQ ID NO 01 and has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0137] In a preferred embodiment of the enzyme of present invention, the amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01 are determined by a multiple sequence alignment tool MAFFT using default algorithm parameters and using the "--reorder" argument for the alignment and using the "--add" argument for the polypeptide sequence of said enzyme. In a more preferred embodiment, the multiple sequence alignment tool MAFFT that is used to determine the amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100- 105, 110-117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01 is MAFFT version 7.505 as released in July 2021 and the default algorithm parameters used in said tool comprise BLOSUM62 as scoring matrix, 1.53 as gap opening penalty and 0.0 as offset value.

[0138] In another and / or additional preferred embodiment of the enzyme of present invention, the enzyme is a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 or 44. More specifically, the enzyme is a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence that is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 or 44 and has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein. The person skilled will understand that the enzyme of present invention is not identical to SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 or 44, but differs from SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 or 44 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) of said SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 or 44, respectively, corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196, and / or 244-254 of SEQ ID NO 01. For the sake of clarity, it is referred to Tables 1-3 wherein the amino acid residue positions of SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 and 44 are indicated in function of the amino acid sequence of SEQ ID NO 01. For example, the amino acid residue at position 83 of SEQ ID NO 01 (i.e. phenylalanine, F) corresponds with position 86 of SEQ ID NO 02, position 67 of SEQ ID NO 03, position 46 of SEQ ID NO 04, position 46 of SEQ ID NO 05, position 45 of SEQ ID NO 06, position 64 of SEQ ID NO 07, position 63 of SEQ ID NO 08, position 73 of SEQ ID NO 09, position 86 of SEQ ID NO 10, position 86 of SEQ ID NO 11, position 73 of SEQ ID NO 12, position 75 of SEQ ID NO 13, position 76 of SEQ ID NO 14, position 45 of SEQ ID NO 15, position 86 of SEQ ID NO 16, position 60 of SEQ ID NO 17 and position 65 of SEQ ID NO 44.

[0139] Table 1. SEQ ID NO 01 and the position of each amino acid residue of SEQ ID NO 01 in SEQ ID NOs 01- 07. means that this amino acid residue is absent in the particular sequence. Amino acid residues shown in bold are related to alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB. Amino acid residues shown in a grey-shaded box are related to alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose.

[0140] Table 2. SEQ ID NO 01 and the position of each amino acid residue of SEQ ID NO 01 in SEQ ID NOs 01, 08-13. means that this amino acid residue is absent in the particular sequence. Amino acid residues shown in bold are related to alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB. Amino acid residues shown in a grey-shaded box are related to alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose.

[0141]

[0142] Table 3. SEQ ID NO 01 and the position of each amino acid residue of SEQ ID NO 01 in SEQ ID NOs 01, 14-17 and 44. means that this amino acid residue is absent in the particular sequence. Amino acid residues shown in bold are related to alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB. Amino acid residues shown in a grey-shaded box are related to alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose.

[0143]

[0144] Preferably, the enzyme of present invention differs from SEQ ID NO 02 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 19-31, 49-60, 84-90, 101-106, 131-140, 175-177, 178-183, and / or 231-241 of SEQ ID NO 02. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 02 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 19-31, 49-60, 84-90, 101-106, 131-140, 175-177, 178-183, and / or 231-241 of SEQ ID NO 02. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein. Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 03 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 37-46, 65-72, 84-89, 93-97, 124-129, 171-173, 174-179, and / or 227-237 of SEQ ID NO 03. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 03 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 37-46, 65-72, 84-89, 93-97, 124-129, 171-173, 174-179, and / or 227-237 of SEQ ID NO 03. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0145] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 04 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 44-50, 61-66, 92-98, 124-126, 128-133, and / or 177-187 of SEQ ID NO 04. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 04 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 44-50, 61-66, 92-98, 124-126, 128-133, and / or 177-187 of SEQ ID NO 04. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0146] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 05 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 44-50, 61-66, 92-98, 124-126, 128-133, and / or 177-187 of SEQ ID NO 05. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 05 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 44-50, 61-66, 92-98, 124-126, 128-133, and / or 177-187 of SEQ ID NO 05. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0147] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 06 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-19, 43-56, 68-74, 100-106, 134-136, 138-143, and / or 194-204 of SEQ ID NO 06. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 06 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-19, 43-56, 68-74, 100-106, 134- 136, 138-143, and / or 194-204 of SEQ ID NO 06. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0148] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 07 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 2-13, 30-41, 62-70, 81-86, 89-92, 118-123, 161-163, 164-169, and / or 217-227 of SEQ ID NO 07. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 07 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 2-13, 30-41, 62-70, 81-86, 89-92, 118-123, 161-163, 164-169, and / or 217-227 of SEQ ID NO 07. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0149] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 08 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 4-15, 32-42, 61-68, 79-84, 89-94, 120-125, 159-161, 162-167, and / or 215-225 of SEQ ID NO 08. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 08 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 4-15, 32-42, 61-68, 79-84, 89-94, 120-125, 159-161, 162-167, and / or 215-225 of SEQ ID NO 08. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0150] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 09 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 7-17, 37-48, 71-78, 90-95, 98-101, 127-132, 170-172, 173-178, and / or 226-236 of SEQ ID NO 09. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 09 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 7-17, 37-48, 71-78, 90-95, 98-101, 127-132, 170-172, 173-178, and / or 226-236 of SEQ ID NO 09. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0151] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 10 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 19-31, 49-60, 84-90, 101-106, 131-140, 175-177, 178-183, and / or 231-241 of SEQ ID NO 10. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 10 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 19-31, 49-60, 84-90, 101-106, 131-140, 175-177, 178-183, and / or 231-241 of SEQ ID NO 10. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0152] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 11 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 19-31, 49-60, 84-90, 101-106, 131-140, 175-177, 178-183, and / or 231-241 of SEQ ID NO 11. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 11 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 19-31, 49-60, 84-90, 101-106, 131-140, 175-177, 178-183, and / or 231-241 of SEQ ID NO 11. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0153] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 12 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 11-22, 41-51, 71-81, 91-96, 101-107, 151-156, 194-196, 197-202, and / or 250- 260 of SEQ ID NO 12. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 12 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 11-22, 41-51, 71-81, 91-96, 101-107, 151-156, 194-196, 197-202, and / or 250-260 of SEQ ID NO 12. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0154] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 13 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 13-23, 32-46, 73-81, 92-97, 123-131, 188-190, 191-196, and / or 240-250 of SEQ ID NO 13. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 13 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 13-23, 32-46, 73-81, 92-97, 123-131,

[0155] 188-190, 191-196, and / or 240-250 of SEQ ID NO 13. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0156] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 14 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 13-24, 33-47, 74-82, 93-98, 124-132, 189-191, 192-197, and / or 241-251 of SEQ ID NO 14. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 14 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 13-24, 33-47, 74-82, 93-98, 124-132,

[0157] 189-191, 192-197, and / or 241-251 of SEQ ID NO 14. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0158] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 15 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 43-49, 59-64, 90-96, 123-125, 128-133, and / or 177-187 of SEQ ID NO 15. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 15 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 43-49, 59-64, 90-96, 123-125, 128-133, and / or 177-187 of SEQ ID NO 15. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein. Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 16 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 19-31, 49-60, 84-90, 101-106, 131-140, 175-177, 178-183, and / or 231-241 of SEQ ID NO 16. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 16 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 19-31, 49-60, 84-90, 101-106, 131-140, 175-177, 178-183, and / or 231-241 of SEQ ID NO 16. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0159] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 17 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 3-17, 29-40, 58-67, 78-83, 88-95, 123-130, 167-169, 170-175, and / or 223-233 of SEQ ID NO 17. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 17 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 3-17, 29-40, 58-67, 78-83, 88-95, 123-130, 167-169, 170-175, and / or 223-233 of SEQ ID NO 17. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein.

[0160] Preferably, the enzyme of present invention differs from SEQ ID NO 44 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 9-19, 33-43, 63-70, 82-101, 106-113, 150-155, 189-191, 192-197, and / or 245-255 of SEQ ID NO 44. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 44 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 9-19, 33-43, 63-70, 82-101, 106-113, 150-155, 189-191, 192- 197, and / or 245-255 of SEQ ID NO 44. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB as described herein. In another and / or additional preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are selected from the list comprising, consisting of or consisting essentially of: a conservative amino acid residue substitution, a non-conservative amino acid residue substitution, a substitution of a hydrophilic amino acid residue into another hydrophilic amino acid residue, a hydrophobic or a neutral amino acid residue, a substitution of a hydrophobic amino acid residue into another hydrophobic amino acid residue, a hydrophilic amino acid residue or a neutral amino acid residue, a substitution of a neutral amino acid residue into another neutral amino acid residue, a hydrophilic amino acid residue or a hydrophobic amino acid residue, a substitution of a polar amino acid residue into a non-polar amino acid residue or vice versa, a substitution of a charged amino acid residue into a non-charged amino acid residue or vice versa, a substitution of an amino acid residue having a positively charged side chain at physiological pH into an amino acid residue having a negatively charged side chain at physiological pH or vice versa, a substitution of an amino acid residue having a positively charged side chain at physiological pH into an amino acid residue having a non-charged side chain at physiological pH or vice versa, a substitution of an amino acid residue having a negatively charged side chain at physiological pH into an amino acid residue having a non-charged side chain at physiological pH or vice versa, and a substitution of an aromatic amino acid residue into a non-aromatic amino acid residue or vice versa.

[0161] In the context of the present invention, a "conservative amino acid residue substitution" is the substitution of an amino acid residue by another structurally related amino acid residue, i.e. an amino acid residue having a side-chain with similar physicochemical properties. In the context of the present invention, throughout the application and claims, the amino acid classification system according to IMGT (ImMunoGeneTics) is used (Pommie et al, 2004, J. Mol. Recognit. 17(1): p. 17-32). The classification of the 20 common amino acids according to IMGT consists of several standardized classes that have been defined by the properties of their side chains (hydropathy, volume, chemical, charge, hydrogen donor or acceptor atoms, and polarity).

[0162] Accordingly, a conservative amino acid residue substitution is preferably the substitution of an amino acid residue from one IMGT class to another amino acid residue from the same IMGT class. Preferably, said IMGT class is hydrophobic class, neutral hydropathy class, hydrophilic class, positively charged class, uncharged class, negatively charged class, polar class, non-polar class or aromatic class.

[0163] Accordingly, in the context of the present invention, a conservative amino acid residue substitution is more preferably the substitution of: an aromatic amino acid residue (i.e. W, Y, F) by another aromatic amino acid residue (i.e. W, Y, F); a polar amino acid residue (i.e. R, N, D, Q, E, H, K, S, T, Y) by another polar amino acid residue (i.e.

[0164] R, N, D, Q, E, H, K, S, T, Y); a non-polar amino acid residue (i.e. A, C, G, I, L, M, F, P, W, V) by another non-polar amino acid residue (i.e. A, C, G, I, L, M, F, P, W, V); a hydrophobic amino acid residue (i.e. I, V, L, F, C, M, A, W) by another hydrophobic amino acid residue (i.e. I, V, L, F, C, M, A, W); a hydrophilic amino acid residue (i.e. N, D, Q, E, K, R) by another hydrophilic amino acid residue (i.e. N, D, Q, E, K, R); an amino acid residue with neither a hydrophobic nor hydrophilic side chain (i.e. G, T, S, Y, P, H) by another amino acid residue with neither a hydrophobic nor hydrophilic side chain (i.e. G, T, S, Y, P, H); an amino acid residue with a positively charged side chain at physiological pH (i.e. R, H, K) by another amino acid residue with a positively charged side chain at physiological pH (i.e. R, H, K); an amino acid residue with a negatively charged side chain at physiological pH (i.e. D, E) by another amino acid residue with a negatively charged side chain at physiological pH (i.e. D, E), or an amino acid residue with a neutral side chain at physiological pH (i.e. A, N, C, Q., G, I, L, M, F, P,

[0165] S, T, W Y, V) by another amino acid residue with an uncharged side chain at physiological pH (A, N, C, Q, G, I, L, M, F, P, S, T, W, Y, V).

[0166] As understood by the skilled person, "a hydrophobic amino acid residue" refers to an amino acid residue having a hydrophobic side chain. Said hydrophobic amino acid residue is I, V, L, F, C, M, A or W (ranked from most hydrophobic, i.e. I, to least hydrophobic, i.e. W).

[0167] As understood by the skilled person, "a hydrophilic amino acid residue" refers to an amino acid residue having a hydrophilic side chain. Said hydrophilic amino acid residue is R, K, E, Q, D or N (ranked from most hydrophilic, i.e. R, to least hydrophilic, i.e. N).

[0168] In the context of the present invention, the terms "amino acid residue with a negatively charged side chain at physiological pH" and "negatively charged amino acid residue" are used interchangeably herein. Likewise, the terms "amino acid residue with a positively charged side chain at physiological pH" and "positively charged amino acid residue" are used interchangeably herein. Likewise, the terms "amino acid residue with a neutral side chain at physiological pH" and "uncharged amino acid residue" are used interchangeably herein.

[0169] The term "neutral amino acid residue" can refer to an amino acid residue having neither a hydrophobic nor a hydrophilic side chain in the context of the property "hydropathy"; or can refer to an amino acid residue having a neutral side chain at physiological pH in the context of the property "charge" as disclosed herein. For clarity reasons, in the context of the present invention, unless specifically stated otherwise, "neutral amino acid residue" refers to an amino acid residue having neither a hydrophobic nor a hydrophilic side chain, whereas "uncharged amino acid residue" refers to an amino acid residue having a neutral side chain at physiological pH.

[0170] In a more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more conservative amino acid residue substitution(s) selected from the list consisting of the substitution of: an aromatic amino acid residue by another aromatic amino acid residue, preferably an F into W or Y; an W into Y and / or an Y into F; a polar amino acid residue by another polar amino acid residue, preferably an R into E or S; an N into E, H, Q, R, S, or Y; an D into H, Q, R or Y; a Q. into E, D, H, K, N, R, S, T, or Y; an H into N, Q, S, or Y; an S into E, H, N, R, or Y; an T into H, S, or Y; and / or an Y into D, E, H, K, N, R, S, T; a non-polar amino acid residue by another non-polar amino acid residue, preferably a C into A or G; a G into A or C; an F into A, G, I, L, P, V, W; an L into A, G; an M into G; a P into A, C, G, I, L, M, V, W; and / or an W into A, C, G, I, L, M, V; a hydrophobic amino acid residue by another hydrophobic amino acid residue, preferably a C into A; an F into A, I, L, V, W; an I into H; an L into A; and / or an W into A, C, I, L, M, V; a hydrophilic amino acid residue by another hydrophilic amino acid residue, preferably a D into Q. or R; an N into E, Q, R; and / or an R into E; an amino acid residue with neither a hydrophobic nor hydrophilic side chain by another amino acid residue with neither a hydrophobic nor hydrophilic side chain, preferably a T into G, H, S, or Y; an S into G, H or Y; an Y into G, H, S, T; a P into G, H, S, T or Y; and / or an H into G, S, or Y; and an amino acid residue with a neutral side chain at physiological pH by another amino acid residue with an uncharged side chain at physiological pH, preferably an A into Y; an N into A, G, Q, S, or Y; a C into A, G, Q, S, or Y; a Q. into A, G, I, L, N, P, S, T, V, W, or Y; a G into A, or C; an I into A, G or S; an L into A, G, Q, S, or Y; an M into G; an F into A, G, I, L, N, P, Q, S, T, V, W, or Y; a P into A, C, F, G, I, L, M, N, Q, S, T, V, W, or Y; a, S into A, G, N, or Y; a T into A, G, S, or Y; a W into A, C, G, I, L, M, S, V, or Y; and / or an Y into A, C, F, G, I, L, N, P, S, T, or V.

[0171] In the context of the invention, a "non-conservative amino acid residue substitution" is an amino acid residue substitution which is not a conservative amino acid residue substitution as described herein. Accordingly, a non-conservative amino acid residue substitution is preferably the substitution of (i) an amino acid residue from one IMGT class to another amino acid residue from a different IMGT class, preferably a different IMGT class within the same property (i.e. hydropathy, volume, chemical, charge, hydrogen donor or acceptor atoms, or polarity; more preferably hydropathy, chemical, charge or polarity; most preferably hydropathy, charge or polarity), or (ii) an aromatic amino acid residue (W, Y, F) into a non-aromatic amino acid residue, preferably an amino acid residue with a side chain that is less hydrophobic than the side chain of the aromatic amino acid residue that is substituted (it is referred to the property hydropathy according to the IMGT system as disclosed herein).

