Novel fucosyltransferase for in vivo synthesis of complex fucosylated human milk oligosaccharide mixtures including LNDFH-III

Abstract

The present invention relates to the production of complex fucosylated human milk oligosaccharides (HMOs), in particular to the production of complex fucosylated HMOs having five or more monosaccharide units, such as LNFP-III, LNFP-VI and LNDFH-III, and mixtures thereof. The invention also relates to genetically engineered cells and alpha-1, 3-fucosyltransferases suitable for use in said production, as well as to a method of producing said fucosylated HMO.

Description

Technical Field

[0001] The present invention relates to the production of complex fucosylated human milk oligosaccharides (HMOs), in particular the production of complex fucosylated HMOs having five or more monosaccharide units, such as LNFP-III, LNFP-VI, and LNDFH-III and mixtures thereof. The present invention also relates to genetically engineered cells suitable for said production. Background Art

[0002] Designing and constructing bacterial cell factories for the production of fucosylated human milk oligosaccharides (HMOs), especially for the production of more complex fucosylated HMOs, is crucial for providing innovative and scalable solutions for future more complex products.

[0003] The production of complex fucosylated HMOs requires multiple enzymes or multi-specific enzymes, such as bispecific glycosyltransferases capable of producing complex difucosylated HMOs. Multi-specific enzymes are preferred because the genetic burden of introducing them into host cells is lower.

[0004] For example, the production of complex fucosylated HMOs is described in WO2019 / 008133, where the α1,3-fucosyltransferase FucT109 is proposed to fucosylate the glucose (Glc) and N-acetylglucosamine (GlcNAc) moieties of lacto-N-neotetraose (LNnT), thereby potentially generating a mixture containing LNnT, LNFP-III, and LNFP-VI (LNnFP-V). However, the production of LNDFH-III is not disclosed.

[0005] Dumon et al., 2004 (Biotechnol. Prog. 2004, 20, 412-419) further describe the α1,3-fucosyltransferases FutA and FutB, and propose that they produce a mixture of LNnT, (LNFP-III in the case of FutB), LNFP-VI, and LNDFH-III.

[0006] In summary, due to the lack of α-1,3-fucosyltransferases with the desired substrate specificity, and the relatively low yield of the desired fucosylated HMOs compared to other HMO products (such as HMO precursor products) present after fermentation, which may require laborious separation procedures, the production of fucosylated HMOs, especially more complex fucosylated HMOs, can be challenging. Summary of the Invention

[0007] The present invention solves the problem of the bispecific glycosyltransferase required for the production of complex difucosylated HMOs with an LNnT backbone (especially the production of LNDFH-III) by identifying a series of α-1,3-fucosyltransferases that show low or no specificity for the galactose moiety in LNnT, which is the substrate for the fucosylation reaction, but have a high substrate specificity for the N-acetylglucosamine (GlcNAc) and glucose (Glu) moieties in LNnT, thereby producing the complex fucosylated HMO LNDFH-III, or an HMO mixture containing LNDFH-III, and having a high total content of fucosylated HMOs. Thus, the α-1,3-fucosyltransferases proposed herein can be used for the production of LNDFH-III. Accordingly, the present invention provides enzymes, mixtures, compositions, uses, genetically engineered cells, and methods for the production of LNDFH-III or an HMO mixture containing LNDFH-III, and having a high total content of fucosylated HMOs.

[0008] In a first aspect, the invention relates to a genetically engineered cell capable of producing LNDFH-III, comprising a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase selected from the group consisting of

[0009] a. Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:1,

[0010] b. Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:2,

[0011] c. Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:3,

[0012] d. Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:4,

[0013] e. Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:5,

[0014] f. Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:6,

[0015] g. Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:7,

[0016] h. CafC comprising or consisting of the amino acid sequence of SEQ ID NO:8, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:8, and

[0017] i. a FutA variant comprising substitutions at positions 128 and 129 corresponding to positions of SEQ ID NO:11, wherein the variant has at least 80% but less than 100% identity with SEQ ID NO:11, and

[0018] wherein the cell further comprises one or more recombinant nucleic acid sequences encoding β-1,4-galactosyltransferase and optionally β-1,3-N-acetylglucosaminyltransferase.

[0019] A second aspect relates to a method for producing one or more fucosylated HMOs, wherein one HMO is LNDFH-III, the method comprising providing and culturing a genetically engineered cell capable of producing LNDFH-III, the cell comprising a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase selected from the following: a) Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:1, b) Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:2, c) Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:3, d) Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:4, e) Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:5, f) Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:6, g) Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:7, h) CafC comprising or consisting of the amino acid sequence of SEQ ID NO:8, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:8, i) FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:10, and j) a FutA variant comprising substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80% sequence identity with SEQ ID NO:11.

[0020] Preferably, the genetic engineering in the method for producing LNDFH-III further comprises one or more recombinant nucleic acid sequences encoding β-1,4-galactosyltransferase and optionally β-1,3-N-acetylglucosaminyltransferase.

[0021] A third aspect relates to the use of an α-1,3-fucosyltransferase in the production of one or more fucosylated HMOs, wherein the enzyme is selected from Osc1, Bgall1, Bbac1, Murba1, Bacfin1, Prev1, Csec1, and CafC, which comprise or consist of the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8, or wherein the enzyme is a FutA variant comprising substitutions at positions 128 and 129 of SEQ ID NO: 11, wherein the variant has at least 80% but less than 100% identity with SEQ ID NO: 11.

[0022] A fourth aspect relates to an HMO mixture produced by the method according to the invention, which consists essentially of:

[0023] a. LNDFH-III and 3FL, or

[0024] b. LNDFH-III, LNFP-III, and 3FL, or

[0025] c. LNDFH-III, LNFP-VI, and 3FL, or

[0026] d. LNDFH-III, LNFP-III, LNFP-VI, and LNnT, or

[0027] e. LNDFH-III, LNFP-III, 3FL, and LNnT, or

[0028] f. LNDFH-III, LNFP-III, LNFP-VI, 3FL, and LNnT.

[0029] A fifth aspect relates to an HMO composition which consists essentially of 20 - 70 mol% of LNDFH-III, 0 - 35 mol% of LNFP-III, 0 - 35 mol% of LNDFP-VI, 0 - 65 mol% of 3FL, 0 - 40% of LNnT, and less than 1% of pLNnH, with a total molar content of 100%.

[0030] The invention also relates to compositions comprising the mixtures of a) - f), including synbiotic mixtures, and to the use of said compositions in infant formula, dietary supplements, or medical nutraceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 . Overview of the synthesis of complex fucosylated HMOs with an LNnT backbone.

[0032] Figure 2 : An experimental setup for evaluating the regeneration and survival rate of freeze-dried probiotics under acidic conditions of pH 3.0.

[0033] Figure 3 : Showing the regeneration and survival rate of freeze-dried Lactobacillus rhamnosus (DSM 33156) incubated at pH 3.0 for 3 hours and plated at four dilution gradients of 1:1000 (E-3), 1:10000 (E-4), 1:100000 (E-5), and 1:1000000 (E-6). A) is the control without HMO; B) is the combination of Lactobacillus rhamnosus (DSM 33156) and an HMO mixture (mixture 1) containing 50% LNDFH-III and 50% 3FL; C) is the combination of Lactobacillus rhamnosus (DSM 33156) and an HMO mixture (mixture 2) containing 70% LNDFH-III, 20% 3FL, and 10% LNFP-III; D) is the combination of Lactobacillus rhamnosus (DSM 33156) and an HMO mixture (mixture 3) containing 60% LNDFH-III, 10% 3FL, 20% LNFP-III, and 10% LNnT; and E) is the combination of Lactobacillus rhamnosus (DSM 33156) and an HMO mixture (mixture 4) containing 45% LNDFH-III, 10% 3FL, 30% LNFP-III, and 15% LNnT. Detailed implementation

[0034] The present invention solves the biotechnological challenges of in vivo HMO production, particularly complex fucosylated HMOs with an LNnT backbone (which contain at least five monosaccharide units, where at least one monosaccharide unit is a fucosyl unit), especially di-fucosylated complex HMOs containing at least six monosaccharide units. Examples of such complex fucosylated HMOs are LNFP-III, LNFP-VI, and LNDFH-III. The present invention provides a specific strain engineering solution for producing specific complex fucosylated HMOs, particularly LNFP-III, LNFP-VI, and / or LNDFH-III, by utilizing the dual-substrate specificity of the α-1,3-fucosyltransferase disclosed herein for the N-acetylglucosamine (GlcNAc) and glucose (Glc) moieties in LNnT (see Figure 1 ), enabling the enzyme to add two fucosyl units in an α-1,3 linkage to the LNnT backbone to form LNDFH-III.

[0035] Complex fucosylated HMOs with an LNnT backbone can be produced from lactose as an initial substrate, but so far, cells are able to internalize, for example, LNT-II or LNnT, which can also serve as initial substrates.

[0036] Production of LNFP-III, LNFP-VI, and / or LNDFH-III

[0037] The advantage of using the α-1,3-fucosyltransferases of the present invention is that they are able to specifically recognize and fucosylate both the GlcNAc and glucose moieties in LNT to generate LNFP-III and / or LNFP-VI, which are then further fucosylated at the GlcNAc or glucose moiety to produce the difucosylated HMO LNDFH-III (see Figure 1 ). In essence, the present invention describes several newly identified enzymes with dual α-1,3-fucosyltransferase activity that are capable of producing the complex fucosylated HMOs LNFP-III, LNFP-VI, and / or LNDFH-II.

[0038] Furthermore, the α-1,3-fucosyltransferases described herein, once introduced into suitable cells, produce different proportions of different complex fucosylated HMOs. Thus, if sufficient amounts of LNnT are available within the genetically engineered cells, different levels of LNFP-III, LNFP-VI, and / or LNDFH-III are produced.

[0039] Therefore, the properties of the α-1,3-fucosyltransferases described herein are very suitable for the high-level industrial production of LNDFH-III and its mixtures containing additional fucosylated species without producing high levels of by-product HMOs such as LNT-II and LNnT.

[0040] Therefore, the α-1,3-fucosyltransferases described herein are very suitable for the production of LNDFH-III and mixtures containing LNDFH-III, LNFP-III, LNFP-VI, LNnT, and / or 3FL. Some of the α-1,3-fucosyltransferases described herein, Bgall1, Bacfin1, Murba1, and FutA_mut2, mainly produce mixtures containing LNDFH-III, LNFP-III, LNnT, and 3FL, wherein the LNnT level is preferably less than 20 mol% of the total HMOs produced, such as less than 15%, such as less than 10%.

[0041] In particular, α-1,3-fucosyltransferases that produce less than 10%, such as less than 5%, of the molar content of the total HMO produced, for example LNFP-III, and less than 10%, such as less than 5%, of LNFP-V are suitable for producing LNDFH-III, because this simplifies the process of purifying LNDFH-III from the culture broth, since it is easier to separate LNFDH-III and 3FL based on their size differences compared to separating LNDFH-III from LNFP-III and / or LNFP-VI. Examples of such α-1,3-fucosyltransferases described herein are Bgall1, Osc1, and Bbac1.

[0042] Some of the α-1,3-fucosyltransferases described herein, Bbac1, Prev1, FucT109, and Csec1, produce HMO mixtures that contain all three complex HMOs, LNFP-III, LNFP-VI, and LNDFH-III, as well as LNnT, and possibly 3FL. In particular, FucT109 appears to be able to produce a mixture of LNFP-III, LNFP-VI, LNDFH-III, and LNnT, where all four HMOs are 15% to 30% of the total molar content of the HMO produced.

[0043] The genetically engineered cells of the present invention express any one or more of the α-1,3-fucosyltransferases disclosed herein, which have high substrate specificity for the GlcNAc and glucose moieties in LNnT, and for the first time are able to produce high titers of LNDFH-III that exceed 25%, such as exceed 28%, such as exceed 35%, such as exceed 40%, such as exceed 45%, such as exceed 50% of the total amount of HMO produced. From the HMO mixture produced by the genetically engineered cells, LNDFH-III, as well as LNFP-III and / or LNFP-VI, can potentially be purified.

[0044] Accordingly, the present invention enables more efficient biotechnological production of more complex fucosylated HMOs, selected from LNFP-III, LNFP-VI, and LNDFH-III, which can be in purified form or in a mixture where the fucosylated HMOs predominate, such as more than 65% of the total HMO produced, preferably the fucosylated HMOs constitute at least 85%, such as at least 90%, such as at least 95% of the total amount of HMO produced.

[0045] In the following sections, the various elements of the present invention are described, in particular the various elements of the genetically engineered cells. It should be understood that these elements can be combined in the various sections.

[0046] Oligosaccharide

[0047] In this text, the term "oligosaccharide" refers to a sugar polymer containing at least three monosaccharide units, i.e., oligosaccharides with three, four, five, six or more monosaccharide units. Oligosaccharides can have a linear or branched structure and contain monosaccharide units linked to each other by glycosidic bonds. Specifically, the oligosaccharide contains a lactose residue at the reducing end and one or more naturally occurring monosaccharides with 5-9 carbon atoms, which are selected from aldoses (such as glucose, galactose, ribose, arabinose, xylose, etc.), ketoses (such as fructose, sorbose, tagatose, etc.), deoxysugars (such as rhamnose, fucose, etc.), deoxyamino sugars (such as N-acetyl-glucosamine, N-acetyl-mannosamine, N-acetyl-galactosamine, etc.), uronic acids and ketoaldonic acids (such as N-acetylneuraminic acid). Preferably, the oligosaccharide is HMO.

[0048] Human milk oligosaccharide (HMO)

[0049] A preferred oligosaccharide of the present invention is human milk oligosaccharide (HMO).

[0050] The term "human milk oligosaccharide" or "HMO" in this text refers to complex carbohydrates found in human breast milk. HMO has a core structure containing a lactose unit at the reducing end, which can be extended by one or more β-N-acetyl-lactosaminyl and / or one or more β-lacto-N-biose units, and the core structure can be substituted by α-L-fucopyranosyl and / or α-N-acetyl-neuraminyl (fucosyl) moieties. The HMO structure is disclosed, for example, by Xi Chen in Chapter 4 of Advances in Carbohydrate Chemistry and Biochemistry 2015 vol 72.

[0051] The present invention focuses on fucosylated HMOs. Examples of fucosylated HMOs include 2'-fucosyllactose (2'-FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucosylhexose I (LNDFH-I), 3-fucosyllactose (3FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucosylhexose III (LNDFH-III), fucosyl-lacto-N-hexose II (FLNH-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-fucopentaose VI (LNFP-VI), lacto-N-difucosylhexose II (LNDFH-II), fucosyl-lacto-N-hexose I (FLNH-I), fucosyl-p-lacto-N-hexose I (FpLNH-I), fucosyl-p-lacto-N-neohexose II (F-pLNnH II), fucosyl-lacto-N-neohexose (FLNnH), 3-fucosyl-3'-fucosyllactose (FSL), fucosyl-LST-a (FLST-a), fucosyl-LSTb (FLSTb), fucosyl-LST-c (FLST-c), fucosyl-LST d (FLST-d), and fucosyl-lacto-N-hexose (SLNH).

[0052] In the context described herein, a complex fucosylated HMO is a fucosylated HMO that contains at least 5 monosaccharide units, wherein at least one monosaccharide unit is a fucosyl unit. Non-limiting examples of complex fucosylated HMOs are fucosylated HMOs composed of 5 monosaccharide units, such as LNFP-I, LNFP-II, LNFP-III, LNFP-V, and LNFP-VI, and complex fucosylated HMOs having 6 monosaccharide units, such as, but not limited to, difucosylated HMOs LNDFH-I, LNDFH-II, and LNDFH-III or sialic acid-fucosylated HMOs FLST-a, FLST-b, FLST-c, and FLST-d. Preferably, a complex fucosylated HMO is one that requires at least three different glycosyltransferase activities for production from lactose as an initial substrate. For example, the formation of LNFP-III or LNFP-VI requires α-1,3-fucosyltransferase, β-1,3-N-acetyl-glucosaminyl-transferase, and β-1,4-galactosyltransferase (see Figure 1), and the formation of LNDFH-II requires at least one α-1,3-fucosyltransferase, β-1,3-N-acetyl-glucosaminyltransferase, and β-1,4-galactosyltransferase class, where at least one α-1,3-fucosyltransferase can be a single α-1,3-fucosyltransferase with dual activity towards both the Glc and GlcNAc moieties of LNnT, or it can be two different α-1,3-fucosyltransferases: one α-1,3-fucosyltransferase specific for the Glc moiety in LNFP-III, and one α-1,3-fucosyltransferase specific for the GlcNAc moiety of LNFP-VI. The enzymes described herein preferably have dual activity towards both the Glc and GlcNAc moieties of LNnT and are thus capable of generating LNDFH-III from LNnT (see Figure 1 ).

[0053] In particular, the fucosylated HMOs produced are selected from complex fucosylated HMOs containing at least five monosaccharide units, where at least one monosaccharide unit is a fucosyl unit. Additionally, in embodiments, the fucosylated HMOs are selected from complex fucosylated HMOs having an LNnT backbone structure, preferably selected from LNFP-III, LNFP-VI, and LNDFH-III. Examples of fucosylated HMOs having an LNnT backbone structure are lacto-N-fucopentaose III (LNFP-II), lacto-N-fucopentaose VI (LNFP-V), lacto-N-difucosylhexose III (LNDFH-III), sialyl-lacto-N-fucopentaose III (S-LNFP-III), mono-fucosyl-lacto-N-hexose III (F-LNH-III), difucosyl-lacto-N-hexose III (DF-LNH-III), trifucosyl-lacto-N-neohexose (TF-LNnH), fucosyl-sialyl-lacto-N-neohexose I (FS-LNnH), and disialyl-fucosyl-lacto-N-neohexose II (DS-F-LNnH-II).

[0054] In an embodiment of the present invention, the α-1,3-fucosyltransferase described herein fucosylates both the N-acetylglucosamine (GlcNAc) and glucose (Glc) moieties of LNnT, and is also capable of fucosylating the glucose (Glc) moiety of lactose. In a further embodiment, the α-1,3-fucosyltransferase described herein fucosylates only the N-acetylglucosamine (GlcNAc) and glucose (Glc) moieties of LNnT. In this regard, one or more fucosylated HMOs are / is preferably LNDFH-III and 3FL, or LNDFH-III, LNFP-III and 3FL, or LNDFH-III, LNFP-VI and 3FL, or LNDFH-III, LNFP-III, LNFP-VI and 3FL.

[0055] In human milk, approximately 60% of the HMO content is fucosylated HMO, so producing a mixture containing a high content of fucosylated HMO is highly desirable for producing a more natural HMO mixture. In an embodiment of the present invention, at least 60 mol%, such as at least 70 mol%, 80 mol%, 85 mol%, 87 mol%, 89 mol%, 90 mol%, 91 mol%, 92 mol%, 93 mol%, 94 mol%, 95 mol%, 96 mol%, 97 mol%, 98 mol%, 99 mol% or at least 99.5 mol% of the produced HMOs are fucosylated HMOs.

[0056] Receptor oligosaccharide

[0057] The genetically engineered cell according to the present invention comprises a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase that is capable of transferring fucose from an activated sugar to the GlcNAc and / or Glc moiety of a receptor oligosaccharide, in an α-1,3 bond on the GlcNAc moiety or in an α-1,3 bond on the Glc moiety.

[0058] As described herein, a receptor oligosaccharide is an oligosaccharide that can serve as a substrate for a glycosyltransferase that is capable of transferring a glycosyl moiety from a glycosyl donor to the receptor oligosaccharide. The glycosyl donor is preferably a nucleotide-activated sugar, as described in the "Glycosyl donor - nucleotide-activated sugar pathway" section. Preferably, the receptor oligosaccharide is a precursor for preparing more complex HMOs, and can also be referred to as a precursor molecule.

[0059] The receptor oligosaccharide can be an intermediate of the present fermentation process, the final product of a separate fermentation process using a separate genetically engineered cell, or can also be an enzymatically or chemically produced molecule.

[0060] In this context, the acceptor oligosaccharide of said α-1,3-fucosyltransferase is preferably lacto-N-neotetraose (LNnT), which is produced from the precursor molecule lacto-N-triose II (LNT-II) (e.g., the acceptor of β-1,3-galactosyltransferase), and this precursor molecule is produced from the initial precursor molecule lactose (e.g., the acceptor of β-1,3-N-acetyl-glucosaminyl-transferase) (see Figure 1 ). In addition, the acceptor oligosaccharide of α-1,3-fucosyltransferase can also be lacto-N-fucopentaose III (LNFP-III) or lacto-N-fucopentaose VI (LNFP-VI), which are produced from the precursor molecule LNnT (e.g., the acceptor of α-1,3-fucosyltransferase). Preferably, the initial precursor molecule is fed into genetically engineered cells, which are capable of producing, for example, LNT-II, LNT, LNFP-III, LNFP-VI, and / or LNDFH-III from the precursor. Most commonly, the initial precursor is lactose, and the genetically engineered cells are capable of producing intermediate precursors (acceptor oligosaccharides such as LNT-II and LNnT) intracellularly. However, the initial precursor can also be LNT-II or LNT if the cells are capable of importing at least one of these compounds.

[0061] Glycosyltransferase

[0062] The genetically engineered cells according to the present invention comprise at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase, such as a fucosyltransferase, capable of transferring a fucosyl residue from a fucosyl donor to an acceptor oligosaccharide to synthesize one or more fucosylated human milk oligosaccharide products, namely fucosyltransferase.

[0063] The genetically engineered cells according to the present invention can comprise one or more additional recombinant nucleic acids encoding one or more recombinant and / or heterologous glycosyltransferases capable of transferring a glycosyl residue from a glycosyl donor to an acceptor oligosaccharide. Preferably, the additional glycosyltransferases enable the genetically engineered cells to synthesize LNnT from precursor molecules such as lactose or LNT-II. In an embodiment, the genetically engineered cells described herein comprise one or more other recombinant nucleic acids encoding one or more recombinant and / or heterologous glycosyltransferases.

