Novel fucosyltransferase for in vivo synthesis of complex fucosylated human milk oligosaccharides

Abstract

The present invention relates to the production of fucosylated human milk oligosaccharides (HMOs), in particular the production of complex fucosylated HMOs having five or more monosaccharide units, such as LNFP-V and LNDFH-II, from precursor oligosaccharides, as well as genetically engineered cells and alpha-1, 3 (4)-fucosyltransferases suitable for use in said production, and methods of producing said fucosylated HMOs.

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-V and LNDFH-II, and 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 human milk oligosaccharides (HMOs), is critical to providing innovative and scalable solutions for future more complex products.

[0003] The production of complex bifucosylated HMOs requires multiple enzymes, or multispecific enzymes, such as bispecific glycosyltransferases capable of producing complex bifucosylated HMOs. Multispecific enzymes are preferred because of the lower genetic burden of introducing them into host cells.

[0004] For example, WO2020 / 115671 proposes that the α1,3-fucosyltransferase FutA and its variants can produce the complex HMO LNFP-V, and WO2019 / 008133 describes that the α1,3-fucosyltransferase FucT109 appears to fucosylate both the glucose (Glc) and N-acetylglucosamine (GlcNAc) portions of lacto-N-neotetraose (LNnT), thereby potentially generating a mixture containing complex fucosylated HMOs LNFP-III and LNFP-VI, but FucT109 appears to only fucosylate the Glc portion of LNT.

[0005] Dumon et al., 2004 (Biotechnol. Prog. 2004, 20, 412-419) further described that the α1,3-fucosyltransferase FutA produces a mixture containing the complex fucosylated HMOs LNDFH-III (LNnDFH-II) and LNFP-VI (LNnFP-V). Dumon et al., 2004 did not disclose difucosylation with an LNT backbone.

[0006] et al. 2015 (Bioorganic & Medicinal Chemistry 23: 6799-6806) disclosed that α-1,4-fucosyltransferase FucTIII can produce LNDFH-II but has no effect on the formation of LNFP-V.

[0007] In summary, the production of fucosylated HMOs, especially the more complex ones, is challenging due to the lack of fucosyltransferases with the required substrate specificity and the low production yield of the desired fucosylated HMOs compared to other HMO products present after fermentation (e.g., HMO precursor products, which may require tedious separation procedures). Summary of the invention

[0008] The present invention solves the need for bispecific glycosyltransferases in the production of complex bifucosylated HMOs with an LNT backbone, in particular, in the production of LNDFH-II, by identifying a group of α-1,3(4)-fucosyltransferases that exhibit low or no specificity for the galactose moiety in LNT as a substrate (acceptor oligosaccharide) for the fucosylation reaction, but have high substrate specificity for the N-acetylglucosamine (GlcNAc) and glucose (Glu) moieties in LNT, thereby producing complex fucosylated HMO LNDFH-II, or a mixture of HMOs containing LNDFH-II, which has a high total content of fucosylated HMOs. Therefore, the α-1,3(4)-fucosyltransferases proposed herein can be used to produce LNDFH-II and a mixture of LNDFH-II and LNFP-V. Thus, provided herein are enzymes, mixtures, compositions, uses, genetically engineered cells and methods for producing LNDFH-II or HMO mixtures comprising LNDFH-II, which mixtures have a high total content of fucosylated HMOs.

[0009] In a first aspect, the present invention relates to a genetically engineered cell capable of producing LNDFH-II, comprising a recombinant nucleic acid sequence encoding a glycosyltransferase selected from the group consisting of:

[0010] a. Bgall1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1,

[0011] b. Murba1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 2, and

[0012] c. Bbac1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 3, and

[0013] d. Para1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 4, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 4.

[0014] When the starting substrate is lactose, preferably the genetically engineered cell further comprises one or more recombinant nucleic acid sequences encoding β-1,3-N-acetylglucosaminyltransferase and β-1,3-galactosyltransferase.

[0015] In a second aspect, the present invention relates to a method for producing one or more fucosylated HMOs, preferably LNDFH-II and / or a mixture of HMOs comprising LNDFH-II, the method comprising culturing a genetically engineered cell capable of producing LNDFH-II, comprising a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase and α-1,4-fucosyltransferase activity (also known as α-1,3(4)-fucosyltransferase), the fucosyltransferase being capable of fucosylating an acceptor oligosaccharide at the GlcNAc moiety and the Glc moiety, wherein the glycosyltransferase is selected from the group consisting of: Bgall1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1; Murba1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1; NO:2 has at least 80% identity; and Bbac1, which comprises or consists of the amino acid sequence according to SEQ ID NO:3, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO:3. Preferably, the cell further comprises one or more recombinant nucleic acid sequences encoding β-1,3-N-acetylglucosaminyltransferase and β-1,3-galactosyltransferase.

[0016] In a third aspect, the present invention relates to the use of an enzyme having α-1,3-fucosyltransferase and α-1,4-fucosyltransferase activity, which is capable of fucosylating an acceptor oligosaccharide at the GlcNAc moiety and the Glc moiety to produce one or more fucosylated HMOs, preferably LNDFH-II and HMO mixtures comprising LNDFH-II.

[0017] In a fourth aspect, the present invention relates to a HMO mixture produced by the process of the present invention, comprising or consisting essentially of:

[0018] a) LNDFH-II, LNFP-V and LNT, or

[0019] b) LNDFH-II, LNFP-V and 3FL, or

[0020] c) LNDFH-II, LNFP-V, LNT and 3FL, or

[0021] d)LNDFH-II, LNT-II, LNFP-V, LNFP-II and LNT

[0022] The invention also relates to a composition comprising a mixture of a) to d) and the use of said composition in infant formula, dietary supplement or medical nutrition. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 . Overview of the synthesis of complex fucosylated HMOs with an LNT-backbone. DETAILED DESCRIPTION

[0024] The present invention addresses the biotechnological challenges of HMO production in vivo, in particular complex fucosylated HMOs with an LNT-backbone comprising at least five monosaccharide units, wherein at least one monosaccharide unit is a fucosyl unit, such as in particular difucosylated complex HMOs with at least six monosaccharide units. Examples of such complex fucosylated HMOs are LNFP-V and LNDFH-II. The present invention provides a specific strain engineering solution for the production of specific complex fucosylated HMOs (in particular LNFP-V and / or LNDFH-II) by utilizing the dual substrate specificity of the α-1,3(4)-fucosyltransferase disclosed herein for the N-acetylglucosamine (GlcNAc) and glucose (Glc) moieties in LNT (see Figure 1 ).

[0025] Complex fucosylated HMOs with an LNT backbone can be produced from lactose as a starting substrate, but as long as the cells are able to internalize, for example, LNT-II or LNT (see, for example, WO2023 / 099680), these can also be used as starting substrates.

[0026] Production of LNFP-V and / or LNDFH-II

[0027] The advantage of using the α-1,3(4)-fucosyltransferases disclosed herein is that they are able to specifically recognize and fucosylate both the GlcNAc and glucose moieties in LNT, thereby generating LNFP-II and / or LNFP-V, which are further fucosylated at the GlcNAc or glucose moieties, thereby generating the difucosylated HMO LNDFH-II (see Figure 1). In essence, the present disclosure describes several newly identified enzymes with dual α-1,3-fucosyltransferase and 1,4-fucosyltransferase activities (α-1,3(4)-fucosyltransferases) that are capable of producing complex fucosylated HMOs LNFP-V and / or LNDFH-II. Thus, when sufficient amounts of LNT are available within the genetically engineered cells to saturate the enzymes, very little, if any, LNFP-II is produced by the preferred α-1,3(4)-fucosyltransferases described in the present disclosure.

[0028] Only one of the α-1,3(4)-fucosyltransferases described herein, Para1, produced similar levels of LNFP-II and LNFP-V when expressed in cells. As can be seen from Example 1, when the expression of Para1 was increased, more LNDFH-II was produced, indicating that higher enzyme expression levels are beneficial for achieving difucosylation.

[0029] Without being bound by theory, the other four α-1,3(4)-fucosyltransferases described herein, Bgall1, Murba1, Bbac1, and FutA, all appear to generate LNDFH-II via LNFP-V as a preferred intermediate (see Figure 1 ), as little or no LNFP-II is produced. Therefore, the properties of these α-1,3(4)-fucosyltransferases are well suited for high-level industrial production of LNDFH-II and / or LNFP-V, or mixtures thereof, without producing high levels of alternative complex fucosylated HMOs (byproducts), such as LNFP-II and / or other byproduct HMOs, such as 3FL, LNT-II, and / or LNnT. pLNH2 is also a potential oligosaccharide byproduct that is not officially reported as an HMO, although it is likely present in small amounts in human breast milk.

[0030] Furthermore, the α-1,3(4)-fucosyltransferase described herein is also very suitable for the production of LNDFH-II and mixtures comprising LNDFH-II, LNFP-V, LNT and / or 3FL.

[0031] The genetically engineered cells of the present disclosure express any one or more α-1,3(4)-fucosyltransferases disclosed herein, with high substrate specificity for GlcNAc and glucose moieties in LNT, and for the first time produce LNDFH-II at a titer exceeding 5%, such as exceeding 10%, such as exceeding 15%, such as exceeding 20%, of the total HMO production. In some cases, two or more copies of nucleic acids encoding α-1,3(4)-fucosyltransferases are required to produce greater than 5% of LNDFH-II. LNDFH-II and LNFP-V can be purified from the HMO mixture produced by the genetically engineered cells.

[0032] Thus, the present disclosure enables more complex fucosylated HMOs, such as LNFP-V and LNDFH-II, to be produced by more efficient biotechnology, either in purified form or as mixtures in which fucosylated HMOs predominate, e.g., greater than 60% of the total HMOs produced.

[0033] In the following sections, the various elements of the present invention, in particular the various elements of the genetically engineered cells, will be described. It will be appreciated that these elements may be combined between the various parts.

[0034] Oligosaccharides

[0035] The term "oligosaccharide" in this context refers to a sugar polymer containing at least three monosaccharide units, i.e., tri, tetra, penta, hexa or higher oligosaccharides. Oligosaccharides can have a linear or branched structure, which contains monosaccharide units interconnected by interglycosidic bonds. Specifically, oligosaccharides include the lactose residue of the reducing end and one or more naturally occurring monosaccharides of 5-9 carbon atoms, and the monosaccharide is selected from aldose (e.g., glucose, galactose, ribose, arabinose, xylose, etc.), ketose (e.g., fructose, sorbose, tagatose, etc.), deoxy sugar (e.g., rhamnose, fucose, etc.), deoxy amino sugar (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acid and ketouronic acid (e.g., N-acetylneuraminic acid). Preferably, oligosaccharides are HMOs.

[0036] Human Milk Oligosaccharides (HMO)

[0037] Preferred oligosaccharides of the present disclosure are human milk oligosaccharides (HMOs).

[0038] The term "human milk oligosaccharide" or "HMO" in this context refers to a complex carbohydrate found in human milk. HMOs have a core structure comprising a lactose unit at the reducing end, which may be elongated by one or more β-N-acetyl-lactosamine units and / or one or more β-lacto-N-disaccharyl units, and which may be substituted by α-L-fucopyranyl and / or α-N-acetyl-neuraminic acid (fucosyl) moieties. The HMO structure is disclosed by, for example, Xi Chen in Chapter 4 of Advances in Carbohydrate Chemistry and Biochemistry 2015 vol 72.

[0039] The present invention focuses on fucosylated HMO. Examples of fucosylated HMO include 2'-fucosyllactose (2'FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-I), lacto-N-difucohexaose II (LNFP-II), lacto-N-difucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-I ... II), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-fucopentaose VI (LNFP-VI), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-p-lacto-N-hexaose I (FpLNH-I), fucosyl-p-lacto-N-neohexaose II (F-pLNNH-I), II), fucosyl-lacto-N-neohexaose (FLNnH), 3-fucosyl-3'-fucosyllactose (FSL), fucosyl-LST-a (FLST-a), fucosyl-LST b (FLST b), fucosyl-LST-c (FLST-c), fucosyl-LST d (FLST-d) and fucosyl-lacto-N-hexaose (SLNH).

[0040] In the context described herein, a complex fucosylated HMO is a fucosylated HMO comprising 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 consisting of 5 monosaccharide units, such as LNFP-I, LNFP-II, LNFP-III, LNFP-V and LNFP-VI, and complex fucosylated HMOs with 6 monosaccharide units, such as but not limited to the difucosylated HMOs LNDFH-I, LNDFH-II and LNDFH-III or the sialyl-fucosyl 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 to be produced from the starting substrate lactose, for example, the formation of LNFP-V requires α-1,3-fucosyltransferase, β-1,3-N-acetylglucosaminyltransferase and β-1,3-galactosyltransferase (see Figure 1 ), the formation of LNFP-II requires α-1,4-fucosyltransferase, β-1,3-N-acetylglucosaminyltransferase, and β-1,3-galactosyltransferase (see Figure 1), and the formation of LNDFH-II requires α-1,3-fucosyltransferase, α-1,4-fucosyltransferase, β-1,3-N-acetylglucosaminyltransferase, and β-1,3-galactosyltransferase, among which α-1,3-fucosyltransferase and α-1,4-fucosyltransferase can be a single fucosyltransferase with dual activity, also known as α-1,3(4)-fucosyltransferase.

[0041] Specifically, the produced fucosylated HMO is selected from complex fucosylated HMOs comprising at least five monosaccharide units, wherein at least one monosaccharide unit is a fucosyl unit. In addition, in an embodiment, the fucosylated HMO is selected from complex fucosylated HMOs having an LNT backbone structure, preferably, selected from LNFP-II, LNFP-V and LNDFH-II. Examples of fucosylated HMOs having an LNT backbone structure are lacto-N-fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose I (LNDFH-I), lacto-N-difucohexaose II (LNDFH-II), sialyl-lacto-N-fucopentaose I (S-LNFP-I or FLST-b), sialyl-lacto-N-fucopentaose II (S-LNFP-II or FLST-a), monofucosyl-lacto-N-hexaose I (F-LNH-I), Monofucosyl-lacto-N-hexaose II (F-LNH-II), monofucosyl-lacto-N-hexaose III (F-LNH-III), difucosyl-lacto-N-hexaose I (DF-LNH-I), difucosyl-lacto-N-hexaose II (DF-LNH-II), difucosyl-lacto-N-hexaose III (DF-LNH-III), trifucosyl-lacto-N-hexaose (TF-LNH), fucosyl-sialo-lacto-N-hexaose I (FS-LNH) and disialyl-fucosyl-lacto-N-hexaose II (DS-F-LNH-II).