[0172] Amino acid residues are classified (according to IMGT classification system) as follows based on the property "hydropathy":

[0173] Hydrophobic: I, V, L, F, C, M, A, W (ranked from most hydrophobic, i.e. I, to least hydrophobic, i.e. W);

[0174] Neutral (neither a hydrophobic nor hydrophilic side chain): G, T, S, Y, P, H (ranked from most hydrophobic, i.e. G, to most hydrophilic, i.e. H);

[0175] Hydrophilic: R, K, E, Q, D, N (ranked from most hydrophilic, i.e. R, to least hydrophilic, i.e. N).

[0176] Preferably, a non-conservative amino acid residue substitution based on the property "hydropathy" is the substitution of a hydrophobic amino acid residue by a hydrophilic amino acid residue or vice versa. A neutral (neither a hydrophobic nor hydrophilic side chain) amino acid residue is preferably substituted by an amino acid residue with a side chain that is either hydrophobic or hydrophilic.

[0177] Amino acid residues are classified (according to IMGT classification system) as follows based on the property "volume":

[0178] Amino acid residues are classified (according to IMGT classification system) as follows based on the property "chemical":

[0179] Amino acid residues are classified (according to IMGT classification system) as follows based on the property "charge":

[0180] Preferably, a non-conservative amino acid residue substitution based on the property "charge" is the substitution of a positively charged amino acid residue by a negatively charged amino acid residue or uncharged amino acid residue; or a negatively charged amino acid residue by a positively charged amino acid residue or uncharged amino acid residue.

[0181] Amino acid residues are classified (according to IMGT classification system) as follows based on the property "hydrogen donor or acceptor atoms":

[0182] Amino acid residues are classified (according to IMGT classification system) as follows based on the property "polarity":

[0183] Accordingly, a non-conservative amino acid residue substitution is more preferably the substitution of (i) an aromatic amino acid residue by a non-aromatic amino acid residue or vice versa; (ii) a non-polar amino acid residue by a polar amino acid residue or vice versa; (iii) a hydrophobic amino acid residue by a hydrophilic amino acid residue or vice versa; (iv) a positively charged amino acid residue by a negatively charged amino acid residue or uncharged amino acid residue; or (v) a negatively charged amino acid residue by a positively charged amino acid residue or uncharged amino acid residue.

[0184] Said "aromatic amino acid residue", non-aromatic amino acid residue", "non-polar amino acid residue", "polar amino acid residue", "hydrophobic amino acid residue", "hydrophilic amino acid residue", "positively charged amino acid residue", "negatively charged amino acid residue" and "uncharged amino acid residue" are as disclosed earlier herein.

[0185] As the skilled person is familiar with, a particular amino acid residue can belong to different IMGT classes depending on the property as disclosed earlier herein. For example, phenylalanine (F) is an aromatic amino acid residue and hence a non-conservative amino acid residue substitution would be the substitution into a non-aromatic amino acid residue. At the same time, phenylalanine (F) is also a nonpolar amino acid and hence a non-conservative amino acid residue substitution would be the substitution into a polar amino acid residue.

[0186] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more non-conservative amino acid residue substitution(s) selected from the list consisting of the substitution of:

[0187] (i) an aromatic amino acid residue by a non-aromatic amino acid residue, preferably a W into A, C, D, E, G, H, I, L, M, S or V; an Y into A, C, D, E, G, H, I, K, L, N, P, R, S, T, or V; and / or an F into E, A, D, G, H, I, L, N, P, Q, R, S, T or V;

[0188] (ii) a non-aromatic amino acid residue by an aromatic amino acid residue, preferably an A into E, H or N; a C into A, G, H, Q, or S; a D into A, H, Q, or R; a G into A, or C; an H into G, N, Q, or S; an I into A, G, H, or S; an L into A, E, G, H, Q, R, or S; an M into G; an N into A, E, G, H, Q, R or S; a P into A, C, D, E, G, H, I, L, M, N, Q, R, S, T, or V; a Q. into E, A, D, G, H, I, K, L, N, P, R, S, T, or V; an R into E, or S; an S into A, E, G, H, N, or R; and / or a T into A, G, H, or S;

[0189] (iii) a non-polar amino acid residue by a polar amino acid residue, preferably an A into E, H, N or Y; a C into H, Q, S, or Y; an I into H, or S; an L into E, H, Q, R, S, or Y; an F into E, D, H, N, Q, R, S, T, or Y; a P into D, E, H, N, Q, R, S, T or Y; and / or an W into D, E, H, S, or Y;

[0190] (iv) a polar amino acid residue by a non-polar amino acid residue, preferably an N into A or G; a D into A; a Q. into A, G, I, L, P, V or W; a H into G; an S into A or G ; a T into A or G; and / or an Y A, G, H, Q, S, or Y into A, C, F, G, I, L, P, or V;

[0191] (v) a hydrophobic amino acid residue by a hydrophilic amino acid residue, preferably an L into E, Q. or R; an F into E, D, N, Q, or R; a C into Q; an A into E or N; and / or a W into D, E;

[0192] (vi) a hydrophilic amino acid residue by a hydrophobic amino acid residue, preferably an N into A; a D into A; and / or a Q. into A, I, L, or W;

[0193] (vii) a positively charged amino acid residue by a negatively charged amino acid residue, preferably an R into E;

[0194] (viii) a positively charged amino acid residue by an uncharged amino acid residue, preferably an R into S; and / or an H into G, N, Q, S, or Y;

[0195] (ix) a negatively charged amino acid residue by a positively charged amino acid residue, preferably a D into H or R; and

[0196] (x) a negatively charged amino acid residue by an uncharged amino acid residue, preferably a D into A or Q.

[0197] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of a hydrophilic amino acid residue into another hydrophilic amino acid residue, a hydrophobic or a neutral amino acid residue, preferably a D into Q, R, A, H or Y; an N into E, Q, R, A, G, H, S, or Y; an R into E or S; and / or a Q. into A, I, L, W, G, H, S, T, or Y.

[0198] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of a hydrophobic amino acid residue into another hydrophobic amino acid residue, a hydrophilic amino acid residue or a neutral amino acid residue, preferably a C into A, Q, G, H, S, or Y; an F into A, I, L, V, W, E, D, N, Q, R, G, H, P, S, T, or Y; an I into H, G, or S; an L into A, E, Q, R, G, H, S, or Y; an W into A, C, I, L, M, V, D, E, G, H, S, or Y; an A into E, N, H, Y; and / or an M into G.

[0199] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of a neutral amino acid residue into another neutral amino acid residue, a hydrophilic amino acid residue or a hydrophobic amino acid residue, preferably a G into A, or C; a T into A, G, H, S, or Y; an S into A, E, G, H, N, R, or Y; an Y into A, C, D, E, F, G, H, I, K, L, N, P, R, S, T, or V; a P into A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, or Y; and / or an H into G, N, Q, S, or Y.

[0200] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of a polar amino acid residue into a non-polar amino acid residue, preferably an R into E or S; an N into E, H, Q, R, S, or Y; an D into H, Q, R or Y; a Q. into E, D, H, K, N, R, S, T, or Y; an H into N, Q, S, or Y; an S into E, H, N, R, or Y; an T into H, S, or Y; and / or an Y into D, E, H, K, N, R, S, T.

[0201] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of a non-polar amino acid residue into a polar amino acid residue, preferably an A into E, H, N or Y; a C into H, Q, S, or Y; an I into H, or S; an L into E, H, Q, R, S, or Y; an F into E, D, H, N, Q, R, S, T, or Y; a P into D, E, H, N, Q, R, S, T or Y; and / or an W into D, E, H, S, or Y.

[0202] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of a charged amino acid residue into a non-charged amino acid residue, preferably an R into S; an H into G, N, Q, S, or Y; and / or a D into A or Q.

[0203] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of a non-charged amino acid residue into a charged amino acid residue, preferably an A into E or H; an N into E, H or R; an C into H; a Q. into E, D, H, K, or R; an I into H; an L into E, H or R; an F into E, D, H or R; a P into D, E, H, or R; an S into E, H, or R; a T into H; a W into D, E or H; and / or an Y into D, E, H, K, or R.

[0204] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of an amino acid residue having a positively charged side chain at physiological pH into an amino acid residue having a negatively charged side chain at physiological pH, preferably an R into E.

[0205] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of an amino acid residue having a negatively charged side chain at physiological pH into an amino acid residue having a positively charged side chain at physiological pH, preferably a D into H or R.

[0206] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of an amino acid residue having a positively charged side chain at physiological pH into an amino acid residue having a non-charged side chain at physiological pH, preferably an R into S; and / or an H into G, N, Q, S, or Y.

[0207] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of an amino acid residue having a non-charged side chain at physiological pH into an amino acid residue having a positively charged side chain at physiological pH, preferably into an A into H; an N into H or R; a C into H; a Q. into H, K or R; an I into H; an L into H or R; an F into H or R; a P into H or R; an S into H or R; a T into H; a W into H; and / or an Y into H, K or R. In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of an amino acid residue having a negatively charged side chain at physiological pH into an amino acid residue having a non-charged side chain at physiological pH, preferably a D into A or Q.

[0208] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of an amino acid residue having a non-charged side chain at physiological pH into an amino acid residue having a negatively charged side chain at physiological pH, preferably an A into E; an N into E; a Q. into D or E; an L into E; an F into D or E; a P into D or E; an S into E; a W into D or E; and / or an Y into D or E.

[0209] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of an aromatic amino acid residue into a non-aromatic amino acid residue, preferably a W into A, C, D, E, G, H, I, L, M, S or V; an Y into A, C, D, E, G, H, I, K, L, N, P, R, S, T, or V; and / or an F into E, A, D, G, H, I, L, N, P, Q, R, S, T or V.

[0210] In another and / or additional more preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are one or more substitution(s) of a non-aromatic amino acid residue into an aromatic amino acid residue, preferably an A into E, H or N; a C into A, G, H, Q, or S; a D into A, H, Q, or R; a G into A, or C; an H into G, N, Q, or S; an I into A, G, H, or S; an L into A, E, G, H, Q, R, or S; an M into G; an N into A, E, G, H, Q, R or S; a P into A, C, D, E, G, H, I, L, M, N, Q, R, S, T, or V; a Q. into E, A, D, G, H, I, K, L, N, P, R, S, T, or V; an R into E, or S; an S into A, E, G, H, N, or R; and / or a T into A, G, H, or S.

[0211] In another and / or additional preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are selected from the list comprising, consisting of or consisting essentially of: an alanine (A) into glutamic acid (E), histidine (H), asparagine (N), or tyrosine (Y), a cysteine (C) into A, glycine (G), H, glutamine (Q), serine (S), or Y, an aspartic acid (D) into A, H, Q, arginine (R), or Y, a phenylalanine (F) into E, A, D, G, H, isoleucine (I), leucine (L), N, proline (P), Q, R, S, threonine (T), valine (V), tryptophan (W), or Y, a G into A, or C, an H into G, N, Q, S, or Y, an I into A, G, H, or S, an L into A, E, G, H, Q, R, S, or Y, a methionine (M) into G, an N into A, E, G, H, Q, R, S, or Y, a P into A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, or Y, - a Q into E, A, D, G, H, I, lysine (K), L, N, P, R, S, T, V, W, or Y, an R into E, or S,

[0212] - an S into A, E, G, H, N, R, or Y, an T into A, G, H, S, or Y,

[0213] - an W into A, C, D, E, G, H, I, L, M, S, V, or Y, and

[0214] - an Y into A, C, D, E, F, G, H, I, K, L, N, P, R, S, T, or V.

[0215] In another and / or additional preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) correspond to position(s) F83, Q148, W54, L144, S188, G102, N104, H189, C82, D55, F61, N52, W53, Y147, A58, P86, P143, T252, Q84, L100, N85, N149, 1251, S59, Y250, R26, T60, H57, S56, W62, S81, R116, L89, L105, M117, 1146, N244 and / or S245 of SEQ ID NO 01.

[0216] In a more preferred embodiment, an amino acid residue substitution corresponding to position F83 of SEQ ID NO 01 is a substitution into E, D, A, G, H, I, L, N, P, Q, R, S, T, V, W, or Y. In an even more preferred embodiment, an amino acid residue substitution corresponding to position F83 of SEQ ID NO 01 is a substitution into E or D.

[0217] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position Q148 of SEQ ID NO 01 is a substitution into E, D, A, G, H, I, K, L, N, P, R, S, T, V, W, or Y. In an even more preferred embodiment, an amino acid residue substitution corresponding to position Q148 of SEQ ID NO 01 is a substitution into E or D.

[0218] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position W54 of SEQ ID NO 01 is a substitution into A, C, D, E, I, H, L, M, S, V, or Y.

[0219] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position L144 of SEQ ID NO 01 is a substitution into A, E, G, or H.

[0220] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position S188 of SEQ ID NO 01 is a substitution into E, H, or Y.

[0221] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position G102 of SEQ ID NO 01 is a substitution into A, or C.

[0222] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position N104 of SEQ ID NO 01 is a substitution into A, G, H, S, or Y.

[0223] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position H189 of SEQ ID NO 01 is a substitution into G, N, Q, or S.

[0224] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position C82 of SEQ ID NO 01 is a substitution into A, G, H, Q, S, or Y.

[0225] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position D55 of SEQ ID NO 01 is a substitution into A, H, Q, R, or Y. In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position F61 of SEQ ID NO 01 is a substitution into A, G, R, or Y.

[0226] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position N52 of SEQ ID NO 01 is a substitution into A, E, H, R, or S.

[0227] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position W53 of SEQ ID NO 01 is a substitution into A, G, H, S, or Y.

[0228] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position Y147 of SEQ ID NO 01 is a substitution into A, C, D, E, F, G, H, I, K, L, N, P, R, S,

[0229] T, or V.

[0230] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position A58 of SEQ ID NO 01 is a substitution into E, H, N, or Y.

[0231] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position P86 of SEQ ID NO 01 is a substitution into C, D, E, F, G, H, I, L, M, N, R, S, T, V,

[0232] W, or Y.

[0233] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position P143 of SEQ ID NO 01 is a substitution into A, G, Q, or S.

[0234] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position T252 of SEQ ID NO 01 is a substitution into A, G, or S.

[0235] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position Q84 of SEQ ID NO 01 is a substitution into A, E, G, H, N, R, or S.

[0236] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position L100 of SEQ ID NO 01 is a substitution into A, H, Q, S, or Y.

[0237] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position N85 of SEQ ID NO 01 is a substitution into G, or S.

[0238] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position N149 of SEQ ID NO 01 is a substitution into E, or Q.

[0239] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position 1251 of SEQ ID NO 01 is a substitution into A, G, H, or S.

[0240] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position S59 of SEQ ID NO 01 is a substitution into A, or H.

[0241] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position Y250 of SEQ ID NO 01 is a substitution into N.

[0242] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position R26 of SEQ ID NO 01 is a substitution into E, or S.

[0243] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position T60 of SEQ ID NO 01 is a substitution into A, H, or Y. In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position H57 of SEQ ID NO 01 is a substitution into G, N, or Y.

[0244] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position S56 of SEQ ID NO 01 is a substitution into A, E, G, H, or R.

[0245] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position W62 of SEQ ID NO 01 is a substitution into Y.

[0246] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position S81 of SEQ ID NO 01 is a substitution into A, or N.

[0247] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position R116 of SEQ ID NO 01 is a substitution into E.

[0248] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position L89 of SEQ ID NO 01 is a substitution into E, or R.

[0249] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position L105 of SEQ ID NO 01 is a substitution into A, or Q.

[0250] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position M117 of SEQ ID NO 01 is a substitution into G.

[0251] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position 1146 of SEQ ID NO 01 is a substitution into H, or S.

[0252] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position N244 of SEQ ID NO 01 is a substitution into R, or S.

[0253] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position S245 of SEQ ID NO 01 is a substitution into Y.

[0254] In another and / or additional preferred embodiment of the enzyme of present invention, the enzyme comprises, consists of or consists essentially of a polypeptide as represented by any one of SEQ ID NOs 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 43 or 45. In another and / or additional preferred embodiment of the enzyme of present invention, the enzyme is SEQ ID NO 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 43 or 45.

[0255] In another and / or additional preferred embodiment of the enzyme of present invention, the enzyme comprises the IPR001503 domain as defined by InterPro 99.0 as released on 28th March 2024.

[0256] In another and / or additional preferred embodiment of the enzyme of present invention, the enzyme has a GDP-fucose binding domain. In another and / or additional preferred embodiment of the enzyme of present invention, the progenitor fucosyltransferase is SEQ ID NO 01 and the mutant fucosyltransferase of present invention differs from said progenitor fucosyltransferase with SEQ ID NO 01 by any one or more amino acid residue substitution(s) selected from the list consisting of F83E, Q148E, F83D, F83S, F83G, F83A, F83Y, F83N, F83Q, F83V, F83T, L144A, F83W, L144G, F83I, Q148N, F83L, Q148A, S188H, P86S, L144E, P143A, T252G, C82A, Q84E, T252S, N104H, L100A, N85S, S188E, Q148L, P86H, L144H, C82S, N52E, N149Q, I251A, I251H, L100H, S59H, Y250N, A58Y, N149E, Q84A, R26S, T60H, H57N, Q84S, N85G, P143Q, P86C, P86E, P86F, P86Y, S56A, W54V, P86M, S56E, L100Q, P86V, Q84G, S59A, P86T, F83R, S56G, T60Y, W54M, W62Y, C82G, F61A, Q84H, S56R, A58E, A58H, Q84R, S81A, Q84N, A58N, H57Y, S56H, N104Y, D55H, L100Y, H189Q, I251S, N104S, R116E, D55Y, H57G, and T60A.