[0064] The additional glycosyltransferases are preferably selected from the group consisting of galactosyltransferases, glucosaminyltransferases, fucosyltransferases, N-acetylglucosaminyltransferases, and sialyltransferases.

[0065] The fucosyltransferase in the genetically engineered cells described herein is an α-1,3-fucosyltransferase. Preferably, the α-1,3-fucosyltransferase is capable of transferring a fucose unit onto the GlcNAc and / or Glc moiety of LNnT, LNFP-III, and / or LNFP-VI.

[0066] In the present invention, the functional enzyme (α-1,3-fucosyltransferase) capable of transferring a fucosyl moiety from a fucosyl donor to a receptor oligosaccharide is selected from a) Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:1, b) Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:2, c) Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:3, d) Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:4, e) Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:5, f) Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:6, g) Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:7, h) CafC comprising or consisting of the amino acid sequence of SEQ ID NO:8, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:8, i) FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:10, and j) a FutA variant comprising substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80% sequence identity with SEQ ID NO:11, and k) FutA_mut2 comprising or consisting of the amino acid sequence of SEQ ID NO:9, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:9. These enzymes can be used, for example, for the production of LNFP-III, LNFP-VI and / or LNDFH-III.

[0067] Without being bound by theory, it is advantageous to have an α-1,3-fucosyltransferase that predominantly produces a mixture of LNDFH-III and LNFP-III and / or LNFP-VI because, when the initial substrate is lactose and the availability of LNnT is not restricted, such an α-1,3-fucosyltransferase would theoretically produce an HMO mixture containing one or more complex fucosylated HMOs (such as LNFP-III and LNDFH-III, or LNDFH-III and LNFP-VI, or LNDFH-III, LNFP-III, and LNFP-VI). In the produced HMO mixture, a large amount of complex fucosylated HMOs would result in easier purification of the produced complex fucosylated HMOs because it would be simpler to purify LNDFH-III, LNFP-III, and / or LNFP-VI from an HMO mixture predominantly containing the desired products, as it is easier to separate complex fucosylated HMOs from smaller HMOs compared to separating different complex fucosylated HMOs of the same or similar size from each other (e.g., separating LNDFH-III from LNFP-III, or separating LNFDH-III from LNFP-VI). Thus, it is considered that a lower initial amount of LNFP-III or LNFP-VI is beneficial for the purification of LNDFH-III when producing LNDFH-III, while a lower initial amount of LNFP-VI is beneficial for the production of a mixture of LNDFH-III and LNFP-III, and a lower starting amount of LNFP-III is beneficial for the production of a mixture of LNDFH-III and LNFP-VI. When a mixture containing LNDFH-III, LNFP-III, and LNFP-VI is desired, a large amount of LNDFH-III, LNFP-III, and LNFP-VI is beneficial because it also simplifies the further purification of the mixture. For example, the α-1,3-fucosyltransferase Osc-1 predominantly produces LNDFH-III as the only complex fucosylated HMO, while the α-1,3-fucosyltransferases Bacfin1, Bgall1, Murba1, and FutA_mut2 predominantly produce LNDFH-III and LNFP-III as the only complex fucosylated HMOs, while the α-1,3-fucosyltransferase CafC predominantly produces LNDFH-III and LNFP-VI as the only complex fucosylated HMO, and the α-1,3-fucosyltransferases FucT109, Prev1, and Csec1 predominantly produce a mixture of LNDFH-III, LNFP-III, and LNFP-VI.

[0068] In a preferred embodiment, the use of the α-1,3-fucosyltransferase according to the invention results in at least 60 mol%, for example at least 70 mol%, 75 mol%, 80 mol%, 85 mol%, 90 mol%, 93 mol%, 95 mol%, 96 mol%, 97 mol% or for example at least 98 mol% of the total molar content of HMOs produced by the cells of the invention being fucosylated HMOs.

[0069] In a preferred embodiment, the use of the α-1,3-fucosyltransferase according to the invention results in at least 20 mol%, for example at least 23 mol%, such as at least 25 mol%, 27 mol%, 30 mol%, 33 mol%, 35 mol%, 38 mol%, 40 mol%, 42 mol%, 45 mol%, 50 mol%, 55 mol% or for example at least 60 mol% of the total molar content of HMOs produced by the cells of the invention being LNDFH-III.

[0070] In a further embodiment, the use of the α-1,3-fucosyltransferase according to the invention results in at least 50 mol%, for example at least 54 mol%, 60 mol%, 70 mol%, 75 mol%, 80 mol%, 85 mol%, 90 mol%, or for example at least 94 mol%, or for example between 50 mol% and 90 mol%, or for example between 70 mol% and 94 mol% of the total molar content of HMOs produced by the cells of the invention being a mixture of LNDFH-III and LNFP-III.

[0071] In a further embodiment, the use of the α-1,3-fucosyltransferase according to the invention results in at least 60 mol%, for example at least 65 mol%, 70 mol%, 75 mol%, 80 mol%, 85 mol%, or for example at least 89 mol%, or for example between 60 mol% and 90 mol% of the total molar content of HMOs produced by the cells of the invention being a mixture of LNDFH-III and LNFP-VI.

[0072] In a further embodiment, the use of the α-1,3-fucosyltransferase according to the invention results in at least 55 mol%, such as at least 59 mol%, 65 mol%, 68 mol%, 70 mol%, 75 mol%, 70 mol%, or such as at least 85 mol%, or such as between 59 mol% and 86 mol% of the total molar content of HMOs produced by the cells of the invention being a mixture of LNDFH-VI, LNFP-III and LNFP-VI.

[0073] In an embodiment, the α-1,3-fucosyltransferase is a FutA variant that contains two substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO: 11, wherein the variant has at least 80% sequence identity with SEQ ID NO: 11, such as at least 85% identity, such as at least 90% identity, such as at least 95% identity, such as 99.5% identity.

[0074] In an embodiment, the α-1,3-fucosyltransferase is a FutA variant that has at least 80% identity with SEQ ID NO: 11, wherein the amino acid corresponding to position 128 of SEQ ID NO: 11 is asparagine or glutamine, and / or the amino acid corresponding to position 129 of SEQ ID NO: 11 is glutamic acid or aspartic acid. Preferably, the FutA variant has at least 85% identity with SEQ ID NO: 11, such as at least 90% identity, such as at least 95% identity, such as 99.5% identity.

[0075] In one embodiment, compared to SEQ ID NO: 11, the FutA_mut2 variant contains the following substitutions A128N or A128Q and H129E or H129D, wherein the variant has at least 80% identity with SEQ ID NO: 11, such as at least 85% identity with SEQ ID NO: 11, such as at least 90% identity, such as at least 95% identity, or such as 99.5% identity.

[0076] In one embodiment, compared to SEQ ID NO: 11, the FutA_mut2 variant contains the following substitutions A128N and H129E, wherein the variant has at least 80% identity with SEQ ID NO: 11, such as at least 85% identity with SEQ ID NO: 11, such as at least 90% identity, such as at least 95% identity, such as 99.5% identity.

[0077] In another embodiment, compared to NCBI reference number WP_000487428.1, the FutA_mut2 variant includes the following substitutions A128N, H129E, D148G, and Y221C, wherein the variant has at least 80% identity with WP_000487928.1, such as at least 85% identity with WP_0000487428.1, such as at least 90% identity, such as at least 95% identity, such as 99.1% identity.

[0078] In an embodiment, the FutA variant is FutA_mut2, which contains or consists of the amino acid sequence of SEQ ID NO: 9.

[0079] In an embodiment, the expression of the α-1,3-fucosyltransferase described herein in a genetically engineered cell is further combined with the expression of one or more other recombinant nucleic acids encoding one or more recombinant and / or heterologous glycosyltransferases.

[0080] In an embodiment, the cell further comprises one or more recombinant nucleic acid sequences encoding a β-1,4-galactosyltransferase.

[0081] In a preferred embodiment, the expression of the α-1,3-fucosyltransferase of the present invention in a genetically engineered cell is combined with the expression of a β-1,4-galactosyltransferase such as galT from Helicobacter pylori. In another embodiment, a third enzyme is expressed, such as a β-1,3-N-acetyl-glucosaminyl-transferase such as LgtA from Neisseria meningitidis.

[0082] In an embodiment, the cell further comprises one or more recombinant nucleic acid sequences encoding an α-2,3-sialyltransferase. In addition to the α-1,3-fucosyltransferases Osc1, Bgall1, Murba1, BAcfin1, Bbac1, Prev1, Csec1, CafC, FutA_mut2, and FucT109, the glycosyltransferase classes further exemplified are preferably selected from the glycosyltransferases described below (Tables 1, 2, 3, and 9).

[0083] α-1,3-fucosyltransferase

[0084] The term "α-1,3-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of a fucosyl group from a donor substrate (such as GDP-fucose) to an acceptor molecule in an α-1,3 linkage (see Figure 1 ). Preferably, the α-1,3-fucosyltransferase used in the present invention is not from the species of the genetically engineered cell, i.e., the gene encoding the α-1,3-fucosyltransferase is of heterologous origin and is selected from the α-1,3-fucosyltransferases identified in Table 1. In the context described herein, the acceptor molecule of the α-1,3-fucosyltransferase is preferably an acceptor oligosaccharide having at least four monosaccharide units with a GlcNAc moiety, such as LNnT. Heterologous α-1,3-fucosyltransferases known in the art that are capable of transferring a fucosyl moiety to LNnT, particularly FutA, have been shown to produce a mixture of LNFP-VI and LNDFH-III (Dumon et al., 2004 Biotechnol. Prog. 20:412-419).

[0085] The α-1,3-fucosyltransferase may be selected from amino acid sequences having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with the amino acid sequence of any one of the α-1,3-fucosyltransferases listed in Table 1.

[0086] Table 1. List of α-1,3(4)-fucosyltransferases capable of producing LNDFH-III and mixtures containing LNDFH-III.

[0087]

[0088] 1 The GenBank ID reflects the full-length enzyme, and truncated, elongated or mutated versions may be used in the present invention, which are represented by the sequences indicated by SEQ ID NO.

[0089] Example 1 discloses the identification of heterologous α-1,3-fucosyltransferases Osc1, Bgall1, Murba1, Bacfin1, Prev1, Csec1, CafC, FutA_mut2, FucT109 and FutA (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11, respectively), which are each capable of producing an HMO mixture containing LNDFH-III when introduced into LNnT-producing cells. Specifically, the enzymes Osc1, Bgall1, Murba1, Bacfin1, Prev1, Csec1, CafC, FutA_mut2, FucT109 and FutA can transfer a fucosyl unit in an α-1,3 bond to the Glc moiety of LNnT and / or LNFP-III to form LNFP-VI and / or LNDFH-III, respectively, and / or in an α-1,3 bond to the GlcNAc moiety of LNnT to form LNFP-III and / or LNDFH-III (see Figure 1 ).

[0090] Furthermore, the experiments conducted in Example 1 showed that as end products, the enzymes Murba1, Bgall1, Bacfin1 and FutA_mut2 did not produce any LNFP-VI, or at least did not produce any detectable amount of LNFP-VI, or produced at least less than 1% of the total amount of HMO produced. Furthermore, the experiments conducted in Example 1 showed that the enzymes FutA and CafC did not produce any LNFP-III, or at least did not produce any detectable amount of LNFP-III.

[0091] The fucosyltransferase described herein is capable of transferring a fucosyl moiety from a fucosyl donor to a recipient oligosaccharide via an α-1,3 bond. This enzyme is also referred to as an α-1,3-fucosyltransferase. In a preferred embodiment, the α-1,3-fucosyltransferase has dual α-1,3-fucosyltransferase activity, meaning that it is capable of fucosylating an oligosaccharide at the GlcNAc moiety and the Glc moiety.

[0092] In an embodiment, the α-1,3-fucosyltransferase is selected from a) Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO:1, b) Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO:2, c) Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO:3, d) Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO:4, e) Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO:5, f) Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO:6, g) Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO:7, h) CafC comprising or consisting of the amino acid sequence of SEQ ID NO:8, or a functional homolog thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO:8, i) FutA_mut2 comprising or consisting of the amino acid sequence of SEQ ID NO:9, or a functional homolog thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO:9, j) a FutA variant comprising substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80% sequence identity with SEQ ID NO:11, and k) FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO:10.

[0093] In a preferred embodiment, the α-1,3-fucosyltransferase is selected from a) Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:1, b) Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:2, c) Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:3. These enzymes can be used, for example, for the production of LNDFH-III with low levels of LNFP-III and LNFP-VI, for example less than 10% each of the total HMO. These enzymes can also be used for the production of an HMO mixture of LNDFH-III and 3FL, where these two HMOs account for at least 80%, for example at least 90%, of the total HMO produced.

[0094] In other preferred embodiments, the α-1,3-fucosyltransferase capable of transferring a fucosyl moiety from a fucosyl donor to an acceptor oligosaccharide is selected from a) Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:2, b) Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:5, c) Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:4, and d) a FutA variant comprising substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80% sequence identity to SEQ ID NO:11. For example, these enzymes can be used for the production of an HMO mixture comprising LNFP-III and LNDFH-III, where LNFP-VI accounts for less than 1% of the total HMO produced.

[0095] In other preferred embodiments, the α-1,3-fucosyltransferase capable of transferring a fucosyl moiety from a fucosyl donor to a recipient oligosaccharide is selected from a) Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:3, b) Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:6, c) FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:10, d) Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:7. These enzymes can be used, for example, for the production of HMO mixtures comprising LNDFH-III and LNFP-III and LNFP-VI.

[0096] In one embodiment, the α-1,3-fucosyltransferase is Osc1 from the bacterium Oscillospiraceae bacterium N12, which comprises or consists of the amino acid sequence of SEQ ID NO:1, or comprises an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO:1 and consists thereof.

[0097] In one embodiment, the α-1,3-fucosyltransferase is Bgall1 from Bacteroides gallinarum, which comprises or consists of the amino acid sequence of SEQ ID NO:2, or comprises an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO:2 and consists thereof.

[0098] In one embodiment, the α-1,3-fucosyltransferase is Bbac1 from a bacterium of the family Bacteroidaceae, which comprises or consists of the amino acid sequence of SEQ ID NO:3, or comprises an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO:3 and consists thereof.

[0099] In one embodiment, the α-1,3-fucosyltransferase is Murba1 from a bacterium of the family Muribaculaceae, which comprises or consists of the amino acid sequence of SEQ ID NO:4, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:4.

[0100] In one embodiment, the α-1,3-fucosyltransferase is Bacfin1 from Bacteroides finegoldii, which comprises or consists of the amino acid sequence of SEQ ID NO:5, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:5.

[0101] In one embodiment, the α-1,3-fucosyltransferase is Prev1 from the genus Prevotella, which comprises or consists of the amino acid sequence of SEQ ID NO:6, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:6.

[0102] In one embodiment, the α-1,3-fucosyltransferase is Csec1 from Faecalibacterium secundum, which comprises or consists of the amino acid sequence of SEQ ID NO:7, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:7.

[0103] In one embodiment, the α-1,3-fucosyltransferase is CafC from Bacteroides nordii, which comprises or consists of the amino acid sequence of SEQID NO:8, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:8.

[0104] In one embodiment, the α-1,3-fucosyltransferase is FucT109 from Bacteroides fragilis NCTC 9343, which comprises or consists of the amino acid sequence of SEQ ID NO:10, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:10.

[0105] In one embodiment, the α-1,3-fucosyltransferase is FutA from Helicobacter pylori, which comprises or consists of the amino acid sequence of SEQ ID NO:11, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:11.

[0106] In one embodiment, the α-1,3-fucosyltransferase is a FutA variant that contains substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as 99.5% sequence identity with SEQ ID NO:11.

[0107] In one embodiment, the α-1,3-fucosyltransferase is the FutA variant FutA_mut2, which comprises or consists of the amino acid sequence of SEQ ID NO:9.

[0108] In one embodiment, the enzyme Osc1 is introduced into a genetically engineered cell that further comprises a β-1,4-galactosyltransferase and preferably also comprises a β-1,3-N-acetyl-glucosaminyltransferase.

[0109] In one embodiment, the enzyme Murba1 is introduced into a genetically engineered cell that further comprises a β-1,4-galactosyltransferase and preferably also comprises a β-1,3-N-acetyl-glucosaminyltransferase.

[0110] In one embodiment, the enzyme Csec1 is introduced into a genetically engineered cell that further comprises a β-1,4-galactosyltransferase and preferably also comprises a β-1,3-N-acetyl-glucosaminyltransferase.

[0111] In one embodiment, the enzyme Bbac1 is introduced into a genetically engineered cell that further comprises a β-1,4-galactosyltransferase and preferably also comprises a β-1,3-N-acetyl-glucosaminyltransferase.

[0112] In one embodiment, the enzyme Bacfin1 is introduced into a genetically engineered cell that further comprises a β-1,4-galactosyltransferase and preferably also comprises a β-1,3-N-acetyl-glucosaminyltransferase.

[0113] In one embodiment, the enzyme Prev1 is introduced into a genetically engineered cell that further comprises a β-1,4-galactosyltransferase and preferably also comprises a β-1,3-N-acetyl-glucosaminyltransferase.

[0114] In one embodiment, the enzyme Bgall1 is introduced into a genetically engineered cell which further comprises β-1,4-galactosyltransferase and preferably also comprises β-1,3-N-acetyl-glucosaminyltransferase.

[0115] In one embodiment, the enzyme CafC is introduced into a genetically engineered cell which further comprises β-1,4-galactosyltransferase and preferably also comprises β-1,3-N-acetyl-glucosaminyltransferase. In one embodiment, the enzyme FutA_mut2 is introduced into a genetically engineered cell which further comprises β-1,4-galactosyltransferase and preferably also comprises β-1,3-N-acetyl-glucosaminyltransferase.

[0116] In one embodiment, the enzyme FucT109 is introduced into a genetically engineered cell which further comprises β-1,4-galactosyltransferase and preferably also comprises β-1,3-N-acetyl-glucosaminyltransferase.

[0117] In one embodiment, the enzyme FutA is introduced into a genetically engineered cell which further comprises β-1,4-galactosyltransferase and preferably also comprises β-1,3-N-acetyl-glucosaminyltransferase.

[0118] β-1,3-N-acetyl-glucosaminyltransferase

[0119] β-1,3-N-acetyl-glucosaminyltransferase is any protein that includes the ability to transfer the N-acetyl-glucosamine of UDP-N-acetyl-glucosamine to lactose or another acceptor molecule in a β-1,3-bond (see Figure 1 ). Preferably, the β-1,3-N-acetyl-glucosaminyltransferase used herein is not from the species of the genetically engineered cell, i.e., the gene encoding β-1,3-N-acetyl-glucosaminyltransferase is of heterologous origin.

[0120] Thus, in an embodiment, the genetically engineered cell further comprises one or more recombinant nucleic acid sequences encoding β-1,3-N-acetyl-glucosaminyltransferase.

[0121] Non-limiting examples of β-1,3-N-acetyl-glucosaminyltransferase are given in Table 2. β-1,3-N-acetyl-glucosaminyltransferase variants may also be useful. Preferably, such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to the amino acid sequence of any of the β-1,3-N-acetyl-glucosaminyltransferases in Table 2.

[0122] Table 2. List of β-1,3-N-acetyl-glucosaminyltransferases

[0123]

[0124]

[0125] In an embodiment, the genetically engineered cell comprises a recombinant nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyltransferase. In one embodiment, the recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosaminyltransferase comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:24 (LgtA, from Neisseria meningitidis) or a functional homolog thereof, the amino acid sequence of which has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:24.

[0126] To produce LNnT from lactose as a substrate, β-1,3-N-acetylglucosaminyltransferase is used to form the LNT-II precursor. In one embodiment, the genetically engineered cell comprises a β-1,3-N-acetylglucosaminyltransferase gene or a functional homolog or fragment thereof to produce the intermediate LNT-II from lactose.

[0127] Some of the following examples use a heterologous β-1,3-N-acetyl-glucosaminyltransferase named LgtA from Neisseria meningitidis or a variant thereof.

[0128] β-1,4-galactosyltransferase

[0129] β-1,4-galactosyltransferase is any protein that includes the ability to transfer the galactose of UDP-galactose to the N-acetyl-glucosaminyl moiety of a receptor molecule via a β-1,4-bond (see Figure 1 ). Preferably, the β-1,4-galactosyltransferase used herein is not from the species of the genetically engineered cell, i.e., the gene encoding β-1,4-galactosyltransferase is of heterologous origin. In the context described herein, the receptor molecule is a receptor sugar, such as LNT-II or a more complex HMO structure.

[0130] The following examples use a heterologous β-1,4-galactosyltransferase GalT or a variant thereof to produce LNnT, which, for example, in combination with the α-1,3-fucosyltransferase described herein, can produce LNFP-III, LNFP-VI, and / or LNDFH-II. Thus, in an embodiment, the genetically engineered cell comprises one or more recombinant nucleic acid sequences encoding β-1,4-galactosyltransferase.

[0131] Non-limiting examples of β-1,4-galactosyltransferases are provided in Table 2. β-1,4-galactosyltransferase variants may also be useful. Preferably, such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to the amino acid sequence of any of the β-1,4-galactosyltransferases in Table 3.

[0132] Table 3. List of β-1,4-glycosyltransferases

[0133] Protein Name GenBankID Source GalT WP_001262061.1 Helicobacter pylori LgtB AAF42257.1 Neisseria meningitidis MC58

[0134] In the embodiments described herein, the β-1,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis, and the β-1,3-galactosyltransferase and / or β-1,4-galactosyltransferase are from Helicobacter pylori, respectively.

[0135] In one embodiment, the recombinant nucleic acid sequence encoding β-1,4-galactosyltransferase comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:25 (galT from Helicobacter pylori) or a functional homolog thereof, the amino acid sequence of which has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO:25.

[0136] To produce LNnT from the LNT-II precursor, β-1,4-galactosyltransferase is required. In one embodiment, the genetically engineered cell comprises a β-1,4-galactosyltransferase gene or a functional homolog or fragment thereof. In an embodiment, the β-1,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis and the β-1,4-galactosyltransferase is from Helicobacter pylori. In a further embodiment, the β1,3-N-acetylglucosaminyltransferase has the amino acid sequence according to SEQ ID NO:24 or a functional homolog thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO:24, and the β-1,4-galactosyltransferase has the amino acid sequence according to SEQ ID NO:25 or a functional homolog thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO:25.