[0042] In an embodiment of the present disclosure, the fucosyltransferase described herein primarily fucosylates the N-acetylglucosamine (GlcNAc) and glucose (Glc) portions of LNT, and is also capable of fucosylating the glucose (Glc) portion of lactose. In this regard, the one or more fucosylated HMOs are preferably 3FL, LNFP-V, LNFP-II and LNDFH-II, or 3FL, LNFP-V and LNDFH-II.

[0043] In other embodiments, the fucosyltransferase disclosed herein only fucosylates the N-acetylglucosamine (GlcNAc) and / or glucose (Glc) moieties of LNT. In this regard, the one or more fucosylated HMOs are preferably LNDFH-II, LNFP-II and LNFP-V or LNDFH-II and LNFP-V.

[0044] In human milk, about 60% of the HMO content is fucosylated HMO, so producing a mixture containing a high content of fucosylated HMO is very advantageous for producing a more natural HMO mixture. In an embodiment of the present disclosure, at least 80 mol%, such as at least 85 mol%, 86 mol%, 87 mol%, 88 mol%, 89 mol%, 90 mol%, 91 mol%, 92 mol%, 93 mol%, 94 mol%, 95 mol%, 96 mol%, 97 mol%, or at least 98 mol% of the produced HMO are fucosylated HMO.

[0045] Receptor oligosaccharide

[0046] The genetically engineered cells described herein comprise a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity capable of transferring fucose from an activated sugar to the GlcNAc and / or Glc portion of an acceptor oligosaccharide with an α-1,4 linkage in the GlcNAc portion or an α-1,3 linkage in the Glc portion.

[0047] In the context described herein, an acceptor 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 acceptor oligosaccharide. The glycosyl donor is preferably a nucleotide activated sugar, as described in the "glycosyl donor-nucleotide activated sugar pathway" section. Preferably, the acceptor oligosaccharide is a precursor for the preparation of a more complex HMO, which can also be referred to as a precursor molecule.

[0048] The acceptor oligosaccharide may be an intermediate product of the present fermentation process, the end product of a separate fermentation process using separate genetically engineered cells, or an enzymatically or chemically produced molecule.

[0049] In the present context, the acceptor oligosaccharide of the α-1,3(4)-fucosyltransferase is preferably lacto-N-tetraose (LNT), which is produced from the precursor molecule lacto-N-triose II (LNT-II) (e.g., the acceptor of β-1,3-galactosyltransferase), which is produced from the initial precursor molecule lactose (e.g., the acceptor of β-1,3-N-acetylglucosaminyltransferase) (cf. Figure 1). In addition, the acceptor oligosaccharide of α-1,3(4)-fucosyltransferase can also be lacto-N-fucopentaose II (LNFP-II) produced by the precursor molecule LNT (e.g., the acceptor of α-1,4-fucosyltransferase) or lacto-N-fucopentaose V (LNFP-V) produced by the precursor molecule LNT (e.g., the acceptor of α-1,3-fucosyltransferase). The initial precursor molecule is preferably given to a genetically engineered cell that is capable of producing, for example, LNT-II, LNT, LNFP-II, LNFP-V and / or LNDFH-II from the precursor. Most commonly, the initial precursor is lactose and the genetically engineered cell is capable of producing intermediate precursors (acceptor oligosaccharides such as LNT-II and LNT) within the cell. However, if the cell is capable of introducing at least one of these compounds, the initial precursor can also be LNT-II or LNT.

[0050] Glycosyltransferase

[0051] The genetically engineered cells described herein comprise at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase, i.e., 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.

[0052] The genetically engineered cells described herein may comprise one or more additional recombinant nucleic acids encoding one or more recombinant and / or heterologous glycosyltransferases capable of transferring glycosyl residues from a glycosyl donor to an acceptor oligosaccharide. Preferably, the additional glycosyltransferases enable the genetically engineered cells to synthesize LNT from a precursor molecule (e.g., lactose or LNT-II). In an embodiment, the genetically engineered cells described herein comprise one or more additional recombinant nucleic acids encoding one or more recombinant and / or heterologous glycosyltransferases.

[0053] The additional glycosyltransferase is preferably selected from the group consisting of galactosyltransferase, glucosaminyltransferase, fucosyltransferase and N-acetylglucosaminyltransferase.

[0054] The fucosyltransferase in the genetically engineered cells described herein is an α-1,3(4)-fucosyltransferase. Preferably, the α-1,3(4)-fucosyltransferase is capable of transferring a fucose unit to the GlcNAc and / or Glc moiety of LNT, LNFP-II and / or LNFP-V.

[0055] In the present disclosure, the functional enzyme (α-1,3(4)-fucosyltransferase) capable of transferring a fucosyl moiety from a fucosyl donor to an acceptor oligosaccharide is selected from: a) Bgall1, which comprises or consists of an amino acid sequence according to 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) Murba1, which comprises or consists of an amino acid sequence according to 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, which comprises or consists of an amino acid sequence according to 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. These enzymes can be used, for example, to produce LNFP-II, LNFP-V and / or LNDFH-II, d) Para1 comprising or consisting of the amino acid sequence according to SEQ ID NO:4, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO:4, and e) FutA comprising or consisting of the amino acid sequence according to SEQ ID NO:5, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO:5.

[0056] In a preferred embodiment, the functional enzyme (α-1,3(4)-fucosyltransferase) capable of transferring a fucosyl moiety from a fucosyl donor to an acceptor oligosaccharide is selected from the group consisting of: a) Bgall1 comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1, b) Murba1 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 2, c) Bbac1 comprising or consisting of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 3. These enzymes can be used, for example, for the production of LNFP-II, LNFP-V and / or LNDFH-II.

[0057] The preference of most α-1,3(4)-fucosyltransferases disclosed herein tends to produce a mixture of LNFP-V and LNDFH-II, with only a small amount or no LNFP-II produced, indicating that these enzymes have a higher specificity for the Glc moiety in LNT and that fucosylation of the GlcNAc moiety occurs mainly on LNFP-V rather than LNT.

[0058] Without being bound by theory, if it is desired to produce less or no alternative complex fucosylated HMOs (e.g., LNFP-II) when the starting substrate is lactose, then α-1,3(4)-fucosyltransferases (e.g., Bgall1, Murba1, Bbac1, and FutA) that produce primarily a mixture of LNFP-V and LNDFH-II, while producing little or no LNFP-II, are advantageous. Reducing the number of alternative complex fucosylated HMOs in turn results in easier purification of the HMOs produced, as it is simpler to purify LNDFH-II and / or LNFP-V from a mixture of HMOs that primarily contain the desired product, as it is easier to separate complex fucosylated HMOs from smaller HMOs than to separate different fucosylated HMOs of the same or similar size from each other, such as LNDFH-II from LNFP-V or LNDFH-II from LNFP-II. Therefore, the presence of only one HMO comprising five monosaccharide units in the production of LNDFH-II, such as only LNFP-II or only LNFP-V, is believed to be beneficial for the purification of LNDFH-II.

[0059] In preferred embodiments, use of the α-1,3(4)-fucosyltransferase described herein results in at least 75 mol%, such as at least 80 mol%, 85 mol%, 90 mol%, 95 mol%, 96 mol%, 97 mol%, or such as at least 98 mol% of the total HMO molar content produced by the cells described herein being fucosylated HMO.

[0060] In further embodiments, use of the α-1,3(4)-fucosyltransferase described herein results in at least 40 mol%, such as at least 50 mol%, 60 mol%, 70 mol%, 80 mol%, or such as at least 90 mol%, or such as between 40 mol% and 90 mol%, or such as between 50 mol% and 80 mol% of the total HMO molar content produced by the cells described herein being a mixture of LNDFH-II and LNFP-V.

[0061] In other preferred embodiments, use of the α-1,3(4)-fucosyltransferase described herein results in at least 1% of the total HMO molar content produced by the cells described herein being LNDFH-II, such as at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, or such as at least 35% of the total HMO molar content produced by the cells described herein being LNDFH-II.

[0062] In other preferred embodiments, use of the α-1,3(4)-fucosyltransferase described herein results in at least 5% of the total molar content of HMO produced by the cells described herein being LNDFH-II.

[0063] In one embodiment, the α-1,3(4)-fucosyltransferase is Bgall1 comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1. Preferably, when expressed in a suitable genetically engineered cell, Bgall1 is capable of producing at least 10%, such as at least 15%, of the total molar content of HMO produced by the cells described herein in LNDFH-II.

[0064] In one embodiment, the α-1,3(4)-fucosyltransferase is Murba1, which comprises or consists of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 2. Preferably, when expressed in sufficient amounts in a suitable genetically engineered cell, Murba1 is capable of producing at least 1%, such as at least 4%, of the total molar content of HMO produced by the cell described herein, of LNDFH-II.

[0065] In one embodiment, the α-1,3(4)-fucosyltransferase is Bbac1, which comprises or consists of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 3. Preferably, when expressed in sufficient amounts in a suitable genetically engineered cell, Bbac1 is capable of producing LNDFH-II at least 5% of the total HMO molar content produced by the cell described herein.

[0066] In one embodiment, the α-1,3(4)-fucosyltransferase is Para1 comprising or consisting of an amino acid sequence according to SEQ ID NO: 4, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 4. Preferably, when expressed in sufficient amounts in a suitable genetically engineered cell, Para1 is capable of producing at least 1%, such as at least 5%, of the total molar content of HMO produced by the cell described herein at LNDFH-II.

[0067] In one embodiment, the α-1,3(4)-fucosyltransferase is FutA comprising or consisting of an amino acid sequence according to SEQ ID NO: 5, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 5. Preferably, when expressed in sufficient amounts in a suitable genetically engineered cell, FutA is capable of producing LNDFH-II at least 5% of the total molar content of HMO produced by the cell described herein.

[0068] In embodiments, expression of the α-1,3(4)-fucosyltransferase described herein in a genetically engineered cell is also combined with expression of one or more other recombinant nucleic acids encoding one or more recombinant and / or heterologous glycosyltransferases.

[0069] In embodiments described herein, the cells further comprise one or more recombinant nucleic acid sequences encoding a β-1,3-galactosyltransferase.

[0070] In a preferred embodiment, expression of the α-1,3(4)-fucosyltransferase described herein in a genetically engineered cell is combined with expression of a β-1,3-galactosyltransferase (e.g., galTK from Helicobacter pylori). In a further embodiment, a third enzyme is expressed, such as a β-1,3-N-acetylglucosaminyltransferase, such as LgtA from Neisseria meningitidis.

[0071] In addition to the α-1,3(4)-fucosyltransferases Bgall1, Bbac1, Para1, Murba1, and FutA, exemplary other glycosyltransferases are preferably selected from the glycosyltransferases described below (Tables 1, 2, and 3).

[0072] α-1,3(4)-fucosyltransferase

[0073] The term "α-1,3(4)-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of a fucosyl group from a donor substrate (e.g., GDP-fucose) to an acceptor molecule via an α-1,3-linkage or an α-1,4-linkage (see Figure 1 ). Preferably, the α-1,3(4)-fucosyltransferase used in the present invention is not derived from a species of genetically engineered cells, i.e., the gene encoding the α-1,3(4)-fucosyltransferase is of heterologous origin and is selected from the α-1,3(4)-fucosyltransferases identified in Table 1. In the context described herein, the acceptor molecule of the α-1,3(4)-fucosyltransferase is preferably an acceptor oligosaccharide of at least four monosaccharide units having at least one glucose and at least one GlcNAc moiety, such as LNT. Heterologous α-1,3(4)-fucosyltransferases capable of transferring a fucosyl moiety to the glucose moiety of LNT are known in the art, in particular FutA having an amino acid sequence as provided in SEQ ID NO: 5 and encoded by a nucleic acid sequence of SEQ ID NO: 10, which is known to be able to produce LNFP-V (WO2020115671).

[0074] The fucosyltransferase may be selected from 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 the amino acid sequence of any one of the α-1,3(4)-fucosyltransferases listed in Table 1.

[0075] Table 1. List of α-1,3(4)-fucosyltransferases capable of producing LNDFH-II.

[0076]

[0077]

[0078] 1 The GenBank ID indicates the full-length enzyme, and truncated or mutant versions may be used in the present invention, which are represented by the sequences shown in SEQ ID NO.

[0079] Example 1 described herein discloses the identification of heterologous α-1,3(4)-fucosyltransferases Bgall1, Murba1 Bbac1, and Para1 (SEQ ID NOs: 1, 2, 3, and 4, respectively), which, when introduced into LNT production cells, are each capable of producing a HMO mixture containing LNDFH-II. Specifically, the three novel enzymes Bgall1, Murba1, and Bbac1 can transfer fucosyl units to the Glc portion of LNT with an α-1,3 linkage to form LNFP-V, and further transfer fucosyl units to the GlcNAc portion of LNFP-V with an α-1,4 linkage to form LNDFH-II to varying degrees. Surprisingly, Example 1 also shows that FutA is capable of transferring fucosyl units to the GlcNAc portion of LNFP-V with an α-1,4 linkage to form LNDFH-II, which, to our knowledge, has never been shown before.

[0080] The heterologous α-1,3(4)-fucosyltransferase Para1 appears to have slightly different specificity from the other four heterologous α-1,3(4)-fucosyltransferases in Table 1 , as it was able to form LNFP-II and LNFP-V from LNT in similar amounts, suggesting that it has similar specificity for the Glc and GlcNAc moieties in LNT and the Glc moiety of LNFP-II and the GlcNAc moiety of LNFP-V, and can produce LNDFH-II from both molecules (see Figure 1 ).

[0081] In summary, Example 1 shows that the enzymes Bgall1, Murba1, Bbac1 and FutA do not produce any LNFP-II, or at least do not produce any detectable amount of LNFP-II as end product. In contrast, the enzyme Para1 from Parabacteroides sp. AM08-6 was found to produce LNFP-II as well as LNFP-V and LNDFH-II as end products.

[0082] The fucosyltransferase described herein is capable of transferring a fucosyl moiety from a fucosyl donor to an acceptor oligosaccharide, forming an α-1,3 bond or an α-1,4 bond. This enzyme is also referred to as an α-1,3(4)-fucosyltransferase. In a preferred embodiment, the fucosyltransferase described herein is a fucosyltransferase having α-1,3-fucosyltransferase and α-1,4-fucosyltransferase activity, which is capable of fucosylation of oligosaccharides at both the GlcNAc moiety and the Glc moiety.

[0083] In an embodiment, the fucosyltransferase having α-1,3(4)-fucosyltransferase activity is selected from: Bgall1, which comprises or consists of the amino acid sequence according to 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; Murba1, which comprises or consists of the amino acid sequence according to 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; and Bbac1, which comprises or consists of the amino acid sequence according to 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.