[0257] In another and / or additional preferred embodiment of the enzyme of present invention, the progenitor fucosyltransferase is SEQ ID NO 01 and the mutant fucosyltransferase of present invention differs from said progenitor fucosyltransferase with SEQ ID NO 01 by one or more amino acid residue substitution(s) selected from the list consisting of C82Y, D55R, F61R, F83P, N52R, W53G, W53S, W54D, W54E, F83E, W54I, Q148E, F83D, W54L, F83W, F83V, F83T, W53A, F83G, F83A, F83S, Y147G, F83N, F83Y, Y147L, F83I, Q148D, F83Q, F83L, D55Q, C82Q, W54V, D55A, A58E, and F83R.

[0258] In another and / or additional preferred embodiment of the enzyme of present invention, the progenitor fucosyltransferase is SEQ ID NO 01 and the mutant fucosyltransferase of present invention differs from said progenitor fucosyltransferase with SEQ ID NO 01 by an amino acid residue substitution at (1) Y147 and Q148, (2) R26 and Q148, (3) R26 and Y147, (4) P86 and Q148, (5) R26 and P86, (6) F83, Y147 and Q148, (7) F83 and Q148, (8) F83 and L144, (9) F83 and S188, (10) F83 and G102, or (11) F83 and H189. In a more preferred embodiment, the amino acid residue substitution at Y147 is any one of Y147A, Y147C, Y147D, Y147E, Y147F, Y147G, Y147H, Y147I, Y147K, Y147N, Y147P, Y147R, Y147S, Y147T, or Y147V. In an alternative and / or additional more preferred embodiment, the amino acid residue substitution at Q148 is any one of Q148E, Q148A, Q148G, Q148H, Q148I, Q148K, Q148L, Q148N, Q148P, Q148R, Q148S, Q148T, Q148V, Q148W, or Q148Y. In an alternative and / or additional more preferred embodiment, the amino acid residue substitution at R26 is R26E. In an alternative and / or additional more preferred embodiment, the amino acid residue substitution at P86 is P86E. In an alternative and / or additional more preferred embodiment, the amino acid residue substitution at F83 is any one of F83E, F83A, F83D, or F83N. In an alternative and / or additional more preferred embodiment, the amino acid residue substitution at L144 is any one of L144A, or L144E. In an alternative and / or additional more preferred embodiment, the amino acid residue substitution at S188 is any one of S188E, or S188H. In an alternative and / or additional more preferred embodiment, the amino acid residue substitution at G102 is G102A. In an alternative and / or additional more preferred embodiment, the amino acid residue substitution at H189 is H189G. In an even more preferred embodiment, the amino acid residue substitution at Y147 and Q148 is any one of Y147H and Q148Y, Y147H and Q148G, Y147N and Q148E, Y147P and Q148K, Y147C and Q148K, Y147F and Q148E, Y147R and Q148A, Y147T and Q148A, Y147V and Q148E, Y147G and Q148E, Y147F and Q148K, Y147N and Q148S, Y147C and Q148H, Y147P and Q148H, Y147C and Q148N, Y147A and Q148R, Y147C and Q148L, Y147F and Q148R, Y147D and Q148V, Y147V and Q148W, Y147P and Q148P, Y147I and Q148Y, Y147A and Q148L, Y147E and Q148R, Y147V and Q148V, Y147K and Q148T, Y147V and Q148I, or, Y147S and Q148Y. In another even more preferred embodiment, the amino acid residue substitution at R26 and Q148 is R26E and Q148E, or, R26E and Q148N. In another even more preferred embodiment, the amino acid residue substitution at R26 and Y147 is R26E and Y147G. In another even more preferred embodiment, the amino acid residue substitution at P86 and Q148 is P86E and Q148E. In another even more preferred embodiment, the amino acid residue substitution at R26 and P86 is R26E and P86E. In another even more preferred embodiment, the amino acid residue substitution at F83, Y147 and Q148 is any one of F83A, Y147V and Q148E; F83N, Y147N and Q148E; F83E, Y147N and Q148E; F83N, Y147V and Q148E; F83E, Y147V and Q148E; F83D, Y147N and Q148E; F83A, Y147N and Q148E; F83D, Y147V and Q148E; F83E, Y147T and Q148A; F83D, Y147T and Q148A; F83N, Y147T and Q148A; or; F83A, Y147T and Q148A. In a most preferred embodiment, the amino acid residue substitution at F83, Y147 and Q148 is any one of F83A, Y147V and Q148E; F83N, Y147N and Q148E; or; F83E, Y147N and Q148E. In another even more preferred embodiment, the amino acid residue substitution at F83 and Q148 is any one of F83D and Q148E; F83E and Q148E; F83A and Q148E; F83N and Q148E; or; F83E and Q148N. In another even more preferred embodiment, the amino acid residue substitution at F83 and L144 is any one of F83E and L144A; or; F83E and L144E. In another even more preferred embodiment, the amino acid residue substitution at F83 and S188 is any one of F83E and S188H; or; F83E and S188E. In another even more preferred embodiment, the amino acid residue substitution at F83 and G102 is F83E and G102A. In another even more preferred embodiment, the amino acid residue substitution at F83 and H189 is F83E and H189G.

[0259] In another and / or additional preferred embodiment of the enzyme of present invention, the amino acid sequence of the mutant fucosyltransferase of present invention has the same length as the amino acid sequence of its progenitor fucosyltransferase. In an alternative preferred embodiment, the amino acid sequence of the mutant fucosyltransferase of present invention is shorter than the amino acid sequence of its progenitor fucosyltransferase. In an alternative preferred embodiment, the amino acid sequence of the mutant fucosyltransferase of present invention is longer than the amino acid sequence of its progenitor fucosyltransferase. In an alternative preferred embodiment, the amino acid sequence of the mutant fucosyltransferase of present invention comprises a secretion signal, a solubility tag and / or a purification tag. In another and / or additional preferred embodiment of the enzyme of present invention, the enzyme has alpha-1, 3-fucosyltransferase activity on lactose. In other words, the enzyme is capable of producing 3- fucosyllactose, preferably the enzyme produces 3-fucosyllactose. In an alternative preferred embodiment of the enzyme of present invention, the enzyme has no alpha-1, 3-fucosyltransferase activity on lactose. In other words, the enzyme is not capable of producing 3-fucosyllactose.

[0260] Surprisingly, the mutant fucosyltransferase as described herein has an improved alpha-1,4 fucosyltransferase activity and / or improved alpha-1,4 fucosyltransferase specificity on the GIcNAc residue of LNB compared to its progenitor fucosyltransferase. More specifically, the expression "mutant fucosyltransferase having an improved alpha-1,4 fucosyltransferase activity on the GIcNAc residue of LNB" is to be understood as a mutant fucosyltransferase as described herein having a higher, adjusted, tweaked and / or faster catalytic alpha-1,4 fucosyltransferase activity on the GIcNAc residue of LNB which results in a higher production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or in a production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein with a higher purity when using said mutant fucosyltransferase compared to its progenitor fucosyltransferase. The expression "mutant fucosyltransferase having an improved alpha-1,4 fucosyltransferase specificity on the GIcNAc residue of LNB" is to be understood as a mutant fucosyltransferase as described herein having a higher, better or increased specific substrate specificity or preference towards the GIcNAc residue of LNB which results in a higher production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or in a production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein with a higher purity when using said mutant fucosyltransferase compared to its progenitor fucosyltransferase.

[0261] Additionally, and surprisingly, the mutant fucosyltransferase as described herein may have a declined alpha-1,3 fucosyltransferase activity and / or declined alpha-1,3 fucosyltransferase specificity on the glucose (Glc) residue of lactose compared to its progenitor fucosyltransferase. Herein, said lactose can reside in free form or can be present in an oligosaccharide like e.g. LNT. More specifically, the expression "mutant fucosyltransferase having a declined alpha-1,3 fucosyltransferase activity on the Glc residue of lactose" is to be understood as a mutant fucosyltransferase as described herein having a lower and / or slower catalytic alpha-1,3 fucosyltransferase activity on the Glc residue of lactose which results in a lower production of 3-FL and / or a fucosylated compound comprising Gal-pi,4-[Fuc-al,3]-Glc and / or in a production of 3-FL and / or a fucosylated compound comprising Gal-pi,4-[Fuc-al,3]-Glc with a lower purity when using said mutant fucosyltransferase compared to its progenitor fucosyltransferase. The expression "mutant fucosyltransferase having a declined alpha-1,3 fucosyltransferase specificity on the Glc residue of lactose" is to be understood as a mutant fucosyltransferase as described herein having a decreased specific substrate specificity or preference towards the Glc residue of lactose which results in a lower production of 3-FL and / or a fucosylated compound comprising Gal-pi,4-[Fuc-al,3]-Glc and / or in a production of 3-FL and / or a fucosylated compound comprising Gal-pi,4-[Fuc-al,3]-Glc with a lower purity when using said mutant fucosyltransferase compared to its progenitor fucosyltransferase.

[0262] According to a second aspect, the invention provides a method for designing a mutant fucosyltransferase having alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of Gal-pi,3-GlcNAc (lacto-N-biose, LNB), wherein the activity and / or specificity for transferring a fucose residue in an alpha-1, 4-glycosidic linkage on the GIcNAc residue of LNB is improved as compared with a progenitor fucosyltransferase of said mutant fucosyltransferase. In a specific embodiment, the method comprises: i. specifying in a progenitor fucosyltransferase one or more amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196 and / or 244- 254 of SEQ ID NO Ol, ii. constructing an amino acid sequence in which the one or more amino acid residue(s) specified in (i) is / are modified by substitution, insertion and / or deletion resulting in said mutant fucosyltransferase, and ill. evaluating the activity and / or substrate specificity with respect to the GIcNAc residue of LNB of the mutant fucosyltransferase obtained in (ii).

[0263] Herein, said LNB can reside in free form or is present as part of a saccharide as described herein. Optionally, the LNB and / or saccharide is / are linked to a peptide, a protein and / or a lipid.

[0264] In the context of present invention, the progenitor fucosyltransferase that is used to design a mutant fucosyltransferase having alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB, wherein the activity and / or specificity for transferring a fucose residue in an alpha-1, 4-glycosidic linkage on the GIcNAc residue of LNB is improved as compared with said progenitor fucosyltransferase, is as described in the first aspect of the invention. Additionally, the mutant fucosyltransferase is an enzyme as described in the first aspect of the invention.

[0265] According to a third aspect, the invention provides a method for the production of a mutant fucosyltransferase having alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of Gal-pi,3-GlcNAc (lacto-N-biose, LNB). In a specific embodiment, the method comprises: i. preparing a nucleic acid molecule encoding an amino acid sequence constructed by the method as described in the second aspect of the invention, ii. expressing the nucleic acid molecule, ill. collecting the mutant fucosyltransferase.

[0266] Herein, said LNB can reside in free form or is present as part of a saccharide as described herein. Optionally, the LNB and / or saccharide is / are linked to a peptide, a protein and / or a lipid. In the context of present invention, the mutant fucosyltransferase is an enzyme as described in the first aspect of the invention.

[0267] According to a fourth aspect, the present invention provides a de novo designed enzyme having alpha- 1,3-fucosyltransferase activity on the glucose (Glc) residue of Gal-pi,4-Glc (lactose) and comprising, consisting of or consisting essentially of when compared to SEQ ID NO 01 one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01. Specifically, said de novo designed enzyme is obtained by modification of a progenitor fucosyltransferase wherein said modification comprises one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190 and / or 191-196 of SEQ ID NO 01. Herein, said progenitor fucosyltransferase is as described in the first aspect of the invention.

[0268] In other words and as understood herein, the enzyme is a mutant fucosyltransferase derived from a progenitor fucosyltransferase wherein the mutant fucosyltransferase differs from said progenitor fucosyltransferase in at least one amino acid residue substitution, insertion and / or deletion compared to the polypeptide sequence of a progenitor fucosyltransferase at amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01. The terms "derived from", "mutated from" and "modified from" are used herein interchangeably and are to be understood as that the polypeptide sequence of the mutant fucosyltransferase differs in at least one amino acid residue substitution, insertion and / or deletion compared to the polypeptide sequence of a progenitor fucosyltransferase at amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190 and / or 191-196 of SEQ ID NO 01.

[0269] An enzyme having alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose is to be understood as that said enzyme is capable of transferring, preferably transfers, a fucose (Fuc) residue from a suitable fucose donor to the Glc residue of lactose in an alpha-1, 3-glycosidic linkage resulting in the production of 3-fucosyllactose (3-FL). Preferably, the enzyme of present invention is capable of using and / or uses GDP- fucose as suitable fucose donor.

[0270] Surprisingly, the de novo designed enzyme of present invention has an improved alpha-1,3 fucosyltransferase activity and / or improved alpha-1,3 fucosyltransferase specificity on the Glc residue of lactose compared to an alpha-1, 3 / alpha-l, 4 fucosyltransferase that does not comprise one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01.

[0271] The expression "improved fucosyltransferase activity of an enzyme" is to be understood as a higher, adjusted, tweaked and / or faster catalytic fucosyltransferase activity of said enzyme on a specific compound which results in a higher production of a specific fucosylated compound and / or in the production of a specific fucosylated compound with a higher purity when using said enzyme compared to a reference fucosyltransferase. Specifically for the enzyme of present invention, the expression "enzyme having an improved alpha-1,3 fucosyltransferase activity on the Glc residue of lactose" is to be understood as an enzyme of present invention having a higher, adjusted, tweaked and / or faster catalytic alpha-1,3 fucosyltransferase activity on the Glc residue of lactose which results in a higher production of 3-FL and / or in a production of 3-FL with a higher purity when using said enzyme of present invention compared to an alpha-1, 3 / alpha-l, 4 fucosyltransferase that does not comprise one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01.

[0272] The expression "improved fucosyltransferase specificity of an enzyme" is to be understood as an increased or preferred specific substrate specificity of said enzyme towards a specific compound to be used in a fucosylation reaction catalyzed by said enzyme which results in a higher production of a specific fucosylated compound and / or in the production of a specific fucosylated compound with a higher purity when using said enzyme compared to a reference fucosyltransferase. Specifically for the enzyme of present invention, the expression "enzyme having an improved alpha-1,3 fucosyltransferase specificity on the Glc residue of lactose" is to be understood as an enzyme of present invention having a higher, better or increased specific substrate specificity or preference towards the Glc residue of lactose which results in a higher production of 3-FL and / or in a production of 3-FL with a higher purity when using said enzyme of present invention compared to an alpha-1, 3 / alpha-l, 4 fucosyltransferase that does not comprise one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01.

[0273] In a specific embodiment, the de novo designed enzyme of present invention is a biologically active enzyme. The skilled person can readily assess whether an enzyme having alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose as described herein is active or not (it is for example referred to Blixt et al, 1999, Glycobiology 9(10): p. 1061-1071; which is incorporated by reference), by bringing the enzyme into contact with (1) a suitable fucose donor, like e.g. GDP-fucose, and (2) lactose under conditions suitable for the transfer of fucose from said donor to the Glc residue of said lactose in an alpha-1,3 glycosidic linkage. Herein, a cellular method or a cell-free method (e.g. enzymatic method) are suitable. Suitable conditions comprise a pH of 5 to 10, i.e. a pH of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, with an optimal pH between 7 to 8, i.e. a pH of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 and a temperature of 20°C to 50°C, i.e. a temperature of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C with an optimal temperature range of 20°C to 30°C, i.e. a temperature of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C. In the context of the invention, an enzyme of present invention is active when at least 10.0 %, preferably at least 20.0%, more preferably at least 25.0%, even more preferably at least 50.0% of lactose is modified by the enzyme, i.e., is modified by the addition of a fucose residue in an alpha-1, 3-linkage on the Glc residue of lactose.

[0274] In an additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01. More specifically, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195, and / or 196 of SEQ ID NO 01. Preferably, said amino acid residue substitution(s), insertion(s) and / or deletion(s) are scattered along the polypeptide sequence of said enzyme. Additionally, and / or alternatively, said amino acid residue substitution(s), insertion(s) and / or deletion(s) can be integrated consecutively in the polypeptide sequence of said enzyme.