[0137] α-2,3-sialyltransferase

[0138] An α-2,3-sialyltransferase refers to a glycosyltransferase that catalyzes the transfer of a sialic acid moiety from a donor substrate (such as CMP-N-acetylneuraminic acid) to an acceptor molecule (such as lactose or LNnT) via an α-2,3-bond. Preferably, the α-2,3-sialyltransferase used herein is not a species derived from a genetically engineered cell, i.e., the gene encoding the α-2,3-sialyltransferase is of heterologous origin and is selected from the α-2,3-sialyltransferases identified in Table 9. Heterologous α2,3-sialyltransferases capable of transferring a sialic acid moiety to lactose are known in the art, and three of them are identified in Table 9.

[0139] The sialyltransferase can be selected from amino acid sequences having at least 80%, such as 80%, such as at least 90%, such as at least 95%, or such as at least 99% identity to the amino acid sequence of any one of the α-2,3-sialyltransferases listed in Table 1.

[0140] Table 9. List of α-2,3-sialyltransferases capable of producing 3’SL.

[0141]

[0142] In one embodiment, the enzyme Osc1 is introduced into a genetically engineered cell further comprising an α-1,3-sialyltransferase, wherein the cell is capable of producing FSL, such as a mixture of 3’SL, 3FL, and FSL.

[0143] In one embodiment, the enzyme FucT109 is introduced into a genetically engineered cell further comprising an α-2,3-sialyltransferase, wherein the cell is capable of producing FSL, such as a mixture of 3’SL, 3FL, and FSL.

[0144] In one embodiment, the enzyme Murba1 is introduced into a genetically engineered cell further comprising an α-2,3-sialyltransferase, wherein the cell is capable of producing FSL, such as a mixture of 3’SL, 3FL, and FSL.

[0145] In one embodiment, the enzyme Bgall1 is introduced into a genetically engineered cell further comprising an α-2,3-sialyltransferase, wherein the cell is capable of producing FSL, such as a mixture of 3’SL, 3FL, and FSL.

[0146] In one embodiment, the enzyme CafC is introduced into a genetically engineered cell further comprising an α-2,3-sialyltransferase, wherein the cell is capable of producing FSL, such as a mixture of 3’SL, 3FL, and FSL.

[0147] In a preferred embodiment, the α-2,3-sialyltransferase is Clari1 from Campylobacter lari (GenBank protein accession number EGK8106227.1) or Poral from Pasteurella oralis (GenBank protein accession number WP_101774487.1).

[0148] Glycosyl donor - nucleotide-activated sugar pathway

[0149] When carrying out the method of the present invention, it is preferred that a glycosyltransferase-mediated glycosylation reaction occurs, in which an activated sugar nucleotide is used as the sugar donor. Activated sugar nucleotides generally have a phosphorylated sugar residue linked to a nucleoside. Specific glycosyltransferases only accept specific sugar nucleotides. Thus, preferably, the following activated sugar nucleotides are involved in glycosyl transfer: glucose - UDP-GlcNAc, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine (GlcNAc), and CMP-N-acetylneuraminic acid.

[0150] The genetically engineered cells according to the present invention can comprise one or more pathways for producing a nucleotide-activated sugar selected from the following: glucose - UDP-GlcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, and CMP-N-acetylneuraminic acid.

[0151] In one embodiment of the present invention, the genetically engineered cells are capable of producing one or more of the above-mentioned activated sugar nucleotides via a de novo pathway. In this regard, the activated sugar nucleotides are prepared by the cells from simple carbon sources such as glycerol, sucrose, fructose, or glucose in a stepwise reaction sequence under the action of enzymes involved in the de novo biosynthetic pathway of the corresponding sugar nucleotides (for a review of monosaccharide metabolism, see, for example, H.H. Freeze and A.D. Elbein: Chapter 4: Glycosylation precursors, in: Essentials of Glycobiology, 2nd Edition (Eds. A. Varki et al.), Cold Spring Harbour Laboratory Press (2009)).

[0152] The enzymes involved in the de novo biosynthetic pathway of activated sugar nucleotides can be naturally present in the cells or introduced into the cells by genetic engineering or recombinant DNA techniques, all of which are part of the general knowledge of those skilled in the art.

[0153] In another embodiment, the genetically engineered cell can use salvaged monosaccharides for sugar nucleotides. In the salvage pathway, monosaccharides derived from degraded oligosaccharides are phosphorylated by kinases and converted to nucleotide sugars by pyrophosphatases. The enzymes involved in this process can be heterologous or native enzymes of the host cell.

[0154] Colanic acid gene cluster

[0155] For the production of fucosylated HMOs, the de novo GDP-fucose pathway is important for ensuring the presence of sufficient GDP-fucose. The colanic acid gene cluster of Escherichia coli encodes selected enzymes (gmd, wcaG, wcaH, wcaI, manB, manC) involved in the de novo synthesis of GDP-fucose, while one or more genes downstream of GDP-L-fucose, such as wcaJ (which is responsible for the production of the extracellular polysaccharide colanic acid, the major oligosaccharide of the bacterial cell wall), can be deleted to prevent the conversion of GDP-fucose to colanic acid.

[0156] To ensure a sufficient amount of GDP-fucose, the promoter of the native colanic acid gene cluster can be replaced with a stronger promoter to generate a recombinant colanic acid gene cluster to drive the additional production of GDP-fucose. In addition, as described in the examples, additional copies of the colanic acid gene cluster or its selected genes can be introduced into the genetically engineered cell.

[0157] In an embodiment, the colanic acid gene cluster can be expressed from its native genomic locus. The expression can be actively modulated. The expression can be modulated by replacing the native promoter with a promoter of interest and / or by expressing the gene cluster from another rather than the native genomic locus, or by episomal expression of the colanic acid gene cluster or its specific genes to increase the copy number of the colanic acid genes encoding the said proteins.

[0158] With respect to the present invention, the term "native genomic locus", with respect to the colanic acid gene cluster, refers to the original and natural position of the gene cluster in the genome of the genetically engineered cell.

[0159] The de novo GDP-fucose pathway genes responsible for forming GDP-fucose include or consist of:

[0160] i) manA, which encodes the protein mannose-6-phosphate isomerase (EC 5.3.1.8, UniProt accession number P00946), which facilitates the interconversion of fructose 6-phosphate (F6P) and mannose-6-phosphate;

[0161] ii) manB, which encodes phosphomannomutase (EC 5.4.2.8, UniProt accession number P24175), which is involved in the biosynthesis of GDP-mannose by catalyzing the conversion of mannose-6-phosphate to mannose-1-phosphate;

[0162] iii) manC, which encodes mannose-1-phosphate guanylyltransferase (EC: 2.7.7.13, UniProt accession number P24174), which is involved in the biosynthesis of GDP-mannose by synthesizing GDP-mannose from GTP and α-D-mannose-1-phosphate;

[0163] iv) gmd, which encodes GDP-mannose-4,6-dehydratase (UniProt accession number P0AC88), which catalyzes the conversion of GDP-mannose to GDP-4-dehydro-6-deoxy-D-mannose;

[0164] v) wcaG (fcl), which encodes GDP-L-fucose synthase (EC 1.1.1.271, UniProt accession number P32055), which catalyzes the two-step NADP-dependent conversion of GDP-4-dehydro-6-deoxy-D-mannose to GDP-fucose.

[0165] Thus, preferably, when producing one or more fucosylated heterologous products, the genetically engineered cells overexpress the entire capsular heteropolysaccharide acid gene cluster and / or one or more genes of the de novo GDP-fucose pathway selected from manA, manB, manC, gmd, and wcaG.

[0166] Lactose permease

[0167] Lactose permease is a membrane protein, which is a member of the major facilitator superfamily and can be classified as a symporter, which uses the proton gradient towards the cell to transport β-galactosides such as lactose into the cell in the same direction. In oligosaccharide production, especially in the production of human milk oligosaccharides (HMOs), lactose is usually the initial substrate that is modified to produce any desired HMO in the biotransformation that occurs inside the cell. Therefore, when producing HMOs, it is desirable to be able to import lactose into the cell, for example, by expressing / or overexpressing a lactose permease such as lacY of Escherichia coli.

[0168] In an embodiment, the lactose permease is as shown in SEQ ID NO:26, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:26, such as at least 85%, 90%, or 95% identity.

[0169] In an embodiment, the expression of lactose permease is regulated by a promoter according to the present invention.

[0170] β-galactosidase

[0171] A host cell suitable for HMO production, such as Escherichia coli, may contain an endogenous β-galactosidase gene or an exogenous β-galactosidase gene. For example, Escherichia coli includes the endogenous lacZ gene (e.g., GenBank accession number V00296 (GI: 41901)). For the purposes of the present invention, when producing HMOs, if lactose is used as the initial substrate for producing complex fucosylated HMOs, it is preferred that the genetically engineered cells do not express functional β-galactosidase to avoid lactose degradation. In an embodiment, the lacZ gene can be inactivated by completely or partially deleting the corresponding nucleic acid sequence from the bacterial genome, or the gene sequence can be mutated in a way that it is not transcribed, or if transcribed, the transcript is not translated, or if translated into a protein (i.e., β-galactosidase), the protein does not have the corresponding enzyme activity. In this way, the bacteria producing HMOs accumulate an increased intracellular lactose pool, which is beneficial for HMO production.

[0172] Input protein

[0173] Most commonly, cells producing HMOs are genetically engineered to use lactose as the initial substrate because, as described above, lactose permease readily takes up lactose. However, it may be desirable to use an initial substrate that requires fewer glycosyltransferases in the cell, as this will reduce the stress on the cell in producing multiple enzymes and can also reduce by-products. For example, if lactose is not used as the initial substrate, cells containing fucosyltransferase will not produce 3FL as a by-product, thus allowing fucose to be used for producing, for example, more LNFP-V and LNDFH-II.

[0174] Examples of suitable LNT-II and LNnT input proteins are described in WO2023099680 and include, for example,

[0175] - Lactose permease (LacY) mutants, such as LacY mutant Y236H or LacY mutant A177V+S306T, wherein the mutation is equivalent to the corresponding position in the SEQ ID NO:14 sequence,

[0176] -ABC transporter complexes, such as the ABC transporters from Bifidobacterium pseudocatenulatum JCM1200 BBPC_1775, 1776, 1777 (NCBI accession numbers BAR04453.1, BAR04454.1, and BAR04455.1, respectively) or the ABC transporters from Bifidobacterium breve UCC2003 BBR_0527 / lntP1, BBR_0528 / lntP2, BBR_0530 / lntS, and BBR_0531 (NCBI accession numbers ABE95224.1, ABE95225.1, ABE95226.1, and ABE95228.1), and / or

[0177] -MFS transporters, such as but not limited to Blon_0962 (NCBI accession number ACJ52061.1).

[0178] Thus, in an embodiment, a nucleic acid or nucleic acid cluster encoding one of these transporters can be introduced into the genetically modified cells described herein. The expression of such a transporter enables the production of complex fucosylated oligosaccharides with LNT-II as the initial substrate.

[0179] Output protein

[0180] Oligosaccharide products, such as HMOs produced by the cells, can accumulate in the intracellular and extracellular matrix. The products can be transported passively into the supernatant, i.e., they diffuse outside the cell membrane. More complex HMO products may remain in the cells, which may ultimately impair cell growth and thus affect the possible total yield of the products from a single fermentation. HMO transport can be facilitated by major facilitator superfamily transporters, which promote the efflux of sugar derivatives from the cells into the supernatant. The output protein can be present exogenously or endogenously and overexpressed under fermentation conditions to enhance the output of the produced oligosaccharide derivatives (HMOs). The specificity for the oligosaccharide products to be secreted can be altered by mutation using known recombinant DNA techniques.

[0181] Thus, the genetically engineered cells according to the present invention can further comprise a nucleic acid sequence encoding an output protein that is capable of outputting one or more fucosylated human milk oligosaccharide products, for example, the transporter can be, for example, a member of the major facilitator superfamily transporters.

[0182] In recent years, several new and efficient major facilitator superfamily transporters have been identified as output proteins for HMOs, each protein being specific for a different recombinantly produced HMO, and the development of recombinant cells expressing such proteins is advantageous for large-scale industrial HMO manufacturing.

[0183] Genetically engineered cells

[0184] In this context, the terms "genetically engineered cells" and "genetically modified cells" can be used interchangeably. As used herein, "genetically engineered cells" refers to host cells whose genetic material has been altered by human intervention using genetic engineering techniques such as, but not limited to, transformation or transfection, e.g., with heterologous and / or recombinant polynucleotide sequences, Crisper / Cas editing, and / or random mutagenesis. In one embodiment, the genetically engineered cells have been transformed or transfected with a recombinant nucleic acid sequence.

[0185] Genetic modification can, for example, be selected from the inclusion of, e.g., glycosyltransferases, and / or the engineered deletion of metabolic pathways of inhibitors or unwanted enzymes, as well as the inclusion of transporters as described in the above sections, and those skilled in the art will know how to combine them into genetically engineered cells capable of producing one or more fucosylated HMOs.

[0186] In one embodiment, the genetically engineered cells capable of producing LNDFH-III comprise a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase selected from a) Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:1, b) Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:2, c) Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:3, d) Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:4, e) Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:5, f) Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:6, g) Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:7, h) CafC comprising or consisting of the amino acid sequence of SEQ ID NO:8, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:8, i) a FutA variant comprising substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80% sequence identity to SEQ ID NO:11, j) futA_mut2 comprising or consisting of the amino acid sequence of SEQ ID NO:9, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:9, k) FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:10.

[0187] Preferably, the fucosyltransferase has α-1,3-fucosyltransferase activity, allowing fucosylation of the oligosaccharide at the 3-position of the GlcNAc moiety and the 3-position of the Glc moiety, while showing limited or no fucosylation at the 2-position of the Gal moiety. Preferably, the Glc moiety is located at the reducing end of the oligosaccharide, and more preferably, the oligosaccharide is LNnT. In one embodiment, a genetically engineered cell capable of producing LNDFH-III comprises a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase selected from a) Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:1, b) Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:2, c) Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:3. These enzymes can be used, for example, for the production of LNDFH-III, where the content of LNFP-III and LNFP-VI is low, for example, each accounting for less than 10% of the total HMO.

[0188] Preferably, the genetically engineered cells expressing these enzymes produce the HMO mixture LNDFH-III and 3FL, where these two HMOs account for at least 80%, such as at least 90%, of the total HMO produced.

[0189] In one embodiment, a genetically engineered cell capable of producing LNDFH-III comprises a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase selected from a) Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:2, b) Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:5, c) Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:4, and d) a FutA variant comprising substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80% sequence identity to SEQ ID NO:11.

[0190] Preferably, the genetically engineered cells expressing these enzymes produce an HMO mixture comprising LNFP-III and LNDFH-III, wherein LNFP-VI accounts for less than 1% of the total HMO produced.

[0191] In one embodiment, the genetically engineered cells capable of producing LNDFH-III comprise a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase selected from a) Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:3, b) Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:6, c) FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:10, d) Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:7.

[0192] Preferably, the genetically engineered cells expressing these enzymes produce an HMO mixture comprising LNDFH-III, LNFP-III, and LNFP-VI.

[0193] In one embodiment, the genetically engineered cells capable of producing LNDFH-III comprise a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having dual α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:1.

[0194] In one embodiment, the genetically engineered cells capable of producing LNDFH-III comprise a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having dual α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:2.

[0195] In one embodiment, the genetically engineered cells capable of producing LNDFH-III comprise a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having dual α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:3.

[0196] In one embodiment, the genetically engineered cells capable of producing LNDFH-III comprise a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having dual α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:4.

[0197] In one embodiment, the genetically engineered cells capable of producing LNDFH-III comprise a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having dual α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:5.

[0198] In one embodiment, the genetically engineered cells capable of producing LNDFH-III comprise a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having dual α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:6.

[0199] In one embodiment, the genetically engineered cells capable of producing LNDFH-III comprise a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having dual α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:7.

[0200] In one embodiment, the genetically engineered cells capable of producing LNDFH-III comprise a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having dual α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is CafC comprising or consisting of the amino acid sequence of SEQ ID NO:8, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO:8.

[0201] In one embodiment, the genetically engineered cells capable of producing LNDFH-III comprise a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having dual α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is futA_mut2 comprising or consisting of the amino acid sequence of SEQ ID NO:9, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO:9.

[0202] In one embodiment, the genetically engineered cells capable of producing LNDFH-III comprise a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having dual α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO:10.

[0203] In one embodiment, the genetically engineered cells capable of producing LNDFH-III comprise a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having dual α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is a FutA variant comprising substitutions at positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80% but less than 100% identity to SEQ ID NO:11.

[0204] In one aspect of the present invention, the genetically engineered cell comprises a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having α-1,3(4)-fucosyltransferase activity, which is capable of producing at least 20 mol% of LNDFH-III of the total molar HMO content produced by the cell. Preferably, at least 25 mol% of the total molar content of HMO produced by the cell is LNDFH-III. In an embodiment, at least 20 mol% of the total molar content of HMO produced by the cell, such as at least 25 mol%, 29 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, or such as at least 65 mol% is LNDFH-III. In a further embodiment, the cell also produces one or more HMOs selected from 3FL, LNnT, LNFP-III, and LNFP-VI.

[0205] In some embodiments, the genetically engineered cells described herein express Osc1, which comprises or consists of the amino acid sequence of SEQ ID NO:1, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO:1, and the mol% content of LNDFH-III produced by the genetically engineered cell is higher than 30 mol% of the total HMO produced, such as higher than 35 mol%, such as higher than 40 mol%, such as higher than 45 mol%, such as higher than 50 mol%, or such as higher than 55 mol%.

[0206] In some embodiments, the genetically engineered cells described herein express Bgall1, which comprises or consists of the amino acid sequence of SEQ ID NO:2, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO:2, and the mol% content of LNDFH-III produced by the genetically engineered cell is higher than 30 mol% of the total HMO produced, such as higher than 40 mol%, such as higher than 50 mol%, such as higher than 55 mol%, such as higher than 60 mol%, or such as higher than 65 mol%.

[0207] In some embodiments, the genetically engineered cells described herein express Bbac1, which comprises or consists of the amino acid sequence of SEQ ID NO:3, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO:3, and the mole % content of LNDFH-III produced by the genetically engineered cells is higher than 30 mole % of the total HMO produced, such as higher than 35 mole %, such as higher than 40 mole % or such as higher than 45 mole %.

[0208] In some embodiments, the genetically engineered cells described herein express Murba1, which comprises or consists of the amino acid sequence of SEQ ID NO:4, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO:4, and the mole % content of LNDFH-III produced by the genetically engineered cells is higher than 30 mole % of the total HMO produced, such as higher than 35 mole %, such as higher than 40 mole % or such as higher than 42 mole %.

[0209] In some embodiments, the genetically engineered cells described herein express Bacfin1, which comprises or consists of the amino acid sequence of SEQ ID NO:5, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO:5, and the mole % content of LNDFH-III produced by the genetically engineered cells is higher than 30 mole % of the total HMO produced, such as higher than 40 mole %, such as higher than 50 mole %, such as higher than 55 mole % or such as higher than 60 mole %.

[0210] In some embodiments, the genetically engineered cells described herein express Prev1, which comprises or consists of the amino acid sequence of SEQ ID NO:6, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO:6, and the mole % content of LNDFH-III produced by the genetically engineered cells is higher than 25 mole % of the total HMO produced, such as higher than 30 mole %, such as higher than 35 mole % or such as higher than 40 mole %.

[0211] In some embodiments, the genetically engineered cells described herein express Csec1, which comprises or consists of the amino acid sequence of SEQ ID NO:7, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO:7, and the molar% content of LNDFH-III produced by the genetically engineered cells is higher than 20 mol% of the total HMO produced, such as higher than 21 mol%, such as higher than 22 mol%, such as higher than 23 mol%, such as higher than 24 mol%, or such as higher than 25 mol%.

[0212] In some embodiments, the genetically engineered cells described herein express CafC, which comprises or consists of the amino acid sequence of SEQ ID NO:8, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO:8, and the molar% content of LNDFH-III produced by the genetically engineered cells is higher than 24 mol% of the total HMO produced, such as higher than 25 mol%, such as higher than 26 mol%, such as higher than 27 mol%, such as higher than 28 mol%, or such as higher than 29 mol%.

[0213] In some embodiments, the genetically engineered cells described herein express a FutA variant that comprises substitutions at positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80% but less than 100% identity to SEQ ID NO:11, and the molar% content of LNDFH-III produced by the genetically engineered cells is higher than 25 mol% of the total HMO produced, such as higher than 30 mol%, such as higher than 35 mol%, or such as higher than 40 mol%.

[0214] In some embodiments, the genetically engineered cells described herein express FutA_mut2, which comprises or consists of the amino acid sequence of SEQ ID NO:9, and the molar% content of LNDFH-III produced by the genetically engineered cells is higher than 25 mol% of the total HMO produced, such as higher than 30 mol%, such as higher than 35 mol%, or such as higher than 40 mol%.

[0215] In some embodiments, the genetically engineered cells described herein express FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO:10, and the molar % content of LNDFH-III produced by the genetically engineered cells is higher than 15 mol% of the total HMOs produced, such as higher than 17 mol%, such as higher than 19 mol%, such as higher than 21 mol% or such as higher than 23 mol%.

[0216] Greater than 60% of the HMOs found in human milk are different types of fucosylated HMOs. Thus, in embodiments, at least 60%, such as at least 75%, 80%, 85%, 90%, 95% or at least 97% of the molar content of the total HMOs produced by the cells is fucosylated. Preferably, the HMOs produced by the cells are selected from 3FL, LNFP-III, LNFP-VI and LNDFH-III.