[0084] In an embodiment, the fucosyltransferase having α-1,3(4)-fucosyltransferase activity is selected from: Bgall1, which comprises or consists of the amino acid sequence according to 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; and Murba1, which comprises or consists of the amino acid sequence according to 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.

[0085] In one embodiment, the fucosyltransferase having α-1,3(4)-fucosyltransferase activity is Bgall1 from Bacteroides gallinarum, 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.

[0086] In another embodiment, the fucosyltransferase having α-1,3(4)-fucosyltransferase activity is Murba1 from a bacterium of the family Murobacteraceae, 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.

[0087] In another embodiment, the fucosyltransferase having α-1,3(4)-fucosyltransferase activity is Bbac1 from a bacterium of the Bacteroidetes family, comprising or consisting of the amino acid sequence of SEQ ID NO:3, or comprising or consisting 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.

[0088] In another embodiment, the fucosyltransferase having α-1,3(4)-fucosyltransferase activity is Para1 from Parabacteroides AM08-6, 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.

[0089] In another embodiment, the fucosyltransferase having α-1,3(4)-fucosyltransferase activity is FutA from Helicobacter pylori ATCC 26695, 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.

[0090] In one embodiment, the enzyme Bgall1 is introduced into a genetically engineered cell which also comprises a β-1,4-galactosyltransferase and preferably also comprises a β-1,3-N-acetylglucosaminyltransferase.

[0091] In one embodiment, the enzyme Murba1 is introduced into a genetically engineered cell which also comprises a β-1,3-galactosyltransferase and preferably also a β-1,3-N-acetylglucosaminyltransferase.

[0092] In one embodiment, the enzyme Bbac1 is introduced into a genetically engineered cell which also comprises a β-1,3-galactosyltransferase and preferably also a β-1,3-N-acetylglucosaminyltransferase.

[0093] In one embodiment, the enzyme Para1 is introduced into a genetically engineered cell which also comprises a β-1,3-galactosyltransferase and preferably also a β-1,3-N-acetylglucosaminyltransferase.

[0094] In one embodiment, the enzyme FutA is introduced into a genetically engineered cell which also comprises a β-1,3-galactosyltransferase and preferably also a β-1,3-N-acetylglucosaminyltransferase.

[0095] β-1,3-N-acetylglucosaminyltransferase

[0096] β-1,3-N-acetylglucosaminyltransferase is any protein that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to lactose or another acceptor molecule via a β-1,3-linkage (see Figure 1 ). Preferably, the β-1,3-N-acetylglucosaminyltransferase used herein is not derived from a species of genetically engineered cells, i.e. the gene encoding the β-1,3-N-acetylglucosaminyltransferase is of heterologous origin. In the context described herein, the acceptor molecule is lactose or an oligosaccharide of at least four monosaccharide units, such as LNT, or a more complex HMO structure.

[0097] Thus, in an embodiment, the genetically engineered cell further comprises one or more recombinant nucleic acid sequences encoding β-1,3-N-acetylglucosaminyltransferase.

[0098] Non-limiting examples of β-1,3-N-acetylglucosaminyltransferases are given in Table 2. β-1,3-N-acetylglucosaminyltransferase 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 β-1,3-N-acetylglucosaminyltransferase in Table 2.

[0099] Table 2. List of β-1,3-N-acetylglucosaminyltransferases

[0100]

[0101] In an embodiment, the genetically engineered cell comprises a recombinant nucleic acid sequence encoding a β-1,3-N-acetylglucosaminyltransferase. In one embodiment, the recombinant nucleic acid sequence encoding a β-1,3-N-acetylglucosaminyltransferase comprises or consists of the amino acid sequence of SEQ ID NO: 12 (LgtA from Neisseria meningitidis), or a functional homolog thereof, the amino acid sequence of the functional homolog 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.

[0102] To produce LNT from the substrate lactose, 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 functional homolog or fragment thereof to produce the intermediate LNT-II from lactose.

[0103] Some of the examples below use a heterologous β-1,3-N-acetylglucosaminyltransferase named LgtA from Neisseria meningitidis or a variant thereof.

[0104] β-1,3-galactosyltransferase

[0105] β-1,3-Galactosyltransferase is any protein that transfers the galactose of UDP-galactose to the N-acetylglucosamine moiety via a β-1,3-linkage and thus to an acceptor molecule (see Figure 1 ). Preferably, the β-1,3-galactosyltransferase used herein is not derived from a genetically engineered cell species, ie, the gene encoding the β-1,3-galactosyltransferase is of heterologous origin. As described herein, the acceptor molecule is an acceptor carbohydrate, such as LNT-II, or a more complex HMO structure.

[0106] The following examples use a heterologous β-1,3-galactosyltransferase named GalTK or a variant thereof to produce LNT, and in combination with the α-1,3(4)-fucosyltransferase described herein, it can produce, for example, LNFP-II, LNFP-V and / or LNDFH-II.

[0107] The β-1,3-galactosyltransferase can be obtained from any of a variety of sources, for example, the galTK gene from Helicobacter pylori (homologous to GenBank protein accession number BD182026.1) as described above, or the WbgO gene from Escherichia coli (E. coli) 055:H7 (GenBank accession number WP_000582563.1), or the jhp0563 gene from Helicobacter pylori (GenBank accession number AEZ55696.1).

[0108] In one embodiment, the recombinant nucleic acid sequence encoding β-1,3-galactosyltransferase comprises or consists of the amino acid sequence of SEQ ID NO:13 (galTK from Helicobacter pylori), or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% identical to SEQ ID NO:13.

[0109] To produce LNT from the LNT-II precursor, a β-1,3-galactosyltransferase is required. In one embodiment, the genetically modified cell comprises a β-1,3-galactosyltransferase gene or a functional homologue or a fragment thereof.

[0110] The following are examples of genetically modified strains described herein having a specific combination of glycosyltransferases that will use lactose as a starting substrate to produce LNDFH-II.

[0111] In one example, LgtA from Neisseria meningitidis was used in combination with galTK from Helicobacter pylori and Bgall1 from Bacteroides gallinarum to produce LNDFH-II starting from lactose as a starting substrate.

[0112] In one example, LgtA from Neisseria meningitidis was used in combination with galTK from Helicobacter pylori and Murba1 from Murbacterium spp. to produce LNDFH-II starting from lactose as a starting substrate.

[0113] In one example, LgtA from Neisseria meningitidis was used in combination with galTK from Helicobacter pylori and Bbac1 from the Bacteroidetes family to produce LNDFH-II starting from lactose as a starting substrate.

[0114] In one example, LgtA from Neisseria meningitidis was used in combination with galTK from Helicobacter pylori and Para1 from Parabacteroides sp. AM08-6 to produce LNDFH-II starting from lactose as a starting substrate.

[0115] In one example, LgtA from Neisseria meningitidis was used in combination with galTK from Helicobacter pylori and FutA from Helicobacter pylori ATCC 26695 to produce LNDFH-II starting from lactose as the acceptor sugar.

[0116] In one example, galTK from Helicobacter pylori was used in combination with Bgall1 from Bacteroides gallinarum to produce LNDFH-II starting from LNT-II as a starting substrate.

[0117] In one example, galTK from Helicobacter pylori was used in combination with Murba1 from the family Murobacterium to produce LNDFH-II starting from LNT-II as a starting substrate.

[0118] In one example, galTK from Helicobacter pylori was used in combination with Bbac1 from the Bacteroidetes family to produce LNDFH-II starting from LNT-II as a starting substrate.

[0119] In one example, galTK from Helicobacter pylori and Para1 from Parabacteroides sp. AM08-6 produced LNDFH-II starting from LNT-II as a starting substrate.

[0120] In one example, galTK from H. pylori was used in combination with FutA from H. pylori ATCC 26695 to produce LNDFH-II starting from LNT-II as a starting substrate.

[0121] Glycosyl donor-nucleotide activated sugar pathway

[0122] When implementing the method of the present invention, preferably glycosylation reaction mediated by glycosyltransferase occurs, wherein activated sugar nucleotides are used as glycosyl donors. Activated sugar nucleotides usually have phosphorylated glycosyl residues attached to nucleosides. Specific glycosyltransferases only accept specific sugar nucleotides. Therefore, preferably the following activated sugar nucleotides participate in glycosyl transfer: glucose-UDP-GlcNAc, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine (GlcNAc) and CMP-N-acetylneuraminic acid.

[0123] The genetically engineered cells described herein may comprise one or more pathways for producing nucleotide activating sugars selected from the group consisting of glucose-UDP-GlcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, and CMP-N-acetylneuraminic acid.

[0124] In one embodiment of the invention, the genetically engineered cells can produce one or more of the above-mentioned activated sugar nucleotides by de novo pathways. In this regard, activated sugar nucleotides are made by cells from simple carbon sources (such as glycerol, sucrose, fructose or glucose) in a stepwise reaction sequence under the action of enzymes participating in the de novo biosynthetic pathways of respective sugar nucleotides (for a review of monosaccharide metabolism, see, for example, HH Freeze and AD Elbein: Chapter 4: Glycosylation Precursors, in: Essentials of Glycobiology, 2nd edition (Eds. A. Varki et al.), Cold Spring Harbour Laboratory Press (2009)).

[0125] The enzymes involved in the de novo biosynthetic pathway of activated sugar nucleotides may be naturally present in the cell or introduced into the cell by means of genetic or recombinant DNA techniques, which are part of the general knowledge of the person skilled in the art.

[0126] In another embodiment, genetically engineered cells can utilize salvaged monosaccharides as sugar nucleotides. In the salvage pathway, monosaccharides from degraded oligosaccharides are phosphorylated by kinases and converted into nucleotide sugars by pyrophosphorylases. The enzymes involved in this process can be heterologous or native enzymes of the host cell.

[0127] Colanic acid gene cluster

[0128] For the production of fucosylated HMOs, the de novo GDP-fucose pathway is important to ensure that sufficient GDP-fucose is present. The colanic acid gene cluster of E. coli encodes selected enzymes (gmd, wcaG, wcaH, wcal, manB, manC) involved in the de novo synthesis of GDP-fucose, while one or more genes downstream of GDP-L-fucose (e.g., wcaJ) are responsible for the production of the extracellular polysaccharide colanic acid, which is the major oligosaccharide of the bacterial cell wall, and these genes can be deleted to prevent the conversion of GDP-fucose to colanic acid.

[0129] In order to ensure a sufficient amount of GDP-fucose, the promoter of the natural colanic acid gene cluster can be exchanged with a stronger promoter to generate a recombinant colanic acid gene cluster to drive additional GDP-fucose production. In addition, additional copies of the colanic acid gene cluster or its selected genes can be introduced into the genetically engineered cells described in the examples.

[0130] In an embodiment, the colanic acid gene cluster can be expressed from its native genomic site. Expression can be actively regulated. Expression can be regulated by exchanging a native promoter with a promoter of interest, and / or by expressing the gene cluster from another non-native genomic site or additionally expressing the colanic acid gene cluster or its specific gene to increase the copy number of the colanic acid gene encoding the protein.

[0131] For purposes of this disclosure, the term "native genomic site" with respect to the colanic acid gene cluster refers to the original and natural location of the gene cluster in the genome of a genetically engineered cell.

[0132] The de novo GDP-fucose pathway genes responsible for GDP-fucose formation include or consist of the following genes:

[0133] i) manA, which encodes the protein mannose-6-phosphate isomerase (EC 5.3.1.8, UniProt accession number P00946), which promotes the interconversion of fructose-6-phosphate (F6P) and mannose-6-phosphate;

[0134] ii) manB, which encodes a protein phosphomannose mutase (EC 5.4.2.8, UniProt accession number P24175), which participates in the biosynthesis of GDP-mannose by catalyzing the conversion of mannose-6-phosphate to mannose-1-phosphate;

[0135] iii) manC, which encodes the protein mannose-1-phosphate guanylate transferase (EC 2.7.7.13, UniProt accession number P24174), which participates in the biosynthesis of GDP-mannose by synthesizing GDP-mannose from GTP and α-D-mannose-1-phosphate;

[0136] iv) gmd, which encodes the protein GDP-mannose-4,6-dehydratase (UniProt accession number P0AC88), which catalyzes the conversion of GDP-mannose to GDP-4-dehydro-6-deoxy-D-mannose;

[0137] v) wcaG (fcl), which encodes the protein 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.

[0138] Therefore, it is preferred that when the genetically engineered cell produces one or more fucosylated heterologous products, the entire colanic acid gene cluster (e.g., as shown in SEQ ID NO: 11 or a functional variant thereof) and / or one or more genes of the de novo GDP-fucose pathway are overexpressed, which are selected from manA, manB, manC, gmd and wcaG.

[0139] Lactose permease

[0140] Lactose permease is a membrane protein that is a member of the major facilitator superfamily and can be classified as a symporter that utilizes the proton gradient toward the cell to transport β-galactosides (e.g., lactose) into the cell in the same direction. In oligosaccharide production, especially in the production of human milk oligosaccharides (HMOs), lactose is often the starting substrate in the biotransformation that occurs inside the cell and is modified to produce any HMO of interest. Therefore, in the production of HMOs, it is desirable to be able to introduce lactose into the cell, for example by expressing and / or overexpressing a lactose permease, such as lacY of E. coli.

[0141] In an embodiment, the lactose permease is as shown in SEQ ID NO: 14, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 14, such as at least 85%, 90% or 95% identical.

[0142] In embodiments, expression of the lactose permease is regulated by a promoter described herein.

[0143] β-Galactosidase

[0144] Host cells suitable for HMO production, such as E. coli, may contain an endogenous β-galactosidase gene or an exogenous β-galactosidase gene, for example, E. coli contains an endogenous lacZ gene (e.g., GenBank Accession No. V00296 (GI:41901)). For the purposes of the present invention, when producing HMOs, if lactose is used as the starting substrate for the production of complex fucosylated HMOs, it is preferred that the genetically engineered cells do not express a functional β-galactosidase to avoid lactose degradation. In embodiments, the lacZ gene may be inactivated by completely or partially deleting the corresponding nucleic acid sequence from the bacterial genome, or the gene sequence is mutated so that it cannot be 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 enzymatic activity. In this way, the bacteria producing the HMOs accumulate an increased intracellular lactose pool, which is beneficial for the production of the HMOs.