[0275] It is to be understood by the skilled person that the enzyme of present invention is not SEQ ID NO 01, i.e. the de novo designed enzyme differs from SEQ ID NO 01 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188- 190, and / or 191-196 of SEQ ID NO 01. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 01 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose as described herein.

[0276] In a preferred embodiment, the enzyme of present invention is a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to the full-length amino acid sequence of SEQ ID NO 01 and having alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein. More specifically, the enzyme is a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence that is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to the full-length amino acid sequences of SEQ ID NO 01 and having alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose as described herein.

[0277] In a specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue substitution(s) at amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01 and has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein. In a preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 only one amino acid residue substitution at an amino acid residue corresponding to position 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 or 196 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 two amino acid residue substitutions at two consecutive or non-consecutive amino acid residues corresponding to position 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 three amino acid residue substitutions at three consecutive or non-consecutive amino acid residues corresponding to position 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 four or more amino acid residue substitutions at four or more consecutive or non-consecutive amino acid residues corresponding to positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01.

[0278] In another and / or additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue insertion(s) at one or more amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01 and has alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose as described herein. In a preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 only one amino acid residue insertion before or after an amino acid residue corresponding to position 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 or 196 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 two consecutive or non-consecutive amino acid residue insertions before and / or after one or two amino acid residue(s) corresponding to positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 three consecutive or non-consecutive amino acid residue insertions before and / or after one, two or three amino acid residue(s) corresponding to positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 four or more consecutive or non-consecutive amino acid residue insertions before and / or after one or more amino acid residue(s) corresponding to positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01.

[0279] In another and / or additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue deletion(s) at amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01 and has alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose as described herein. In a preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 only one amino acid residue deletion at an amino acid residue corresponding to position 52, 53, 54, 55, 56, 57,

[0280] 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,

[0281] 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 or 196 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 two amino acid residue deletions at two consecutive or non-consecutive amino acid residues corresponding to positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 three amino acid residue deletions at three consecutive or non-consecutive amino acid residues corresponding to positions 52, 53, 54, 55, 56, 57, 58,

[0282] 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,

[0283] 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01. In another preferred embodiment, the enzyme of present invention comprises when compared to SEQ ID NO 01 four or more amino acid residue deletions at four or more consecutive or non-consecutive amino acid residues corresponding to positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01. In another and / or additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue substitution(s) and one or more amino acid residue insertion(s) at amino acid residue(s) corresponding to positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01 and has alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose as described herein.

[0284] In another and / or additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue substitution(s) and one or more amino acid residue deletion(s) at amino acid residue(s) corresponding to positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01 and has alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose as described herein.

[0285] In another and / or additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue insertions(s) and one or more amino acid residue deletion(s) at amino acid residue(s) corresponding to positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01 and has alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose as described herein.

[0286] In another and / or additional specific embodiment of present invention, the de novo designed enzyme comprises when compared to SEQ ID NO 01 one or more amino acid residue substitution(s), one or more amino acid residue insertion(s) and one or more amino acid residue deletion(s) at amino acid residue(s) corresponding to positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 81, 82, 83, 84, 85, 86, 87, 88, 89, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 188, 189, 190, 191, 192, 193, 194, 195 and / or 196 of SEQ ID NO 01 and has alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose as described herein.

[0287] In a preferred embodiment of the enzyme of present invention, the amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01 are determined by a multiple sequence alignment tool MAFFT using default algorithm parameters and using the "--reorder" argument for the alignment and using the "--add" argument for the polypeptide sequence of said enzyme. In a more preferred embodiment, the multiple sequence alignment tool MAFFT that is used to determine the amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01 is MAFFT version 7.505 as released in July 2021 and the default algorithm parameters used in said tool comprise BLOSUM62 as scoring matrix, 1.53 as gap opening penalty and 0.0 as offset value. In another and / or additional preferred embodiment of the enzyme of present invention, the enzyme is a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, or 44. More specifically, the enzyme is a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence that is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 or 44 and has alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose as described herein. The person skilled will understand that the enzyme of present invention is not identical to SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 or 44, but differs from SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 or 44 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) of said SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 or 44, respectively, corresponding to positions 52-62, 81-89, 100-117 and / or 188-196 of SEQ ID NO 01. The amino acid positions of SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 and 44 are indicated in function of the amino acid sequence of SEQ ID NO 01 in Tables 1-3.

[0288] Preferably, the enzyme of present invention differs from SEQ ID NO 02 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 49-60, 84-90, 101-106, 175-177, and / or 178-183 of SEQ ID NO 02. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 02 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 49-60, 84-90, 101-106, 175-177, and / or 178-183 of SEQ ID NO 02. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose.

[0289] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 03 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 37-46, 65-72, 84-97, 171-173, and / or 174-179 of SEQ ID NO 03. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 03 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 37-46, 65-72, 84-97, 171-173, and / or 174-179 of SEQ ID NO 03. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0290] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 04 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 44-50, 61-66, 124-126, and / or 128-133 of SEQ ID NO 04. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 04 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 44-50, 61-66, 124-126, and / or 128-133 of SEQ ID NO 04. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0291] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 05 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 44-50, 61-66, 124-126, and / or 128-133 of SEQ ID NO 05. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 05 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 44-50, 61-66, 124-126, and / or 128-133 of SEQ ID NO 05. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0292] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 06 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-19, 43-56, 68-74, 134-136, and / or 138-143 of SEQ ID NO 06. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 06 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-19, 43-56, 68-74, 134-136, and / or 138-143 of SEQ ID NO 06. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0293] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 07 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 30-41, 62-70, 81-92, 161-163, and / or 164-169 of SEQ ID NO 07. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 07 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 30-41, 62-70, 81-92, 161-163, and / or 164-169 of SEQ ID NO 07. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0294] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 08 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 32-42, 61-68, 79-94, 159-161, and / or 162-167 of SEQ ID NO 08. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 08 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 32-42, 61-68, 79-94, 159-161, and / or 162-167 of SEQ ID NO 08. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0295] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 09 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 37-48, 71-78, 90-101, 170-172, and / or 173-178 of SEQ ID NO 09. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 09 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 37-48, 71-78, 90-101, 170-172, and / or 173-178 of SEQ ID NO 09. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0296] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 10 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 49-60, 84-90, 101-106, 175-177, and / or 178-183 of SEQ ID NO 10. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 10 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 49-60, 84-90, 101-106, 175-177, and / or 178-183 of SEQ ID NO 10. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0297] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 11 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 49-60, 84-90, 101-106, 175-177, and / or 178-183 of SEQ ID NO 11. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 11 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 49-60, 84-90, 101-106, 175-177, and / or 178-183 of SEQ ID NO 11. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0298] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 12 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 41-51, 71-81, 91-107, 194-196, and / or 197-202 of SEQ ID NO 12. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 12 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 41-51, 71-81, 91-107, 194-196, and / or 197-202 of SEQ ID NO 12. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0299] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 13 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 32-46, 73-81, 92-97, 188-190, and / or 191-196 of SEQ ID NO 13. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 13 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 32-46, 73-81, 92-97, 188-190, and / or 191-196 of SEQ ID NO 13. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0300] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 14 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 33-47, 74-82, 93-98, 189-191, and / or 192-197 of SEQ ID NO 14. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 14 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 33-47, 74-82, 93-98, 189-191, and / or 192-197 of SEQ ID NO 14. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0301] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 15 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 43-49, 59-64, 123-125, and / or 128-133 of SEQ ID NO 15. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 15 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 10-21, 43-49, 59-64, 123-125, and / or 128-133 of SEQ ID NO 15. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0302] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 16 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 49-60, 84-90, 101-106, 175-177, and / or 178-183 of SEQ ID NO 16. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 16 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 49-60, 84-90, 101-106, 175-177, and / or 178-183 of SEQ ID NO 16. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0303] Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 17 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 29-40, 58-67, 78-95, 167-169, and / or 170-175 of SEQ ID NO 17. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 17 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 29-40, 58-67, 78-95, 167-169, and / or 170-175 of SEQ ID NO 17. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein. Alternatively, and / or additionally, the enzyme of present invention preferably differs from SEQ ID NO 44 in at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 33-43, 63-70, 82-113, 189-191, and / or 192-197 of SEQ ID NO 44. Additionally, the enzyme of present invention can comprise one or more other modification(s) compared to said SEQ ID NO 44 in addition to said at least one amino acid residue substitution, insertion and / or deletion at amino acid residue(s) corresponding to positions 33-43, 63-70, 82-113, 189-191, and / or 192-197 of SEQ ID NO 44. Said modification(s) can comprise but is / are not limited to e.g. fusion to a chaperone protein, a solubility tag, a secretory signal, a cleavable linker, a tag for antibody detection, a tag for fluorescence detection. Despite said possible additional modification(s) the enzyme of present invention has alpha-1, 3- fucosyltransferase activity on the Glc residue of lactose as described herein.

[0304] In another and / or additional preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s) is / are selected from the list comprising, consisting of or consisting essentially of an E into N; a G into H; a H into Q; an N into G, or S; a P into H; a Q into S; and a T into A, E, or S.

[0305] In another and / or additional preferred embodiment of the enzyme of present invention, the one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) correspond to position(s) E109, N107,

[0306] T106, P115, Q84, G192, and / or H57 of SEQ ID NO 01.

[0307] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position E109 of SEQ ID NO 01 is a substitution into N.

[0308] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position N107 of SEQ ID NO 01 is a substitution into G, or S.

[0309] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position T106 of SEQ ID NO 01 is a substitution into A, E, or S.

[0310] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position P115 of SEQ ID NO 01 is a substitution into H.

[0311] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position Q84 of SEQ ID NO 01 is a substitution into S.

[0312] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position G192 of SEQ ID NO 01 is a substitution into H.

[0313] In another and / or additional more preferred embodiment, an amino acid residue substitution corresponding to position H57 of SEQ ID NO 01 is a substitution into Q.

[0314] In another and / or additional preferred embodiment of the enzyme of present invention, the enzyme comprises, consists of or consists essentially of a polypeptide as represented by any one of SEQ ID NOs 37, 38, 39, 40, 41, or 42. In another and / or additional preferred embodiment of the enzyme of present invention, the enzyme is SEQ ID NO 37, 38, 39, 40, 41, or 42.

[0315] Surprisingly, the mutant fucosyltransferase as described herein has an improved alpha-1,3 fucosyltransferase activity and / or improved alpha-1,3 fucosyltransferase specificity on the Glc residue of lactose compared to its progenitor fucosyltransferase. More specifically, the expression "mutant fucosyltransferase having an improved alpha-1,3 fucosyltransferase activity on the Glc residue of lactose" is to be understood as a mutant fucosyltransferase as described herein having a higher, adjusted, tweaked and / or faster catalytic alpha-1,3 fucosyltransferase activity on the Glc residue of lactose which results in a higher production of 3-FL and / or in a production 3-FL with a higher purity when using said mutant fucosyltransferase compared to its progenitor fucosyltransferase. The expression "mutant fucosyltransferase having an improved alpha-1,3 fucosyltransferase specificity on the Glc residue of lactose" is to be understood as a mutant fucosyltransferase as described herein having a higher, better or increased specific substrate specificity or preference towards the Glc residue of lactose which results in a higher production of 3-FL and / or in a production of 3-FL as described herein with a higher purity when using said mutant fucosyltransferase compared to its progenitor fucosyltransferase.

[0316] According to a fifth aspect, the invention provides a method for designing a mutant fucosyltransferase having alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose, wherein said mutant fucosyltransferase is derived from a progenitor fucosyltransferase having alpha-1, 3-fucosyltransferase activity and / or alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB. The method comprises: i. specifying in said progenitor fucosyltransferase one or more amino acid residue(s) corresponding to positions 52-62, 81-89, 100-117, 188-190, and / or 191-196 of SEQ ID NO 01, ii. constructing a mutant fucosyltransferase with an amino acid sequence in which the one or more amino acid residue(s) specified in (i) is / are modified by substitution, insertion and / or deletion resulting in said mutant fucosyltransferase, and ill. evaluating the activity and / or substrate specificity with respect to the Glc residue of lactose of the mutant fucosyltransferase obtained in (ii).

[0317] In the context of present invention, the progenitor fucosyltransferase that is used to design a mutant fucosyltransferase having alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose is as described in the first aspect of the invention. Additionally, the mutant fucosyltransferase is an enzyme as described in the fourth aspect of the invention.

[0318] According to a sixth aspect, the invention provides a method for the production of a mutant fucosyltransferase having alpha-1, 3-fucosyltransferase activity on the Glc residue of lactose. In a specific embodiment, the method comprises the steps of: i. preparing a nucleic acid molecule encoding a mutant fucosyltransferase with an amino acid sequence constructed by the method as described in the fifth aspect of present invention, ii. expressing the nucleic acid molecule, ill. collecting the mutant fucosyltransferase.

[0319] In the context of present invention, the mutant fucosyltransferase is an enzyme as described in the fourth aspect of the invention.

[0320] Within the context of present invention, LNB resides in free form or is part of a saccharide. Preferably, said saccharide is a saccharide comprising LNB wherein said LNB is glycosidically linked to one or more monosaccharide residues as defined herein excluding a fucose residue that is glycosidically linked to the GIcNAc residue of said LNB in an alpha-1, 4-linkage. More preferably, said LNB of said saccharide is linked to two or more monosaccharide residues as defined herein excluding a fucose residue that is glycosidically linked to the GIcNAc residue of said LNB in an alpha-1, 4-linkage. Even more preferably, said LNB of said saccharide is linked to three or more monosaccharide residues as defined herein excluding a fucose residue that is glycosidically linked to the GIcNAc residue of said LNB in an alpha-1, 4-linkage. The one or more monosaccharide residues may be glycosidically linked to said Gal residue of said LNB. Alternatively, and / or additionally, said one or more monosaccharide residues may be glycosidically linked to said GIcNAc residue of said LNB. Said saccharide may be a saccharide with a linear structure. Alternatively, said saccharide may be a saccharide with a branched structure. Said saccharide may be an oligosaccharide, a polysaccharide or a glycan as defined herein.

[0321] In another and / or additional preferred embodiment, the saccharide is a saccharide comprising a formula Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Rg]-GlcNAc-Rd, wherein the galactose (Gal) residue of said Gal-pi,3-GlcNAc is glycosidically linked to an Ra, Rb, Re and / or an Rf group, and / or wherein the GIcNAc residue of said Gal- pi,3-GlcNAc is glycosidically linked to an Rg and / or an Rd group, and wherein any one of said Ra, Rb, Rg, Rd, Re and Rf groups is selected from the list comprising a monosaccharide excluding the Rg group being a fucose residue that is glycosidically linked to the GIcNAc residue of said Gal-pi,3-GlcNAc in an alpha-1, 4- linkage, a disaccharide and an oligosaccharide.

[0322] In another and / or additional preferred embodiment, the saccharide is an oligosaccharide with the formula Gal-pi,3-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-GlcNAc-Rd is glycosidically linked to an Rd group and wherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide.

[0323] In another and / or additional preferred embodiment, the saccharide is an oligosaccharide with the formula Gal-pi,3-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-GlcNAc is linked with a beta-glycosidic bond to said Rd group and wherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide.

[0324] In another more preferred embodiment, the saccharide is a saccharide comprising the formula Gal-pi,3- [Rg]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-[Rg]-GlcNAc-Rd is linked to 1) an Rg group via an alpha-glycosidic or a beta-glycosidic linkage and 2) an Rd group via an alpha-glycosidic or a beta- glycosidic linkage, and wherein said Rg group and Rd group are selected from the list consisting of a monosaccharide excluding said Rg group being a fucose that is bound in an alpha-1, 4-glycosidic linkage to said GIcNAc, a disaccharide and an oligosaccharide. In an even more preferred embodiment, the saccharide is a saccharide comprising the formula Gal-pi,3-[Rg]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-[Rg]-GlcNAc-Rd is linked to 1) an Rg group via an alpha-glycosidic linkage and 2) an Rd group via a beta-glycosidic linkage, and wherein said Rg group and Rd group are selected from the list consisting of a monosaccharide excluding said Rg group being a fucose that is bound in an alpha-1, 4- glycosidic linkage to said GIcNAc, a disaccharide and an oligosaccharide. In a most preferred embodiment, the saccharide is an oligosaccharide with the formula Gal-pi,3-[Rg]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-[Rg]-GlcNAc-Rd is linked to 1) an Rg group via an alpha-glycosidic linkage and 2) an Rd group via a beta-glycosidic linkage, and wherein said Rg group and Rd group are selected from the list consisting of a monosaccharide excluding said Rg group being a fucose that is bound in an alpha-1, 4- glycosidic linkage to said GIcNAc, a disaccharide and an oligosaccharide.