[0217] In some embodiments, the genetically engineered cells described herein express Osc1, which comprises or consists of the amino acid sequence of SEQ ID NO:1, or comprises an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO:1, and the molar % content of fucosylated HMOs produced by the genetically engineered cells is higher than 85 mol% of the total HMOs produced, such as higher than 90 mol%, such as higher than 95 mol%, such as higher than 97 mol%, such as higher than 98 mol% or such as higher than 99 mol%.

[0218] In some embodiments, the genetically engineered cells described herein express Osc1, which comprises or consists of the amino acid sequence of SEQ ID NO:1, or comprises an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO:1, and produces only fucosylated HMOs.

[0219] In some embodiments, the genetically engineered cells described herein express Bgall1, which comprises or consists of the amino acid sequence of SEQ ID NO:2, or comprises an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO:2, and the molar % content of fucosylated HMOs produced by the genetically engineered cells is higher than 85 mol% of the total HMOs produced, such as higher than 90 mol%, such as higher than 95 mol%, such as higher than 97 mol%, such as higher than 98 mol% or such as higher than 99 mol%.

[0220] In some embodiments, the genetically engineered cells described herein express Bgall1, which comprises or consists of the amino acid sequence of SEQ ID NO:2, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:2, and produce only fucosylated HMOs.

[0221] In some embodiments, the genetically engineered cells described herein express Bbac1, which comprises or consists of the amino acid sequence of SEQ ID NO:3, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:3, and the molar % content of fucosylated HMOs produced by the genetically engineered cells is higher than 80 mol% of the total HMOs produced, such as higher than 85 mol%, such as higher than 90 mol%, such as higher than 93 mol%, or such as higher than 95 mol%.

[0222] In some embodiments, the genetically engineered cells described herein express Murba1, which comprises or consists of the amino acid sequence of SEQ ID NO:4, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:4, and the molar % content of fucosylated HMOs produced by the genetically engineered cells is higher than 70 mol% of the total HMOs produced, such as higher than 75 mol%, such as higher than 80 mol%, such as higher than 82 mol%, or such as higher than 84 mol%.

[0223] In some embodiments, the genetically engineered cells described herein express Bacfin1, which comprises or consists of the amino acid sequence of SEQ ID NO:5, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:5, and the molar % content of fucosylated HMOs produced by the genetically engineered cells is higher than 75 mol% of the total HMOs produced, such as higher than 80 mol%, such as higher than 85 mol%, such as higher than 87 mol%, or such as higher than 89 mol%.

[0224] In some embodiments, the genetically engineered cells described herein express Prev1, which comprises or consists of the amino acid sequence of SEQ ID NO:6, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:6, and the molar % content of fucosylated HMO produced by the genetically engineered cells is higher than 75 mol% of the total HMO produced, such as higher than 80 mol%, such as higher than 85 mol%, such as higher than 87 mol%, or such as higher than 90 mol%.

[0225] In some embodiments, the genetically engineered cells described herein express Csec1, which comprises or consists of the amino acid sequence of SEQ ID NO:7, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:7, and the molar % content of fucosylated HMO produced by the genetically engineered cells is higher than 50 mol% of the total HMO produced, such as higher than 55 mol%, such as higher than 57 mol%, such as higher than 59 mol% or such as higher than 61 mol%.

[0226] In some embodiments, the genetically engineered cells described herein express CafC, which comprises or consists of the amino acid sequence of SEQ ID NO:8, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:8, and the molar % content of fucosylated HMO produced by the genetically engineered cells is higher than 85 mol% of the total HMO produced, such as higher than 90 mol%, such as higher than 95 mol%, such as higher than 97 mol%, such as higher than 98 mol% or such as higher than 99 mol%.

[0227] In some embodiments, the genetically engineered cells described herein express a FutA variant that comprises substitutions at positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80% but less than 100% identity with SEQ ID NO:11, and the molar % content of fucosylated HMO produced by the genetically engineered cells is higher than 80 mol% of the total HMO produced, such as higher than 85 mol%, such as higher than 90 mol%, such as higher than 93 mol%, such as higher than 95 mol%, or such as higher than 96 mol%.

[0228] In some embodiments, the genetically engineered cells described herein express FutA_mut2, which comprises or consists of the amino acid sequence of SEQ ID NO:9, and the molar percentage content of fucosylated HMO produced by the genetically engineered cells is higher than 80 mol% of the total HMO produced, such as higher than 85 mol%, such as higher than 90 mol%, or higher than 93 mol%, higher than 95 mol% or higher than 96 mol%.

[0229] In some embodiments, the genetically engineered cells described herein express FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:10, and the molar percentage content of fucosylated HMO produced by the genetically engineered cells is higher than 60 mol% of the total HMO produced, such as higher than 63 mol%, such as higher than 65 mol%, such as higher than 67 mol% or such as higher than 69 mol%.

[0230] In a preferred embodiment, the fucosylated HMOs produced by the cells described herein are selected from the group consisting of LNDFH-II and the group consisting of 3FL, LNFP-III, and LNFP-VI.

[0231] The genetically engineered cells described herein preferably express genes encoding key enzymes for fucosylated HMO biosynthesis. In addition, it is advantageous if the genetically engineered cells express the genes required for producing LNnT, whether from lactose or LNT-II as the initial substrate (see Figure 1 ), and / or alternatively the cells express the input proteins of LNT-II or LNnT.

[0232] In an embodiment, the genetically engineered cells comprise one or more additional glycosyltransferases. The one or more additional glycosyltransferases are preferably selected from the group consisting of galactosyltransferases, glucosaminyltransferases, fucosyltransferases, and N-acetylglucosaminyltransferases.

[0233] In some embodiments, the genetically engineered cells comprise one or more recombinant nucleic acid sequences encoding β-1,4-galactosyltransferase and optionally β-1,3-N-acetylglucosaminyltransferase. In some embodiments, the β-1,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis and the β-1,4-galactosyltransferase is from Helicobacter pylori.

[0234] In some embodiments, the genetically engineered cells described herein further express the de novo GDP-fucose pathway genes manA, manB, manC, gmd, and wcaG responsible for forming GDP-fucose. By introducing a nucleic acid construct encoding the capsular polysaccharide acid gene cluster (CA) (including the genes gmd, wcaG, wcaH, wcaI, manC, and manB of Escherichia coli), as shown in SEQ ID NO: 23, overexpressing one or more of these genes and / or upregulating CA may be advantageous, allowing the formation of GDP-fucose, which enables the cells to produce higher levels of fucosylated oligosaccharides from one or more intermediate oligosaccharide substrates, such as lactose or LNnT, LNFP-III, and / or LNFP-VI. Depending on the intended use of the substrate, one or more additional glycosyltransferases and pathways for producing nucleotide-activated sugars (such as glucose-UDP-GlcNAc, CMP-N-acetylneuraminic acid, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, and / or CMP-N-acetylneuraminic acid) may also be present in the genetically engineered cells.

[0235] It is further understood that the genetically engineered cells described herein may also contain any of the above modifications, such as additional glycosyltransferases, appropriate import proteins, overexpression of lactose permease, LNT-II, or LNT import protein, inactivation of β-galactosidase, especially if lactose is used as the initial substrate, and appropriate export proteins for the complex fucosylated HMOs produced by the cells.

[0236] HMO mixture produced by the cells

[0237] Genetically engineered cells containing the α-1,3-fucosyltransferase with dual fucosyltransferase specificity described herein will generally produce an HMO mixture, which is the result of a multi-step process within the cells towards the final HMO product LNDFH-III (see Figure 1 ). In the process of producing LNDFH-III using lactose as the initial substrate, it is expected that the cells will produce a certain amount of 3FL (fucosylated lactose), LNT-II, LNnT, LNFP-III, and LNFP-VI, some of which species are only produced as synthetic intermediates and do not exist in the final mixture produced by the cells.

[0238] The mole % of individual HMO components supported by the experimental data of the examples shows an exemplary HMO composition range, wherein the mixture of the final HMO products consists essentially of LNDFH-III and one or more HMOs selected from 3-FL, LNT-II, LNT, LNFP-III, and LNFP-VI. Preferably, the amount of LNT-II is very low or not present in detectable amounts.

[0239] In an embodiment, the cell produces a final mixture consisting essentially of LNDFH-III and 3FL. In another embodiment, the cell produces a final mixture consisting essentially of LNDFH-III, LNFP-III and 3FL. In another embodiment, the cell produces a final mixture consisting essentially of LNDFH-III, LNFP-VI and 3FL. In another embodiment, the cell produces a final mixture consisting essentially of LNDFH-III, LNFP-III, LNFP-VI and 3FL. In another embodiment, the cell produces a final mixture consisting essentially of LNDFH-III, LNFP-III, 3FL and LNnT. In another embodiment, the cell produces a final mixture consisting essentially of LNDFH-III, LNFP-III, LNFP-VI, 3FL and LNnT.

[0240] In an embodiment, the cell produces an HMO mixture consisting essentially of 20-70 mol% of LNDFH-III, 0-35 mol% of LNFP-III, 0-35 mol% of LNDFP-VI, 0-65 mol% of 3FL, 0-40% of LNnT and up to 1% of pLNnH, with a total of 100% molar content.

[0241] In some embodiments, the genetically engineered cells described herein express Osc1, which comprises or consists of the amino acid sequence of SEQ ID NO:1, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:1, and the mixture produced consists essentially of 30-60 mol% of LNDFH-III and 40-65 mol% of 3FL, with a total of 100% molar content.

[0242] In some embodiments, the genetically engineered cells described herein express Bgall1, which comprises or consists of the amino acid sequence of SEQ ID NO:2, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:2, and the mixture produced consists essentially of 50-80 mol% of LNDFH-III, 1-10 mol% of LNFP-III and 15-50% of 3FL, with a total of 100% molar content.

[0243] In some embodiments, the genetically engineered cells described herein express Bbac1, which comprises or consists of the amino acid sequence of SEQ ID NO:3, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO:3, and the mixture produced consists essentially of 40 - 50 mol% of LNDFH-III, 5 - 15 mol% of LNFP-VI, 3 - 8 mol% of LNFP-III, and 2 - 12 mol% of LNnT and 30 - 40 mol% of 3FL, for a total of 100% molar content.

[0244] In some embodiments, the genetically engineered cells described herein express Murba1, which comprises or consists of the amino acid sequence of SEQ ID NO:4, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO:4, and the mixture produced consists essentially of 40 - 50 mol% of LNDFH-III, 25 - 35 mol% of LNFP-VI, 10 - 20 mol% of LNnT, and 5 - 15 mol% of 3FL, for a total of 100% molar content.

[0245] In some embodiments, the genetically engineered cells described herein express Bacfin1, which comprises or consists of the amino acid sequence of SEQ ID NO:5, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO:5, and the mixture produced consists essentially of 55 - 65 mol% of LNDFH-III, 17 - 27 mol% of LNFP-III, and 6 - 16 mol% of LNnT and 1 - 11 mol% of 3FL, for a total of 100% molar content.

[0246] In some embodiments, the genetically engineered cells described herein express Prev1, which comprises or consists of the amino acid sequence of SEQ ID NO:6, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO:6, and the mixture produced consists essentially of 36 - 46 mol% of LNDFH-III, 25 - 35 mol% of LNFP-VI, 10 - 20 mol% of LNFP-III, and 2 - 12 mol% of LNnT and 2 - 12 mol% of 3FL, for a total of 100% molar content.

[0247] In some embodiments, the genetically engineered cells described herein express Csec1, which comprises or consists of the amino acid sequence of SEQ ID NO:7, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO:7, and the mixture produced consists essentially of 25-35 mol% of LNDFH-III, 15-25 mol% of LNFP-VI, 8-18 mol% of LNFP-III and 33-43 mol% of LNnT, for a total of 100% molar content.

[0248] In some embodiments, the genetically engineered cells described herein express CafC, which comprises or consists of the amino acid sequence of SEQ ID NO:8, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO:8, and the mixture produced consists essentially of 25-35 mol% of LNDFH-III, 29-39 mol% of LNFP-VI and 31-41% of 3FL, for a total of 100% molar content.

[0249] In some embodiments, the genetically engineered cells described herein express FutA_mut2, which comprises or consists of the amino acid sequence of SEQ ID NO:9, and the mixture produced consists essentially of 37-47 mol% of LNDFH-III, 26-36 mol% of LNFP-III, 19-29 mol% of 3FL and less than 5% of LNnT, for a total of 100% molar content.

[0250] In some embodiments, the genetically engineered cells described herein express FucT109, which comprises or consists of the amino acid sequence of SEQ ID NO:10, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO:10, and the mixture produced consists essentially of 19-29 mol% of LNDFH-III, 13-23 mol% of LNFP-VI, 22-32 mol% of LNFP-III and 24-34 mol% of LNnT and less than 5 mol% of 3FL, for a total of 100% molar content.

[0251] In some embodiments, the genetically engineered cells described herein express a FutA variant that contains substitutions at positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80% but less than 100% identity to SEQ ID NO:11, and the resulting mixture consists essentially of 37 - 47 mol% LNDFH-III, 26 - 36 mol% LNFP-III, 19 - 29 mol% 3FL, and less than 5% LNnT, for a total of 100 mol%.

[0252] host cell

[0253] In embodiments, the engineered cells are microorganisms. The genetically engineered cells are preferably microbial cells, such as prokaryotic or eukaryotic cells. Suitable microbial cells that can be used as host cells include bacterial cells, archaeal cells, algal cells, and fungal cells.

[0254] The genetically engineered cells can be, for example, bacterial or yeast cells. In a preferred embodiment, the genetically engineered cells are bacterial cells.

[0255] There is no restriction in principle regarding the bacterial host cell; they can be eubacteria (Gram-positive or Gram-negative) or archaea, provided that they allow genetic manipulation for insertion of the gene of interest and can be cultured on a manufacturing scale. Preferably, the host cell has properties allowing cultivation to high cell density. Non-limiting examples of bacterial host cells suitable for the recombinant industrial production of HMOs according to the invention can be members of the Enterobacteriaceae family, preferably of the genus Escherichia, more preferably of the species Escherichia coli. Other examples of suitable host cells are Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Campylobacter sp, Pantoea citrea, Pectobacterium carotovorum or Xanthomonas campestris. Bacteria of the genus Bacillus can also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus and Bacillus circulans.Similarly, the methods of the invention can be used to engineer bacteria of the genera Lactobacillus and Lactococcus, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species. Useful species also include strains, such as those engineered as described herein, from the genus Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), the genus Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), the genus Sporolactobacillus spp., the genus Micromomospora spp., the genus Micrococcus spp., the genus Rhodococcus spp., and the genus Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa).

[0256] Non-limiting examples of fungal host cells suitable for recombinant industrial production of heterologous products are, for example, yeast cells such as Komagataella, Kluyveromyces, Yarrowia, Pichia, Saccaromyces, Schizosaccharomyces or Hansenula, or filamentous fungi selected from the genus Aspergillus, Fusarium or Thricoderma.

[0257] In one or more exemplary embodiments, the genetically engineered cells are selected from the group consisting of Escherichia sp., Bacillus sp., Lactobacillus sp., Corynebacterium sp. and Campylobacter sp.

[0258] In one or more exemplary embodiments, the genetically engineered cells are selected from Escherichia coli, Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris and Saccharomyces cerevisiae.

[0259] In one or more exemplary embodiments, the genetically engineered cell is Bacillus subtilis.

[0260] In one or more exemplary embodiments, the genetically engineered cell is Saccharomyces cerevisiae or Pichia pastoris.

[0261] In one or more exemplary embodiments, the genetically engineered cell is Escherichia coli.

[0262] In one or more exemplary embodiments, the present invention relates to a genetically engineered cell, wherein the cell is derived from the Escherichia coli K-12 strain or DE3.

[0263] Recombinant nucleic acid sequence

[0264] The present invention relates to a genetically engineered cell comprising a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having dual α-1,3-fucosyltransferase activity, such enzymes being selected from Osc1, Bgall1, Bbac1, Murba1, Bacfin1, Prev1, Csec1, CafC, FucT109 and FutA_mut2, wherein said cell produces human milk oligosaccharides (HMOs). In particular, the molar percentage content of at least one fucosylated HMO, preferably LNDFH-III, is higher than 25%, such as higher than 50%, of the total HMOs produced.

[0265] In this context, the terms "recombinant nucleic acid sequence", "recombinant gene / nucleic acid / nucleotide sequence / DNA encoding...", or "encoding nucleic acid sequence" are used interchangeably and are intended to refer to an artificial nucleic acid sequence (i.e., produced in vitro using standard laboratory methods for making nucleic acid sequences) that comprises a set of contiguous, non-overlapping triplets (codons) that, when under the control of an appropriate control sequence (i.e., a promoter sequence), are transcribed into mRNA and translated into a protein.

[0266] The boundaries of the coding sequence are typically determined by a ribosome binding site upstream of the open reading frame at the 5' end of the mRNA, a transcription start codon (AUG, GUG or UUG), and a translation stop codon (UAA, UGA or UAG). The coding sequence can include, but is not limited to, genomic DNA, cDNA, synthetic and recombinant nucleic acid sequences.

[0267] The term "nucleic acid" includes RNA, DNA and cDNA molecules. It should be understood that due to the degeneracy of the genetic code, a large number of nucleic acid sequences encoding a given protein can be produced.

[0268] The recombinant nucleic acid sequence can be a coding DNA sequence such as a gene, or a non-coding DNA sequence such as regulatory DNA, such as a promoter sequence or other non-coding regulatory sequences.

[0269] Furthermore, the recombinant nucleic acid sequence can be heterologous. As used herein, "heterologous" refers to a polypeptide, amino acid sequence, nucleic acid sequence or nucleotide sequence that is exogenous to a cell or organism, i.e., a polypeptide, amino acid sequence, nucleic acid molecule or nucleotide sequence that does not naturally occur in said cell or organism is exogenous.

[0270] The present invention also relates to nucleic acid constructs which comprise a recombinant DNA sequence encoding a nucleic acid sequence, i.e. a gene of interest (such as the α-1,3-fucosyltransferase gene), and a non-coding regulatory DNA sequence, such as a promoter DNA sequence, such as a recombinant promoter sequence derived from a promoter sequence of the lac operon or the glp operon, or a promoter sequence derived from another genomic promoter DNA sequence, or a synthetic promoter sequence, wherein the coding and promoter sequences are operably linked.

[0271] The term "operably linked" refers to the functional relationship between two or more nucleic acid (e.g. DNA) fragments. It refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, a promoter sequence is operably linked to a coding sequence if the promoter sequence stimulates or regulates the transcription of the coding sequence in a suitable host cell or other expression system.

[0272] Typically, a promoter sequence operably linked to a transcriptional sequence is physically contiguous with the transcriptional sequence, i.e. they are cis-acting.

[0273] In one exemplary embodiment, the nucleic acid construct of the present invention can be part of a vector DNA, and in another embodiment, the construct is an expression cassette / cassettes integrated into the genome of the host cell.

[0274] Thus, the term "nucleic acid construct" refers to an artificially constructed nucleic acid fragment, particularly a DNA fragment, which is intended to be inserted into a target cell, such as a bacterial cell, to modify the expression of a genomic gene or the expression of a gene / coding DNA sequence that may be included in the construct. Thus, in an embodiment, the present invention relates to a nucleic acid construct comprising a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase, wherein the recombinant nucleic acid sequence is selected from nucleic acid sequences encoding Osc1, Bgall1, Bbac1, Murba1, Bacfin1, Prev1, Csec1, CafC, FutA_mut2, FucT109, such as nucleic acid sequences according to SEQ ID NO:12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 or functional variants thereof.

[0275] The genetically engineered cells according to the present invention may also contain multiple copies of a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase. Example 1 shows that increasing the copy number of α-1,3-fucosyltransferase alters the proportion of HMOs produced. Specifically, studies have shown that increasing the copy number of Bgall1 by introducing two genomic copies results in an increase in 3FL production and a substantial decrease in LNDFH-III production.

[0276] Thus, copy number variations can be used in production to customize specific HMO mixtures, in which case, depending on the needs of the specific product, the mixture contains different proportions of 3FL, LNFP-III, LNFP-VI, and / or LNDFH-III.

[0277] Thus, in an embodiment, the genetically engineered cell herein contains one, two, three, or more genomic copies of a recombinant nucleic acid sequence encoding a glycosyltransferase selected from Osc1, Bgall1, Bbac1, Murba1, Bacfin1, Prev1, Csec1, CafC, FutA_mut2, FucT109, which consists of or is composed of the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0278] In one embodiment, the genetically engineered cell contains two, three, or more genomic copies and / or plasmid-borne copies of a recombinant nucleic acid sequence encoding a FutA variant that contains substitutions at positions 128 and 129 of SEQ ID NO: 11, wherein the variant has at least 80% but less than 100% identity with SEQ ID NO: 11.

[0279] In a further embodiment, the plasmid is a high-copy number plasmid, preferably the pUC57 or pBB-B9 plasmid.

[0280] One embodiment of the present invention relates to a nucleic acid construct comprising a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase, wherein the recombinant nucleic acid sequence is selected from a) Osc1, which comprises the nucleic acid sequence of SEQ ID NO:12 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:12 or consisting thereof; b) Bgall1, which comprises the nucleic acid sequence of SEQ ID NO:13 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:13 or consisting thereof; c) Bbac1, which comprises the nucleic acid sequence of SEQ ID NO:14 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:14 or consisting thereof; d) Murba1, which comprises the nucleic acid sequence of SEQ ID NO:15 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:15 or consisting thereof; e) Bacfin1, which comprises the nucleic acid sequence of SEQ ID NO:16 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:16 or consisting thereof; f) Prev1, which comprises the nucleic acid sequence of SEQ ID NO:17 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:17 or consisting thereof; g) Csec1, which comprises the nucleic acid sequence of SEQ ID NO:18 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:18 or consisting thereof; h) CafC, which comprises the nucleic acid sequence of SEQ ID NO:19 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:19 or consisting thereof; i) FutA_mut2, which comprises the nucleic acid sequence of SEQ ID NO:20 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:20 or consisting thereof;and / or j) FucT109, comprising or consisting of a nucleic acid sequence of SEQ ID NO:21 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO:21;

[0281] Preferably, the α-1,3-fucosyltransferase coding sequence is under the control of a promoter sequence selected from the promoter sequences having the nucleic acid sequences identified in Table 4.