[0145] Importin

[0146] The most common HMO producing cells are genetically engineered to use lactose as a starting substrate because, as mentioned above, lactose is readily taken up by the lactose permease. However, it may be desirable to use a starting substrate that requires less glycosyltransferase to be present in the cell because this will reduce the stress on the cell in terms of producing multiple enzymes and in addition reduce the byproduct spectrum, e.g. if lactose is not used as a starting substrate, then cells containing fucosyltransferases will not produce the byproduct 3FL, allowing the use of fucose to produce, for example, more LNFP-V and LNDFH-II. WO2022 / 242860 proposes how to identify LNT-II import proteins. WO2023 / 099680 also proposes a number of potential LNT and LNT-II import proteins. Examples of suitable LNT-II import proteins are:

[0147] - a lactose permease (LacY) mutant, such as LacY mutant Y236H or LacY mutant A177V+S306T, wherein the mutation is identical to the corresponding position in the sequence of SEQ ID NO: 14,

[0148] - an ABC transporter complex, for example the ABC transporters from B. pseudocatenulatum JCM1200BBPC_1775, 1776, 1777 (NCBI accession numbers BAR04453.1, BAR04454.1 and BAR04455.1, respectively) or the ABC transporters from B. breve UCC2003BBR_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

[0149] - MFS transporter, such as but not limited to Blon_0962 (NCBI accession number ACJ52061.1).

[0150] Thus, in embodiments, a nucleic acid or nucleic acid cluster encoding one of these transporters can be introduced into a genetically modified cell as described herein. Expression of such transporters enables the production of complex fucosylated oligosaccharides with LNT-II as a starting substrate.

[0151] Export protein

[0152] Oligosaccharide products, such as HMO produced by cells, can accumulate in the cell and in the extracellular matrix. The product can be transported to the supernatant in a passive manner, that is, it diffuses through the cell membrane to the outside. More complex HMO products may remain in the cell, which is likely to eventually damage cell growth, thereby affecting the total yield of products that can be produced in a single fermentation. HMO transport can be facilitated by major facilitator superfamily transport proteins, which promote the outflow of sugar derivatives from cells to the supernatant. Export proteins can be exogenous or endogenous, and are overexpressed under fermentation conditions to increase the output of produced oligosaccharide derivatives (HMOs). Mutations by known recombinant DNA techniques can change the specificity of the oligosaccharide products to be secreted.

[0153] Thus, the genetically engineered cells described herein may further comprise a nucleic acid sequence encoding an exporter protein capable of exporting a fucosylated human milk oligosaccharide product, such an exporter protein may be, for example, a member of the major facilitator superfamily of transporters.

[0154] In recent years, several new and efficient major facilitator superfamily transporters have been identified as HMO exporters, each specific for a different recombinantly produced HMO, and the development of recombinant cells expressing said proteins has facilitated large-scale industrial HMO manufacturing.

[0155] Genetically engineered cells

[0156] In this context, the terms "genetically engineered cells" and "genetically modified cells" are used interchangeably. As used herein, "genetically engineered cells" refer 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, such as using heterologous and / or recombinant polynucleotide sequences, Crisper / Cas editing and / or random mutations. In one embodiment, the genetically engineered cells have been transformed or transfected by recombinant nucleic acid sequences.

[0157] The genetic modification may, for example, be selected from the group consisting of addition of glycosyltransferases, and / or metabolic pathway engineering, deletion of inhibitory factors or undesired enzymes and addition of transporters, as described above, and the skilled person will know how to combine these into a genetically engineered cell capable of producing one or more fucosylated HMOs.

[0158] In one embodiment, the genetically engineered cell capable of producing LNDFH-II comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is selected from: a) Bgall1, which comprises or consists of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1, b) Murba1, which comprises or consists of the amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 2, c) Bbac1, which comprises or consists of the amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 3, d) Para1, which comprises or consists of the amino acid sequence according to SEQ ID NO: 4, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: NO:4 has at least 80% identity, and e) FutA, which comprises or consists of the amino acid sequence according to SEQ ID NO:5, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO:5.

[0159] Preferably, the fucosyltransferase has dual α-1,3-fucosyltransferase and α-1,4-fucosyltransferase activities, allowing the oligosaccharide to be fucosylated at position 4 of the GlcNAc moiety and position 3 of the Glc moiety, while showing limited or no fucosylation at position 2 of the Gal moiety. Preferably, the Glc moiety is located at the reducing end of the oligosaccharide, more preferably, the oligosaccharide is LNT.

[0160] In one embodiment, the genetically engineered cell capable of producing one or more fucosylated HMOs comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is selected from: a) Bgall1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1, b) Murba1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 2, c) Bbac1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 3.

[0161] In another embodiment, the genetically engineered cell capable of producing one or more fucosylated HMOs, at least one of which is LNDFH-II, comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is Para1 comprising or consisting of the amino acid sequence according to SEQ ID NO:4, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO:4.

[0162] In another embodiment, the genetically engineered cell capable of producing one or more fucosylated HMOs, at least one of which is LNDFH-II, comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is FutA comprising or consisting of an amino acid sequence according to SEQ ID NO: 5, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO: 5. In some embodiments, genetically modified cells with high expression of FutA are advantageous, which can be achieved, for example, by having at least two copies of a recombinant nucleic acid sequence encoding FutA (SEQ ID NO: 5) or a functional variant thereof in the genome, or by introducing a high copy number plasmid encoding FutA (SEQ ID NO: 5) or a functional variant thereof and / or by controlling expression from a strong or very strong promoter.

[0163] In a preferred embodiment, the genetically engineered cell capable of producing LNDFH-II comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is selected from a) Bgall1, which comprises or consists of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1, b) Murba1, which comprises or consists of the amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 2, c) Bbac1, which comprises or consists of the amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 3.

[0164] In a currently preferred embodiment, the genetically engineered cell capable of producing LNDFH-II comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is Bgall1, which comprises or consists of the amino acid sequence according to SEQ ID NO: 1, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO: 1 [Bgall1 amino acid].

[0165] In one aspect of the invention, the genetically engineered cell comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity capable of producing at least 1 mol% of LNDFH-II of the total HMO molar content produced by the cell. Preferably, at least 5 mol% of the total HMO molar content produced by the cell is LNDFH-II. In an embodiment, at least 5 mol%, such as at least 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 20 mol%, 25 mol%, 30 mol%, or such as at least 35 mol% of the total HMO molar content produced by the cell is LNDFH-II. In another embodiment, the cell also produces one or more HMOs selected from 3FL, LNT and LNFP-V.

[0166] In some embodiments, the genetically engineered cells described herein express Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or comprising or consisting 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 genetically engineered cells produce LNDFH-II at a molar % content higher than 10 molar %, such as higher than 15 molar %, such as higher than 17 molar %, such as higher than 19 molar %, such as higher than 20 molar %, or such as higher than 23 molar % of the total HMO produced.

[0167] In some embodiments, the genetically engineered cells described herein express Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, or comprising or consisting 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 genetically engineered cells produce LNDFH-II at a molar % content higher than 1 molar %, such as higher than 3 molar %, such as higher than 4 molar %, such as higher than 5 molar %, or such as higher than 6 molar % of the total HMOs produced.

[0168] In some embodiments, the genetically engineered cells described herein express Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or comprising or consisting 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 genetically engineered cells produce LNDFH-II at a mol % content greater than 1 mol %, such as greater than 3 mol %, such as greater than 4 mol %, such as greater than 5 mol %, or such as greater than 6 mol % of the total HMOs produced.

[0169] In some embodiments, the genetically engineered cells described herein express Para1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or comprising or consisting 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 genetically engineered cells produce LNDFH-II at a molar % content greater than 1 molar %, such as greater than 3 molar %, such as greater than 4 molar %, such as greater than 5 molar %, or such as greater than 6 molar % of the total HMOs produced.

[0170] In some embodiments, the genetically engineered cells described herein express FutA comprising or consisting of the amino acid sequence of SEQ ID NO: 5, or comprising or consisting 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 genetically engineered cells produce LNDFH-II at a mol% content higher than 4 mol%, such as higher than 10 mol%, such as higher than 15 mol%, such as higher than 20 mol%, such as higher than 25 mol%, or such as higher than 35 mol% of the total HMO produced.

[0171] Greater than 60% of the HMOs found in human milk are fucosylated HMOs of different species. Thus, in an embodiment, 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 cell are fucosylated. Preferably, the fucosylated HMOs produced by the cell are selected from 3FL, LNFP-V and LNDFH-II. 1. In one embodiment, the genetically engineered cells described herein produce a mixture of 3FL, LNFP-V and LNDFH-II, wherein the mixture accounts for at least 90 mole % of the total HMOs produced by the cell.

[0172] In some embodiments, the genetically engineered cells described herein express Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or comprising or consisting 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 genetically engineered cells produce fucosylated HMOs at a mol % content higher than 85 mol %, such as higher than 87 mol %, such as higher than 89 mol %, such as higher than 90 mol %, such as higher than 93 mol %, or such as higher than 96 mol % of the total HMOs produced.

[0173] In some embodiments, the genetically engineered cells described herein express Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or comprising or consisting 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 genetically engineered cells produce fucosylated HMOs at a mol % content higher than 75 mol %, such as higher than 77 mol %, such as higher than 80 mol %, such as higher than 85 mol %, such as higher than 90 mol %, or such as higher than 95 mol %.

[0174] In some embodiments, the genetically engineered cells described herein express Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or comprising or consisting 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 genetically engineered cells produce fucosylated HMOs in a mol % content greater than 85 mol %, such as greater than 87 mol %, such as greater than 89 mol %, such as greater than 90 mol %, such as greater than 93 mol %, or such as greater than 96 mol % of the total HMOs produced.

[0175] In some embodiments, the genetically engineered cells described herein express FutA comprising or consisting of the amino acid sequence of SEQ ID NO:5, or comprising or consisting 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 mol% content of fucosylated HMOs produced by the genetically engineered cells is higher than 85 mol%, such as higher than 87 mol%, such as higher than 89 mol%, such as higher than 90 mol%, such as higher than 93 mol%, or such as higher than 96 mol% of the total HMOs produced.

[0176] In a preferred embodiment, the fucosylated HMO produced by the cells described herein is selected from the group consisting of 3FL, LNFP-V and LNDFH-II.

[0177] The genetically engineered cells described herein preferably express genes encoding key enzymes for the biosynthesis of fucosylated HMOs. In addition, if the genetically engineered cells express genes required for the production of LNT, starting with lactose or LNT-II as the starting substrate (see Figure 1 ), and / or the cells express LNT-II or an importin of LNT, then it is advantageous.

[0178] In an embodiment, the genetically engineered cell comprises one or more additional glycosyltransferases. The additional one or more glycosyltransferases are preferably selected from the group consisting of galactosyltransferase, glucosaminyltransferase, fucosyltransferase and N-acetylglucosaminyltransferase.

[0179] In some embodiments, the genetically engineered cell comprises one or more recombinant nucleic acid sequences encoding a β-1,3-galactosyltransferase and optionally a β-1,3-N-acetylglucosaminyltransferase. In some embodiments, the β-1,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis and the β-1,3-galactosyltransferase is from Helicobacter pylori.

[0180] In some embodiments, the genetically engineered cells described herein also express the de novo GDP-fucose pathway genes responsible for the formation of GDP-fucose manA, manB, manC, gmd and wcaG. Overexpression of one or more of these genes and / or upregulation of the colanic acid gene cluster (CA) from Escherichia coli, including genes gmd, wcaG, wcaH, wcaI, manC and manB, by introducing a nucleic acid construct encoding CA as shown in SEQ ID NO: 11, is advantageous, GDP-fucose can be formed, which enables the cell to produce higher levels of fucosylated oligosaccharides from one or more intermediate oligosaccharide substrates (e.g., lactose or LNT, LNFP-II and / or LNFP-V). 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.

[0181] It is further understood that the genetically engineered cells described herein may also comprise any of the modifications described above, such as additional glycosyltransferases, overexpression of suitable import proteins such as lactose permease, LNT-II or LNT import protein, inactivation of β-galactosidase (particularly if lactose is used as a starting substrate), and export proteins suitable for the production of complex fucosylated HMOs by the cell.

[0182] Cell-produced HMO mixtures

[0183] Genetically engineered cells containing the α-1,3(4)-fucosyltransferases described herein with dual fucosyltransferase specificity typically produce a mixture of HMOs as a result of a multistep process within the cell to form the HMO product LNDFH-II (see Figure 1 ). In the process of producing LNDFH-II using lactose as the starting substrate, it is expected that the cells will produce a certain amount of 3FL (fucosylated lactose), LNT-II, LNT, LNFP-V and possibly LNFP-II.

[0184] The mole percentages (%) of the various HMO components supported by the experimental data in the examples show exemplary HMO composition ranges, wherein the mixture of the final HMO product consists essentially of 3FL, LNT-II, LNT, LNFP-II, LNFP-V and LNDFH-II 3FL or LNT-II, LNT, LNFP-V and LNDFH-II, wherein the content of 3FL may be very low or not present at all.

[0185] In an embodiment, the cell produces a final mixture comprising LNDFH-II, LNFP-V and LNT. In another embodiment, the cell produces an HMO mixture consisting essentially of a mixture selected from the group consisting of i) LNDFH-II, LNFP-V and LNT, ii) LNDFH-II, LNFP-V and 3FL, iii) LNDFH-II, LNFP-V, LNT and 3FL, iv) LNDFH-II, LNFP-V, LNFP-II, LNT and LNT-II, and v) LNDFH-II, LNFP-V, LNFP-II, LNT, LNT-II and 3FL. In another embodiment, the cell produces a mixture consisting essentially of LNDFH-II, LNFP-V and 3FL. In another embodiment, the cell produces a mixture consisting essentially of LNDFH-II, LNFP-V, LNT and 3FL. In another embodiment, the cell produces a mixture consisting essentially of LNDFH-II, LNFP-V, LNFP-II, LNT, and LNT-II.

[0186] In an embodiment, the cell produces an HMO mixture consisting essentially of 1-40 mol% LNDFH-II, 2-85 mol% LNFP-V, 0-10 mol% LNFP-II, 0-65 mol% 3FL, 0-550% LNT, and 0-50 mol% LNT-II, which add up to a total of 100 mol%.

[0187] In some embodiments, the genetically engineered cells described herein express Bgall1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or comprising or consisting 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 mixture produced consists essentially of 3FL, LNT, LNFP-V, and LNDFH-II, wherein 15-40% of the total molar content of HMOs in the mixture is LNDFH-II, 20-60% of the total molar content of HMOs in the mixture is LNFP-V, 0-15% of the total molar content of HMOs in the mixture is LNT, and 15-60% of the total molar content of HMOs in the mixture is 3FL, which add up to a total of 100% molar content.