[0325] In another more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-Gal- pi,3-GlcNAc, wherein the Gal residue of said Ra-Gal-pi,3-GlcNAc is linked to an Ra group via an alpha- glycosidic or a beta-glycosidic linkage and wherein said Ra is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide. In an even more preferred embodiment, said saccharide is an oligosaccharide with the formula Ra-Gal-pi,3-GlcNAc, wherein the Gal residue of said Ra- Gal-pi,3-GlcNAc is linked to an Ra group via an alpha-glycosidic linkage and wherein said Ra is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide.

[0326] In another more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-[Rb]- Gal-pi,3-GlcNAc, wherein the Gal residue of said Ra-[Rb]-Gal-pi,3-GlcNAc is linked to an 1) Ra group via an alpha-glycosidic or a beta-glycosidic linkage and 2) an Rb group via an alpha-glycosidic or a beta- glycosidic linkage, and wherein said Ra group and Rb group are selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide.

[0327] In another more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-[Rb]- [Re]-Gal-pi,3-GlcNAc, wherein the Gal residue of said Ra-[Rb]-[Re]-Gal-pi,3-GlcNAc is linked to an 1) Ra group via an alpha-glycosidic or a beta-glycosidic linkage, 2) an Rb group via an alpha-glycosidic or a beta- glycosidic linkage and 3) an Re group via an alpha-glycosidic or a beta-glycosidic linkage, and wherein said Ra group, Rb group and Re group are selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide.

[0328] In another more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-[Rb]- [Re]-[Rf]-Gal-pi,3-GlcNAc, wherein the Gal residue of said Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-GlcNAc is linked to an 1) Ra group via an alpha-glycosidic or a beta-glycosidic linkage, 2) an Rb group via an alpha-glycosidic or a beta-glycosidic linkage, 3) an Re group via an alpha-glycosidic or a beta-glycosidic linkage, and 4) an Rf group via an alpha-glycosidic or a beta-glycosidic linkage, and wherein said Ra group, Rb group, Re and Rf group are selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide.

[0329] In another more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-Gal- pi,3-GlcNAc-Rd, wherein the Gal residue of said Ra-Gal-pi,3-GlcNAc-Rd is linked to an Ra group via an alpha-glycosidic or a beta-glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal-pi,3-GlcNAc- Rd is linked to an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group and Rd group are selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide. In an even more preferred embodiment, the saccharide is an oligosaccharide with the formula Ra-Gal-pi,3-GlcNAc-Rd, wherein the Gal residue of said Ra-Gal-pi,3-GlcNAc-Rd is linked to an Ra group via an alpha-glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal-pi,3-GlcNAc-Rd is linked to an Rd group via a beta-glycosidic linkage and wherein said Ra group and Rd group are selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide. In a most preferred embodiment, the saccharide is an oligosaccharide with the formula Ra-Gal-pi,3-GlcNAc-Rd, wherein the Gal residue of said Ra-Gal-pi,3-GlcNAc-Rd is linked to an Ra group via an alpha-glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal-pi,3-GlcNAc-Rd is linked to an Rd group via a beta-glycosidic linkage and wherein said Ra group is a monosaccharide and wherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide. In another even more preferred embodiment, the saccharide is an oligosaccharide with the formula Ra-Gal-pi,3-GlcNAc-Rd, wherein the Gal residue of said Ra-Gal-pi,3-GlcNAc-Rd is linked to an Ra group via an alpha-glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal-pi,3-GlcNAc-Rd is linked to an Rd group via a beta- 1,3-glycosidic linkage and wherein said Ra group and Rd group are selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide. In another even more preferred embodiment, the saccharide is an oligosaccharide with the formula Ra-Gal-pi,3-GlcNAc-pi,3-Gal-X comprising an X group that is glycosidically linked, and wherein the Gal residue of said Ra-Gal-pi,3-GlcNAc-pi,3-Gal-X is linked to an Ra group via an alpha-glycosidic linkage and wherein said Ra group and X group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In another even more preferred embodiment, the saccharide is an oligosaccharide with the formula Ra- Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-X comprising an X group that is glycosidically linked, and wherein the Gal residue of said Ra-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-X is linked to an Ra group via an alpha-glycosidic linkage and wherein said Ra group and X group are selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide. In another more preferred embodiment, the saccharide is Fuc-al,2- Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc. In another more preferred embodiment, the saccharide is Neu5Ac- a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc.

[0330] In another more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-[Rb]- Gal-pi,3-GlcNAc-Rd wherein the Gal residue of said Ra-[Rb]-Gal-pi,3-GlcNAc-Rd is linked to 1) an Ra group via an alpha-glycosidic or a beta-glycosidic linkage and 2) an Rb group via an alpha-glycosidic or a beta- glycosidic linkage and wherein the GIcNAc residue of said Ra-[Rb]-Gal-pi,3-GlcNAc-Rd is linked to an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group, Rb group and Rd group are selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide. In a more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-[Rb]-Gal-pi,3- GIcNAc-Rd wherein the Gal residue of said Ra-[Rb]-Gal-pi,3-GlcNAc-Rd is linked to 1) an Ra group via an alpha-glycosidic linkage and 2) an Rb group via an alpha-glycosidic linkage and wherein the GIcNAc residue of said Ra-[Rb]-Gal-pi,3-GlcNAc-Rd is linked to an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group and said Rb group are a monosaccharide and wherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide.

[0331] In another more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-[Rb]- [Re]-Gal-pi,3-GlcNAc-Rd wherein the Gal residue of said Ra-[Rb]-[Re]-Gal-pi,3-GlcNAc-Rd is linked to 1) an Ra group via an alpha-glycosidic or a beta-glycosidic linkage, 2) an Rb group via an alpha-glycosidic or a beta-glycosidic linkage and 3) an Re group via an alpha-glycosidic or a beta-glycosidic linkage and wherein the GIcNAc residue of said Ra-[Rb]-[Re]-Gal-pi,3-GlcNAc-Rd is linked to an Rd group via an alpha- glycosidic or a beta-glycosidic linkage and wherein said Ra group, Rb group, Re group and Rd group are selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide.

[0332] In another more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-[Rb]- [Re]-[Rf]-Gal-pi,3-GlcNAc-Rd wherein the Gal residue of said Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-GlcNAc-Rd is linked to 1) an Ra group via an alpha-glycosidic or a beta-glycosidic linkage, 2) an Rb group via an alpha- glycosidic or a beta-glycosidic linkage, 3) an Re group via an alpha-glycosidic or a beta-glycosidic linkage and 4) an Rf group via an alpha-glycosidic or a beta-glycosidic linkage, and wherein the GIcNAc residue of said Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-GlcNAc-Rd is linked to an Rd group via an alpha-glycosidic or a beta- glycosidic linkage and wherein said Ra group, Rb group, Re group, Rf group and Rd group are selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide.

[0333] In another more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-Gal- pi,3-[Rg]-GlcNAc-Rd wherein the Gal residue of said Ra-Gal-pi,3-[Rg]-GlcNAc-Rd is linked to an Ra group via an alpha-glycosidic or a beta-glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal-pi,3- [Rg]-GlcNAc-Rd is linked to 1) an Rg group via an alpha-glycosidic or a beta-glycosidic linkage and 2) an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group, Rg group and Rd group are selected from the list consisting of a monosaccharide excluding said Rg group being a fucose that is bound in an alpha-1, 4-glycosidic linkage to said GIcNAc, a disaccharide and an oligosaccharide. In an even more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-Gal-pi,3- [Rg]-GlcNAc-Rd wherein the Gal residue of said Ra-Gal-pi,3-[Rg]-GlcNAc-Rd is linked to an Ra group via an alpha-glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal-pi,3-[Rg]-GlcNAc-Rd is linked to 1) an Rg group via an alpha-glycosidic linkage and 2) an Rd group via an alpha-glycosidic or a beta- glycosidic linkage and wherein said Ra group and Rg group are a monosaccharide excluding said Rg group being a fucose that is bound in an alpha-1, 4-glycosidic linkage to said GIcNAc, and wherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide. In an even more preferred embodiment, the saccharide substrate is a saccharide comprising the formula Ra-Gal- pi,3-[Rg]-GlcNAc-Rd wherein the Gal residue of said Ra-Gal-pi,3-[Rg]-GlcNAc-Rd is linked to an Ra group via an alpha-glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal-pi,3-[Rg]-GlcNAc-Rd is linked to 1) an Rg group via an alpha-glycosidic linkage and 2) an Rd group via a beta-glycosidic linkage and wherein said Ra group and Rg group are a monosaccharide excluding said Rg group being a fucose that is bound in an alpha-1, 4-glycosidic linkage to said GIcNAc, and wherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide.

[0334] In another more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-[Rb]- Gal-pi,3-[Rg]-GlcNAc-Rd wherein the Gal residue of said Ra-[Rb]-Gal-pi,3-[Rg]-GlcNAc-Rd is linked to 1) an Ra group via an alpha-glycosidic or a beta-glycosidic linkage and 2) an Rb group via an alpha-glycosidic or a beta-glycosidic linkage and wherein the GIcNAc residue of said Ra-[Rb]-Gal-pi,3-[Rg]-GlcNAc-Rd is linked to 1) an Rg group via an alpha-glycosidic or a beta-glycosidic linkage and 2) an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group, Rb group, Rg group and Rd group are selected from the list consisting of a monosaccharide excluding said Rg group being a fucose that is bound in an alpha-1, 4-glycosidic linkage to said GIcNAc, a disaccharide and an oligosaccharide.

[0335] In another more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-[Rb]- [Re]-Gal-pi,3-[Rg]-GlcNAc-Rd wherein the Gal residue of said Ra-[Rb]-[Re]-Gal-pi,3-[Rg]-GlcNAc-Rd is linked to 1) an Ra group via an alpha-glycosidic or a beta-glycosidic linkage, 2) an Rb group via an alpha- glycosidic or a beta-glycosidic linkage and 3) an Re group via an alpha-glycosidic or a beta-glycosidic linkage and wherein the GIcNAc residue of said Ra-[Rb]-[Re]-Gal-pi,3-[Rg]-GlcNAc-Rd is linked to 1) an Rg group via an alpha-glycosidic or a beta-glycosidic linkage and 2) an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group, Rb group, Re group, Rg group and Rd group are selected from the list consisting of a monosaccharide excluding said Rg group being a fucose that is bound in an alpha-1, 4-glycosidic linkage to said GIcNAc, a disaccharide and an oligosaccharide.

[0336] In another more preferred embodiment, the saccharide is a saccharide comprising the formula Ra-[Rb]- [Re]-[Rf]-Gal-pi,3-[Rg]-GlcNAc-Rd wherein the Gal residue of said Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Rg]-GlcNAc- Rd is linked to 1) an Ra group via an alpha-glycosidic or a beta-glycosidic linkage, 2) an Rb group via an alpha-glycosidic or a beta-glycosidic linkage, 3) an Re group via an alpha-glycosidic or a beta-glycosidic linkage and 4) an Rf group via an alpha-glycosidic or a beta-glycosidic linkage and wherein the GIcNAc residue of said Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Rg]-GlcN Ac-Rd is linked to 1) an Rg group via an alpha-glycosidic or a beta-glycosidic linkage and 2) an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group, Rb group, Re group, Rf group, Rg group and Rd group are selected from the list consisting of a monosaccharide excluding said Rg group being a fucose that is bound in an alpha-1, 4- glycosidic linkage to said GIcNAc, a disaccharide and an oligosaccharide.

[0337] In another and / or additional preferred embodiment, the saccharide is an oligosaccharide, a mammalian milk oligosaccharide (MMO), a human milk oligosaccharide (HMO), a negatively charged oligosaccharide, a sialylated oligosaccharide, a non-charged oligosaccharide, Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc (LNT, lacto-N-tetraose) or Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-Glc (lacto-N-fucopentaose V, LNFP-V).

[0338] According to another aspect, the invention provides the use of an enzyme of present invention for the production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc. Optionally, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is linked to a peptide, a protein and / or a lipid. Throughout the application, the term "fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc" may be replaced by the term "fucosylated compound" and vice versa.

[0339] In the context of present invention, the fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc preferably is the trisaccharide Gal-pi,3-[Fuc-al,4]-GlcNAc, which is also known as 4-fucosyllacto-N-biose or 4-FLNB. Alternatively, the fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc preferably is a saccharide comprising Gal-pi,3-[Fuc-al,4]-GlcNAc wherein said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to one or more monosaccharide residues. Said one or more monosaccharide residues is / are selected from the list comprising, consisting of or consisting essentially of N-acetyl-L- rhamnosamine, N-acetyl-D-fucosamine, N-acetyl-L-pneumosamine, N-acetylmuramic acid, N-acetyl-L- quinovosamine, glucose (Glc), galactose (Gal), N-acetylglucosamine (GIcNAc), glucosamine (Glen), mannose (Man), xylose (Xyl), N-acetylmannosamine (ManNAc), a sialic acid, Neu5Ac, Neu5Gc, N- acetylgalactosamine (GalNAc), galactosamine (Gain), fucose (Fuc), rhamnose (Rha), glucuronic acid, gluconic acid, fructose (Fru) and polyols. In a more preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising Gal-pi,3-[Fuc-al,4]-GlcNAc wherein said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to two or more monosaccharide residues. In another more preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc is a saccharide comprising Gal-pi,3-[Fuc-al,4]-GlcNAc wherein said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to three or more monosaccharide residues. Said one or more monosaccharide residues may be glycosidically linked to said Gal residue of the Gal-pi,3-[Fuc-al,4]-GlcNAc of said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc. Alternatively and / or additionally, said one or more monosaccharide residues may be glycosidically linked to said GIcNAc residue of the Gal-pi,3- [Fuc-al,4]-GlcNAc of said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc. Said fucosylated compound may be a saccharide with a linear structure. Alternatively, said fucosylated compound may be a saccharide with a branched structure. Said saccharide may be an oligosaccharide, a polysaccharide or a glycan as defined herein.

[0340] More preferably, the fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising a formula Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd, wherein the galactose (Gal) residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to an Ra, Rb, Re and / or an Rf group, and / or wherein the GIcNAc residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to an Rc and / or an Rd group. In the scope of the invention, any one of said Ra, Rb, Rc, Rd, Re and Rf groups is selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide, as described herein.

[0341] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-[Fuc- al,4]-GlcNAc-Rd is linked to an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Rd group is selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In an even more preferred embodiment, said fucosylated compound comprising Gal- pi,3-[Fuc-al,4]-GlcNAc is an oligosaccharide with the formula Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to an Rd group and wherein said Rd group is selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In another even more preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is an oligosaccharide with the formula Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd wherein the GIcNAc residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is linked with a beta-glycosidic bond to said Rd group. In a most preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc is selected from the list comprising Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-Glc (LNFP-II, lacto-N-fucopentaose II) and Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-Glc (LNDFH-II, lacto- N-difucohexaose II).

[0342] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3- [Fuc-al,4]-[Rc]-GlcNAc-Rd is linked to 1) an Rc group via an alpha-glycosidic or a beta-glycosidic linkage and 2) an Rd group via an alpha-glycosidic or a beta-glycosidic linkage, and wherein said Rc group and Rd group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In an even more preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd is linked to 1) an Rc group via an alpha-glycosidic linkage and 2) an Rd group via a beta-glycosidic linkage, and wherein said Rc group and Rd group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In a most preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is an oligosaccharide with the formula Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-[Fuc- al,4]-[Rc]-GlcNAc-Rd is linked to 1) an Rc group via an alpha-glycosidic linkage and 2) an Rd group via a beta-glycosidic linkage, and wherein said Rc group and Rd group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide.

[0343] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc, wherein the Gal residue of said Ra-Gal-pi,3-[Fuc- al,4]-GlcNAc is linked to an Ra group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra is selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In an even more preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is an oligosaccharide with the formula Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc, wherein the Gal residue of said Ra- Gal-pi,3-[Fuc-al,4]-GlcNAc is linked to an Ra group via an alpha-glycosidic linkage and wherein said Ra is selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide.

[0344] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-[Rb]-Gal-pi,3-[Fuc-al,4]-GlcNAc, wherein the Gal residue of said Ra-[Rb]-Gal- pi,3-[Fuc-al,4]-GlcNAc is linked to an 1) Ra group via an alpha-glycosidic or a beta-glycosidic linkage and 2) an Rb group via an alpha-glycosidic or a beta-glycosidic linkage, and wherein said Ra group and Rb group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide.

[0345] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-[Rb]-[Re]-Gal-pi,3-[Fuc-al,4]-GlcNAc, wherein the Gal residue of said Ra-[Rb]- [Re]-Gal-pi,3-[Fuc-al,4]-GlcNAc is linked to an 1) Ra group via an alpha-glycosidic or a beta-glycosidic linkage, 2) an Rb group via an alpha-glycosidic or a beta-glycosidic linkage and 3) an Re group via an alpha- glycosidic or a beta-glycosidic linkage, and wherein said Ra group, Rb group and Re group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide.