[0282] Table 4 - Selected promoter sequences

[0283]

[0284]

[0285] * Promoter activity was evaluated in the LacZ assay described below, where the PglpF promoter served as a positive reference in the same assay. To compare different assays, the activity relative to the PglpF promoter was calculated, and the range indicates the results from multiple assays.

[0286] The promoter can be of heterologous origin, the native promoter of a genetically modified cell, or can be a recombinant promoter combining heterologous and / or native elements.

[0287] One way to increase product yield can be to regulate the production of the desired enzyme activity for product production, such as glycosyltransferases or enzymes involved in the biosynthetic pathway of glycosyl donors.

[0288] Increasing the promoter strength that drives the expression of the desired enzyme may be a way to achieve this goal. The strength of the promoter can be evaluated using the lacZ enzyme assay, in which β-galactosidase activity is assayed as described previously (see Miller J.H. Experiments in molecular genetics, Cold spring Harbor Laboratory Press, NY, 1972). Briefly, cells are diluted in Z buffer and permeabilized with sodium dodecyl sulfate (0.1%) and chloroform. The LacZ assay is performed at 30 °C. The samples are pre-warmed, the assay is initiated by adding 200 μl of o-nitrophenyl-β-galactosidase (4 mg / ml), and the assay is stopped by adding 500 μl of 1 M Na2CO3 when the sample turns slightly yellow. The release of o-nitrophenol is then determined as the change in optical density at 420 nm. The specific activity is reported in Miller units (MU) [A420 / (min*ml*A600)]. Regulatory elements with an activity higher than 10,000 MU are considered strong, those with an activity lower than 3,000 MU are considered weak, and those in between have medium strength. An example of a strong regulatory element is the PglpF promoter, which has an activity of approximately 14,000 MU, and an example of a weak promoter is Plac, which has an activity of approximately 2300 MU when induced with IPTG. In a preferred embodiment, the expression of the nucleic acid sequence is under the control of a strong promoter selected from SEQ ID NO 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 and 38.

[0289] In an embodiment, the expression of the nucleic acid sequence described herein is under the control of: the PglpF (SEQ ID NO:39) or Plac (SEQ ID NO:48) promoter or PmglB_UTR70 (SEQ ID NO:36) or PglpA_70UTR (SEQ ID NO:37) or PglpT_70UTR (SEQ ID NO:38) or variants thereof, such as the promoters identified in Table 4, in particular the PglpF_SD4 variant of SEQ ID NO:34 or the Plac_70UTR variant of SEQ ID NO:30, or the PmglB_70UTR variants of SEQ ID NO:27, 28, 29, 31, 32, 33, 35 and 36. Other suitable variants of the PglpF, PglpA_70UTR, PglpT_70UTR and PmglB_70UTR promoter sequences are described in WO2019 / 123324 and WO2020 / 255054 (incorporated herein by reference).

[0290] In a preferred embodiment, the recombinant nucleic acid sequence is individually under the control of one or more promoters selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NO: 39, 48, 36, 37 and 38, respectively) and variants thereof.

[0291] Integration of the nucleic acid construct of interest contained in the construct (expression cassette) into the bacterial genome can be achieved by conventional methods, such as by using a linear cassette containing flanking sequences homologous to specific sites on the chromosome, as described for the attTn7-site (Waddell C.S. and Craig N.L., Genes Dev. (1988) Feb; 2(2):137-49.); methods for genomic integration of nucleic acid sequences, in which recombination is mediated by the Red recombinase function of bacteriophage λ or the RecE / RecT recombinase function of the Rac prophage (Murphy, J Bacteriol. (1998); 180(8):2063-7; Zhang et al., Nature Genetics (1998) 20:123-128; Muyrers et al., EMBO Rep. (2000) 1(3):239-243); methods based on Red / ET recombination (Wenzel et al., Chem Biol. (2005), 12(3):349-56; Vetcher et al., ApplEnviron Microbiol. (2005); 71(4):1829-35); or positive clones, i.e., clones carrying the expression cassette, can be selected, for example, by loss or gain of a marker gene or gene function.

[0292] In one or more exemplary embodiments, the invention relates to one or more recombinant nucleic acid sequences shown in SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 [nucleic acid sequences encoding Osc1, Bgall1, Bbac1, Murba1, Bacfin1, Prev1, Csec1, CafC, FutA_mut2 and FucT109].

[0293] In particular, the present invention relates to one or more recombinant nucleic acid sequences and / or their functional homologs, which have at least 70% identity, such as at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least, at least 95% identity, at least 98% identity, or 100% identity, with SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 [nucleic acids encoding Osc1, Bgall1, Bac1, Murba1, Bacfin1, Prev1, Csec1, CafC, FutA_mut2 and FucT109, respectively].

[0294] Sequence identity

[0295] The term "sequence identity" as used herein describes the relatedness between two amino acid sequences or between two nucleotide sequences based on their paired comparison, i.e., a candidate sequence (e.g., a sequence of the present invention) and a reference sequence (e.g., a prior art sequence). For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), which is implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or higher (available at https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30BLOSUM62) substitution matrix. The output of Needle labeled "identity" (obtained using the -nobrief option) is used as the percentage identity. Generally, sequence identity can be calculated as follows: (number of identical residues x 100) / (length of the alignment region).

[0296] For the purposes of the present invention, sequence identity between two nucleotide sequences was determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or higher. The parameters used were a gap open penalty of 10, a gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "identity" (obtained using the -nobrief option) was used as the percentage identity. Generally sequence identity can be calculated as follows: (identical deoxyribonucleotides x 100) / (length of the alignment region).

[0297] Functional homolog

[0298] A functional homolog or functional variant of a protein / nucleic acid sequence as described herein is a protein / nucleic acid sequence with an altered genetic code that retains its original function. Functional homologs can be obtained by mutagenesis or can be naturally occurring variants from the same or other species. Compared to the function of a protein / nucleic acid sequence, a functional homolog should have at least 50%, such as at least 60%, 70%, 80%, 90% or 100% of the remaining function.

[0299] A functional homolog of any of the disclosed amino acid or nucleic acid sequences may also have higher functionality. A functional homolog of any of the amino acid sequences shown in Table 1 or a recombinant nucleic acid encoding any of the sequences of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 should desirably be capable of participating in the production of fucosylated HMOs in terms of increased HMO production, output of HMO products from cells or input of substrates for HMO production, such as acceptor oligosaccharides of at least three monosaccharide units, improved purity / by-product formation, reduced biomass formation, viability of the genetically engineered cells, robustness of the genetically engineered cells according to the present invention or reduced consumption goods required for production.

[0300] Use of genetically engineered cells or enzymes

[0301] The present invention also relates to any commercial use of the enzymes, genetically engineered cells or nucleic acid constructs disclosed herein, such as but not limited to in a method for producing one or more fucosylated human milk oligosaccharides (HMOs), preferably LNDFH-III.

[0302] Accordingly, the present invention also relates to the use of an α-1,3-fucosyltransferase in the preparation of a fucosylated product comprising LNDFH-III, wherein the α-1,3-fucosyltransferase is selected from Osc1, Bgall1, Bbac1, Murba1, Bacfin1, Prev1, Csec1, CafC, FutA_mut2 and FucT109 comprising or consisting of the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a further embodiment, the α-1,3-fucosyltransferase for producing the fucosylated product is a FutA variant which comprises substitutions at positions 128 and 129 of SEQ ID NO: 11, wherein the variant has at least 80% but less than 100% identity with SEQ ID NO: 11.

[0303] In a further embodiment, the α-1,3-fucosyltransferase for producing the fucosylated product is selected from a) Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1, b) Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 2, c) Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 3, wherein the fucosylated product comprises LNDFH-III and has low levels of LNFP-III and LNFP-VI, such as each less than 10% of the total HMO. Preferably, the α-1,3-fucosyltransferase produces an HMO mixture of LNDFH-III and 3FL, wherein these two HMOs account for at least 80%, such as at least 90%, of the total HMO produced.

[0304] In a further embodiment, the α-1,3-fucosyltransferase for producing a fucosylated product is selected from a) Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:2, b) Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:5, c) Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:4, and d) a FutA variant comprising substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80% sequence identity with SEQ ID NO:11, wherein the fucosylated product comprises LNFP-III and LNDFH-III, and wherein LNFP-VI accounts for less than 1% of the total HMOs produced.

[0305] In a further embodiment, the α-1,3-fucosyltransferase for producing a fucosylated product is selected from a) Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:3, b) Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:6, c) FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:10, and d) Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:7, wherein the fucosylated product comprises LNDFH-III, LNFP-III and LNFP-VI.

[0306] In an embodiment, the α-1,3-fucosyltransferase described herein is also used to make a fucosylated product, wherein the fucosylated product comprises one or more fucosylated oligosaccharides, including LNDFH-III.

[0307] In an exemplary embodiment, the genetically engineered cells and / or nucleic acid constructs described herein are used to manufacture HMOs. Preferably, in the manufacture of an HMO mixture, the molar percentage content of LNDFH-III produced by the genetically engineered cells is higher than 20% of the total amount of HMOs produced. Preferably, in the manufacture of HMOs, a fucosylated HMO mixture containing LNDFH-III is used as the main product, and the molar percentage content of LNDFH-III produced by the genetically engineered cells is higher than 20% of the total amount of HMOs produced, such as higher than 25%, such as higher than 30%, such as higher than 35%, such as higher than 40%, such as higher than 45%, such as higher than 50%, such as higher than 55%, such as higher than 60%, or such as higher than 65%.

[0308] In an embodiment, the α-1,3-fucosyltransferase described herein is also used to manufacture a fucosylated product, wherein the fucosylated product is one or more fucosylated oligosaccharides, such as one or more HMOs, preferably an HMO mixture, wherein at least 60% of the mixture consists of LNDFH-III in combination with LNFP-III, LNFP-VI, and / or 3FL.

[0309] In an exemplary embodiment, the genetically engineered cells and / or nucleic acid constructs according to the invention are used to manufacture one or more fucosylated HMOs, preferably LNDFH-III.

[0310] The production of these HMOs may require the presence of two or more glycosyltransferase activities.

[0311] Method for producing fucosylated human milk oligosaccharides (HMOs)

[0312] The invention also relates to a method for producing one or more fucosylated human milk oligosaccharides (HMOs), preferably LNDFH-III, the method comprising culturing the genetically engineered cells according to the invention.

[0313] The invention relates to a method for producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing genetically engineered cells, the cells comprising:

[0314] A recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase, wherein the fucosyltransferase is selected from:

[0315] a. Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:1,

[0316] b. Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:2,

[0317] c. Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:3,

[0318] d. Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:4,

[0319] e. Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:5,

[0320] f. Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:6, and

[0321] g. Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:7.

[0322] In an embodiment, the genetically engineered cells are cultured in a suitable medium providing a suitable carbon source and in the presence of an initial substrate selected from lactose or LNT-II. Preferably, the initial substrate is lactose. Another embodiment is a method for producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing genetically engineered cells that comprise

[0323] a. a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase, wherein the enzyme is selected from:

[0324] i. Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:1,

[0325] ii. Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:2,

[0326] iii. Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:3,

[0327] iv. Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:4,

[0328] v. Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:5,

[0329] vi. Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:6,

[0330] vii. Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:7,

[0331] viii. CafC comprising or consisting of the amino acid sequence of SEQ ID NO:8, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:8,

[0332] ix. Fut_mut2 comprising or consisting of the amino acid sequence of SEQ ID NO:9, x. FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:10, and

[0333] xi. A FutA variant comprising substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80% sequence identity with SEQ ID NO:11, and

[0334] b. A recombinant nucleic acid sequence encoding an enzyme having β-1,4-galactosyltransferase activity, and

[0335] c. Optionally, a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetyl-glucosaminyltransferase activity, and

[0336] d. Culturing the cells in a suitable medium in the presence of an initial substrate, and

[0337] At least one of the fucosylated HMOs is LNDFH-III. The fucosylated HMOs prepared by the above method may further include fucosylated HMOs selected from 3FL, LNFP-III, and LNFP-VI, and may also include non-fucosylated HMOs such as LNnT.

[0338] Another embodiment relates to a method for producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing genetically engineered cells that comprise

[0339] a. a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase, wherein the enzyme is selected from:

[0340] i) Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:1, ii) Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:2,

[0341] iii) Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:3,

[0342] iv) Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:4,

[0343] v) Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:5,

[0344] vi) Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:6, and

[0345] vii) Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:7,

[0346] viii) CafC comprising or consisting of the amino acid sequence of SEQ ID NO:8, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:8,

[0347] ix) FutA_mut2 comprising or consisting of the amino acid sequence of SEQ ID NO:9, and

[0348] b. A recombinant nucleic acid sequence encoding an enzyme having β-1,4-galactosyltransferase activity;

[0349] c. Optionally, a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0350] d. Culturing said cells in a suitable medium in the presence of an initial substrate, and

[0351] wherein at least 25%, such as at least 28%, of the molar content of the HMO prepared by this method is LNDFH-III.

[0352] In an embodiment, the initial substrate is selected from lactose or LNT-II. If the initial substrate is LNT-II, the cells express an enzyme having β-1,3-N-acetylglucosaminyltransferase activity. Preferably, the initial substrate is lactose and the cells express an enzyme having β-1,3-N-acetylglucosaminyltransferase activity and an enzyme having β-1,4-galactosyltransferase activity.

[0353] Another embodiment of the invention relates to a method for producing LNDFH-III and one or more additional HMOs, which comprises

[0354] a. Providing a genetically engineered cell capable of producing LNDFH-III, which comprises a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having dual α-1,3-fucosyltransferase specificity, which transferase is capable of fucosylating an oligosaccharide at the GlcNAc moiety and the Glu moiety, wherein the glycosyltransferase is selected from:

[0355] i. Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:7,

[0356] ii. CafC comprising or consisting of the amino acid sequence of SEQ ID NO:8, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:8,

[0357] iii. a FutA variant comprising a substitution at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein the variant has at least 80% but less than 100% identity with SEQ ID NO:11, and

[0358] iv. FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof, having an amino acid sequence with at least 80% identity to SEQ ID NO:10,

[0359] wherein the cell further comprises one or more recombinant nucleic acid sequences encoding a β-1,3-N-acetylglucosaminyltransferase and / or a β-1,4-galactosyltransferase, and

[0360] b. culturing the cell in a suitable medium, and

[0361] wherein at least 20 mol%, such as at least 25 mol%, of the total HMO produced is LNDFH-III, and less than 45 mol% of the total HMO produced is LNFP-VI.

[0362] In an embodiment, the fucosylated HMO is LNDFH-III. In a further embodiment, one or more HMOs selected from 3FL, LNFP-III, LNFP-VI, LNDFH-III, LNnT, LNT-II, and pLNnH are prepared by the method of the invention.

[0363] The method comprises culturing a genetically engineered cell that produces a fucosylated HMO, and further comprises culturing the genetically engineered cell in the presence of a carbon source (energy source) selected from glucose, sucrose, fructose, xylose, and glycerol.

[0364] The method particularly comprises culturing a genetically engineered cell that produces a fucosylated HMO, wherein the content of LNDFH-III produced by the cell is at least 25%, such as at least 28%, of the total HMO content produced by the cell.

[0365] The method particularly comprises culturing a genetically engineered cell that produces a fucosylated HMO, wherein at least 60 mol%, such as at least 70 mol%, 75 mol%, 80 mol%, 85 mol%, 90 mol%, 93 mol%, 95 mol%, 96 mol%, 97 mol%, or such as at least 98 mol%, of the total molar content of HMO produced by the cell is a fucosylated HMO.

[0366] The method particularly includes culturing genetically engineered cells that produce fucosylated HMOs, wherein at least 50 mol%, such as at least 54 mol%, 60 mol%, 70 mol%, 75 mol%, 80 mol%, 85 mol%, 90 mol%, or for example at least 94 mol%, or for example between 50 mol% and 90 mol%, or for example between 70 mol% and 94 mol% of the total HMOs produced by the cells according to the invention is a mixture of LNDFH-III and 3FL. In one embodiment, the fucosylated HMOs produced are mainly LNDFH-III and 3FL, and the sum of other fucosylated HMOs is less than 15%, such as less than 10%, of the total molar content of the HMOs produced.

[0367] The method particularly includes culturing genetically engineered cells that produce fucosylated HMOs, wherein at least 50 mol%, such as at least 54 mol%, 60 mol%, 70 mol%, 75 mol%, 80 mol%, 85 mol%, 90 mol% or for example at least 94 mol%, or for example between 50 mol% and 90 mol%, or for example between 70 mol% and 94 mol% of the total HMOs produced by the cells according to the invention is a mixture of LNDFH-III and LNFP-III.

[0368] The method particularly includes culturing genetically engineered cells that produce fucosylated HMOs, wherein at least 60 mol%, such as at least 65 mol%, 70 mol%, 75 mol%, 80 mol%, 85 mol% or for example at least 89 mol%, or for example between 60 mol% and 90 mol% of the total HMOs produced by the cells according to the invention is a mixture of LNDFH-III and LNFP-VI.

[0369] The method particularly includes culturing genetically engineered cells that produce fucosylated HMOs, wherein at least 55 mol%, such as at least 59 mol%, 65 mol%, 68 mol%, 70 mol%, 75 mol%, 70 mol% or such as at least 85 mol%, or such as between 59 mol% and 86 mol% of the total HMOs produced by the cells according to the invention is a mixture of LNDFH-III, LNFP-III and LNFP-VI.

[0370] In an embodiment, an HMO mixture is produced by the method according to the invention, wherein the produced HMO mixture is substantially free of LNFP-III.

[0371] In an embodiment, an HMO mixture is produced by the method according to the invention, wherein the produced HMO mixture is substantially free of LNFP-VI.

[0372] In an embodiment, an HMO mixture is produced by the method according to the invention, wherein the produced HMO mixture is substantially free of LNnT.

[0373] In an embodiment, an HMO mixture is produced by the method according to the invention, wherein the produced HMO mixture is substantially free of LNT-II.

[0374] In an embodiment, an HMO mixture is produced by the method according to the invention, wherein the produced HMO mixture is substantially free of 3FL.

[0375] The HMO mixtures prepared by the methods disclosed herein can be described by their ratios in the HMO mixture. The "ratio" as described herein shall be understood as the ratio between two HMO amounts, for example but not limited to the amount of one HMO divided by the amount of another HMO, i.e., the ratio of LNDFH-III:LNFP-III is 2:1, indicating that LNDFH-III in the mixture is 2 times that of LNFP-III.

[0376] In an embodiment, an HMO mixture having a ratio of LNDFH-III:LNFP-III from 1:1 to 3:1 is produced by the method according to the invention. In an embodiment, an HMO mixture having a ratio of LNDFH-III:LNFP-VI higher than 1.3, such as higher than 1.5, such as higher than 5, such as higher than 50, such as higher than 100, such as higher than 200 is produced by the method according to the invention. In one embodiment, an HMO mixture is produced by the method according to the invention, which is selected from i) LNDFH-III and 3FL, ii) LNDFH-III, LNFP-III and 3FL, iii) LNDFH-III, LNFP-VI and 3FL, iv) LNDFH-III, LNFP-III, 3FL and LNnT, v) LNDFH-III, LNFP-III, LNFP-VI and 3FL, and vi) LNDFH-III, LNFP-III, LNFP-VI, 3FL and LNnT.

[0377] The methods described herein include providing a glycosyl donor, which is synthesized alone by one or more genetically engineered cells and / or added exogenously to the culture medium from an alternative source. Preferably, the glucosyl donor is produced by an endogenous or recombinant de novo pathway in the genetically engineered cells.

[0378] In one aspect, the method described herein further includes providing a receptor sugar as an initial substrate for HMO formation, the receptor sugar comprising at least two monosaccharide units, which is added exogenously to the culture medium and / or produced by separate microbial fermentation. As an alternative to adding an initial substrate for HMO production to the fermentation medium, the genetically modified cells can be further engineered to produce the initial substrate intracellularly (see, for example, WO2015 / 150328).

[0379] In one aspect, the method described herein includes providing a receptor sugar, which comprises at least two monosaccharide units, selected from lactose, LNT-II, and LNnT, and adding it before and / or during the genetically modified cell culture. In a preferred embodiment, the initial substrate for HMO formation is lactose, which is added to the culture during the fermentation of the genetically engineered cells.

[0380] Recover fucosylated human milk oligosaccharides (HMOs) from the culture medium and / or the genetically engineered cells.

[0381] Culturing / fermentation

[0382] Culturing (culturing, cultivating) or fermenting (fermenting, fermentation) (used interchangeably herein) in a controlled bioreactor generally includes (a) a first stage of exponential cell growth in a culture medium ensured by a carbon source, and (b) a second stage of cell growth in a culture medium operating under carbon limitation, where the carbon source is continuously added together with a receptor oligosaccharide (such as lactose), thereby allowing the formation of HMO products at this stage. Carbon (sugar) limitation refers to the stage in fermentation where the growth rate is kinetically controlled by the concentration of the carbon source (sugar) in the culture broth, and the concentration of the carbon source (sugar) is in turn determined by the carbon addition rate (sugar feed rate) to the fermenter.

[0383] The terms "manufacturing" or "manufacturing scale" or "large-scale production" or "large-scale fermentation" are used interchangeably and, in the context of the present invention, define a fermentation with a minimum volume of 100 L, such as 1000 L, such as 10,000 L, 100,000 L, 200,000 L of culture medium. Generally, the "manufacturing scale" process is defined as being able to process the target HMO product in large volume yields, e.g., in the case of a therapeutic compound or composition, to meet the requirements of toxicity testing, clinical trials, and market supply. In addition to large volume, different from simple laboratory-scale methods such as shake flask culture, the manufacturing scale method is characterized by a technical system using a bioreactor (fermenter) equipped with devices for agitation, aeration, nutrient feeding, and monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.). To a large extent, the behavior of the expression system in laboratory-scale methods, such as shake flasks, bench-top bioreactors, or the deep well format described in the examples of the present invention, does allow prediction of the behavior of the system in the complex environment of a bioreactor.