[0188] In some embodiments, the genetically engineered cells described herein express Murba1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or comprising or consisting 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 mixture produced consists essentially of 3FL, LNT, LNFP-V, and LNDFH-II, wherein 1-15% of the total molar content of HMOs in the mixture is LNDFH-II, 35-70% of the total molar content of HMOs in the mixture is LNFP-V, 0-25% of the total molar content of HMOs in the mixture is LNT, and 5-55% of the total molar content of HMOs in the mixture is 3FL, which add up to a total of 100% molar content.

[0189] In some embodiments, the genetically engineered cells described herein express Bbac1 comprising or consisting of the amino acid sequence of SEQ ID NO:3, or comprising or consisting 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 produce a mixture consisting essentially of 3FL, LNT, LNFP-V, and LNDFH-II, wherein 2-15% of the total molar content of HMOs in the mixture is LNDFH-II, 60-75% of the total molar content of HMOs in the mixture is LNFP-V, 0-10% of the total molar content of HMOs in the mixture is LNT, and 15-30% of the total molar content of HMOs in the mixture is 3FL, which add up to a total of 100% molar content.

[0190] In some embodiments, the genetically engineered cells described herein express Para1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or comprising or consisting 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 produce a mixture consisting essentially of 3FL, LNT, LNFP-II, LNFP-V, and LNDFH-II, wherein 2-15% of the total molar content of HMOs in the mixture is LNDFH-II, 0-10% of the total molar content of HMOs in the mixture is LNFP-V, 0-10% of the total molar content of HMOs in the mixture is LNFP-II, 30-50% of the total molar content of HMOs in the mixture is LNT-II, 30-60% of the total molar content of HMOs in the mixture is LNT, and 0-50% of the total molar content of HMOs in the mixture is 3FL, adding up to a total of 100% molar content.

[0191] In some embodiments, the genetically engineered cells described herein express FutA comprising or consisting of the amino acid sequence of SEQ ID NO:5, or comprising or consisting 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 produce a mixture consisting essentially of 3FL, LNT, LNFP-V, and LNDFH-II, wherein 2-40% of the total molar content of HMOs in the mixture is LNDFH-II, 35-85% of the total molar content of HMOs in the mixture is LNFP-V, 0-25% of the total molar content of HMOs in the mixture is LNT, and 0-20% of the total molar content of HMOs in the mixture is 3FL, adding up to a total of 100% molar content.

[0192] Host cells

[0193] In an embodiment, the engineered cell is a microorganism. The genetically engineered cell is preferably a microbial cell, such as a prokaryotic cell or a eukaryotic cell. Suitable microbial cells that can be used as host cells include bacterial cells, archaeal cells, algae cells and fungal cells.

[0194] The genetically engineered cell can be, for example, a bacterial or yeast cell. In a preferred embodiment, the genetically engineered cell is a bacterial cell.

[0195] Regarding bacterial host cells, there are no restrictions in principle; they can be true bacteria (Gram-positive or Gram-negative) or archaea, as long as they allow genetic manipulation to insert the gene of interest and can be cultured on a manufacturing scale. Preferably, the host cell has properties that allow cultivation to high cell densities. Non-limiting examples of bacterial host cells suitable for recombinant industrial production of HMOs according to the present invention can be members of the order Enterobacterales, preferably the genus Escherichia coli, more preferably the species Escherichia coli. Other suitable host cell examples are Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Campylobacter sp, Pantoea citrea, Pectobacterium carotovorum or Xanthomonas campestris. Bacillus bacteria, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans, can also be used.Similarly, bacteria of the genera Lactobacillus and Lactococcus can be engineered using the methods of the invention, 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 species of bacteria suitable for use in the invention described herein. Also included as useful species as part of the invention are strains engineered as described herein from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomosporas spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa). aeruginosa)).

[0196] Non-limiting examples of fungal host cells suitable for the recombinant industrial production of heterologous products are, for example, yeast cells, such as Komagataella, Kluyveromyces, Yarrowia, Pichia, Saccaromyces, Schizosaccharomyces or Hansenula, or filamentous fungi from the genera Aspargillus, Fusarium or Thricoderma.

[0197] In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of Escherichia coli, Bacillus, Lactobacillus, Corynebacterium sp., and Campylobacter sp.

[0198] In one or more exemplary embodiments, the genetically engineered cell is selected from Escherichia coli, Bacillus subtilis, lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

[0199] In one or more exemplary embodiments, the genetically engineered cell is Bacillus subtilis.

[0200] In one or more exemplary embodiments, the genetically engineered cell is Saccharomyces cerevisiae or Pichia pastoris.

[0201] In one or more exemplary embodiments, the genetically engineered cell is Escherichia coli.

[0202] In one or more exemplary embodiments, the present invention relates to a genetically engineered cell, wherein the cell is derived from E. coli K-12 strain or DE3.

[0203] Recombinant nucleic acid sequence

[0204] The present invention relates to a genetically engineered cell comprising a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, such as an enzyme selected from Bgall1, Bbac1, Murba1, Para1 and FutA, wherein the cell produces human milk oligosaccharides (HMOs), in particular at least one fucosylated HMO, and preferably having a molar percentage (%) content of the above-mentioned LNDFH-II, or accounting for at least 5% of the total HMOs produced.

[0205] In this context, the terms "recombinant nucleic acid sequence", "recombinant gene / nucleic acid / nucleotide sequence / DNA code" or "coding nucleic acid sequence" are used interchangeably and refer to an artificial nucleic acid sequence (i.e., a sequence produced in vitro using standard experimental methods for preparing nucleic acid sequences) that contains a set of consecutive, non-overlapping triplets (codons) that are transcribed into mRNA and translated into protein when under the control of appropriate control sequences (i.e., promoter sequences).

[0206] The boundaries of the coding sequence are usually determined by the ribosome binding site, transcription start codon (AUG, GUG or UUG) and translation stop codon (UAA, UGA or UAG) located upstream of the open reading frame at the 5' end of the mRNA. The coding sequence may include, but is not limited to, genomic DNA, cDNA, synthetic and recombinant nucleic acid sequences.

[0207] The term "nucleic acid" includes RNA, DNA and cDNA molecules. It is understood that due to the degeneracy of the genetic code, a large number of nucleic acid sequences can be produced that encode a given protein.

[0208] 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.

[0209] The recombinant nucleic acid sequence can also be heterologous. "Heterologous" as used herein refers to a polypeptide, amino acid sequence, nucleic acid sequence or nucleotide sequence that is foreign to a cell or organism, i.e., a polypeptide, amino acid sequence, nucleic acid molecule or nucleotide sequence that does not naturally occur in the cell or organism.

[0210] The present invention also relates to a nucleic acid construct comprising a coding nucleic acid sequence, i.e. a recombinant DNA sequence of a target gene (e.g. a fucosyltransferase gene), and a non-coding regulatory DNA sequence, such as a promoter DNA sequence, e.g. a recombinant promoter sequence derived from a promoter sequence of a lac operon or a glp operon, or a promoter sequence derived from another genomic promoter DNA sequence, or a synthetic promoter sequence, wherein the coding sequence and the promoter sequence are operably linked.

[0211] The term "operably linked" refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. It refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter sequence is operably linked to a coding sequence if it stimulates or regulates transcription of the coding sequence in a suitable host cell or other expression system.

[0212] Typically, promoter sequences that are operably linked to a transcribed sequence are physically adjacent to the transcribed sequence, ie, they are cis-acting.

[0213] In one exemplary embodiment, the nucleic acid construct of the present invention may be part of a vector DNA, in another embodiment, the construct is an expression cassette / cassette that is integrated into the host cell genome.

[0214] Therefore, the term "nucleic acid construct" refers to an artificially constructed nucleic acid fragment, in particular 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 contained in the construct. Therefore, in an embodiment, the present invention relates to a nucleic acid construct comprising a recombinant nucleic acid sequence encoding a fucosyltransferase, wherein the recombinant nucleic acid sequence is selected from the nucleic acid sequences encoding Bgall1, Murba1, Bbac1, Para1 and FutA, for example, according to SEQ ID NO: 6, 7, 8, 9 or 10, or a functional variant thereof.

[0215] The genetically engineered cells described herein can also include multiple copies of the recombinant nucleic acid sequence encoding fucosyltransferase. In Example 1, it is shown that the copy number of fucosyltransferase is increased to change the ratio of HMO produced. Specifically, the results show that by first introducing two genomic copies, and then introducing a high copy number plasmid (pUC57-Bgall1-PglpF-amp) to increase the copy number of Bgall1, first 3FL and LNDFH-II are increased by 1.5 to 1.8 times, while the LNFP-V produced is reduced by 1.5 times. The overexpression of the plasmid causes a slight increase in the output of 3FL, while the LNFP-V and LNDFH-II produced are reduced. In addition, the copy number of Murba1 is increased to two genomic copies, and further including a high copy plasmid (pUC57-Murba1-PglpF-amp), the relative output of LNDFH-II and 3FL is increased, while the output of LNT and LNFP-V is reduced. Increasing the Para1 copy number from one to two copies resulted in a five-fold increase in the relative production of LNDFH-II, with a concomitant increase in the relative amount of LNT-II and a decrease in LNT. Increasing the FutA copy number unexpectedly resulted in a 2.5-fold increase in the relative production of LNDFH-II when one additional copy was added, and an almost eight-fold increase when expressed from a plasmid, with a concomitant increase in the relative production of 3FL and a decrease in the relative production of LNT and LNFP-V.

[0216] Therefore, copy number variation can be used to produce customized HMO mixtures, in this case containing different ratios of 3FL, LNT-II, LNT, LNFP-V, and LNDFH-II, depending on the needs of a specific product.

[0217] Thus, in an embodiment, the genetically engineered cell described herein comprises one, two, three or more genomic copies of a recombinant nucleic acid sequence encoding a glycosyltransferase selected from the group consisting of Bgall1, Murba1, Bbac1, Para1 and FutA, comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, 2, 3, 4 or 5, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1, 2, 3, 4 or 5. In a further embodiment, the recombinant nucleic acid sequence encoding a glycosyltransferase selected from the group consisting of Bgall1, Murba1, Bbac1, Para1 and FutA, comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, 2, 3, 4 or 5, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1, 2, 3, 4 or 5. In additional embodiments, the plasmid is a high copy number plasmid, preferably a pUC57 or pBB-B9 plasmid.

[0218] In additional embodiments, the genetically engineered cells described herein comprise at least two genomic copies of a recombinant nucleic acid sequence encoding a fucosyltransferase FutA, the recombinant nucleic acid sequence comprising or consisting of an amino acid sequence according to SEQ ID NO: 5, or a functional homolog thereof, the amino acid sequence of the functional homolog having at least 80% identity to SEQ ID NO: 5. In additional embodiments, the cell may further comprise at least one plasmid-borne copy of a recombinant nucleic acid encoding a fucosyltransferase FutA.

[0219] In a further embodiment, the genetically engineered cell described herein comprises one, two, three or more genomic copies and / or plasmid-borne copies of a recombinant nucleic acid sequence encoding a glycosyltransferase selected from Bgall1, Murba1 and Bbac1, which comprises or consists of an amino acid sequence according to SEQ ID NO: 1, 2 or 3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1, 2 or 3.

[0220] One embodiment relates to a nucleic acid construct comprising a recombinant nucleic acid sequence encoding a fucosyltransferase, wherein the recombinant nucleic acid sequence is selected from a) Bgall1, which comprises or consists of a nucleic acid sequence of SEQ ID NO:6, or comprises or consists of a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or for example at least 99% sequence identity with SEQ ID NO:6; b) Murba1, which comprises or consists of a nucleic acid sequence of SEQ ID NO:7, or comprises or consists of a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or for example at least 99% sequence identity with SEQ ID NO:7; c) Bbac1, which comprises or consists of a nucleic acid sequence of SEQ ID NO:8, or comprises or consists of a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or for example at least 99% sequence identity with SEQ ID NO:8, and / or d) Para1, which comprises or consists of SEQ ID NO: ID NO:9, or comprises or consists of 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:9, and e) FutA, which comprises or consists of a nucleic acid sequence of SEQ ID NO:10, or comprises or consists of 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:10.

[0221] Preferably, the fucosyltransferase coding sequence is under the control of a promoter sequence selected from the promoter sequences having the nucleic acid sequence identified in Table 3.

[0222] Table 3 - Selected promoter sequences

[0223]

[0224] * Promoter activity was assessed in the LacZ assay described below, with the PglpF promoter as a positive reference in the same assay. To compare different assays, activity was calculated relative to the PglpF promoter, and ranges represent results from multiple assays.

[0225] The promoter may be heterologous, a promoter native to the genetically engineered cell, or it may be a recombinant promoter, combining heterologous and / or native elements.

[0226] One approach to increasing product yields may be to modulate the activity of an enzyme required for production of the product, such as a glycosyltransferase or an enzyme involved in the glycosyl donor biosynthetic pathway.

[0227] Increasing the promoter strength that drives the expression of the desired enzyme can be a method to achieve this purpose. The strength of the promoter can be evaluated using the lacZ enzyme test method, wherein the method for determining the activity of β-galactosidase is as described above (e.g., with reference to Miller JH Experiments in molecular Genetics, Cold spring Harbor Laboratory Press, NY, 1972). In short, the cells are diluted in Z buffer and permeabilized with sodium dodecyl sulfate (0.1%) and chloroform. The LacZ test method is carried out at 30°C. Preheat the sample, start the test by adding 200 μl of o-nitrophenyl-β-galactosidase (4 mg / ml), and stop the test by adding 500 μl of 1M Na2CO3 when the sample turns slightly yellow. The release of o-nitrophenol is subsequently determined based on the change in optical density at 420 nm. Specific activity is expressed in Miller units (MU) [A420 / (min*ml*A600)]. A regulatory element with an activity higher than 10,000MU is considered strong, a regulatory element with an activity lower than 3,000MU is considered weak, and those in between have medium strength. An example of a strong regulatory element is the PglpF promoter with an activity of about 14.000MU, and an example of a weak promoter is Plac, which has an activity of about 2300MU when induced with IPTG. In a preferred embodiment, the expression of the nucleic acid sequence is controlled by a strong promoter selected from SEQ ID NOs 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26.

[0228] In an embodiment, the expression of the nucleic acid sequence described herein is controlled by the PglpF (SEQ ID NO: 27) or Plac (SEQ ID NO: 36) promoter or PmglB_UTR70 (SEQ ID NO: 24) or PglpA_70UTR (SEQ ID NO: 25) or PglpT_70UTR (SEQ ID NO: 26) or variants thereof (e.g., the promoters identified in Table 3), particularly the PglpF_SD4 variant of SEQ ID NO: 22 or the Plac_70UTR variant of SEQ ID NO: 18, or the PmglB_70UTR variants of SEQ ID NO: 15, 16, 17, 19, 20, 21, 23 and 24. Further suitable variants of the PglpF, PglpA_70UTR, PglpT_70UTR and PmglB_70UTR promoter sequences are described in WO2019 / 123324 and WO2020 / 255054, respectively (incorporated herein for reference).