[0346] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Fuc-al,4]-GlcNAc, wherein the Gal residue of said Ra- [Rb]-[Re]-[Rf]-Gal-pi,3-[Fuc-al,4]-GlcNAc is linked to an 1) Ra group via an alpha-glycosidic or a beta- glycosidic linkage, 2) an Rb group via an alpha-glycosidic or a beta-glycosidic linkage, 3) an Re group via an alpha-glycosidic or a beta-glycosidic linkage, and 4) an Rf group via an alpha-glycosidic or a beta- glycosidic linkage, and wherein said Ra group, Rb group, Re and Rf group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide.

[0347] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd, wherein the Gal residue of said Ra-Gal-pi,3- [Fuc-al,4]-GlcNAc-Rd is linked to an Ra group via an alpha-glycosidic or a beta-glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd is linked to an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group and Rd group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In an even more preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is an oligosaccharide with the formula Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd, wherein the Gal residue of said Ra-Gal-pi,3-[Fuc- al,4]-GlcNAc-Rd is linked to an Ra group via an alpha-glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd is linked to an Rd group via a beta-glycosidic linkage and wherein said Ra group and Rd group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In a most preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is an oligosaccharide with the formula Ra-Gal-pi,3-[Fuc-al,4]- GIcNAc-Rd, wherein the Gal residue of said Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd is linked to an Ra group via an alpha-glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd is linked to an Rd group via a beta-glycosidic linkage and wherein said Ra group is a monosaccharide and wherein said Rd group is selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In another even more preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is an oligosaccharide with the formula Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd, wherein the Gal residue of said Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd is linked to an Ra group via an alpha- glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd is linked to an Rd group via a beta-1, 3-glycosidic linkage and wherein said Ra group and Rd group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In another even more preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is an oligosaccharide with the formula Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-X comprising an X group that is glycosidically linked, and wherein the Gal residue of said Ra-Gal-pi,3-[Fuc-ocl,4]-GlcNAc-pi,3-Gal-X is linked to an Ra group via an alpha-glycosidic linkage and wherein said Ra group and X group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide.

[0348] Even more preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is an oligosaccharide with the formula Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-X comprising an X group that is glycosidically linked, and wherein the Gal residue of said Ra-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal- pi,4-X is linked to an Ra group via an alpha-glycosidic linkage and wherein said Ra group and X group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In another more preferred embodiment, said fucosylated compound is Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3- Gal-pi,4-Glc (LNDFH-I, lacto-N-difucohexaose I). In another more preferred embodiment, said fucosylated compound is Neu5Ac-a2,3-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-Glc.

[0349] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-[Rb]-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd wherein the Gal residue of said Ra-[Rb]- Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd is linked to 1) an Ra group via an alpha-glycosidic or a beta-glycosidic linkage and 2) an Rb group via an alpha-glycosidic or a beta-glycosidic linkage and wherein the GIcNAc residue of said Ra-[Rb]-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd is linked to an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group, Rb group and Rd group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In a more preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-[Rb]-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd wherein the Gal residue of said Ra-[Rb]-Gal-pi,3-[Fuc- al,4]-GlcNAc-Rd is linked to 1) an Ra group via an alpha-glycosidic linkage and 2) an Rb group via an alpha- glycosidic linkage and wherein the GIcNAc residue of said Ra-[Rb]-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd is linked to an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group and said Rb group are a monosaccharide and wherein said Rd group is selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In a most preferred embodiment, said fucosylated compound is a saccharide comprising the blood group A type I (difucosyl) epitope GalNAc- al,3-[Fuc-al,2]-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,x-R wherein R is a monosaccharide, disaccharide, oligosaccharide and wherein x is 3, 4 or 6. In another most preferred embodiment, said fucosylated compound is the A antigen-heptasaccharide, also known as A-Hepta or GalNAc-al,3-[Fuc-al,2]-Gal-pi,3- [Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-Glc.

[0350] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-[Rb]-[Re]-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd wherein the Gal residue of said Ra- [Rb]-[Re]-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd is linked to 1) an Ra group via an alpha-glycosidic or a beta- glycosidic linkage, 2) an Rb group via an alpha-glycosidic or a beta-glycosidic linkage and 3) an Re group via an alpha-glycosidic or a beta-glycosidic linkage and wherein the GIcNAc residue of said Ra-[Rb]-[Re]- Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd is linked to an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group, Rb group, Re group and Rd group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide.

[0351] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd wherein the Gal residue of said Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd is linked to 1) an Ra group via an alpha-glycosidic or a beta-glycosidic linkage, 2) an Rb group via an alpha-glycosidic or a beta-glycosidic linkage, 3) an Re group via an alpha-glycosidic or a beta-glycosidic linkage and 4) an Rf group via an alpha-glycosidic or a beta- glycosidic linkage, and wherein the GIcNAc residue of said Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Fuc-al,4]-GlcNAc- Rd is linked to an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group, Rb group, Re group, Rf group and Rd group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide.

[0352] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd wherein the Gal residue of said Ra-Gal- pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd is linked to an Ra group via an alpha-glycosidic or a beta-glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd is linked to 1) an Rc group via an alpha-glycosidic or a beta-glycosidic linkage and 2) an Rd group via an alpha-glycosidic or a beta- glycosidic linkage and wherein said Ra group, Rc group and Rd group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In an even more preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra- Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd wherein the Gal residue of said Ra-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc- Rd is linked to an Ra group via an alpha-glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal- pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd is linked to 1) an Rc group via an alpha-glycosidic linkage and 2) an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group and Rc group are a monosaccharide, and wherein said Rd group is selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In an even more preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-Gal-pi,3-[Fuc-al,4]- [Rc]-GlcNAc-Rd wherein the Gal residue of said Ra-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd is linked to an Ra group via an alpha-glycosidic linkage and wherein the GIcNAc residue of said Ra-Gal-pi,3-[Fuc-al,4]-[Rc]- GIcNAc-Rd is linked to 1) an Rc group via an alpha-glycosidic linkage and 2) an Rd group via a beta- glycosidic linkage and wherein said Ra group and Rc group are a monosaccharide, and wherein said Rd group is selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide. In a most preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the disialyl lewis a (Lea) epitope Neu5Ac-a2,3-Gal-pi,3-[Neu5Ac-a2,6]-[Fuc-al,4]- GlcNAc-pi,x-R wherein R is a monosaccharide, a disaccharide or an oligosaccharide and x is 3, 4 or 6. In another most preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is fucosyldisialyllacto-N-tetraose I, also known as DS-LNF 11, FDS-LNT I or Neu5Ac-a2,3-Gal-pi,3-[Neu5Ac- a2,6]-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-Glc.

[0353] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-[Rb]-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd wherein the Gal residue of said Ra- [Rb]-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd is linked to 1) an Ra group via an alpha-glycosidic or a beta- glycosidic linkage and 2) an Rb group via an alpha-glycosidic or a beta-glycosidic linkage and wherein the GIcNAc residue of said Ra-[Rb]-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd is linked to 1) an Rc group via an alpha- glycosidic or a beta-glycosidic linkage and 2) an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group, Rb group, Rc group and Rd group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide.

[0354] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-[Rb]-[Re]-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd wherein the Gal residue of said Ra-[Rb]-[Re]-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd is linked to 1) an Ra group via an alpha-glycosidic or a beta-glycosidic linkage, 2) an Rb group via an alpha-glycosidic or a beta-glycosidic linkage and 3) an Re group via an alpha-glycosidic or a beta-glycosidic linkage and wherein the GIcNAc residue of said Ra-[Rb]- [Re]-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd is linked to 1) an Rc group via an alpha-glycosidic or a beta- glycosidic linkage and 2) an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group, Rb group, Re group, Rc group and Rd group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide.

[0355] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising the formula Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd wherein the Gal residue of said Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd is linked to 1) an Ra group via an alpha- glycosidic or a beta-glycosidic linkage, 2) an Rb group via an alpha-glycosidic or a beta-glycosidic linkage, 3) an Re group via an alpha-glycosidic or a beta-glycosidic linkage and 4) an Rf group via an alpha-glycosidic or a beta-glycosidic linkage and wherein the GIcNAc residue of said Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Fuc-al,4]- [Rc]-GlcNAc-Rd is linked to 1) an Rc group via an alpha-glycosidic or a beta-glycosidic linkage and 2) an Rd group via an alpha-glycosidic or a beta-glycosidic linkage and wherein said Ra group, Rb group, Re group, Rf group, Rc group and Rd group are selected from the list comprising a monosaccharide, a disaccharide and an oligosaccharide.

[0356] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is an oligosaccharide, a mammalian milk oligosaccharide (MMO) or a human milk oligosaccharide (HMO).

[0357] In another preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a negatively charged molecule or a neutral molecule. In a more preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a sialylated molecule.

[0358] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a negatively charged or a neutral oligosaccharide. In a more preferred embodiment, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a sialylated oligosaccharide.

[0359] More preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide comprising an epitope selected from the list comprising Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,x-R, Gal-pi,3- [Fuc-al,4]-GlcNAc-(pi,3)-Gal-R; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,x-R; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,x-R; HSO3(-3)-Gal-pi,3-[Fuc-al,4]- GlcNAc-(pi,x)-R; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-GalNAc-R; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-[Gal-pi,4- GlcNAc-pi,6]-Gal-pi,3-GalNAc-R; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-[Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6]- Gal-pi,3-GlcNAc-pi,3-Gal-pi,3-GalNAc-R; Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,x-R; Neu5Ac-a2,3- Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,x-R; Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,3-[Fuc-al,4]- GlcNAc-pi,x-R; Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,3-GalNAc-al,x-R; Neu5Ac-a2,3-Gal- pi,3-[Neu5Ac-a2,6]-[Fuc-al,4]-GlcNAc-pi,x-R and GalNAc-al,3-[Fuc-al,2]-Gal-pi,3-[Fuc-al,4]-GlcNAc- pi,x-R, wherein x is selected from the list comprising 3, 4 and 6, and wherein R is selected from the list comprising a monosaccharide, disaccharide and oligosaccharide.

[0360] Even more preferably, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is an oligosaccharide selected from the list comprising: Gal-pi,3-[Fuc-al,4]-GlcNAc; Gal-pi,3-[Fuc-al,4]- GlcNAc-pi,3-Gal-pi,4-Glc; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-Glc; Gal-pi,3-[Fuc- al,4]-GlcNAc-pi,3-[Gal-pi,4-GlcNAc-pi,6]-Gal-pi,4-Glc; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-[Gal-pi,4- [Fuc-al,3]-GlcNAc-pi,6]-Gal-pi,4-Glc; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-[Neu5Ac-a2,6-Gal-pi,4-GlcNAc- pi,6]-Gal-pi,4-Glc; Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-GlcNAc-pi,6-[Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal- pi,4-Glc; Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6-[Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal- pi,4-Glc; Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6-[Gal-pi,3-[Fuc-al,4]- GlcNAc-pi,3]-Gal-pi,4-Glc; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6-[Gal-pi,4- GlcNAc-pi,3]-Gal-pi,4-Glc; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6-[Gal-pi,4- [Fuc-al,3]-GlcNAc-pi,3]-Gal-pi,4-Glc; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-GlcNAc- pi,6-[Gal-pi,3-GlcNAc-pi,3]-Gal-pi,4-Glc; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-GlcNAc- pi,6-[Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal-pi,4-Glc; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-[Gal-pi,4- GlcNAc-pi,6]-Gal-pi,4-GlcNAc-pi,6-[Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal-pi,4-Glc; Gal-pi,4-[Fuc- al,3]-GlcNAc-pi,6-[Gal-pi,3-GlcNAc-pi,3]-Gal-pi,4-GlcNAc-pi,6-[Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3]- Gal-pi,4-Glc; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-[Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6]-Gal-pi,4-GlcNAc-pi,6- [Gal-pi,3-GlcNAc-pi,3]-Gal-pi,4-Glc; Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6-[Gal-pi,3-[Fuc-al,4]-GlcNAc- pi,3]-Gal-pi,4-GlcNAc-pi,6-[Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal-pi,4-Glc; Gal-pi,3-[Fuc-al,4]- GlcNAc-pi,3-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]- GlcNAc-pi,3-Gal-pi,4-Glc; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,3-Gal-pi,4- [Fuc-al,3]-Glc; Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-GlcNAc[6S]-pi,3-Gal-pi,4-Glc; Gal- piA[Fuc-al,3]-GlcNAc-pi,6-[Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal-piAGIcNAc-pi,6-[Fuc-al,2-Gal- pi,3-GlcNAc-pi,3]-Gal-pi,4-Glc; Neu5Ac-a273-Gal-pi73-[Fuc-al74]-GlcNAc; Neu5Ac-a2,3-Gal-pi,3-[Fuc- al,4]-GlcNAc-pi,3-Gal; Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-Glc; Fuc-al,2-Gal-pi,3-[Fuc- al,4]-GlcNAc-pi,3-[Gal-pi,4-GlcNAc-pi,6]-Gal-pi,4-Glc; Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3- [Gal-piA[Fuc-al,3]-GlcNAc-pi,6]-Gal-piAGIc; Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-[Neu5Ac- a2,6-Gal-pi,4-GlcNAc-pi,6]-Gal-pi,4-Glc; Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc- al,3]-GlcNAc-pi,3-Gal-pi,4-Glc; Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]- GlcNAc[6S]-pi,3-Gal-pi,4-Glc; Neu5Ac-a276-Gal-pi74-GlcNAc-pi73-Gal-pi74-GlcNAc-pi76-[Fuc-al72-Gal- pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal-pi,4-Glc; Neu5Ac-a273-Gal-pi73-[Fuc-al74]-GlcNAc-pi73-[Gal-pi74- [Fuc-al,3]-GlcNAc-pi,6]-Gal-pi,4-Glc; Gal-pi73-GlcNAc-pi73-Gal-pi74-GlcNAc-pi76-[Neu5Ac-a273-Gal- pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal-pi,4-Glc; Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-GlcNAc-pi,6-[Neu5Ac-a2,3- Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal-piAGIc; Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6- [Neu5Ac-a2,3-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal-pi,4-Glc; Neu5Ac-a273-Gal-pi73-[Fuc-al74]- GlcNAc-pi,3-[Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,6]-Gal-pi,4-Glc; Neu5Ac-a273-Gal-pi73-[Fuc-al74]- GlcNAc-pi,3-[Gal-pi,4-GlcNAc-pi,6]-Gal-pi,4-Glc; Neu5Ac-a273-Gal-pi73-[Fuc-al74]-GlcNAc-pi73-Gal- pi,4-Glc; Gal-piA[Fuc-al,3]-GlcNAc-pi,3-Gal-piAGIcNAc-pi,6-[Neu5Ac-a2,3-Gal-pi,3-[Fuc-al,4]- GlcNAc-pi,3]-Gal-pi,4-Glc; Neu5Ac-a276-Gal-pi74-GlcNAc-pi73-Gal-pi74-[Fuc-al73]-GlcNAc-pi76-[Fuc- al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal-pi,4-Glc; Fuc-al72-Gal-pi73-GlcNAc-pi73-Gal-pi74-GlcNAc- pi76-[Fuc-al72-Gal-pi73-[Fuc-al74]-GlcNAc-pi73]-Gal-pi74-Glc; Fuc-al72-Gal-pi73-GlcNAc-pi73-Gal- pi74-[Fuc-al73]-GlcNAc-pi76-[Fuc-al72-Gal-pi73-[Fuc-al74]-GlcNAc-pi73]-Gal-pi74-Glc; Fuc-al72-Gal- pi73-[Fuc-al74]-GlcNAc-pi73-Gal-pi74-[Fuc-al73]-GlcNAc-pi76-[Fuc-al72-Gal-pi73-[Fuc-al74]-GlcNAc- pi,3]-Gal-pi,4-Glc; Gal-pi73-[Fuc-al74]-GlcNAc-pi73-Gal-pi74-GlcNAc-pi76-[Neu5Ac-a273-Gal-pi73-[Fuc- al,4]-GlcNAc-pi,3]-Gal-pi,4-Glc; Fuc-al72-Gal-pi73-GlcNAc-pi73-Gal-pi74-[Fuc-al73]-GlcNAc-pi76- [Neu5Ac-a2,3-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal-pi,4-Glc; Fuc-al72-Gal-pi73-[Fuc-al74]-GlcNAc- pi,3-Gal-piA[Fuc-al,3]-GlcNAc-pi,3-Gal-piA[Fuc-al,3]-GlcNAc-pi,3-Gal-pi,4-Glc; Gal-pi74-[Fuc- al73]-GlcNAc-pi76-[Fuc-al72-Gal-pi73-[Fuc-al74]-GlcNAc-pi73]-Gal-pi74-GlcNAc-pi76-[Gal-pi73-GlcNAc- pi,3]-Gal-pi,4-Glc; Gal-pi73-[Fuc-al74]-GlcNAc-pi73-[Gal-pi74-GlcNAc-pi76]-Gal-pi74-GlcNAc-pi76-[Fuc- al,2-Gal-pi,3-GlcNAc-pi,3]-Gal-pi,4-Glc; Fuc-al72-Gal-pi73-[Fuc-al74]-GlcNAc-pi73-[Gal-pi74-GlcNAc- pi,6]-Gal-piAGIcNAc-pi,6-[Gal-pi,3-GlcNAc-pi,3]-Gal-pi,4-Glc; Gal-pi73-GlcNAc-pi73-[Gal-pi74- GlcNAc-pi,6]-Gal-piAGIcNAc-pi,6-[Fuc-al,2-Gal-pi3-[Fuc-al,4]-GlcNAc-pi,3]-Gal-piAGIc; Gal-pi73- [Fuc-al74]-GlcNAc-pi73-[Gal-pi74-[Fuc-al73]-GlcNAc-pi76]-Gal-pi74-GlcNAc-pi76-[Fuc-al72-Gal-pi73- GlcNAc-pi,3]-Gal-pi,4-Glc; Fuc-al72-Gal-pi73-[Fuc-al74]-GlcNAc-pi73-[Gal-pi74-GlcNAc-pi76]-Gal-pi74- GlcNAc-pi76-[Fuc-al72-Gal-pi73-GlcNAc-pi73]-Gal-pi74-Glc; Fuc-al72-Gal-pi73-[Fuc-al74]-GlcNAc-pi73- [Gal-piA[Fuc-al,3]-GlcNAc-pi,6]-Gal-piAGIcNAc-pi,6-[Gal-pi,3-GlcNAc-pi,3]-Gal-piAGIc; Gal-pi73- GlcNAc-pi73-[Gal-pi74-GlcNAc-pi76]-Gal-pi74-[Fuc-al73]-GlcNAc-pi76-[Fuc-al72-Gal-pi73-[Fuc-al74]- GlcNAc-pi,3]-Gal-pi,4-Glc; Fuc-al,2-Gal-pi,3-GlcNAc-pi,3-[Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6]-Gal-pi,4- GlcNAc-pi,6-[Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal-pi,4-Glc; Gal-pi,3-[Fuc-al,4]-GlcNAc- pi,3-[Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6]-Gal-pi,4-GlcNAc-pi,6-[Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc- pi,3]-Gal-pi,4-Glc; Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-[Gal-pi,4-[Fuc-al,3]-GlcNAc-pi,6]-Gal- pi GIcNAc-pi,6-[Fuc-al,2-Gal-pi,3-GlcNAc-pi,3]-Gal-pi,4-Glc; Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc- pi,3-[Gal-pi,4-GlcNAc-pi,6]-Gal-pi,4-GlcNAc-pi,6-[Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3]-Gal- pi,4-Glc; Neu5Ac-a2,3-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-[Gal-pi,4-GlcNAc-pi,6-Gal-pi,4-GlcNAc-pi,6]- Gal-pi,4-Glc; Neu5Ac-a2,3-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-[Fuc-al,2-Gal-pi,4-GlcNAc-pi,6-Gal-pi,4- GlcNAc-pi,6]-Gal-pi,4-Glc; Neu5Ac-a2,3-Gal-pi,3-[Neu5Ac-a2,6]-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-Glc; and GalNAc-al,3-[Fuc-al,2]-Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-Glc.