[0384] There is no limitation regarding the suitable cell culture medium used in the fermentation process. The culture medium can be semi-defined, i.e., containing complex culture medium compounds (such as yeast extract, soy peptone, casein amino acids, etc.), or it can be chemically defined without any complex compounds. The carbon source can be selected from glucose, sucrose, fructose, xylose, and glycerol. In one or more exemplary embodiments, the culture medium is supplemented with one or more energy sources and carbon sources selected from glycerol, sucrose, and glucose. In additional embodiments, lactose is added as a substrate for HMO formation during the cultivation of genetically engineered cells.

[0385] In one or more exemplary embodiments, the culture medium contains sucrose as the sole carbon and energy source. In one or more exemplary embodiments, the genetically engineered cells contain one or more heterologous nucleic acid sequences encoding one or more heterologous polypeptides capable of utilizing sucrose as the sole carbon and energy source for the genetically engineered cells.

[0386] In one or more exemplary embodiments, the genetically engineered cells contain a PTS-dependent sucrose utilization system, which also contains the scrYA and scrBR operons as described in WO2015 / 197082 (incorporated herein by reference).

[0387] After carrying out the method of the present invention, the produced fucosylated HMO can be collected from the cell culture or fermentation broth in a conventional manner.

[0388] Recovery / Harvest

[0389] Recovering fucosylated human milk oligosaccharides (HMOs) from the culture medium and / or genetically engineered cells. In this context, the term "recover" is used interchangeably with the term "harvest". Both "recover" and "harvest" in the context refer to collecting the produced HMOs from the culture / culture broth after fermentation termination. In one or more exemplary embodiments, it may include collecting HMOs included in both the biomass (i.e., host cells) and the culture medium, i.e., before / without separating the fermentation broth from the biomass. In other embodiments, the produced HMOs can be collected separately from the biomass and the fermentation broth, i.e., after separating the biomass from the culture medium (i.e., fermentation broth).

[0390] Separation of cells from the culture medium can be carried out by any method known to those skilled in the art, such as any suitable type of centrifugation or filtration. Separation of cells from the culture medium can be carried out immediately after harvesting the fermentation broth, or at a later stage after storing the fermentation broth under appropriate conditions. Recovering the produced HMOs from the remaining biomass (or total fermentation broth) includes extracting HMOs from the biomass (i.e., production cells).

[0391] After recovery from the fermentation product, the HMOs can be used for further processing and purification.

[0392] The HMOs can be purified according to procedures known in the art, such as those described in WO2017 / 152918, WO2017 / 182965 or WO2015 / 188834, where the latter describes the purification of fucosylated HMOs. The purified HMOs can be used as nutritional products, drugs or for any other purpose, such as for research.

[0393] At the end of the culture, the oligosaccharides can accumulate as products inside and outside the cell matrix.

[0394] The method according to the invention includes culturing genetically engineered microbial cells in a culture medium designed to support the growth of the microorganisms and containing one or more carbohydrate sources or only a carbon source, such as selected from glucose, sucrose, fructose, xylose and glycerol. In one or more exemplary embodiments, the culture medium is supplemented with one or more energy and carbon sources selected from glycerol, sucrose and glucose.

[0395] Manufactured product

[0396] The term "manufactured product" refers to one or more HMOs, i.e., one or more product HMOs, or the composition of a mixture of HMOs. Preferably, the product HMO or composition is produced using the genetically engineered cells described herein by the methods described herein.

[0397] Accordingly, one embodiment of the invention relates to a mixture of HMOs consisting essentially of the following components:

[0398] a. LNDFH-III and 3FL, or

[0399] b. LNDFH-III, LNFP-III and 3FL, or

[0400] c. LNDFH-III, LNFP-VI and 3FL, or

[0401] d. LNDFH-III, LNFP-III, 3FL and LNnT, or

[0402] e. LNDFH-III, LNFP-III, LNFP-VI, 3FL and LNnT.

[0403] One embodiment relates to an HMO composition that consists essentially of 20 - 70 mol% of LNDFH-III, 0 - 35 mol% of LNFP-III, 0 - 35 mol% of LNDFP-VI, 0 - 65 mol% of 3FL, 0 - 40% of LNnT, and up to 1% of pLNnH, with a total of 100% molar content.

[0404] Another embodiment relates to an HMO composition that consists essentially of 35 - 60 mol% of LNDFH-III and 40 - 65 mol% of 3FL, with a total of 100% molar content.

[0405] Another embodiment relates to an HMO composition that consists essentially of 40 - 50 mol% of LNDFH-III, 25 - 35 mol% of LNFP-VI, 10 - 20 mol% of LNnT, and 5 - 15 mol% of 3FL, with a total of 100% molar content.

[0406] Another embodiment relates to an HMO composition that consists essentially of 25 - 35 mol% of LNDFH-III, 15 - 25 mol% of LNFP-VI, 8 - 18 mol% of LNFP-III, and 33 - 43 mol% of LNnT, with a total of 100% molar content.

[0407] Another embodiment relates to an HMO composition that consists essentially of 40 - 50 mol% of LNDFH-III, 5 - 15 mol% of LNFP-VI, 3 - 8 mol% of LNFP-III, 2 - 12 mol% of LNnT, and 30 - 40 mol% of 3FL, with a total of 100% molar content.

[0408] Another embodiment relates to an HMO composition that consists essentially of 55 - 65 mol% of LNDFH-III, 17 - 27 mol% of LNFP-III, 6 - 16 mol% of LNnT, and 1 - 11 mol% of 3FL, totaling 100 mol%. In a preferred embodiment, the HMO mixture consists of 60 mol% of LNDFH-I, 20 mol% of LNFP-III, 10 mol% of LNnT, and 10 mol% of 3FL.

[0409] Another embodiment relates to an HMO composition that consists essentially of 36 - 46 mol% of LNDFH-III, 25 - 35 mol% of LNFP-VI, 10 - 20 mol% of LNFP-III, 2 - 12 mol% of LNnT, and 2 - 12 mol% of 3FL, totaling 100 mol%.

[0410] Another embodiment relates to an HMO composition that consists essentially of 50 - 70 mol% of LNDFH-III, 1 - 13 mol% of LNFP-III, and 20 - 50% of 3FL, totaling 100 mol%. In a preferred embodiment, the HMO mixture consists of 70 mol% of LNDFH-I, 10 mol% of LNFP-III, and 20 mol% of 3FL.

[0411] Another embodiment relates to an HMO composition that consists essentially of 25 - 35 mol% of LNDFH-III, 29 - 39 mol% of LNFP-VI, and 31 - 41% of 3FL, totaling 100 mol%.

[0412] Another embodiment relates to an HMO composition that consists essentially of 36 - 46 mol% of LNDFH-III, 10 - 40 mol% of LNFP-III, 2 - 20 mol% of LNnT, and 2 - 15 mol% of 3FL, totaling 100 mol%. In a preferred embodiment, the HMO mixture consists of 45 mol% of LNDFH-I, 30 mol% of LNFP-III, 15 mol% of LNnT, and 10 mol% of 3FL.

[0413] Another embodiment relates to an HMO composition that consists essentially of 37 - 47 mol% of LNDFH-III, 26 - 36 mol% of LNFP-III, 19 - 29 mol% of 3FL, and less than 5% of LNnT, totaling 100 mol%.

[0414] Another embodiment relates to an HMO composition consisting essentially of 19 - 29 mol% LNDFH-III, 13 - 23 mol% LNFP-VI, 22 - 32 mol% LNFP-III and 24 - 34 mol% LNnT and less than 5 mol% 3FL, totaling 100% molar content.

[0415] In a further embodiment, the composition or mixture consists essentially of fucosylated HMOs, i.e., at least 80 mol%, such as at least 90 mol%, such as at least 95 mol%, for example at least 98 mol% of the total HMOs in the composition are fucosylated.

[0416] In an embodiment, the fucosylated composition or mixture consists essentially of LNFP-III, LNFP-VI and / or 3FL in addition to LNDFH-III.

[0417] Advantageously, the methods disclosed herein provide valuable mixtures of HMOs with highly fucosylated HMOs including the complex HMO LNDFH-III. Some of the genetically engineered cells described herein produce sufficient LNDFH-III, e.g., above 20% of the total HMO, to facilitate its purification from the HMO mixtures produced by culture. Genetically engineered cells producing LNFP-III and / or LNFP-VI at less than 10% of the total HMO molar% are particularly suitable for purifying LNDFH-III.

[0418] The manufactured product can be a powder, composition, suspension or gel containing one or more HMOs.

[0419] Use of HMO composition or mixture

[0420] HMOs are naturally present in breast milk. After thousands of years of evolution, HMO research (clinical and preclinical) now shows that supplementing with the correct levels of specific HMOs can provide unique health benefits. Since fucosylated HMOs account for more than 60% of the total HMOs in human milk, mixtures with a high content of fucosylated HMOs are more desirable.

[0421] Therefore, LNDFH-III and HMO mixtures containing LNDFH-III are highly relevant as nutritional supplements or therapeutic agents.

[0422] Clinical data in infants show that human milk oligosaccharide supplements may help cultivate the desired microbiota by serving as a food source for beneficial gut bacteria. In particular, human milk oligosaccharide supplements may help support immunity and gut health and have a potential role in cognitive development, which may open up future innovation opportunities.

[0423] One aspect of the present invention relates to the use of the mixtures or compositions disclosed herein in infant nutritionals.

[0424] The present invention also relates to the use of the mixtures or compositions disclosed herein as dietary supplements or medical nutraceuticals or pharmaceutical compositions.

[0425] The HMO mixture or composition can be used to enhance beneficial bacteria in the gut microbiome. Beneficial bacteria are, for example, Bifidobacterium sp., Lactobacillus sp. or Barnesiella sp. The enhancement of beneficial bacteria can in turn lead to an increased production of short-chain fatty acids (SCFAs) such as acetate, propionate and butyrate, which have been shown to have many benefits in infants and toddlers, such as inhibiting pathogenic bacteria, preventing infections and diarrhea, and reducing the risk of allergies and metabolic disorders (see, for example, WO2006 / 130205, WO2017 / 129644, WO2017 / 129649).

[0426] The HMO mixture or composition produced according to the method described herein can be used to reduce the abundance of unwanted viruses and bacteria in the gut microbiome. Examples of pathogenic bacteria and viruses that can be reduced by the HMO mixture described herein include Candida albicans, Clostridium difficile, Enterococcus faecium, Escherichia coli, Helicobacter pylori, Streptococcus agalactiae, Shigella dysenteriae, Staphylococcus aureus, norovirus and rotavirus. Each composition described herein can also be used to treat and / or reduce the risk of a wide range of bacterial infections in humans.

[0427] The HMO mixtures or compositions produced according to the methods described herein can be used to increase the regeneration and viability of lyophilized probiotics (including probiotics of the genera Bifidobacterium and Lactobacillus), in particular to increase the regeneration and / or viability and / or shelf life in an acidic environment (such as the stomach or acidic foods), which is an advantage of using the HMO mixtures described herein. Examples of Bifidobacterium that may have increased regeneration and viability are Bifidobacterium animalis lactis BB12 DSM 32269, Bifidobacterium animalis BIF6, Bifidobacterium longum DSM 32946, Bifidobacterium longum BB536, Bifidobacterium bifidum DSMZ 32403, Bifidobacterium infantis, Bifidobacterium breve DSM 33789, Bifidobacterium infantis SP37 DSM 32687, Bifidobacterium adolescentis DSM 34065, and / or Bifidobacterium animalis ssp. animalis DSM 16284. Examples of Lactobacillus that may have increased regeneration and viability are Lactobacillus rhamnosus GG DSM 32550, Lactobacillus rhamnosus 19070-2 DSM 26357, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus LBrGG ATCC53103, Lactobacillus DSM 33156, Lactobacillus reuteri DSM 12246, Lactobacillus plantarum TIFN101, Lactobacillus gasseri Lg-36 200B FloraFit Danisco, Lactobacillus casei DSM 32382, Lactobacillus paracasei, Lactobacillus paracasei L26-CBS116412, Lactobacillus plantarum PS128, Lactobacillus plantarum (Sacco) DSM32383, Lactococcus lactis PAREVE, and / or Limosilactobacillus reuteri S12 DSM 33752.

[0428] In the context of the present application, "regeneration" means the process of regaining / restoring the viability of dried bacteria (i.e., "regenerating" the bacterial cells by rehydration, where "rehydration" means restoring fluid). This process is sometimes also referred to as "reconstitution".

[0429] In the context of the present application, "viability" is the ability of bacterial cells to survive and function as living cells. One way to determine the viability of bacterial cells is by spreading them on an agar plate with a suitable growth medium and counting the number of colonies formed after incubation for a predetermined time (plate count). Alternatively, FACS analysis can be used.

[0430] In the context of the present application, "improving the regeneration of Bifidobacterium and / or Lactobacillus bacteria" means an increase in the amount (quantity) of Bifidobacterium and / or Lactobacillus bacteria, and the bacteria successfully regenerate / recover, compared to the corresponding control (i.e., the amount / quantity of Bifidobacterium and / or Lactobacillus bacteria without the addition of HMO).

[0431] One embodiment of the present invention is to use an HMO composition consisting essentially of one of the following mixtures to regenerate or revive probiotic strains of Bifidobacterium and / or Lactobacillus:

[0432] a. LNDFH-III and 3FL, or

[0433] b. LNDFH-III, LNFP-III and 3FL, or

[0434] c. LNDFH-III, LNFP-III, 3FL and LNnT, or

[0435] Preferably, the probiotic strains are selected from the genus Lactobacillus rhamnosus, the genus Lactobacillus paracasei and / or the genus Bifidobacterium adolescentis, such as Lactobacillus rhamnosus DSM 33156, Lactobacillus rhamnosus ATCC53103, Lactobacillus paracasei L26-CBS116412 and / or Bifidobacterium adolescentis DSM 34065.

[0436] In the context of the present application, "improving the viability of Bifidobacterium and / or Lactobacillus bacteria" means an increase in the amount (quantity) of viable Bifidobacterium and / or Lactobacillus bacteria, compared to the corresponding control (i.e., the amount / quantity of Bifidobacterium and / or Lactobacillus bacteria without the addition of HMO).

[0437] In the context of the present application, "acidic" means a pH below 7.0 (e.g., pH ≤ 6.0, or ≤ 5.0, or ≤ 4.0, or ≤ 3.0, or in the range of 1.0 - 6.0, such as 2.0 to 5.0). The pH measured in the stomach is in the range of about 1.5 - 3.5. The pH measured in a healthy vagina is in the range of about 3.8 - 5.0. The pH of fruit juice is in the range of about 2.0 - 4.5.

[0438] The HMO mixtures or compositions produced according to the methods described herein or otherwise described herein can be used to extend the shelf life of probiotics (such as Bifidobacterium and / or Lactobacillus).

[0439] Compositions containing a combination of prebiotics (such as HMO) and probiotics are commonly referred to as synbiotics or synbiotic compositions. Preferably, compared to the effects of individual prebiotics and probiotics, synbiotic compositions provide additional, preferably synergistic, effects.

[0440] One embodiment of the present invention is a synbiotic composition comprising one or more probiotics and an HMO mixture, as described herein, particularly in the "manufactured product" section. Preferably, the one or more probiotics are of the genus Bifidobacterium and / or Lactobacillus, such as any of the specific species mentioned above.

[0441] One embodiment of the present invention is a synbiotic composition comprising an HMO mixture substantially composed of one of the following mixtures in combination with a probiotic strain selected from Bifidobacterium and / or Lactobacillus species:

[0442] a. LNDFH-III and 3FL, or

[0443] b. LNDFH-III, LNFP-III and 3FL, or

[0444] c. LNDFH-III, LNFP-III, 3FL and LNnT, or

[0445] Preferably, the probiotic strain is selected from one or more of the genus Lactobacillus rhamnosus, the genus Lactobacillus paracasei and / or the genus Bifidobacterium adolescentis, such as Lactobacillus rhamnosus DSM 33156, Lactobacillus rhamnosus ATCC53103, Lactobacillus paracasei L26-CBS116412 and / or Bifidobacterium adolescentis DSM 34065.

[0446] In another embodiment, the synbiotic composition comprises or consists of a probiotic selected from the genus Lactobacillus rhamnosus, the genus Lactobacillus paracasei and / or the genus Bifidobacterium adolescentis and an HMO mixture, the HMO mixture being substantially composed of 40 - 70 mol% of LNDFH-III, 0 - 35 mol% of LNFP-III, 0 - 35 mol% of LNFP-VI, 5 - 55 mol% of 3FL, 0 - 20% of LNnT and less than 1% of pLNnH, with the total molar content of HMO in the composition reaching 100%.

[0447] In one embodiment, the synbiotic composition comprises or consists of a probiotic selected from the genus Lactobacillus rhamnosus, the genus Lactobacillus paracasei and / or the genus Bifidobacterium adolescentis and an HMO mixture selected from:

[0448] a. A mixture substantially composed of 35 - 60 mol% of LNDFH-III and 40 - 65 mol% of 3FL, with a total molar content of 100%,

[0449] b. A mixture consisting essentially of 50 mol% of LNDFH-III and 50 mol% of 3FL,

[0450] c. A mixture consisting essentially of 40 - 50 mol% of LNDFH-III, 25 - 35 mol% of LNFP-VI, 10 - 20 mol% of LNnT, and 5 - 15 mol% of 3FL, totaling 100% molar content,

[0451] d. A mixture consisting essentially of 25 - 35 mol% of LNDFH-III, 15 - 25 mol% of LNFP-VI, 8 - 18 mol% of LNFP-III, and 33 - 43 mol% of LNnT, totaling 100% molar content,

[0452] e. A mixture consisting essentially of 40 - 50 mol% of LNDFH-III, 5 - 15 mol% of LNFP-VI, 3 - 8 mol% of LNFP-III, 2 - 12 mol% of LNnT, and 30 - 40 mol% of 3FL, totaling 100% molar content,

[0453] f. A mixture consisting essentially of 55 - 65 mol% of LNDFH-III, 17 - 27 mol% of LNFP-III, 6 - 16 mol% of LNnT, and 1 - 11 mol% of 3FL, totaling 100% molar content,

[0454] g. A mixture consisting essentially of 60 mol% of LNDFH-III, 20 mol% of LNFP-III, 10 mol% of LNnT, and 10 mol% of 3FL,

[0455] h. A mixture consisting essentially of 36 - 46 mol% of LNDFH-III, 25 - 35 mol% of LNFP-VI, 10 - 20 mol% of LNFP-III, 2 - 12 mol% of LNnT, and 2 - 12 mol% of 3FL, totaling 100% molar content,

[0456] i. A mixture consisting essentially of 50 - 70 mol% of LNDFH-III, 1 - 13 mol% of LNFP-III, and 20 - 50% of 3FL, totaling 100% molar content,

[0457] j. A mixture consisting essentially of 70 mol% of LNDFH-III, 10 mol% of LNFP-III, and 20 - 50% of 3FL,

[0458] k. A mixture consisting essentially of 36 - 46 mol% of LNDFH-III, 25 - 35 mol% of LNFP-VI, 10 - 20 mol% of LNFP-III, 2 - 12 mol% of LNnT, and 2 - 12 mol% of 3FL, with a total of 100 mol% content,

[0459] l. A mixture consisting essentially of 45 mol% of LNDFH-III, 30 mol% of LNFP-III, 10 mol% of 3FL, and 15% of LNnT,

[0460] m. A mixture consisting essentially of 25 - 35 mol% of LNDFH-III, 29 - 39 mol% of LNFP-VI, and 31 - 41 mol% of 3FL, with a total of 100 mol% content,

[0461] n. A mixture consisting essentially of 37 - 47 mol% of LNDFH-III, 26 - 36 mol% of LNFP-III, 19 - 29 mol% of 3FL, and less than 5% of LNnT, with a total of 100 mol% content, and

[0462] o. A mixture consisting essentially of 19 - 29 mol% of LNDFH-III, 13 - 23 mol% of LNFP-VI, 22 - 32 mol% of LNFP-III, 24 - 34 mol% of LNnT, and less than 5 mol% of 3FL, with a total of 100 mol% content.

[0463] Preferably, the probiotic is selected from the following strains: Lactobacillus rhamnosus 33156, Lactobacillus rhamnosus LBrGG-ATCC53103, Lactobacillus paracasei L26-CBS116412, and / or Bifidobacterium adolescentis DSM34065.

[0464] The HMO mixtures or compositions described herein can be used to improve the flowability of powders or reduce the viscosity of liquids.

[0465] The compositions and mixtures of HMOs described in the "Manufactured Product" section may also form part of compositions containing other parts, such as active pharmaceutical ingredients, food supplements, excipients, surfactants, etc.

[0466] Accordingly, the embodiments described herein relate to the use of compositions containing HMO mixtures produced according to the present invention as dietary supplements or medical nutraceuticals. In a further embodiment, the compositions for infant formula, dietary supplements, or medical nutraceuticals contain LNDFH-III, 3FL, LNFP-III, and LNFP-VI.

[0467] The HMO mixtures or compositions described herein are used in nutritional compositions. Nutritional compositions are, for example, infant formula powders, rehydration solutions or dietary maintenance, medical nutritional products or supplements for the elderly or immunocompromised individuals. Macronutrients such as edible fats, carbohydrates and proteins may also be included in such anti-infection compositions. Edible fats include, for example, coconut oil, soybean oil and glycerol monoesters and glycerol diesters. Carbohydrates include, for example, glucose, edible lactose and hydrolyzed corn starch. Proteins include, for example, soy protein, whey and skim milk. Vitamins and minerals (such as calcium, phosphorus, potassium, sodium, chlorine, magnesium, manganese, iron, copper, zinc, selenium, iodine and vitamins A, E, D, C and B complex) may also be included in such anti-infection compositions.

[0468] In an embodiment, the composition comprising the HMO mixture produced according to the present invention is a pharmaceutical composition.

[0469] The present invention also relates to the use of the mixture or composition according to the present invention as a dietary supplement and / or medical nutritional product.