[0229] In a preferred embodiment, the recombinant nucleic acid sequence is individually controlled by one or more promoters selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 27, 36, 24, 25 and 26, respectively) and variants thereof.

[0230] The nucleic acid construct of interest contained in the construct (expression cassette) can be integrated into the bacterial genome by conventional methods, for example, by using a linear fragment containing flanking sequences homologous to specific sites on the chromosome, such as described for the attTn7 site (Waddell CS and Craig NL, Genes Dev. (1988) February; 2(2): 137-49.); methods for the genomic integration of recombinant nucleic acid sequences 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. (2003); 1(4): 237-243; Biol. (2005), 12(3):349-56.; Vetcher et al., Appl Environ Microbiol. (2005); 71(4):1829-35); or positive clones, ie clones carrying the expression cassette, can be selected by, for example, loss or gain of marker gene or gene function.

[0231] In one or more exemplary embodiments, the present disclosure relates to one or more recombinant nucleic acid sequences as shown in SEQ ID NOs: 6, 7, 8, 9 and 10 [nucleic acids encoding Bgall1, Murba1, Bbac1, Para1 and FutA, respectively].

[0232] In particular, the present disclosure relates to one or more recombinant nucleic acid sequences and / or functional homologs thereof, the sequences of 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 95% identity, at least 98% identity or 100% identity to SEQ ID NO: 6, 7, 8, 9 and 10 [nucleic acids encoding Bgall1, Murba1, Bbac1, Para1 and FutA, respectively].

[0233] Sequence identity

[0234] The term "sequence identity" as used herein describes the relevance of two amino acid sequences or two nucleotide sequences based on their paired comparisons, i.e., a candidate sequence (e.g., a sequence of the present invention) and a reference sequence (e.g., a prior art sequence). For purposes disclosed herein, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo / . Biol. 48: 443-453), 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 gap open penalty (gap open penalty) 10, gap extension penalty (gap extension penalty) 0.5, and EBLOSUM62 (EMBOSS version of 30BLOSUM62) substitution matrix. The Needle output (obtained using the -nobrief option) labeled as "identity" is used as percentage identity. Usually, sequence identity can be calculated as follows: (same residue × 100) / (length of comparison region).

[0235] For the purpose disclosed herein, the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), 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), 10 preferred versions 5.0.0 or higher. The parameters used are gap open penalty 10, gap extension penalty 0.5 and DNAFULL (EMBOSS version is NCBI NUC4.4) substitution matrix. The Needle output (obtained using the -nobrief option) labeled as "identity" is used as percentage identity. Typically, sequence identity can be calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment region).

[0236] Functional homologs

[0237] Functional homologues or functional variants of protein / nucleic acid sequences described herein are protein / nucleic acid sequences with altered genetic codes that retain their original functions. Functional homologues can be obtained by mutation, or can be natural variants from the same or other species. Compared with the function of the protein / nucleic acid sequence, functional homologues should have at least 50% of the remaining function, such as at least 60%, 70%, 80%, 90% or 100%.

[0238] Functional homologs of any of the disclosed amino acid or nucleic acid sequences may also have higher functions. Functional homologs of any of the amino acid sequences shown in Table 1 or recombinant nucleic acids encoding any of the sequences in SEQ ID NOs: 6, 7, 8, 9 and 10 should ideally be able to participate in the production of fucosylated HMOs, including increasing HMO production, exporting HMO products out of cells or importing substrates for producing HMOs (e.g., acceptor oligosaccharides of at least three monosaccharide units), improving purity / byproduct formation, reducing biomass formation, the viability of genetically engineered cells, the robustness of genetically engineered cells according to the present disclosure, or reducing the consumables required for production.

[0239] Uses of genetically engineered cells or enzymes

[0240] The present disclosure 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 of producing one or more fucosylated human milk oligosaccharides (HMOs), preferably LNDFH-II.

[0241] Therefore, the present disclosure also relates to the use of a fucosyltransferase having α-1,3(4)-fucosyltransferase activity in the production of a fucosylated product comprising LNDFH-II, wherein the fucosyltransferase is selected from Bgall1, Murba1, Bbac1, Para1 and FutA, which comprises or consists of an amino acid sequence according to SEQ ID NO: 1, 2, 3, 4 or 5, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1, 2, 3, 4 or 5.

[0242] In a further embodiment, the fucosyltransferase used to produce the fucosylated product is selected from Bgall1, Murba1 and Bbac1, which comprises or consists of the amino acid sequence according to SEQ ID NO: 1, 2 or 3, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1, 2 or 3.

[0243] In embodiments, the fucosyltransferases described herein are also used to produce a fucosylated product, wherein the fucosylated product comprises one or more fucosylated oligosaccharides that include LNDFH-II.

[0244] In an exemplary embodiment, the genetically engineered cells and / or nucleic acid constructs described herein are used to produce HMOs. Preferably, when producing a mixture of HMOs, the mole % content of LNDFH-II produced by the genetically engineered cells is higher than 5% of the total amount of HMOs produced. Preferably, when producing HMOs, the fucosylated HMO mixture containing LNDFH-II is intended as the main product, and the mole % content of LNFP-III produced by the genetically engineered cells is higher than 5% of the total amount of HMOs produced, such as higher than 10%, such as higher than 15%, such as higher than 20%, such as higher than 25%, such as higher than 30%, or such as higher than 35%.

[0245] In a further embodiment, the fucosyltransferase used to produce the fucosylated product is selected from Bgall1, Murba1, Bbac1, Para1, comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, 2, 3 or 4, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO: 1, 2, 3 or 4. In an embodiment, the fucosyltransferase described herein is also used to produce a fucosylated product, wherein the fucosylated product is one or more fucosylated oligosaccharides, such as one or more HMOs, preferably a mixture of HMOs, wherein at least 60%, such as at least 75%, such as at least 85%, such as at least 90%, such as at least 95% of the mixture consists of LNDFH-II in combination with LNFP-V and / or 3FL.

[0246] In an exemplary embodiment, the genetically engineered cells, α-1,3(4)-fucosyltransferases and / or nucleic acid constructs described herein are used to produce one or more fucosylated HMOs, preferably LNDFH-II or LNDF-II and LNFP-V.

[0247] The production of these HMOs may require the presence of two or more glycosyltransferase activities.

[0248] A method for producing fucosylated human milk oligosaccharides (HMO)

[0249] The present disclosure also relates to a method for producing one or more fucosylated human milk oligosaccharides (HMOs), preferably LNDFH-II, comprising culturing the genetically engineered cells described herein.

[0250] One aspect is a method for producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising the steps of:

[0251] a) providing a genetically modified cell as described herein; and

[0252] b) culturing the cell according to (a) in a suitable cell culture medium to produce said one or more fucosylated HMOs, and

[0253] c) optionally, purifying said one or more fucosylated HMOs.

[0254] The present disclosure relates to a method of producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically engineered cell comprising:

[0255] A recombinant nucleic acid sequence encoding a fucosyltransferase having alpha-1,3(4)-fucosyltransferase activity, wherein the fucosyltransferase is selected from the group consisting of:

[0256] a. Bgall1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1, [Bgall1 amino acid];

[0257] b. Murba1, which comprises or consists of the amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 2, [Murba1 amino acid];

[0258] c. Bbac1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 3, [Bbac1 amino acid], and

[0259] d. Para1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 4, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 4, [Para1 amino acid].

[0260] The present disclosure also relates to a method of producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically engineered cell comprising:

[0261] A recombinant nucleic acid sequence encoding a fucosyltransferase having alpha-1,3(4)-fucosyltransferase activity, wherein the fucosyltransferase is selected from the group consisting of:

[0262] a. Bgall1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1, [Bgall1 amino acid];

[0263] b. Murba1, which comprises or consists of the amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 2, [Murba1 amino acid];

[0264] c. Bbac1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 3, [Bbac1 amino acid], and

[0265] d. Para1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 4, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 4, [Para1 amino acid], and

[0266] Wherein at least one fucosylated HMO is LNDFH-II. The fucosylated HMO produced by the above method may also include a fucosylated HMO selected from 3FL, LNFP-II and LNFP-V, and may also include a non-fucosylated HMO, such as LNT and / or LNT-II.

[0267] Another embodiment is directed to a method of producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically engineered cell comprising:

[0268] a. A recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the fucosyltransferase is selected from:

[0269] i. Bgall1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1, [Bgall1 amino acid];

[0270] ii. Murba1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 2, [Murba1 amino acid];

[0271] iii. Bbac1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 3, [Bbac1 amino acid],

[0272] iv. Para1 comprising or consisting of the amino acid sequence according to SEQ ID NO:4, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO:4, [Para1 amino acid], and

[0273] v. FutA comprising or consisting of the amino acid sequence according to SEQ ID NO: 5, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 5, [FutA amino acid], and

[0274] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-galactosyltransferase activity, and

[0275] c. Optionally, a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity, and

[0276] Wherein the at least one fucosylated HMO is LNDFH-II.

[0277] In an embodiment, the genetically engineered cells are cultured in a suitable medium providing a suitable carbon source and in the presence of a starting substrate selected from lactose or LNT-II. Preferably, the starting substrate is lactose and the cells express an enzyme having β-1,3-N-acetylglucosaminyltransferase activity.

[0278] Another embodiment relates to a method of producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising

[0279] a) culturing a genetically engineered cell comprising

[0280] a. A recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is selected from:

[0281] i. Bgall1, which comprises or consists of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1, [Bgall amino acid];

[0282] ii. Murba1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 2, [Murba1 amino acid]; and

[0283] iii. Bbac1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 3, [Bbac1 amino acid],

[0284] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-galactosyltransferase activity; and

[0285] c. Optionally, a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0286] b) cultivating said cells in a suitable medium in the presence of a starting substrate and wherein at least 5% of the molar content of the HMO produced by the method is LNDFH-II.

[0287] Another embodiment relates to a method for producing LNDFH-II and one or more additional fucosylated HMOs, comprising

[0288] a) providing a genetically engineered cell capable of producing LNDFH-II, comprising

[0289] a. at least two genomic copies of a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase and α-1,4-fucosyltransferase activity, the fucosyltransferase being capable of fucosylating oligosaccharides at the GlcNAc moiety and the Glc moiety, and wherein the fucosyltransferase is selected from:

[0290] i) Bgall1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1; and

[0291] ii) FutA comprising or consisting of the amino acid sequence according to SEQ ID NO: 5, or a functional homologue thereof, the amino acid sequence of which is at least 95% identical to SEQ ID NO: 5; and

[0292] b. a recombinant nucleic acid sequence encoding β-1,3-galactosyltransferase, and

[0293] c. optionally a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosaminyltransferase, and

[0294] b) cultivating the cells in a suitable medium in the presence of a starting substrate and wherein at least 15%, such as at least 20%, of the molar content of the HMO produced by the method is LNDFH-II.

[0295] In an embodiment, the starting substrate is selected from lactose or LNT-II. Preferably, the starting substrate is lactose and the cell expresses an enzyme having β-1,3-N-acetylglucosaminyltransferase activity.

[0296] In an embodiment, the fucosylated HMO is LNDFH-II. In a further embodiment, one or more HMOs selected from the group consisting of 3FL, LNT, LNFP-II and LNFP-V are produced by the method of the present invention.

[0297] The method specifically includes culturing a genetically engineered cell that produces fucosylated HMOs, wherein the LNDFH-II content produced by the cell is at least 5% of the total HMO content produced by the cell. The method specifically includes culturing a genetically engineered cell that produces fucosylated HMOs, wherein the LNDFH-II and LNFP-V content produced by the method is at least 35% of the total HMO content produced by the cell.

[0298] 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), such as a carbon source selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.

[0299] In one embodiment, the methods disclosed herein produce a mixture of HMOs, wherein at least 50%, e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% by molar content of the total amount of HMOs produced are fucosylated HMOs.

[0300] In one embodiment, the methods disclosed herein produce a mixture of HMOs, wherein at least 5%, such as at least 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, or such as at least 35% of the total amount of HMOs produced is LNDFH-II in a molar content.

[0301] In one embodiment, the method disclosed herein produces a mixture of HMOs, wherein at least 75%, such as at least 80%, 85%, 90%, 95% or at least 97% of the total HMOs produced by the method have a molar content of fucosylated HMOs selected from 3FL, LNFP-II, LNFP-V and LNDFH-II.

[0302] In one embodiment, the methods disclosed herein produce a mixture of HMOs, wherein at least 65%, such as at least 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the total HMOs produced by the method have a molar content of fucosylated HMOs selected from 3FL, LNFP-V and LNDFH-II.

[0303] In one embodiment, the methods disclosed herein produce a mixture of HMOs, wherein the produced mixture of HMOs is substantially free of LNFP-II.

[0304] In one embodiment, the methods disclosed herein produce a mixture of HMOs, wherein the produced HMO mixture is substantially free of 3FL.

[0305] In one embodiment, the methods disclosed herein produce a mixture of HMOs, wherein the cells produce a mixture of HMOs comprising 3FL, LNT, LNFP-V, and LNDFH-II.

[0306] In one embodiment, the methods disclosed herein produce a mixture of HMOs, wherein the cells produce a mixture of HMOs comprising LNT, LNT-II, LNFP-II, LNFP-V, and LNDFH-II.

[0307] In one embodiment, the methods disclosed herein produce LNDFH-II.

[0308] In embodiments, the genetic cells used in the methods described herein are genetically engineered cells having one or more of the modifications previously described.

[0309] The methods disclosed herein include providing a glycosyl donor that is synthesized by one or more genetically engineered cells alone and / or added exogenously to the culture medium from other sources. Preferably, the glycosyl donor is produced in the genetically engineered cells by endogenous or recombinant de novo pathways.

[0310] One embodiment disclosed herein further comprises providing an acceptor sugar as a starting substrate for HMO formation, the acceptor sugar comprising at least two monosaccharide units, which is exogenously added to the culture medium and / or produced by a separate microbial fermentation. As an alternative to adding the starting substrate for producing HMO to the fermentation medium, the genetically modified cells can be further engineered to produce the starting substrate intracellularly (see, for example, WO2015 / 150328).