[0361] According to another and / or additional aspect, the invention provides the use of an enzyme of present invention for the production of a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc as described herein. Optionally, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein is an oligosaccharide mixture. In a preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises (1) a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and (2) at least one disaccharide and / or at least one oligosaccharide. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3- [Fuc-al,4]-GlcNAc as described herein comprises charged and non-charged (neutral) oligosaccharides. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises sialylated and non-charged (neutral) oligosaccharides. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises only noncharged (neutral) oligosaccharides. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises at least one fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and at least one sialylated di- and / or oligosaccharide. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein is an oligosaccharide mixture comprising at least one fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein, sialic acid, lactose and / or at least one sialylated di- and / or oligosaccharide. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises, consists of or consists essentially of LNFP-II and LNDFH-II. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc as described herein comprises, consists of or consists essentially of LNFP-II, LNFP-V and LNDFH-II. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises, consists of or consists essentially of LNFP-II and 3-FL. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises, consists of or consists essentially of LNFP-V and 3-FL. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises, consists of or consists essentially of LNFP-II, LNDFH-II and 3-FL. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc as described herein comprises, consists of or consists essentially of LNFP-V, LNDFH-II and 3-FL. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises, consists of or consists essentially of LNFP-II, LNFP-V, LNDFH-II and 3-FL. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises, consists of or consists essentially of lactose, LNFP-II and LNDFH-II. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc as described herein comprises, consists of or consists essentially of lactose, LNFP-II, LNFP-V and LNDFH-II. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises, consists of or consists essentially of lactose, LNFP-II and 3-FL. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises, consists of or consists essentially of lactose, LNFP-V and 3-FL. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc as described herein comprises, consists of or consists essentially of lactose, LNFP-II, LNDFH-II and 3-FL. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises, consists of or consists essentially of lactose, LNFP-V, LNDFH-II and 3-FL. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises, consists of or consists essentially of lactose, LNFP-II, LNFP-V, LNDFH-II and 3-FL. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein, 2'FL, 3-FL, 3'-SL and 6'-SL. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3- [Fuc-al,4]-GlcNAc as described herein comprises a fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc as described herein and LNT. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and lactose. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises 2'FL, 3-FL, 3'-SL, 6'-SL, LNFP-I, LNFP-II, LNFP-III and LSTc. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises 2'FL, 3-FL, DiFL, 3'-SL, 6'-SL, LSTc, LNFP-I, LNFP-II, LNFP-III, LNT, LNnT and LNDFH-I. In another and / or additional preferred embodiment, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein comprises 2'FL, 3-FL, DiFL, 3'-SL, 6'-SL, LSTa, LSTc, LSTd, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNT, LNnT, LNDFH-I, LNDFH-II, LNnFP-l and LNnDFH.

[0362] According to another aspect, the present invention provides the use of an enzyme of present invention for the production of 3-FL. According to another aspect, the present invention provides the use of an enzyme of present invention for the production of an oligosaccharide mixture comprising 3-FL and a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein.

[0363] According to another aspect, the present invention provides an isolated nucleic acid molecule comprising, consisting of, consisting essentially of, and / or encoding an enzyme of present invention. Another aspect of the present invention provides the use of an isolated nucleic acid molecule encoding an enzyme of present invention for the production of said enzyme in a biologically active form. Another aspect of the present invention provides the use of an isolated nucleic acid molecule encoding an enzyme of present invention for the production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or for the production of 3-FL. Another aspect of the present invention provides the use of an isolated nucleic acid molecule encoding an enzyme of present invention for the production of a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or a mixture comprising 3-FL. Optionally, said mixture is an oligosaccharide mixture. Preferably, said mixture comprises a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and 3-FL.

[0364] According to another aspect, the present invention provides a vector comprising an isolated nucleic acid molecule that comprises, consists of, consists essentially of, and / or encodes an enzyme of present invention. A further aspect of the present invention provides the use of a vector comprising an isolated nucleic acid molecule encoding an enzyme of present invention for the production of said enzyme in a biologically active form. Another aspect of the present invention provides the use of a vector comprising an isolated nucleic acid molecule encoding an enzyme of present invention for the production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or for the production of 3-FL. Another aspect of the present invention provides the use of a vector comprising an isolated nucleic acid molecule encoding an enzyme of present invention for the production of a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or a mixture comprising 3-FL. Optionally, said mixture is an oligosaccharide mixture. Preferably, said mixture comprises a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and 3-FL.

[0365] According to another aspect, the present invention provides a method for the production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein. The method comprises the steps of: a) providing i. a fucose donor, ii. LNB and / or a saccharide comprising LNB, ill. an enzyme as described herein, b) contacting said enzyme and said fucose donor with said LNB and / or saccharide comprising LNB under conditions wherein the enzyme catalyses the transfer of a fucose residue from said fucose donor to the GIcNAc residue of said LNB and / or said LNB as part of said saccharide comprising LNB in an alpha-1, 4-glycosydic linkage resulting in the production of said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein.

[0366] Optionally, the method further comprises separating and / or purifying said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein. Preferably, said fucose donor is GDP-fucose. Optionally, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide. Optionally, said LNB, saccharide comprising LNB and / or fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc is / are linked to a peptide, a protein and / or a lipid as described herein. Optionally, said mixture is an oligosaccharide mixture. Preferably, said mixture is a mixture as described herein.

[0367] According to another aspect, the present invention provides a method for the production of 3-FL and / or a mixture comprising 3-FL. Optionally, said mixture is an oligosaccharide mixture. The method comprises the steps of: a) providing i. GDP-fucose, ii. lactose, ill. an enzyme as described herein, b) contacting said enzyme and GDP-fucose with said lactose under conditions wherein the enzyme catalyses the transfer of a fucose residue from said GDP-fucose to the Glc residue of said lactose in an alpha-1, 3-glycosydic linkage resulting in the production of said 3-FL and / or a mixture comprising 3-FL.

[0368] Optionally, the method further comprises separating and / or purifying said 3-FL and / or said mixture comprising 3-FL.

[0369] In a preferred embodiment of the method of present invention, the fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, 3-FL, the mixture comprising a fucosylated compound comprising Gal-pi,3- [Fuc-al,4]-GlcNAc as described herein and / or the mixture comprising 3-FL is produced in a cell-free system. In an alternative preferred embodiment of the method of present invention, the fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, 3-FL, the mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or the mixture comprising 3-FL is produced by a cell. In a more preferred embodiment of the method of present invention, the fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, 3-FL, the mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or the mixture comprising 3-FL is produced by a single cell.

[0370] In a preferred embodiment of the present invention, the method comprises the steps of i) providing a cell expressing an enzyme as described herein, ii) providing a fucose donor, preferably said fucose donor is GDP-fucose, iii) providing LNB and / or a saccharide comprising LNB, iv) cultivating and / or incubating said cell under conditions permissive to express said enzyme resulting in the production of said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein, v) optionally, separating and / or purifying said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein from the cultivation and / or incubation. Optionally, the cell is capable to produce said fucose donor, LNB, and / or saccharide comprising LNB. Additionally, the cell is optionally cultivated and / or incubated under conditions permissive to produce said fucose donor, LNB and / or said saccharide comprising LNB.

[0371] In a preferred embodiment of the present invention, the method comprises the steps of i) providing a cell expressing an enzyme as described herein, ii) providing GDP-fucose, iii) providing lactose, iv) cultivating and / or incubating said cell under conditions permissive to express said enzyme resulting in the production of said 3-FL and / or said mixture comprising 3-FL, v) optionally, separating and / or purifying said 3-FL and / or said mixture comprising 3-FL from the cultivation and / or incubation. Optionally, the cell is capable of producing said GDP-fucose and / or said lactose. Additionally, the cell is optionally cultivated and / or incubated under conditions permissive to produce said GDP-fucose and / or said lactose.

[0372] According to the invention, said method for the production of a fucosylated compound comprising Gal- pi,3-[Fuc-al,4]-GlcNAc, 3-FL, the mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc as described herein and / or the mixture comprising 3-FL can make use of a non-metabolically engineered cell or can make use of a metabolically engineered cell as disclosed herein. In a preferred embodiment of the method of present invention, the fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc is produced by a cell comprising a pathway for production of said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or for production of said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein. In another and / or additional preferred embodiment of the method of present invention, 3-FL is produced by a cell comprising a pathway for production of said 3-FL and / or for production of said mixture comprising 3-FL.

[0373] In another and / or additional preferred embodiment of the method of present invention, the fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, 3-FL, the mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or the mixture comprising 3-FL is produced by a metabolically engineered cell. In another and / or additional preferred embodiment of the method of present invention, the fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or the mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc as described herein is produced by a cell of present invention.

[0374] In another and / or additional preferred embodiment of the method of present invention, 3-FL and / or the mixture comprising 3-FL is produced by a cell of present invention.

[0375] Throughout the application, unless explicitly stated otherwise, the terms "genetically engineered cell", "metabolically engineered cell", "cell genetically engineered for" and "cell metabolically engineered for" are used interchangeably and preferably refer to a cell which is genetically or metabolically engineered, respectively, for 1) expression or overexpression of an enzyme of present invention, and / or 2) the production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein, 3-FL, a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or a mixture comprising 3-FL according to the invention.

[0376] In another aspect, the present invention provides a cell metabolically engineered for the production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein, 3-FL, a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or a mixture comprising 3-FL. In the context of the invention, said fucosylated compound comprising Gal-pi,3- [Fuc-al,4]-GlcNAc, 3-FL, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc as described herein and / or said mixture comprising 3-FL is preferably not produced by the wild-type progenitor of said cell.

[0377] A metabolically engineered cel I, preferably a single cell, is provided which is capable to express, preferably expresses an enzyme as described herein.

[0378] In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell is modified with one or more expression modules. Said expression modules are also known as transcriptional units and comprise polynucleotides for expression of recombinant genes including coding gene sequences and appropriate transcriptional and / or translational control signals that are operably linked to the coding genes. Said control signals comprise promoter sequences, untranslated regions, ribosome binding sites, terminator sequences. Said expression modules can contain elements for expression of one single recombinant gene but can also contain elements for expression of more recombinant genes or can be organized in an operon structure for integrated expression of two or more recombinant genes. Said polynucleotides may be produced by recombinant DNA technology using techniques well-known in the art. Methods which are well known to those skilled in the art to construct expression modules include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989 and yearly updates).

[0379] The expression of each of said expression modules can be constitutive or is created by a natural or chemical inducer. As used herein, constitutive expression should be understood as expression of a gene that is transcribed continuously in an organism. Expression that is created by a natural inducer should be understood as a facultative or regulatory expression of a gene that is only expressed upon a certain natural condition of the host (e.g. organism being in labour, or during lactation), as a response to an environmental change (e.g. including but not limited to hormone, heat, cold, pH shifts, light, oxidative or osmotic stress / signalling), or dependent on the position of the developmental stage or the cell cycle of said host cell including but not limited to apoptosis and autophagy. Expression that is created by a chemical inducer should be understood as a facultative or regulatory expression of a gene that is only expressed upon sensing of external chemicals (e.g. IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose) via an inducible promoter or via a genetic circuit that either induces or represses the transcription or translation of said polynucleotide to a polypeptide.

[0380] The expression modules can be integrated in the genome of said cell or can be presented to said cell on a vector. Said vector can be present in the form of a plasmid, cosmid, phage, liposome, or virus, which is to be stably transformed / transfected into said metabolically engineered cell. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. These vectors may contain selection markers such as but not limited to antibiotic markers, auxotrophic markers, toxinantitoxin markers, RNA sense / antisense markers. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and / or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., see above. For recombinant production, cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the cell can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986), and Sambrook et al., 1989, supra.

[0381] As used herein an expression module comprises polynucleotides for expression of at least one recombinant gene. Said recombinant gene is involved in the expression of a polypeptide acting in the synthesis of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein, 3-FL, a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein and / or a mixture comprising 3-FL; or said recombinant gene is linked to other pathways in said cell that are not involved in the synthesis of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc as described herein, 3-FL, a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc as described herein and / or a mixture comprising 3-FL. Said recombinant genes encode endogenous proteins with a modified expression or activity, preferably said endogenous proteins are overexpressed; or said recombinant genes encode heterologous proteins that are heterogeneously introduced and expressed in said modified cell, preferably overexpressed. The endogenous proteins can have a modified expression in the cell which also expresses a heterologous protein.

[0382] In a preferred embodiment, the expression of each of said expression modules present in said metabolically engineered cell is constitutive or tuneable as described herein.

[0383] In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell comprises a polynucleotide sequence encoding an enzyme as described herein and operably linked to control sequences recognized by the cell, wherein said polynucleotide sequence is foreign to the cell, said polynucleotide sequence further i) being integrated in the genome of said cell and / or ii) presented to said cell on a vector. In a more preferred embodiment, the cell comprises two or more polynucleotide sequences, each encoding an enzyme as described herein and operably linked to control sequences recognized by the cell. Preferably, said two or more polynucleotide sequences each encode a different enzyme as described herein. In other words, the cell is modified to express different mutant fucosyltransferases as described herein. Alternatively, said two or more polynucleotide sequences enc...