[0470] In an embodiment, the present invention relates to the use of the mixture or composition according to the present invention in infant nutrition.

[0471] Item

[0472] The following items describe various embodiments of the present invention

[0473] 1. A genetically engineered cell capable of producing LNDFH-III, comprising a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase selected from the following,

[0474] a. Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:1,

[0475] b. Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:2,

[0476] c. Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:3,

[0477] d. Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:4,

[0478] e. Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:5,

[0479] f. Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:6,

[0480] g. Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:7,

[0481] h. CafC comprising or consisting of the amino acid sequence of SEQ ID NO:8, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:8, and

[0482] i. A FutA variant comprising substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein said variant has at least 80% sequence identity with SEQ ID NO:11, and

[0483] wherein said cell further comprises one or more recombinant nucleic acid sequences encoding β-1,4-galactosyltransferase and optionally β-1,3-N-acetylglucosaminyltransferase.

[0484] 2. The genetically engineered cell according to any one of item 1, wherein at least 28% of the molar content of the total HMO produced by said cell is LNDFH-III.

[0485] 3. The genetically engineered cell according to item 1 or 2, wherein said cell further produces one or more HMOs selected from 3FL, LNnT, LNFP-III and LNFP-VI.

[0486] 4. The genetically engineered cell according to any one of the preceding claims, wherein said cell further comprises one or more recombinant nucleic acid sequences encoding β-1,3-N-acetylglucosaminyltransferase and / or β-1,4-galactosyltransferase.

[0487] 5. The genetically engineered cell according to any one of the preceding items, wherein said cell further comprises a substrate importer selected from lactose importer, lacto-N-triose-II (LNT-II) importer or LNnT importer.

[0488] 6. The genetically engineered cell according to any one of the foregoing, wherein the recombinant nucleic acid sequence is respectively under the control of one or more promoters selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR, and PglpT_70UTR (SEQ ID NO: 39, 48, 36, 37, and 38) and variants thereof.

[0489] 7. The genetically engineered cell according to any one of the foregoing, wherein the engineered cell is a microorganism.

[0490] 8. The genetically engineered cell, wherein the engineered cell is a prokaryotic cell or a eukaryotic cell.

[0491] 9. The genetically engineered cell according to item 7 or 8, wherein the engineered cell is a fungal host selected from yeast cells such as Komagataella, Kluyveromyces, Yarrowia, Pichia, Saccaromyces, Schizosaccharomyces, or Hansenula, or a filamentous fungus such as Aspargillus, Fusarium, or Thricoderma.

[0492] 10. The genetically engineered cell according to item 7 or 8, wherein the engineered cell is a bacterial cell selected from the genera Escherichia, Bacillus, Lactobacillus, Corynebacterium, and Campylobacter.

[0493] 11. The genetically engineered cell according to item 7 or 8, wherein the engineered cell is selected from Escherichia coli, Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

[0494] 12. A method for producing one or more fucosylated HMOs, comprising providing and culturing a genetically engineered cell comprising a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase selected from

[0495] a. Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1,

[0496] b. Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 2,

[0497] c. Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:3,

[0498] d. Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:4,

[0499] e. Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:5,

[0500] f. Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:6,

[0501] g. Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:7,

[0502] h. CafC comprising or consisting of the amino acid sequence of SEQ ID NO:8, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:8,

[0503] i. FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:10, and

[0504] j. A FutA variant comprising substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein said variant has at least 80% sequence identity with SEQ ID NO:11, and

[0505] wherein at least one of said fucosylated HMOs is LNDFH-III.

[0506] 13. The method according to item 12, said method comprising providing and culturing a genetically engineered cell according to any one of items 1 to 11.

[0507] 14. The method according to item 12 or 13, wherein the cultivation is carried out in a suitable cell culture medium to produce the one or more fucosylated HMOs, and at least one of the fucosylated HMOs is LNDFH-III.

[0508] 15. The method according to items 12 to 14, wherein the one or more fucosylated HMOs are purified.

[0509] 16. The method according to any one of items 12 to 15, wherein at least 25% of the molar content of the total HMOs produced by the method is LNDFH-III.

[0510] 17. The method according to any one of items 12 to 16, wherein at least one additional fucosylated HMO selected from 3FL, LNFP-III, and LNFP-VI is prepared.

[0511] 18. The method according to any one of items 12 to 17, wherein the produced LNDFH-III:LNFP-VI ratio is higher than 1.3.

[0512] 19. The method according to any one of items 12 to 17, wherein the LNDFH-III:LNFP-III ratio is between 1:1 and 3:1.

[0513] 20. The method according to any one of items 12 to 17, wherein less than 10% of the molar content of the total HMOs produced is LNFP-III, and less than 10% of the molar content of the total HMOs produced is LNFP-VI.

[0514] 21. The method according to any one of items 12 to 17, wherein the produced fucosylated HMOs are mainly LNDFH-III and 3FL, and the sum of the other fucosylated HMOs is less than 15%, such as less than 10%, of the molar content of the total HMOs produced.

[0515] 22. The method according to any one of items 12 to 21, wherein the genetically engineered cells are cultured in the presence of acceptor substrates selected from lactose, LNT-II, and LNnT.

[0516] 23. Use of an α-1,3-fucosyltransferase in the production of one or more fucosylated HMOs, wherein the enzyme is selected from Osc1, Bgall1, Bbac1, Murba1, Bacfin1, Prev1, Csec1, CafC and FutA_mut2 comprising or consisting of the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9, or a FutA variant comprising substitutions at positions 128 and 129 of SEQ ID NO: 11, wherein the variant has at least 80% but less than 100% identity to SEQ ID NO: 11.

[0517] 24. An HMO mixture prepared by the method according to any one of items 12 to 22, which consists essentially of:

[0518] a. LNDFH-III and 3FL, or

[0519] b. LNDFH-III, LNFP-III and 3FL, or

[0520] c. LNDFH-III, LNFP-VI and 3FL, or

[0521] d. LNDFH-III, LNFP-III, LNFP-VI and LNnT, or

[0522] e. LNDFH-III, LNFP-III, 3FL and LNnT, or

[0523] f. LNDFH-III, LNFP-III, LNFP-VI, 3FL and LNnT.

[0524] 25. An HMO composition consisting essentially of 20 - 70 mol% of LNDFH-III, 0 - 35 mol% of LNFP-III, 0 - 35 mol% of LNFP-VI, 0 - 65 mol% of 3FL, 0 - 40% of LNnT and less than 1% of pLNnH, with a total molar content of 100%.

[0525] 26. The composition according to item 25, wherein the composition comprises an HMO mixture selected from:

[0526] a. A mixture consisting essentially of 35 - 60 mol% of LNDFH-III and 40 - 65 mol% of 3FL, with a total molar content of 100%,

[0527] b. A mixture consisting essentially of 50 mol% of LNDFH-III and 50 mol% of 3FL,

[0528] c. A mixture consisting essentially of 40 - 50 mol% of LNDFH-III, 25 - 35 mol% of LNFP-VI, 10 - 20 mol% of LNnT, and 5 - 15 mol% of 3FL, with a total of 100 mol%,

[0529] d. A mixture consisting essentially of 25 - 35 mol% of LNDFH-III, 15 - 25 mol% of LNFP-VI, 8 - 18 mol% of LNFP-III, and 33 - 43 mol% of LNnT, with a total of 100 mol%,

[0530] e. A mixture consisting essentially of 40 - 50 mol% of LNDFH-III, 5 - 15 mol% of LNFP-VI, 3 - 8 mol% of LNFP-III, 2 - 12 mol% of LNnT, and 30 - 40 mol% of 3FL, with a total of 100 mol%,

[0531] f. A mixture consisting essentially of 55 - 65 mol% of LNDFH-III, 17 - 27 mol% of LNFP-III, 6 - 16 mol% of LNnT, and 1 - 11 mol% of 3FL, with a total of 100 mol%,

[0532] g. A mixture consisting essentially of 60 mol% of LNDFH-III, 20 mol% of LNFP-III, 10 mol% of LNnT, and 10 mol% of 3FL,

[0533] h. A mixture consisting essentially of 36 - 46 mol% of LNDFH-III, 25 - 35 mol% of LNFP-VI, 10 - 20 mol% of LNFP-III, 2 - 12 mol% of LNnT, and 2 - 12 mol% of 3FL, with a total of 100 mol%,

[0534] i. A mixture consisting essentially of 50 - 70 mol% of LNDFH-III, 1 - 13 mol% of LNFP-III, and 20 - 50% of 3FL, with a total of 100 mol%,

[0535] j. A mixture consisting essentially of 70 mol% of LNDFH-III, 10 mol% of LNFP-III, and 20 - 50% of 3FL,

[0536] k. A mixture consisting essentially of 36 - 46 mol% of LNDFH-III, 25 - 35 mol% of LNFP-VI, 10 - 20 mol% of LNFP-III, 2 - 12 mol% of LNnT, and 2 - 12 mol% of 3FL, with a total of 100% molar content,

[0537] l. A mixture consisting essentially of 45 mol% of LNDFH-III, 30 mol% of LNFP-III, 10 mol% of 3FL, and 15% of LNnT,

[0538] m. A mixture consisting essentially of 25 - 35 mol% of LNDFH-III, 29 - 39 mol% of LNFP-VI, and 31 - 41 mol% of 3FL, with a total of 100% molar content,

[0539] n. A mixture consisting essentially of 37 - 47 mol% of LNDFH-III, 26 - 36 mol% of LNFP-III, 19 - 29 mol% of 3FL, and less than 5% of LNnT, with a total of 100% molar content, and

[0540] o. A mixture consisting essentially of 19 - 29 mol% of LNDFH-III, 13 - 23 mol% of LNFP-VI, 22 - 32 mol% of LNFP-III, 24 - 34 mol% of LNnT, and less than 5 mol% of 3FL, with a total of 100% molar content.

[0541] 27. A synbiotic composition comprising a probiotic strain selected from one or more Bifidobacterium and / or Lactobacillus species and an HMO mixture according to item 24 or an HMO composition according to item 25 or 26.

[0542] 28. The synbiotic composition according to item 27, wherein the probiotic strain is selected from one or more of the genus Lactobacillus rhamnosus, the genus Lactobacillus paracasei, and / or the genus Bifidobacterium adolescentis.

[0543] 29. The synbiotic composition according to item 27 or 28, wherein the probiotic strain is selected from one or more of Lactobacillus rhamnosus DSM 33156, Lactobacillus rhamnosus ATCC53103, Lactobacillus paracasei L26-CBS116412, and / or Bifidobacterium adolescentis DSM 34065.

[0544] 30. The synbiotic composition according to items 27 to 29, wherein the pH of the composition is less than 5.0.

[0545] 31. Use of the mixture according to item 24, the HMO composition according to item 25 or 26, or the synbiotic composition according to any one of items 27 to 30 in infant formula, dietary supplements and / or medical nutraceuticals.

[0546] Sequence

[0547] This application contains a sequence listing in text format and electronic format, which is hereby incorporated by reference.

[0548] An overview of the SEQ ID NOs used in this application can be found in Table 1 (α-1,3-fucosyltransferase protein sequences (SEQ ID NO: 1-11 and 51)) and Table 4 (promoter sequences SEQ ID NO: 27-50). Other sequences described in this application are DNA sequences encoding α-1,3-fucosyltransferase (SEQ ID NO: 12 to 22 and 52), DNA sequences encoding the capsular exopolysaccharide acid gene cluster of Escherichia coli (SEQ ID NO: 23), and β-1,3-N-acetylglucosaminyltransferase LgtA from Neisseria meningitidis (SEQ ID NO: 24), β-1,4-galactosyltransferase galT from Helicobacter pylori (SEQ ID NO: 25) and lactose permease LacY (SEQ ID NO: 26).

[0549] Example

[0550] Method

[0551] Unless otherwise specified, standard techniques, vectors, control sequence elements, and other expression system elements known in the field of molecular biology are used for nucleic acid manipulation, transformation, and expression. Such standard techniques, vectors, and elements can be found in the following references: Ausubel et al., Current Protocols in Molecular Biology (1995) (John Wiley & Sons); Sambrook, Fritsch, & Maniatis, Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, NY); Berger & Kimmel, Methods in Enzymology 152: Guide to Molecular Cloning Techniques (1987) (Academic Press); Bukhari et al., DNA Insertion Elements, Plasmids and Episomes (1977) (Cold Spring Harbor Laboratory Press, NY); Miller, J.H. Experiments in molecular genetics (1972.) (Cold spring Harbor Laboratory Press, NY).

[0552] The embodiments described below are chosen to illustrate the invention and not to limit the invention in any way.

[0553] Enzymes:

[0554] After screening 50 enzymes with fucosyltransferase activity, ten enzymes with hitherto unproven dual α-1,3-fucosyltransferase activity were found. These enzymes have α-1,3-fucosyltransferase activity towards the glucose of LNnT and α-1,3-fucosyltransferase activity towards the N-acetylglucosamine moiety, thereby producing the complex difucosylated HMO LNDFH-III. Table 5 provides the GenBank IDs and sources of the ten α-1,3-fucosyltransferases and the prior art 3-fucosyltransferase FutA.

[0555] Table 5. List of enzymes tested within the framework of the present invention

[0556] Enzyme Name GenBank ID^ SEQ ID NO: Source Osc1 MBC8592593.1 1 Oscillospiraceae bacterium N12 Bgall1* WP_204430034.1 2 Bacteroides gallinarum Bbac1 MBR5851405.1 3 Bacteroidaceae bacterium Murba1 MBP3303338.1 4 Muribaculaceae bacterium Bacfin1 WP_195630400.1 5 Bacteroides finegoldii Prev1 MBP3220331.1 6 Prevotella sp. Csec1 KHM48694.1 7 Faecalibacterium secundum CafC** WP_007483358.1 8 Bacteroides nordii FutA_mut2*** - 9 Artificial construct FucT109^^ CAH09151.1 10 Bacteroides fragilis NCTC 9343 FutA^^^ 11 Helicobacter pylori FutB^^^ WP_000487430.1 51 Helicobacter pylori

[0557] Compared with the GenBank sequences, the sequences used in the present application can be truncated at the N- or C-terminus, and these sequences are represented by SEQ ID NO.

[0558] *Chen et al., 2022 Arg Food Chem has shown that Bgall1 (FutM2) is used for the production of 3FL

[0559] **WO2016 / 040531 has proposed that CafC be used for the production of 3FL, DFL, and LNFP-III

[0560] ***WO2020 / 115671 has shown that FutA_mut2 is used for the production of LNFP-V

[0561] ^^WO 2019 / 008133 has proposed that FucT109 be used for the production of a mixture of LNFP-III and LNFP-VI

[0562] ^^^Dumon et al., 2004 (α-1,3-fucosyltransferase, Biotechnol. Prog. 2004, 20, 412-419) has shown that FutA and FutB are used for the production of LNDFH-III.

[0563] Strain

[0564] The strain (genetically engineered cell) constructed in the present application is based on Escherichia coli K-12 DH1 with the following genotype: Fˉ, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. Additional modifications were made to the Escherichia coli K-12 DH1 strain to generate the MDO strain with the following modifications: lacZ: deletion of 1.5 kbp, lacA: deletion of 0.5 kbp, nanKETA: deletion of 3.3 kbp, melA: deletion of 0.9 kbp, wcaJ: deletion of 0.5 kbp, mdoH: deletion of 0.5 kbp, and insertion of the Plac promoter upstream of the gmd gene.

[0565] The method of inserting a gene of interest into the Escherichia coli genome is well known to those skilled in the art. A specific selectable marker gene and screening method can be used to insert the gene cassette into the Escherichia coli chromosome using gene gorging (see, for example, Herring and Blattner 2004 J. Bacteriol. 186:2673-81 and Warming et al. 2005 Nucleic Acids Res. 33(4):e36).

[0566] To obtain a strain for producing LNnT, the MDO strain was further engineered by chromosomal integration of β-1,3-GlcNAc transferase (LgtA, from Neisseria meningitidis, homologous to NCBI accession number WP_033911473.1 and shown as SEQ ID NO:24) and β-1,4-galactosyltransferase (GalT from Helicobacter pylori, homologous to GenBank ID WP_001262061.1 and shown as SEQ ID NO:25), both under the control of the PglpF promoter (SEQ ID NO:39). This engineered strain is called the LNnT strain.

[0567] A codon-optimized DNA sequence encoding a single α-1,3-fucosyltransferase was genomically integrated into the LNnT strain.

[0568] Table 6 provides the genotypes of the background strain (MDO), the LNnT strain, and the α-1,3 / 4-fucosyltransferase-expressing strains capable of producing LNDFH-III and its mixtures.

[0569] Table 6. Genotypes of strains capable of producing LNDFH-III used in this example.

[0570]

[0571] *1,3FT is the abbreviation of α-1,3-fucosyltransferase, and its DNA sequence was inserted into the genome of the host strain.

[0572] 1 lgtA-PglpF - Two genomic insertion copies of the gene encoding β-1,3-N-acetyl-glucosaminyltransferase (SEQ ID NO:24) under the control of the PglpF promoter.

[0573] 2 galT-PglpF - One genomic insertion gene encoding β-1,4-galactosyltransferase (SEQ ID NO:25) under the control of the PglpF promoter.

[0574] 3 CA = An additional capsular exopolysaccharide acid gene cluster (gmd-wcaG-wcaH-wcaI-manC-manB, SEQ ID NO:23) located at a locus different from the native locus under the control of the PglpF promoter.

[0575] deep well assay

[0576] The deep-well assays in this example were performed as originally described by Lv et al. (Bioprocess Biosyst Eng 20(2016)39:1737—1747) and optimized for the purposes of this invention. More specifically, the strains disclosed in this example were screened in 96 deep-well plates using a 4-day protocol. During the first 24 hours, the pre-cultures were grown to high density (OD600 up to 5), and then transferred to a medium allowing for induction of gene expression and product formation.

[0577] More specifically, during the first day, fresh pre-cultures were prepared using a basal minimal medium (BMM) (pH 7.0) supplemented with magnesium sulfate (0.12 g / L), thiamine (0.004 g / L), and glucose (5.5 g / L). The basal minimal medium had the following composition: NaOH (1 g / L), KOH (2.5 g / L), KH2PO4 (7 g / L), NH4H2PO4 (7 g / L), citric acid (0.5 g / l), trace mineral solution (5 mL / L). The trace mineral stock solution contained: ZnSO4·7H2O 0.82 g / L, citric acid 20 g / L, MnSO4·H2O 0.98 g / L, FeSO4·7H2O 3.925 g / L, CuSO4·5H2O 0.2 g / L. The pH of the basal minimal medium was adjusted to 7.0 with 5N NaOH and autoclaved. The pre-cultures were incubated at 34 °C and 1000 rpm shaking for 24 hours, and then further transferred to 0.75 mL of fresh BMM (pH 7.5) to start the main culture. The fresh BMM was supplemented with magnesium sulfate (0.12 g / L), thiamine (0.02 g / L), a dose of glucose solution (0.1 - 0.15 g / L), and a dose of lactose solution (5 - 20 g / L). In addition, a 20% stock solution of sucrose (40 - 45 g / L) or maltodextrin (19 - 20 g / L) was provided as a carbon source, and specific hydrolases, sucrase, or glucoamylase were added respectively to release glucose at a rate suitable for carbon-limited growth and similar to a typical fed-batch fermentation process. The main culture was incubated at 28 °C and 1000 rpm shaking for 72 hours. To analyze the total fermentation broth, the 96-well plates were boiled at 100 °C, then centrifuged, and finally the supernatant was analyzed by HPLC.

[0578] Fermentation

[0579] Escherichia coli strains were cultured in a 250 mL fermenter (Ambr250 HT Bioreactor system, Sartorius) starting from 100 mL of a mineral medium consisting of 30 g / L glucose and NH4H2PO4, KH2PO4, MgSO4·7H2O, KOH, NaOH, citric acid, trace element solution, antifoam, and thiamine. The dissolved oxygen level was maintained at 20% by first stirring and then starting aeration at 700 rpm (maximum 4500 rpm) and 1 VVM (maximum 3 VVM). The pH was maintained at 6.8 by titration with 8.5% NH4OH solution. The culture was started with a 2% (v / v) inoculum from a preculture containing 10 g / L glucose, (NH4)2HPO4, KH2PO4, MgSO4·7H2O, KOH, NaOH, citric acid, trace element solution, antifoam, and thiamine. After the glucose contained in the basal minimal medium was exhausted, a feed solution containing glucose, MgSO4·7H2O, H3PO4, and trace mineral solution was continuously added to the fermenter at a rate to maintain carbon-limited conditions. The temperature was initially 33 °C but decreased linearly at a rate of 3 °C per hour to 30 °C 12 hours after the start of feeding. Lactose was added in the form of a bolus addition of a 25% lactose monohydrate solution 6 hours after the start of feeding and then every 19 hours to prevent lactose from becoming a rate-limiting factor. The growth, metabolic activity, and metabolic state of the cells were monitored by online measurement of stirring, dissolved oxygen tension, reflectance, NH4OH base addition, O2 uptake rate, and CO2 evolution rate. During the entire fermentation process, HPLC sampling was used to determine the concentrations of HMO products, lactose, and other minor by-products.

[0580] Example 1 - In Vivo LNDFH-III Synthesis

[0581] Genetically modified cells expressing a single α-1,3-fucosyltransferase (listed in Table 6) were screened for their ability to produce fucosylated complex HMO LNDFH-III and its mixtures.

[0582] We collected ten enzymes (Table 7) for testing, which, to our knowledge, had not been previously described as being able to synthesize LNDFH-III when introduced into genetically modified cells producing LNnT and GDP-fucose. Some enzymes are known to be used for the production of other fucosylated HMOs, namely FucT109a, CafC, FutA_mut2, FutA, and FutB, and it has been suggested in the prior art that these enzymes are able to modify the glucose moiety of lactose, LNnT, or LNT or the N-acetylglucosamine moiety of LNnT.