[0311] In one embodiment, the method disclosed herein comprises providing an acceptor sugar comprising at least two monosaccharide units selected from lactose, LNT-II and LNT, and adding it before and / or during culturing the genetically modified cells. In a preferred embodiment, the starting substrate for HMO formation is lactose, which is added to the culture during fermentation of the genetically engineered cells.

[0312] Fucosylated human milk oligosaccharides (HMOs) are recovered from the culture, from the culture medium or / and from genetically engineered cells.

[0313] Cultivation / fermentation

[0314] Cultivation, cultivation process, fermentation or fermentation process (used interchangeably herein) in a controlled bioreactor generally includes (a) a first phase of exponential growth of cells in a medium ensured by a carbon source, and (b) a second phase of cell growth in a medium operated under carbon limitation, wherein the carbon source is continuously added together with an acceptor oligosaccharide (e.g., lactose) to allow the formation of HMO products during this phase. Carbon (sugar) limitation refers to the phase in the fermentation process where the growth rate is kinetically controlled by the carbon source (sugar) concentration in the culture broth, which in turn is controlled by the rate of carbon addition (sugar feed rate) in the fermentor.

[0315] The terms "manufacturing" or "manufacturing scale" or "large-scale production" or "large-scale fermentation" are used interchangeably and in the meaning of the present invention define a fermentation with a minimum volume of 100 L, such as 1000 L, such as 10.000 L, such as 100.000 L, such as 200.000 L of culture broth. Typically, a "manufacturing scale" process is defined as one that is able to process large volumes of HMO products, e.g., in the case of therapeutic compounds or compositions, to meet the needs of toxicity testing, clinical trials and market supply. In addition to the large volumes, unlike simple laboratory scale methods such as shake flask cultures, manufacturing scale methods are characterized by technological systems using bioreactors (fermenters) equipped with stirring, aeration, nutrient feeding, monitoring and control devices for 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, benchtop bioreactors or the deep well format described in the examples of the present disclosure, does allow to predict the behavior of the system in the complex environment of the bioreactor.

[0316] There are no restrictions on suitable cell culture media used in the fermentation process. The culture medium can be semi-defined, i.e., containing complex culture media compounds (e.g., yeast extract, soy peptone, casamino acids, etc.), or 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 and carbon sources selected from glycerol, sucrose and glucose. In other embodiments, lactose is added during the cultivation of genetically engineered cells as a substrate for HMO formation.

[0317] 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.

[0318] In one or more exemplary embodiments, the genetically engineered cell comprises a PTS-dependent sucrose utilization system further comprising the scrYA and scrBR operons described in WO2015 / 197082 (incorporated herein by reference).

[0319] 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.

[0320] Recycling / Harvesting

[0321] Recovering fucosylated human milk oligosaccharides (HMO) from culture medium and / or genetically engineered cells. In the context, the term "recovery" is used interchangeably with the term "harvest". Both "recovery" and "harvesting" in the context refer to collecting the produced HMO from the culture / fermentation broth after the fermentation is terminated. In one or more exemplary embodiments, it may include collecting the HMO contained in the biomass (i.e., host cells) and the culture medium, i.e., before / without separation of the fermentation broth from the biomass. In other embodiments, the produced HMO may be collected separately from the biomass and the fermentation broth, i.e., after / subsequently separation of the biomass from the culture medium (i.e., the fermentation broth).

[0322] The separation of cells from the culture medium can be performed by any of the methods known to those skilled in the art, such as any suitable type of centrifugation or filtration. The separation of cells from the culture medium can be performed immediately after harvesting the fermentation broth, or can be performed at a later stage when the fermentation broth is stored under appropriate conditions. Recovering the produced HMO from the remaining biomass (or the total fermentation broth) includes extracting the HMO from the biomass (i.e., the production cells).

[0323] After recovery from fermentation, HMOs can be used for further processing and purification.

[0324] HMOs can be purified according to methods known in the art, such as described in WO2017 / 152918, WO2017 / 182965 or WO2015 / 188834, the latter of which describes the purification of fucosylated HMOs. The purified HMOs can be used as nutraceuticals, pharmaceuticals or for any other purpose, such as for research.

[0325] At the end of the culture, oligosaccharides can accumulate as products both within cells and in the extracellular matrix.

[0326] The methods described herein include culturing genetically engineered microbial cells in a culture medium designed to support the growth of the microorganism and comprising one or more carbohydrate sources or only carbon sources, 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.

[0327] Manufactured products

[0328] The term "manufactured product" refers to one or more HMOs that are intended as a composition of one or more product HMOs or a mixture of HMOs. Preferably, the product HMO or composition is produced by the methods described herein using the genetically engineered cells described herein.

[0329] One embodiment relates to an HMO composition consisting essentially of LNDFH-II, LNFP-V, LNT and 3FL, wherein the content of LNT-II and LNFP-II is relatively low, for example, less than 10% of the total molar content of HMO in the composition.

[0330] Another embodiment is directed to an HMO composition consisting essentially of 15-30 mol% LNDFH-II, 20-60 mol% LNFP-V, 0-15 mol% LNT, and 15-60% 3FL, which add up to a total of 100 mol%.

[0331] Another embodiment is directed to an HMO composition consisting essentially of 1-10 mol% LNDFH-II and 35-70 mol% LNFP-V, 0-25 mol% LNT and 5-55% 3FL, which add up to a total of 100 mol%.

[0332] Another embodiment is directed to an HMO composition consisting essentially of 2-12 mol% LNDFH-II and 62-72 mol% LNFP-V, 0-10 mol% LNT, and 17-27% 3FL, which add up to a total of 100 mol%.

[0333] Another embodiment is directed to an HMO composition consisting essentially of 1-15 mol% LNDFH-II and 2-10 mol% LNFP-V, 2-10 mol% LNFP-II, 30-55 mol% LNT, and 35-50% LNT-II, adding up to a total of 100 mol%.

[0334] Another embodiment is directed to an HMO composition consisting essentially of 2-40 mol% LNDFH-II and 40-82 mol% LNFP-V, 2-25 mol% LNT and 0-20% 3FL, which add up to a total of 100 mol% content.

[0335] Advantageously, the methods disclosed herein provide valuable HMO mixtures having high levels of fucosylated HMOs including the complex HMO LNDFH-II. Some of the genetically engineered cells described herein produce sufficient LNDFH-II, e.g., greater than 20% of the total HMOs, to facilitate its purification from the HMO mixture produced by culture.

[0336] The manufactured product may be a powder, composition, suspension or gel comprising one or more HMOs.

[0337] Use of HMO compositions or mixtures

[0338] HMOs are naturally present in human milk and have evolved over thousands of years, and existing studies (clinical and preclinical) on HMOs have shown that supplementing with appropriate levels of specific HMOs can provide us with unique health benefits. Since fucosylated HMOs account for greater than 60% of the total HMOs in human milk, such as greater than 75%, such as greater than 85%, such as greater than 90%, such as greater than 95%, a mixture with a high content of fucosylated HMOs is more natural.

[0339] Therefore, LNDFH-II and HMO mixtures containing LNDFH-II are of high relevance as either nutritional supplements or therapeutic agents.

[0340] Clinical data in infants suggest that human milk oligosaccharide supplementation may help develop a desirable microbiota by acting as a food source for beneficial gut bacteria. In particular, human milk oligosaccharide supplementation may help support immunity and gut health, with a potential role in cognitive development, which could open the door to future innovation opportunities.

[0341] HMO mixtures or compositions can be used to enhance beneficial bacteria in the intestinal microbiota. Beneficial bacteria are, for example, bacteria of the genus Bifidobacterium, Lactobacillus, or Barnesiella sp. The enhancement of beneficial bacteria may in turn lead to increased production of short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, which have been shown to have many benefits for infants and young children, such as inhibiting pathogens, preventing infection and diarrhea, and reducing the risk of allergies and metabolic disorders (see, for example, WO2006 / 130205, WO2017 / 129644, WO2017 / 129649).

[0342] The HMO mixtures or compositions produced according to the methods described herein can be used to reduce the number of harmful viruses and bacteria in the intestinal microbiota. Examples of pathogenic bacteria and viruses that can be reduced by the HMO mixtures described herein include Candida albicans, Clostridium difficile, Enterococcus faecium, Escherichia coli, Helicobacter pylori, Streptococcus agalactiae, Shigella dysenteriae, Staphylococcus aureus, nora virus, and rotavirus. Each of the compositions described herein can also be used to treat and / or reduce the risk of infection of humans with a variety of bacteria.

[0343] Probiotics can be consumed as live bacteria or dried (e.g., freeze-dried) products. Regardless of the drying method, rehydration is an important step in the recovery of dehydrated bacteria; an insufficient rehydration / regeneration step may result in poor cell viability and ultimately low survival. Therefore, rehydration is a key step in the recovery of freeze-dried cultures. For both live and rehydrated bacteria, bacterial survival under acidic conditions is critical because they need to pass through the acidic environment of the stomach and may also face storage (shelf life) in acidic foods.

[0344] The HMO mixture or composition produced according to the methods described herein can be used to improve the regeneration and vitality of freeze-dried probiotics including probiotics of the genus Bifidobacterium and / or Lactobacillus. Examples of Bifidobacteria that may have improved regeneration and vitality are Bifidobacterium animals lactis BB12 DSM 32269, Bifidobacterium animals lactis 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 subsp. animalis DSM 16284. Examples of Lactobacillus species 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, Lactobacillus reuteri DSM 12246, Lactobacillus plantarum TIFN101, Lactobacillus gasseri Lg-36 200B FloraFit Danisco, Lactobacillus casei DSM 32382, Lactobacillus paracasei, Lactobacillus plantarum PS128, Lactobacillus plantarum (Sacco) DSM 32383, Lactococcus lactis PAREVE, Lactobacillus paracaseis sp. Paracasei and / or Lactobacillus Lactobacillus reuteri S12 DSM 33752.

[0345] In the context of this application, "regeneration" refers to the process of regaining / restoring the vitality of dried bacteria (i.e. "reviving" bacterial cells by rehydration, where "rehydration" means restoring liquid). This process is sometimes also called "reconstitution".

[0346] In the context of the present application, "viability" is the ability of bacterial cells to survive and function as living cells. One method of determining bacterial cell viability is to plate them on agar plates with a suitable growth medium and count the number of colonies formed after incubation for a predetermined time (plate count). Alternatively, FACS analysis can be used.

[0347] In the context of the present application, "improved regeneration" of Bifidobacterium and / or Lactobacillus bacteria means an increase in the number (number) of successfully regenerated / resuscitated Bifidobacterium and / or Lactobacillus bacteria compared to a corresponding control (i.e. the number / number of Bifidobacterium and / or Lactobacillus bacteria without the addition of HMO).

[0348] In the context of the present application, "improved viability" of Bifidobacterium and / or Lactobacillus bacteria means an increase in the number (number) of Bifidobacterium and / or Lactobacillus bacteria compared to a corresponding control (i.e. the number / number of Bifidobacterium and / or Lactobacillus bacteria without addition of HMO).

[0349] The HMO mixtures or compositions produced according to the methods described herein can be used to extend the shelf life of probiotics, such as Bifidobacterium and / or Lactobacillus.

[0350] One embodiment of the invention is a composition comprising a HMO mixture as described herein (particularly in the "HMO mixture" section) and one or more probiotics. Preferably, the probiotics are of the genus Bifidobacterium and / or Lactobacillus, such as any of the specific species mentioned above.

[0351] The HMO mixtures or compositions produced according to the methods described herein can be used to improve the flow properties of powders or to reduce the viscosity of liquids.

[0352] The HMO compositions and mixtures described in the "Product of Manufacture" section may also constitute part of a composition comprising other parts, such as active pharmaceutical ingredients, food supplements, excipients, surfactants, and the like.

[0353] Thus, embodiments relate to the use of the HMO compositions or mixtures described herein as dietary supplements or medical nutrition. In further embodiments, a composition for infant formula, dietary supplements or medical nutrition comprises LNDFH-II, 3FL and LNFP-V.

[0354] The HMO mixtures or compositions described herein are used in nutritional compositions. The nutritional compositions are, for example, infant formula, rehydration, or dietary maintenance, medical nutrition, or supplements for the elderly or immunocompromised population. Such anti-infective compositions may also include macronutrients such as edible fats, carbohydrates, and proteins. Edible fats include, for example, coconut oil, soybean oil, and mono- and diglycerides. Carbohydrates include, for example, glucose, edible lactose, and hydrolyzed corn starch. Proteins include, for example, soy protein, whey, and skim milk. Such anti-infective compositions may also include vitamins and minerals (e.g., calcium, phosphorus, potassium, sodium, chlorine, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E, D, C, and B complex).

[0355] In an embodiment, a composition comprising a mixture of HMOs produced as described herein is a pharmaceutical composition.

[0356] The present invention also relates to the use of a mixture or composition as described herein as a dietary supplement and / or medical nutrition.

[0357] In an embodiment, the present invention relates to the use of a mixture or composition as described herein in infant nutrition.

[0358] sequence

[0359] This application contains a Sequence Listing in both text and electronic formats, which is hereby incorporated by reference.

[0360] An overview of the SEQ ID NOs used in the present application can be found in Table 1 (α-1,3(4)-fucosyltransferase protein sequence) and Table 3 (promoter sequence), additional sequences described in the present application are the DNA sequences encoding α-1,3(4)-fucosyltransferase (SEQ ID NOs: 6 to 10), the DNA sequence encoding the colanic acid gene cluster from Escherichia coli (SEQ ID NO: 11) and the β-1,3-N-acetylglucosaminyltransferase LgtA from Neisseria meningitidis (SEQ ID NO: 12), the β-1,3-galactosyltransferase galTK from Helicobacter pylori (SEQ ID NO: 13), the lactose permease LacY (SEQ ID NO: 14), the FucTIII α-1,3-fucosyltransferase (SEQ ID NO: 39) and the DNA sequence encoding it (SEQ ID NO: 40).

[0361] project

[0362] 1. A genetically engineered cell capable of producing LNDFH-II, comprising a recombinant nucleic acid sequence encoding a fucosyltransferase, wherein the glycosyltransferase is selected from:

[0363] a. Bgall1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1,

[0364] b. Murba1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 2,

[0365] c. Bbac1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 3, and

[0366] d. Para1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 4, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 4.

[0367] 2. The genetically engineered cell according to item 1, wherein the fucosyltransferase has α-1,3(4)-fucosyltransferase activity.

[0368] 3. The genetically engineered cell according to item 1 or 2, wherein the fucosyltransferase is capable of fucosylating the N-acetylglucosamine (GlcNAc) moiety and the glucose (Glc) moiety in the acceptor oligosaccharide.

[0369] 4. The genetically engineered cell according to items 1 to 3, wherein the fucosyltransferase shows low or no specificity for the galactose moiety in LNT as the acceptor oligosaccharide.