Claims

1. Claims1. A de novo designed enzyme having alpha-1, 4-fucosyltransferase activity on the N-acetylglucosamine (GIcNAc) residue of Gal-pi,3-GlcNAc (lacto-N-biose, LNB), wherein said de novo designed enzyme is obtained by modification of a progenitor fucosyltransferase wherein said modification comprises one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) at amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191-196 and / or 244-254 of SEQ ID NO 01, wherein said progenitor fucosyltransferase: comprises the IPR001503 domain as defined by InterPro 99.0 as released on 28th March 2024, has a GDP-fucose binding domain, has alpha-1,3 fucosyltransferase activity and / or alpha-1,4 fucosyltransferase activity, and comprises, consists of or consists essentially of a polypeptide as represented by any one of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17 or 44.

2. Enzyme according to claim 1, wherein said LNB is part of a saccharide.

3. Enzyme according to any one of claim 1 or 2, wherein said enzyme: comprises the IPR001503 domain as defined by InterPro 99.0 as released on 28th March 2024, has a GDP-fucose binding domain, is a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to the full-length amino acid sequences as represented by SEQ ID NOs 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, or 44, and / or comprises, consists of or consists essentially of a polypeptide as represented by any one of SEQ ID NOs 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 43 or 45.

4. Enzyme according to any one of previous claims, wherein said de novo designed enzyme is a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to the full-length amino acid sequence of said progenitor fucosyltransferase.

5. Enzyme according to any one of previous claims, wherein said progenitor fucosyltransferase has a prokaryotic or eukaryotic origin.

6. Enzyme according to any one of previous claims, wherein said one or more amino acid residue substitution(s), insertion(s) and / or deletion(s) correspond to position(s) F83, Q148, W54, L144, S188, G102, N104, H189, C82, D55, F61, N52, W53, Y147, A58, P86, P143, T252, Q84, L100, N85, N149, 1251, S59, Y250, R26, T60, H57, S56, W62, S81, R116, L89, L105, M117, 1146, N244 and / or S245 of SEQ ID7. Enzyme according to claim 6, wherein said amino acid residue substitution corresponding to position:- F83 of SEQ ID NO 01 is a substitution into E, D, A, G, H, I, L, N, P, Q, R, S, T, V, W, or Y, preferably E or D,- Q148 of SEQ ID NO 01 is a substitution into E, D, A, G, H, I, K, L, N, P, R, S, T, V, W, or Y, preferablyE or D,- W54 of SEQ ID NO 01 is a substitution into A, C, D, E, I, H, L, M, S, V, or Y,L144 of SEQ ID NO 01 is a substitution into A, E, G, or H,S188 of SEQ ID NO 01 is a substitution into E, H, or Y,G102 of SEQ ID NO 01 is a substitution into A, or C,N104 of SEQ ID NO 01 is a substitution into A, G, H, S, or Y,H189 of SEQ ID NO 01 is a substitution into G, N, Q, or S,C82 of SEQ ID NO 01 is a substitution into A, G, H, Q, S, or Y,D55 of SEQ ID NO 01 is a substitution into A, H, Q, R, or Y,F61 of SEQ ID NO 01 is a substitution into A, G, R, or Y,N52 of SEQ ID NO 01 is a substitution into A, E, H, R, or S,W53 of SEQ ID NO 01 is a substitution into A, G, H, S, or Y,- Y147 of SEQ ID NO 01 is a substitution into A, C, D, E, F, G, H, I, K, L, N, P, R, S, T, or V,A58 of SEQ ID NO 01 is a substitution into E, H, N, or Y,- P86 of SEQ ID NO 01 is a substitution into C, D, E, F, G, H, I, L, M, N, R, S, T, V, W, or Y,P143 of SEQ ID NO 01 is a substitution into A, G, Q, or S,T252 of SEQ ID NO 01 is a substitution into A, G, or S,Q84 of SEQ ID NO 01 is a substitution into A, E, G, H, N, R, or S,L100 of SEQ ID NO 01 is a substitution into A, H, Q, S, or Y,N85 of SEQ ID NO 01 is a substitution into G, or S,N149 of SEQ ID NO 01 is a substitution into E, or Q,1251 of SEQ ID NO 01 is a substitution into A, G, H, or S,S59 of SEQ ID NO 01 is a substitution into A, or H,Y250 of SEQ ID NO 01 is a substitution into N,R26 of SEQ ID NO 01 is a substitution into E, or S,T60 of SEQ ID NO 01 is a substitution into A, H, or Y,H57 of SEQ ID NO 01 is a substitution into G, N, or Y,S56 of SEQ ID NO 01 is a substitution into A, E, G, H, or R,W62 of SEQ ID NO 01 is a substitution into Y,S81 of SEQ ID NO 01 is a substitution into A, or N,R116 of SEQ ID NO 01 is a substitution into E,L89 of SEQ ID NO 01 is a substitution into E, or R,L105 of SEQ ID NO 01 is a substitution into A, or Q,M117 of SEQ ID NO 01 is a substitution into G,1146 of SEQ ID NO 01 is a substitution into H, or S,N244 of SEQ ID NO 01 is a substitution into R, or S, and / orS245 of SEQ ID NO 01 is a substitution into Y.

8. Enzyme according to any one of claims 2 to 7 , wherein said saccharide is: a saccharide comprising LNB wherein said LNB is glycosidically linked to one or more monosaccharide residues excluding a fucose residue that is glycosidically linked to the GIcNAc residue of said LNB in an alpha-1, 4-linkage, a saccharide comprising a formula Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Rg]-GlcNAc-Rd, wherein the galactose (Gal) residue of said Gal-pi,3-GlcNAc is glycosidically linked to an Ra, Rb, Re and / or an Rf group, and / or wherein the GIcNAc residue of said Gal-pi,3-GlcNAc is glycosidically linked to an Rg and / or an Rd group, and wherein any one of said Ra, Rb, Rg, Rd, Re and Rf groups is selected from the list comprising a monosaccharide excluding the Rg group being a fucose residue that is glycosidically linked to the GIcNAc residue of said Gal-pi,3-GlcNAc in an alpha-1, 4-linkage, a disaccharide and an oligosaccharide, an oligosaccharide with the formula Gal-pi,3-GlcNAc-Rd, wherein the GIcNAc residue of said Gal- pi,3-GlcNAc is glycosidically linked to an Rd group and wherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide, an oligosaccharide with the formula Gal-pi,3-GlcNAc-Rd, wherein the GIcNAc residue of said Gal- pi,3-GlcNAc is linked with a beta-glycosidic bond to said Rd group and wherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide, and / or an oligosaccharide, a mammalian milk oligosaccharide (MMO), a human milk oligosaccharide (HMO), a negatively charged oligosaccharide, a sialylated oligosaccharide, a non-charged oligosaccharide, Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc (LNT, lacto-N-tetraose) or Gal-pi,3-GlcNAc- pi,3-Gal-pi,4-[Fuc-al,3]-Glc (lacto-N-fucopentaose V, LNFP-V).

9. Enzyme according to any one of previous claims, wherein said enzyme has alpha-1, 3- fucosyltransferase activity on lactose.

10. Use of an enzyme according to any one of claims 1 to 9 for the production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, optionally said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is an oligosaccharide mixture.

11. Use according to claim 10, wherein said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc is:Gal-pi,3-[Fuc-al,4]-GlcNAc (4-fucosyllacto-N-biose, 4-FLNB),a saccharide comprising Gal-pi,3-[Fuc-al,4]-GlcNAc wherein said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to one or more monosaccharide residues, a saccharide comprising a formula Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd, wherein the galactose (Gal) residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to an Ra, Rb, Re and / or an Rf group, and / or wherein the GIcNAc residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to an Rc and / or an Rd group, and wherein any one of said Ra, Rb, Rc, Rd, Re and Rf groups is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide, an oligosaccharide with the formula Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to an Rd group and wherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide, an oligosaccharide with the formula Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is linked with a beta-glycosidic bond to said Rd group and wherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide, and / or an oligosaccharide, a mammalian milk oligosaccharide (MMO), a human milk oligosaccharide (HMO), a negatively charged oligosaccharide, a sialylated oligosaccharide, a non-charged oligosaccharide, Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-Glc (LNFP-II, lacto-N-fucopentaose II) or Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-Glc (LNDFH-II, lacto-N-difucohexaose II).

12. A cell possessing, being capable of expressing, expressing and / or metabolically engineered for expression or overexpression of an enzyme according to any one of claims 1 to 9.

13. A cell comprising a pathway for production of a fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc and / or a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc, characterized in that said cell possesses, is capable of expressing, expresses and / or is metabolically engineered for expression or overexpression of an enzyme according to any one of claims 1 to 9, optionally said cell is metabolically engineered for production of said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc.

14. Cell according to claim 13, wherein said pathway for production of said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a fucosylation pathway.

15. Cell according to claim 14, wherein said fucosylation pathway comprises at least one enzyme selected from the list comprising, consisting of or consisting essentially of a mannose-6-phosphate isomerase, a phosphomannomutase, a mannose-l-phosphate guanylyltransferase, a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, a fucose permease, a fucose kinase, a fucose-l-phosphate guanylyltransferase and a fucosyltransferase.

16. Cell according to any one of claims 12 to 15, wherein said cell comprises a pathway for production of LNB and / or a saccharide comprising LNB.

17. Cell according to any one of claims 13 to 16, wherein said fucosylated compound comprising Gal-pi,3- [Fuc-al,4]-GlcNAc is:Gal-pi,3-[Fuc-al,4]-GlcNAc (4-fucosyllacto-N-biose, 4-FLNB), a saccharide comprising Gal-pi,3-[Fuc-al,4]-GlcNAc wherein said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to one or more monosaccharide residues, a saccharide comprising a formula Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd, wherein the galactose (Gal) residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to an Ra, Rb, Re and / or an Rf group, and / or wherein the GIcNAc residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to an Rc and / or an Rd group, and wherein any one of said Ra, Rb, Rc, Rd, Re and Rf groups is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide, an oligosaccharide with the formula Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to an Rd group and wherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide, an oligosaccharide with the formula Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is linked with a beta-glycosidic bond to said Rd group and wherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide, and / or an oligosaccharide, a mammalian milk oligosaccharide (MMO), a human milk oligosaccharide (HMO), a negatively charged oligosaccharide, a sialylated oligosaccharide, a non-charged oligosaccharide, Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-Glc (LNFP-II, lacto-N-fucopentaose II) or Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-Glc (LNDFH-II, lacto-N-difucohexaose II).

18. A method for the production of a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or a mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, said method comprising the steps of: a) providing i. a fucose donor, preferably said fucose donor is GDP-fucose, ii. Gal-pi,3-GlcNAc (lacto-N-biose, LNB) and / or a saccharide comprising LNB, ill. a de novo designed enzyme according to any one of claims 1 to 9, b) contacting said enzyme and said fucose donor with said LNB and / or saccharide comprising LNB under conditions wherein the enzyme catalyses the transfer of a fucose residue from said fucosedonor to the GIcNAc residue of said LNB and / or said LNB as part of said saccharide comprising LNB in an alpha-1, 4-glycosydic linkage resulting in the production of said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, c) optionally, separating and / or purifying said fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc, optionally, said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is a saccharide, optionally, said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc is an oligosaccharide mixture.

19. Method according to claim 18, wherein said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]- GIcNAc is produced (1) in a cell-free system or (2) by: a cell, a single cell, a metabolically engineered cell, a cell metabolically engineered for expression or overexpression of said enzyme, a cell comprising a pathway for production of said fucosylated compound comprising Gal-pi,3- [Fuc-al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3- [Fuc-al,4]-GlcNAc, and / or a cell metabolically engineered for production of said fucosylated compound comprising Gal-pi,3- [Fuc-al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3- [Fuc-al,4]-GlcNAc.

20. Method according to claim 19, wherein said method comprises the steps of: i. providing a cell expressing said enzyme, ii. providing a fucose donor, preferably said fucose donor is GDP-fucose, optionally said GDP-fucose is produced by said cell, ill. providing said LNB and / or said saccharide comprising LNB, optionally said LNB and / or saccharide comprising LNB is / are produced by said cell, iv. cultivating and / or incubating said cell under conditions permissive to express said enzyme, optionally permissive to produce said fucose donor, LNB and / or saccharide comprising LNB, resulting in the production of said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc,v. optionally, separating and / or purifying said fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc- al,4]-GlcNAc from the cultivation and / or incubation.

21. Method according to any one of claim 19 or 20, wherein said cell is any one of claims 12 to 17.

22. Method according to any one of claims 18 to 21, wherein said conditions comprise: use of a cultivation and / or incubation medium comprising at least one precursor for the production of said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc, and / or adding to the cultivation and / or incubation medium at least one precursor feed for the production of said fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc and / or said mixture comprising a fucosylated compound comprising Gal-pi,3-[Fuc-al,4]-GlcNAc.

23. Method according to claim 22, wherein said precursor is selected from the list comprising, consisting of or consisting essentially of GIcNAc, glucose, galactose, fucose, fructose, glucose-l-phosphate, glucose-6-phosphate, fructose-6-phosphate, glucosamine-6-phosphate, glucosamine-l-phosphate, N-acetylglucosamine-6-phosphate, galactose-l-phosphate, sialic acid, UDP-glucose, UDP-galactose, GDP-fucose, CMP-sialic acid, LNB, said saccharide comprising LNB, lactose, lacto-N-triose, LNT and LNFP-V, LSTa, LSTb.

24. Method according to any one of claims 18 to 23, wherein said fucosylated compound comprising Gal- pi,3-[Fuc-al,4]-GlcNAc is:Gal-pi,3-[Fuc-al,4]-GlcNAc (4-fucosyllacto-N-biose, 4-FLNB), a saccharide comprising Gal-pi,3-[Fuc-al,4]-GlcNAc wherein said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to one or more monosaccharide residues, a saccharide comprising a formula Ra-[Rb]-[Re]-[Rf]-Gal-pi,3-[Fuc-al,4]-[Rc]-GlcNAc-Rd, wherein the galactose (Gal) residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to an Ra, Rb, Re and / or an Rf group, and / or wherein the GIcNAc residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to an Rc and / or an Rd group, and wherein any one of said Ra, Rb, Rc, Rd, Re and Rf groups is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide, an oligosaccharide with the formula Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is glycosidically linked to an Rd group and wherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide, an oligosaccharide with the formula Gal-pi,3-[Fuc-al,4]-GlcNAc-Rd, wherein the GIcNAc residue of said Gal-pi,3-[Fuc-al,4]-GlcNAc is linked with a beta-glycosidic bond to said Rd group andwherein said Rd group is selected from the list consisting of a monosaccharide, a disaccharide and an oligosaccharide, or an oligosaccharide, a mammalian milk oligosaccharide (MMO), a human milk oligosaccharide (HMO), a negatively charged oligosaccharide, a sialylated oligosaccharide, a non-charged oligosaccharide, Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-Glc (LNFP-II, lacto-N-fucopentaose II) or Gal-pi,3-[Fuc-al,4]-GlcNAc-pi,3-Gal-pi,4-[Fuc-al,3]-Glc (LNDFH-II, lacto-N-difucohexaose II).

25. A method for designing a mutant fucosyltransferase having alpha-1, 4-fucosyltransferase activity on the N-acetylglucosamine (GIcNAc) residue of Gal-pi,3-GlcNAc (lacto-N-biose, LNB), wherein the activity and / or specificity for transferring a fucose residue in an alpha-1, 4-glycosidic linkage on the GIcNAc residue LNB is improved as compared with a progenitor fucosyltransferase of said mutant fucosyltransferase, the method comprising: i. specifying in said progenitor fucosyltransferase one or more amino acid residue(s) corresponding to positions 22-33, 52-62, 81-89, 100-105, 110-117, 143-150, 188-190, 191- 196 and / or 244-254 of SEQ ID NO 01, ii. constructing an amino acid sequence in which the one or more amino acid residue(s) specified in (i) is / are modified by substitution, insertion and / or deletion resulting in said mutant fucosyltransferase, and ill. evaluating the activity and / or substrate specificity with respect to the GIcNAc residue of LNB of the mutant fucosyltransferase obtained in (ii).

26. Method according to claim 25, wherein said LNB is part of a saccharide.

27. Method according to any one of claim 25 or 26, wherein said progenitor fucosyltransferase: comprises the IPR001503 domain as defined by InterPro 99.0 as released on 28th March 2024, has a GDP-fucose binding domain, has alpha-1,3 fucosyltransferase activity and / or alpha-1,4 fucosyltransferase activity, and comprises, consists of or consists essentially of a polypeptide as represented by any one of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, or 44.

28. Method according to any one of claims 25 to 27, wherein said mutant fucosyltransferase: comprises the IPR001503 domain as defined by InterPro 99.0 as released on 28th March 2024, has a GDP-fucose binding domain, is a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to the full-length amino acid sequences as represented by SEQ ID NOs 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, or 44 and having alpha-1, 4-fucosyltransferase activity on the GIcNAc residue of LNB, and / orcomprises, consists of or consists essentially of a polypeptide as represented by any one of SEQ.ID NOs 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 43 or 45.

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