[0583] Specifically, FucT109 (also known as CafA) has been shown to be used for the production of LNFP-III, LNFP-VI, 3FL, and DFL (WO2019008133 and WO2016040531), CafC has been shown to be used for the production of 3FL and DFL (WO2016 / 040531), and FutA_mut2 has been shown to be used for the production of LNFP-V when introduced into an LNT-producing strain (WO2020 / 115671). Chen et al. have shown in 2022 ArgFood Chem that Bgall1 (FutM2) is used for the production of 3FL. However, none of these have been shown to be used for the production of LNDFH-III.

[0584] Dumon et al., 2004 (Biotechnol. Prog. 2004, 20, 412-419) have shown that FutA and FutB are used for the production of LNDFH-II and are used as reference α-1,3-fucosyltransferases.

[0585] As described in the "Methods" section, genetically modified strains expressing 12 individual α-1,3-fucosyltransferases were generated (Table 5). Cells were screened in a deep-well assay device as described in the "Methods" section.

[0586] Table 6 lists the genotypes of the strains capable of producing LNDFH-III. The molar content of the individual HMOs produced by the strains was measured by HPLC.

[0587] The results of LNDFH-III-producing cells are shown in Table 7 as a fraction (percentage, %) of the total HMO molar content produced by each strain.

[0588] Table 7: Percentage (%) of the individual HMO content produced by each strain relative to the total HMO molar (mM) content.

[0589] Enzyme FT Copy Number LNDFH-III LNFP-VI LNFP-III LNnT 3FL pLNnH Bgall1-1 1 67.4 0.3 7.8 0.1 24.4 0.0 Bgall1-2 2 52.1 0.0 2.7 0.0 45.3 0.0 Bacfin1 1 61.4 0.3 22.1 10.5 5.7 0.0 Osc1-1 1 57.0 0.3 0.5 0.1 42.1 0.0 Osc1-2 2 36.8 0.0 0.0 0.0 63.2 0.0 Murba1 1 43.9 0.8 31.1 14.8 9.2 0.3 Bbac1 1 46.3 8.9 4.3 4.9 35.7 0.0 Prev1 1 41.3 29.8 14.9 7.3 6.7 0.0 FutA_mut2 1 42.0 0.0 30.8 3.0 24.3 0.0 CafC 1 29.5 33.7 0.0 0.7 36.0 0.0 FucT109 1 23.9 18.0 26.6 28.7 2.0 0.8 Csec1 1 29.1 19.2 12.8 37.7 0.9 0.2 FutA 1 25.0 64.6 0.0 9.7 0.6 0.0 FutB 1 0.0 0.5 6.2 78.9 1.3 13.2

[0590] It is believed that HMOs present in amounts less than 3%, such as less than 2% or less than 1%, do not exist in significant amounts. From the data provided in Table 7, it can be seen that six new enzymes in the production of fucosylated HMOs, Osc1, Murba1, Csec1, Bbac1, Bacfin1, and Prev1, as well as the enzymes known to produce less complex fucosylated HMOs, Bgall1, CafC, and FutA_mut2, can transfer fucose units in an α-1,3 bond to the Glu and GlcNAc moieties of LNnT at the Glu moiety and the GlcNAc moiety, forming LNDFH-III, the level of which is higher than 25% of the total HMO. The enzyme FucT109, known from the prior art to produce LNFP-III and LNFP-VI, surprisingly also shows that in addition to producing 18% of LNFP-VI, 27% of LNFP-III, and 29% of LNnT respectively, it is also capable of producing 24% of the total HMO of LNDFH-III. In fact, FucT109 seems to be the only enzyme that produces all four HMO species at levels higher than 15%. Therefore, if it is desired to produce an HMO mixture in which LNDFH-III, LNFP-III, LNFP-VI, and LNnT all exist at levels higher than 15%, this is indeed an interesting enzyme.

[0591] Interestingly, among the prior art enzymes reported to produce LNDFH-III, FutA is the only enzyme capable of producing LNDFH-III, resulting in 25% of the total HMO produced being LNDFH-III. In addition, FutA seems to produce significantly more LNFP-VI, resulting in an LNDFH-III:LNFP-VI ratio of 0.4. Conversely, the LNDFH-III:LNFP-V1 ratio of all new LNDFH-III producers is higher than 1.3. FutA also does not produce any LNPF-III at all, indicating that FutA can only fucosylate the GlcNAc moiety of the LNnT backbone with the fucose moiety present on the Glc moiety, that is, FutA can only fucosylate the GlcNAc moiety of LNFP-VI but not LNnT.

[0592] In the current assay, FutB does not produce any LNDFH-III, and under the current conditions, since the main products produced by the FutB strain are non-fucosylated LNnT and pLNnH HMOs, it generally appears to be a very poor fucosyltransferase.

[0593] As can be seen from Table 7, in cells expressing Osc1, the production of LNFP-III and LNFP-VI was negligible, indicating that Osc1 is highly effective in fucosylating both the Glc and GlcNAc moieties of LNnT. However, Osc1 also had a relatively high activity towards lactose, resulting in 42% 3FL. However, since 3FL is more easily separable from LNDFH-III than LNFP-III and LNFP-VI, this strain would be advantageous if the production of pure LNDFH-III is desired. In addition, it can be seen that increasing the copy number of Osc1 to two genetic copies led to a slight increase in 3FL production while completely eliminating the production of LNFP-III and LNFP-VI in the strain, indicating that Osc1 may prefer lactose over LNnT as a substrate.

[0594] Bgall1 was the best producer of LNDFH-III, with LNDFH-III accounting for 67% of the total HMO produced, but the level of LNFP-III in Bgall1 was slightly higher compared to Osc1. Similar to Osc1, increasing the copy number of Bgall1 to two genomic copies slightly decreased the relative amount of LNDFH-III produced from 67% of the total HMO content to 52%, while significantly decreasing the relative amount of LNFP-III produced from 8% to 3% and increasing the amount of 3FL produced from 24% to 45%. Thus, Bgall1 seems to prefer lactose over LNnT as a substrate.

[0595] Therefore, changes in the copy number of Bgall1 and Osc1 can be used to customize specific HMO mixtures, which, in this case, depending on the needs of the specific product, consist of different proportions of LNDFH-III and 3FL.

[0596] As described above, if the purification of the produced LNDFH-III is desired, a lower amount or absence of alternative fucosylated species of the same or similar size in the produced mixture is highly advantageous and preferred, and thus Bgall1 and Osc1 are particularly suitable for this purpose.

[0597] It was found that the enzymes Murba1, Bacfin1, and FutA_mut2 produce a mixture of LNDFH-III, LNFP-III, LNnT, and 3FL. These enzymes may be particularly useful if a mixture with an LNDFH-III:LNFP-III ratio of 1:1 to 1:3 is desired. It was found that Prev1, Bbac1, Csec1, and FucT109 produce a highly complex HMO mixture, including LNDFH-III, LNFP-III, LNFP-VI, LNT, and 3FL, thus producing a total of 5 different HMOs from a single cell, where both Csec1 and FucT109 produce a relatively low amount of 3FL (between 1% and 2% respectively). It was found that the enzyme CafC produces an HMO mixture that essentially contains fucosylated HMOs, namely LNDFH-III, LNFP-VI, and 3FL, where the 3FL produced is less than 1% of LNnT.

[0598] Example 2 - Fermentation using Bgall1 or Osc1 α-1,3 / 4-fucosyltransferase strains to produce LNDFH-III

[0599] To confirm the HMO profiles observed in the deep-well assays, particularly the content of LNDFH-III in the total HMOs produced, the Osc1-1 and Bgall1-1 strains of Example 1, which contain a single genomic copy of Osc1 or Bgall1, were fermented as described in the "Methods" section above. The results are shown in Table 8.

[0600] Table 8: Percentage (%) of the content of individual HMOs produced by the strains in the total HMO content

[0601] Strain Number 3FL LNT-II LNFP-III LNFP-VI LNDFH-III LNnT Bgall1-1 18.3 0.4 6.9 0.0 74.2 0.2 Osc1-1 50.8 0.6 0.7 0.0 47.8 0.1

[0602] From the data in Table 8, it can be seen that the LNDFH-III fractions of the Osc1-1 and Bgall1 strains in fermentation are similar. As shown by the results in Example 1, Bgall1 tends to form more LNDFH-III and less 3FL, while Osc1 forms more 3FL than LNDFH-III. In addition, both strains show the ability and applicability of Bgall1 and Osc1 to produce LNDFH-III, with only low yields of alternative complex fucosylated HMOs (LNFP-III and LNFP-VI) in fermentation. In particular, Osc1 has very low levels of both LNFP-III and LNFP-VI as well as LNnT, and there is a chance to obtain very pure LNDFH-III when the culture broth from this strain is purified.

[0603] Example 3 - Regeneration and survival rate of Lactobacillus species in freeze-drying

[0604] Probiotics can be consumed as live bacteria or as dried (e.g., lyophilized) products. Independent of the drying method, rehydration involves an important step in restoring dehydrated bacteria; an insufficient rehydration / regeneration step may result in poor cell viability and low final survival rates. Therefore, rehydration is a very critical step in the reactivation of lyophilized cultures. For live and rehydrated bacteria, the survival rate of bacteria under acidic conditions is crucial because they need to pass through the acidic environment of the stomach and may also face storage (shelf life) in acidic foods.

[0605] In this example, it was tested whether an HMO mixture similar to that produced by the strains described in Examples 1 and 2 could provide benefits in terms of probiotic rehydration (regeneration) and survival rate. This test was conducted under acidic conditions similar to those under which bacteria must survive when passing through the stomach or when administered in acidic beverages.

[0606] Three different Lactobacillus strains were tested

[0607] Lactobacillus rhamnosus 33156

[0608] Lactobacillus rhamnosus LBrGG-ATCC53103

[0609] Lactobacillus paracasei L26-CBS116412

[0610] As shown in Table 9, the lyophilized probiotics were added to test tubes either alone (control) or in combination with an HMO mixture (5% w / v) (0.4 mg / ml). The compositions were dissolved in sterile phosphate-buffered saline (PBS, pH = 3), heated to 37 °C and mixed vigorously for about 30 seconds until no visible lumps remained. The test tubes were incubated at 37 °C for 3 hours. The samples were further diluted and 100 μl was plated in duplicate on MRS agar plates, which were then incubated at 37 °C in an anaerobic chamber. The experimental setup is shown in Figure 2 .

[0611] Table 9: HMO composition tested in this example

[0612] HMO C Mixture 1 Mixture 2 Mixture 3 Mixture 4 LNDFH-III 0 50 70 60 45 LNFP-III 0 0 10 20 30 3FL 0 50 20 10 10 LNnT 0 0 0 10 15

[0613] CFU / ml was calculated based on the colonies counted after 48 hours of incubation (average of two plates). Figure 3 Photograph of the plate showing colonies of Lactobacillus rhamnosus DSM 33156 after 48 hours of incubation. Plates of the other two strains are not shown, but they look similar. Table 10 summarizes the results for all three strains.

[0614] Table 10: Average CFU / ml of the designated strains after 3 hours of acid treatment, followed by 48 hours of incubation at 37 °C

[0615]

[0616]

[0617] Compared to the control without the HMO mixture, all lyophilized Lactobacillus strains solubilized with the HMO mixtures described herein showed enhanced regeneration and survival capabilities. These data clearly demonstrate that in the presence of any HMO mixture, the regeneration and survival rates of Lactobacillus rhamnosus and Lactobacillus paracasei strains can be improved after exposure to low pH conditions such as in the stomach or in acidic beverages.

[0618] For Lactobacillus rhamnosus -DSM 33156, it was also seen that replacing some of the 3FL in Mixture 1 with LNnT and LNFP-III was even more effective in enhancing regeneration and survival rates than observed for Mixtures 1 and 2.

[0619] For Lactobacillus rhamnosus LBrGG, using only the mixture of LNDFH-III and 3FL (Mixture 1) seems to be beneficial, although Mixture 4 indicates that LNFP-III can replace some of the 3FL. It is also worth noting that Lactobacillus rhamnosus LBrGG cannot survive the acidic treatment without the HMO mixture.

[0620] For Lactobacillus paracasei L26, Mixture 3, which contains all 4 HMOs and where 80% of the mixture is LNDFH-III and LNFP-III, each supplemented with 10% of LNnT and 3FL, seems to have the greatest benefit for regeneration and survival rates.

[0621] To our knowledge, there was no previous evidence indicating that the tested mixtures provide the benefit of improving the regeneration and survival capabilities of Lactobacillus strains in an acidic environment.

[0622] Example 4 - Regeneration and Survival Rates of Lyophilized Bifidobacterium Species

[0623] As shown in Example 3 above, the ability of the HMO mixtures in Table 9 to improve the regeneration and survival capabilities of Bifidobacterium adolescentis (DSM 34065) in an acidic environment was also tested.

[0624] As shown in Table 9, freeze-dried probiotic Bifidobacterium adolescentis (DSM 34065) was dissolved alone or in combination with an HMO mixture (5% w / v) in sterile water at pH 3.0 (0.4 mg / ml), heated to 37°C, and vigorously mixed for approximately 30 seconds until no visible clumps remained. The tubes were incubated at 37°C for 30 minutes. Then, 100 μl was plated in duplicate on MRS cysteine agar plates and incubated at 37°C in an anaerobic chamber for 72 hours. The regeneration and survival rate of the probiotic were determined by counting the colonies on the plates after 72 hours of incubation.

[0625] CFU / ml was calculated based on the colonies counted on the undiluted plates after 72 hours of incubation (average of two plates). The results are shown in Table 11.

[0626] Table 11: Mean CFU / ml of Bifidobacterium adolescentis (DSM 34065) after 30 minutes of acid treatment followed by 72 hours of incubation at 37°C

[0627]

[0628] As can be seen from Table 11, compared to the control without HMO, these mixtures enabled some Bifidobacterium adolescentis (DSM 34065) strains to survive the acid treatment. Mixture 2, which had the largest amount of LNDFH-III combined with the other two fucosylated HMOs 3FL and LNFP-III, seemed to have the greatest impact on the regeneration and survival ability of Bifidobacterium adolescentis (DSM 34065).

Claims

1. A genetically engineered cell capable of producing LNDFH-III, comprising a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase selected from the following, a. Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof, having an amino acid sequence with at least 80% identity to SEQ ID NO:1, b. Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof, having an amino acid sequence with at least 80% identity to SEQ ID NO:2, c. Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof, having an amino acid sequence with at least 80% identity to SEQ ID NO:3, d. Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof, having an amino acid sequence with at least 80% identity to SEQ ID NO:4, e. Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof, having an amino acid sequence with at least 80% identity to SEQ ID NO:5, f. Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof, having an amino acid sequence with at least 80% identity to SEQ ID NO:6, g. Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof, having an amino acid sequence with at least 80% identity to SEQ ID NO:7, h. CafC comprising or consisting of the amino acid sequence of SEQ ID NO:8, or a functional homolog thereof, having an amino acid sequence with at least 80% identity to SEQ ID NO:8, and i. a FutA variant comprising substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein said variant has at least 80% sequence identity to SEQ ID NO:11, and wherein said cell further comprises one or more recombinant nucleic acid sequences encoding β-1,4-galactosyltransferase and optionally β-1,3-N-acetylglucosaminyltransferase.

2. The genetically engineered cell according to claim 1, wherein at least 25% of the molar content of the total HMO produced by said cell is LNDFH-III.

3. The genetically engineered cell according to claim 1 or 2, wherein said cell further produces one or more HMOs selected from 3FL, LNnT, LNFP-III and LNFP-VI.

4. The genetically engineered cell according to any one of the preceding claims, wherein said cell further comprises one or more recombinant nucleic acid sequences encoding β-1,3-N-acetylglucosaminyltransferase and / or β-1,4-galactosyltransferase.

5. The genetically engineered cell according to any one of the preceding claims, wherein the cell further comprises a substrate importer selected from lactose importer, lacto-N-triose-II (LNT-II) importer or LNnT importer.

6. The genetically engineered cell according to any one of the preceding claims, wherein the engineered cell is selected from Escherichia coli, Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris and Saccharomyces cerevisiae.

7. A method for producing one or more fucosylated HMOs, comprising the steps of: a. Providing and culturing a genetically engineered cell comprising a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase selected from: i. Osc1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:1; ii. Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:2; iii. Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:3; iv. Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:4; v. Bacfin1 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:5; vi. Prev1 comprising or consisting of the amino acid sequence of SEQ ID NO:6, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:6; vii. Csec1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:7; viii. CafC comprising or consisting of the amino acid sequence of SEQ ID NO:8, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:8; ix. FucT109 comprising or consisting of the amino acid sequence of SEQ ID NO:10, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:10, and x. a FutA variant comprising substitutions at positions corresponding to positions 128 and 129 of SEQ ID NO:11, wherein said variant has at least 80% sequence identity with SEQ ID NO:11, and b. culturing the cells according to (a) in a suitable cell culture medium to produce said one or more fucosylated HMOs, wherein at least one of the fucosylated HMOs is LNDFH-III, and c. optionally purifying said one or more fucosylated HMOs.

8. The method according to claim 7, wherein at least 25% of the molar content of the total HMOs produced by said method is LNDFH-III.

9. The method according to any one of claims 7 or 8, wherein at least one additional fucosylated HMO selected from 3FL, LNFP-III and LNFP-VI is produced.

10. The method according to any one of claims 7 to 9, wherein the genetically engineered cells are cultured in the presence of a receptor substrate selected from lactose, LNT-II and LNnT.

11. Use of an α-1,3-fucosyltransferase in the production of one or more fucosylated HMOs, wherein at least one of the fucosylated HMOs is LNDFH-III, and wherein the enzyme is selected from Osc1, Bgall1, Bbac1, Murba1, Bacfin1, Prev1, Csec1, CafC and FutA_mut2 comprising or consisting of the amino acid sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8 or 9, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8 or 9, or a FutA variant comprising substitutions at positions 128, 129 of SEQ ID NO:11, wherein said variant has at least 80% but less than 100% identity with SEQ ID NO:

11.

12. An HMO mixture produced by the method according to any one of claims 7 to 10, said mixture consisting essentially of: a. LNDFH-III and 3FL, or b. LNDFH-III, LNFP-III and 3FL, or c. LNDFH-III, LNFP-VI and 3FL, or d. LNDFH-III, LNFP-III, LNFP-VI and LNnT, or e. LNDFH-III, LNFP-III, 3FL and LNnT, or f. LNDFH-III, LNFP-III, LNFP-VI, 3FL and LNnT.

13. An HMO composition consisting essentially of 20 - 70 mol% of LNDFH-III, 0 - 35 mol% of LNFP-III, 0 - 35 mol% of LNFP-VI, 0 - 65 mol% of 3FL, 0 - 40% of LNnT, and less than 1% of pLNnH, with a total of 100 mol%.

14. The composition according to claim 13, wherein the composition comprises an HMO mixture selected from the following: a. A mixture consisting essentially of 35 - 60 mol% of LNDFH-III and 40 - 65 mol% of 3FL, with a total of 100 mol%. b. A mixture consisting essentially of 50 mol% of LNDFH-III and 50 mol% of 3FL. c. A mixture consisting essentially of 40 - 50 mol% of LNDFH-III, 25 - 35 mol% of LNFP-VI, 10 - 20 mol% of LNnT, and 5 - 15 mol% of 3FL, with a total of 100 mol%. d. A mixture consisting essentially of 25 - 35 mol% of LNDFH-III, 15 - 25 mol% of LNFP-VI, 8 - 18 mol% of LNFP-III, and 33 - 43 mol% of LNnT, with a total of 100 mol%. e. A mixture consisting essentially of 40 - 50 mol% of LNDFH-III, 5 - 15 mol% of LNFP-VI, 3 - 8 mol% of LNFP-III, 2 - 12 mol% of LNnT, and 30 - 40 mol% of 3FL, with a total of 100 mol%. f. A mixture consisting essentially of 55 - 65 mol% of LNDFH-III, 17 - 27 mol% of LNFP-III, 6 - 16 mol% of LNnT, and 1 - 11 mol% of 3FL, with a total of 100 mol%. g. A mixture consisting essentially of 60 mol% of LNDFH-III, 20 mol% of LNFP-III, 10 mol% of LNnT, and 10 mol% of 3FL. h. A mixture consisting essentially of 36 - 46 mol% of LNDFH-III, 25 - 35 mol% of LNFP-VI, 10 - 20 mol% of LNFP-III, 2 - 12 mol% of LNnT, and 2 - 12 mol% of 3FL, with a total of 100 mol%. i. A mixture consisting essentially of 50 - 70 mol% of LNDFH-III, 1 - 13 mol% of LNFP-III, and 20 - 50% of 3FL, with a total of 100 mol%. j. A mixture consisting essentially of 70 mol% of LNDFH-III, 10 mol% of LNFP-III, and 20 - 50% of 3FL. k. A mixture consisting essentially of 36 - 46 mol% of LNDFH-III, 25 - 35 mol% of LNFP-VI, 10 - 20 mol% of LNFP-III, 2 - 12 mol% of LNnT, and 2 - 12 mol% of 3FL, with a total of 100 mol%, l. A mixture consisting essentially of 45 mol% of LNDFH-III, 30 mol% of LNFP-III, 10 mol% of 3FL, and 15% of LNnT, m. A mixture consisting essentially of 25 - 35 mol% of LNDFH-III, 29 - 39 mol% of LNFP-VI, and 31 - 41% of 3FL, with a total of 100 mol%, n. A mixture consisting essentially of 37 - 47 mol% of LNDFH-III, 26 - 36 mol% of LNFP-III, 19 - 29 mol% of 3FL, and less than 5% of LNnT, with a total of 100 mol%, and o. A mixture consisting essentially of 19 - 29 mol% of LNDFH-III, 13 - 23 mol% of LNFP-VI, 22 - 32 mol% of LNFP-III, 24 - 34 mol% of LNnT, and less than 5 mol% of 3FL, with a total of 100 mol%.

15. A composition comprising a probiotic strain selected from one or more Bifidobacterium and / or Lactobacillus species and the HMO mixture according to claim 12 or the HMO composition according to claim 13 or 14.

16. Use of the mixture according to claim 12 or the composition according to any one of claims 13 to 15 in infant formula, dietary supplements, and / or medical nutraceuticals.

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