[0370] 5. The genetically engineered cell according to item 3 or 4, wherein for α-1,3(4)-fucosyltransferase, the acceptor oligosaccharide is selected from LNT, LNFP-V and LNFP-II.

[0371] 6. The genetically engineered cell according to any of the preceding items, wherein the genetically engineered cell comprises one or more additional recombinant nucleic acids encoding one or more heterologous glycosyltransferases.

[0372] 7. The genetically engineered cell according to any one of the preceding items, wherein the cell further comprises a recombinant nucleic acid sequence encoding β-1,3-galactosyltransferase.

[0373] 8. The genetically engineered cell according to item 6 or 7, wherein the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosaminyltransferase.

[0374] 9. The genetically engineered cell according to any one of items 7 or 8, wherein the β-1,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis and the β-1,3-galactosyltransferase is from Helicobacter pylori.

[0375] 10. The genetically engineered cell according to items 6 to 9, wherein the 1,3-N-acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO: 12, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 12, and the β-1,3-galactosyltransferase has an amino acid sequence according to SEQ ID NO: 13, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 13.

[0376] 11. The genetically engineered cell according to any one of the preceding items, wherein the cell further comprises a substrate importin selected from the group consisting of lactose importin, lacto-N-triose-II (LNT-II) importin or LNT importin.

[0377] 12. The genetically engineered cell according to any of the preceding items, wherein the cell also produces greater than 1 mol % of LNDFH-II, preferably greater than 5 mol %, preferably greater than 10 mol %, preferably greater than 15 mol %, preferably greater than 20 mol % of LNDFH-II, based on the total amount of HMOs produced by the cell.

[0378] 13. The genetically engineered cell according to any one of the preceding items, wherein the cell also produces one or more HMOs selected from 3FL, LNT-II, LNT, pLNH2, LNFP-II and LNFP-V.

[0379] 14. The genetically engineered cell according to any of the preceding items, wherein the cell produces or consists of a fucosylated HMO mixture comprising LNFP-V and LNDFH-II.

[0380] 15. The genetically engineered cell according to any of the preceding items, wherein the cell further produces an HMO mixture comprising, or consisting essentially of

[0381] a. LNDFH-II, LNFP-V and LNT, or

[0382] b. LNDFH-II, LNFP-V and 3FL, or

[0383] c.LNDFH-II, LNFP-V, LNT and 3FL, or

[0384] d.LNDFH-II, LNT-II, LNFP-V, LNFP-II and LNT.

[0385] 16. The genetically engineered cell according to any of the preceding items, wherein the cell produces more than 75 mol%, such as more than 85 mol%, such as more than 90 mol%, such as more than 95 mol% of the total HMO produced by the cell.

[0386] 17. The genetically engineered cell according to any of the preceding items, wherein the cell produces a mixture of 3FL, LNFP-V and LNDFH-II, wherein the mixture accounts for at least 90 mol% of the total HMOs produced by the cell.

[0387] 18. A genetically engineered cell according to any one of the preceding items, wherein the recombinant nucleic acid sequence is individually controlled by one or more promoters selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NO: 27, 36, 24, 25 and 26) and variants thereof as disclosed in Table 3.

[0388] 19. The genetically engineered cell according to item 18, wherein the promoter is a strong promoter selected from SEQ ID NO 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27.

[0389] 20. A genetically engineered cell according to any of the preceding items, wherein the cell comprises two, three or more genomic copies and / or plasmid-borne copies of a recombinant nucleic acid sequence encoding a glycosyltransferase selected from the group consisting of Bgall1, Murba1, Bbac1 and Para1, wherein Bgall1, Murba1, Bbac1 and Para1 comprise or consist of an amino acid sequence according to SEQ ID NO: 1, 2, 3 or 4, or a functional homologue thereof, wherein the amino acid sequence of the functional homologue has at least 80% identity with SEQ ID NO: 1, 2, 3 or 4.

[0390] 21. The genetically engineered cell according to item 20, wherein the cell comprises two, three or more genomic copies of Bgall1, Murba1, bac1 or Para1.

[0391] 22. The genetically engineered cell according to item 20, wherein the cell comprises a high copy number plasmid encoding Bgall1, Murba1, bac1 or Para1.

[0392] 23. The genetically engineered cell according to any of the preceding items, wherein the cell overexpresses at least one enzyme in the de novo GDP-fucose pathway that is responsible for GDP-fucose formation.

[0393] 24. The genetically engineered cell according to item 23, wherein the one or more enzymes are selected from mannose-6-phosphate isomerase (manA), phosphomannose mutase (manB), mannose-1-phosphate guanylate transferase (manC), GDP-mannose-4,6-dehydratase (gmd) and GDP-L-fucose synthase (wcaG).

[0394] 25. The genetically engineered cell according to any of the preceding items, wherein the cell comprises a recombinant nucleic acid sequence according to SEQ ID NO: 11 encoding the colanic acid (CA) gene cluster.

[0395] 26. The genetically engineered cell according to any one of the preceding items, wherein the engineered cell is a microorganism.

[0396] 27. The genetically engineered cell according to any one of the preceding items, wherein the engineered cell is a bacterium or a fungus.

[0397] 28. The genetically engineered cell according to item 27, wherein the fungus is selected from yeast cells of the genera Komagata, Kluyveromyces, Yarrowia, Pichia, Saccharomyces, Schizosaccharomyces or Hansenula, or a filamentous fungus selected from the genera Aspergillus, Fusarium or Trichoderma.

[0398] 29. The genetically engineered cell according to item 27, wherein the bacterium is selected from the group consisting of Escherichia coli, Bacillus, Lactobacillus, Corynebacterium and Campylobacter.

[0399] 30. The genetically engineered cell according to any one of items 26 to 29, wherein the engineered cell is selected from Escherichia coli, Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris and Saccharomyces cerevisiae.

[0400] 31. The genetically engineered cell according to item 29 or 30, wherein the engineered cell is an Escherichia coli microorganism.

[0401] 32. A method for producing one or more fucosylated HMOs, the method comprising culturing a genetically engineered cell comprising a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the fucosyltransferase is selected from the group consisting of:

[0402] a. Bgall1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1,

[0403] b. Murba1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 2,

[0404] c. Bbac1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 3,

[0405] d. Para1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 4, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 4, and

[0406] e. FutA comprising or consisting of the amino acid sequence according to SEQ ID NO: 5, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 5, and

[0407] Wherein the at least one fucosylated HMO is LNDFH-II.

[0408] 33. The method of claim 32, wherein at least 5% of the total molar content of HMOs produced by the genetically engineered cells cultured in said method is LNDFH-II.

[0409] 34. A method according to item 32 or 33, wherein at least 65%, such as at least 70%, 80%, 90%, 95% or at least 97% of the total HMO molar content produced by the method is fucosylated HMO selected from 3FL, LNFP-V and LNDFH-II.

[0410] 35. The method according to any one of items 32 to 34, wherein the genetically engineered cell is a cell according to any one of items 1 to 31.

[0411] 36. The method according to item 35, wherein the method comprises culturing the genetically engineered cell in the presence of an energy source selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.

[0412] 37. The method according to any one of items 32 to 36, wherein the genetically engineered cell is cultured in the presence of an acceptor substrate selected from lactose, LNT-II or LNT.

[0413] 38. The method according to items 32 to 37, wherein the fucosylated HMO produced by said method is purified to a purity of at least 95%.

[0414] 39. Use of a fucosyltransferase having α-1,3 / 4-fucosyltransferase activity in the production of one or more fucosylated HMOs, wherein the enzyme is selected from the group consisting of Bgall1, Murba1, Bbac1, Para1 and FutA or a functional homologue thereof, wherein the Bgall1, Murba1, Bbac1, Para1 and FutA comprises or consists of an amino acid sequence according to SEQ ID NO: 1, 2, 3, 4 or 5, the amino acid sequence of the functional homologue having at least 80% identity with SEQ ID NO: 1, 2, 3, 4 or 5, wherein one of the HMOs produced is LNDFH-II.

[0415] 40. An HMO mixture produced using the method of any one of items 32 to 38, consisting essentially of:

[0416] a. LNDFH-II, LNFP-V and LNT, or

[0417] b. LNDFH-II, LNFP-V and 3FL, or

[0418] c.LNDFH-II, LNFP-V, LNT and 3FL, or

[0419] d.LNDFH-II, LNT-II, LNFP-V, LNFP-II and LNT.

[0420] 41. An HMO composition consisting essentially of 1-40 mol% LNDFH-II, 2-85 mol% LNFP-V, 0-10 mol% LNFP-II, 0-65 mol% 3FL,

[0421] 0-55 mol% LNT and 0-50 mol% LNT-II, which add up to a total of 100 mol% content.

[0422] 42. The composition according to item 41, wherein the mixture is selected from:

[0423] a. A mixture consisting essentially of 15-40 mol % LNDFH-II and 20-60 mol % LNFP-V, 0-15 mol % LNT and 15-60 mol % 3FL, the total molar content added being 100%;

[0424] b. A mixture consisting essentially of 15-40 mol % LNDFH-II and 15-40 mol % LNFP-V, 0-5 mol % LNT and 25-60 mol % 3FL, the total molar content being added to 100%;

[0425] c. A mixture consisting essentially of 1-15 mol % LNDFH-II and 35-70 mol % LNFP-V, 0-25 mol % LNT and 5-55% 3FL, the total molar content being added to 100%;

[0426] d. A mixture consisting essentially of 2-15 mol % LNDFH-II and 60-75 mol % LNFP-V, 0-10 mol % LNT and 15-30 mol % 3FL, adding up to a total of 100 mol %; and

[0427] e. A mixture consisting essentially of 2-15 mol % LNDFH-II and 0-10 mol % LNFP-V, 0-10 mol % LNFP-II, 30-50 mol % LNT-II, 30-60 mol % LNT and 0-5 mol % 3FL, the total molar content being 100%, and

[0428] f. A mixture consisting essentially of 2-40 mol % LNDFH-II and 35-85 mol % LNFP-V, 0-25 mol % LNT and 0-20 mol % 3FL, adding up to a total of 100 mol % content.

[0429] 43. Use of the HMO mixture or composition according to items 40 to 42 in infant formula, dietary supplements and / or medical nutrition.

[0430] Example

[0431] method

[0432] Unless otherwise indicated, nucleic acid manipulation, conversion and expression are performed using standard techniques, vectors, control sequence elements and other expression system elements known in the field of molecular biology. Such standard techniques, vectors and elements can be found in the following literature: Ausubel et al. (write), Current Protocols in Molecular Biology (1995) (John Wiley & Sons); Sambrook, Fritsch, & Maniatis (write), 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. (write), DNA Insertion Elements, Plasmids and Episomes (1977) (Cold Spring Harbor Laboratory Press, NY); Miller, JH Experiments in molecular genetics (1972.) (Cold Spring Harbor Laboratory Press, NY).

[0433] The embodiments described below are chosen to illustrate the present invention and are not intended to limit the present invention in any way.

[0434] Enzymes:

[0435] Screening of 50 enzymes with fucosyltransferase activity revealed that three enzymes unexpectedly exhibited dual fucosyltransferase activity, with α-1,3-fucosyltransferase activity acting on glucose and α-1,4-fucosyltransferase activity acting on the N-acetylglucosamine portion of LNT, resulting in the production of the complex dually fucosylated HMO LNDFH-II. Table 4 provides the GenBank IDs and sources of these eight fucosyltransferases.

[0436] Table 4. List of enzymes tested within the framework of the present disclosure

[0437]

[0438] ^Compared with the GenBank sequences, the sequences used in this application may be truncated at the N-terminus or C-terminus, and these sequences are represented by SEQ ID NO.

[0439] *Bgall1 (FutM2) has been shown to produce 3FL in Chen et al., 2022 Arg Food Chem and CN114107152.

[0440] **FutA has been shown to produce LNFP-V in WO2020115671.

[0441] ***exist FucTIII has been shown to produce LNDFH-II in et al. 2015 (Bioorganic & Medicinal Chemistry 23:6799–6806).

[0442] strains

[0443] The strain (genetically engineered cell) constructed in this application is based on Escherichia coli K-12DH1, and its genotype is: Fˉ, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. The E. coli K-12DH1 strain was additionally modified to generate the MDO strain with the following modifications: lacZ: 1.5 kbp deletion, lacA: 0.5 kbp deletion, nanKETA: 3.3 kbp deletion, melA: 0.9 kbp deletion, wcaJ: 0.5 kbp deletion, mdoH: 0.5 kbp deletion, and the Plac promoter was inserted upstream of the gmd gene.

[0444] Methods for inserting a target gene into the E. coli genome are well known to those skilled in the art. A gene cassette can be inserted into the E. coli chromosome using gene gorging using specific selection marker genes and screening methods (see, for example, Herring and Blattner 2004 J. Bacteriol. 186: 2673-81 and Warming et al. 2005 Nucleic Acids Res. 33 (4): e36).

[0445] To obtain an LNT-producing strain, the MDO strain was further modified by chromosomal integration of β-1,3-GlcNAc transferase (LgtA from Neisseria meningitidis, homologous to NCBI Accession No. WP_033911473.1, and as shown in SEQ ID NO: 12) and β-1,3-galactosyltransferase (GalTK from Helicobacter pylori, homologous to GenBank Accession No. BD182026.1, and as shown in SEQ ID NO: 13), both under the control of the PglpF promoter (SEQ ID NO: 27), and the strain was named LNT strain.

[0446] A codon-optimized DNA sequence encoding a single α-1,3 / 4-fucosyltransferase was genomically integrated into the LNT strain.

[0447] Table 5 provides the genotypes of the background strain (MDO), the LNT strain, and the α-1,3 / 4-fucosyltransferase expressing strain capable of producing LNDFH-II, and mixtures thereof.

[0448] Table 5. Genotypes of strains capable of producing LNNDFH-II used in this example.

[0449]

[0450] *1,3FT is the abbreviation of α-1,3-fucosyltransferase, and the DNA sequence is inserted into the genome of the host strain.

[0451] 1 lgtA-PglpF - two genomically inserted copies of a gene encoding β-1,3-N-acetylglucosaminyltransferase (SEQ ID NO: 12) under the control of the PglpF promoter.

[0452] 2 galTK-PglpF - a genomic insertion of a gene encoding β-1,3-galactosyltransferase (SEQ ID NO: 13) under the control of the PglpF promoter (SEQ ID NO: 27).

[0453] 3 CA = additional colanic acid gene cluster (gmd-wcaG-wcaH-wcaI-manC-manB, SEQ ID NO: 11) located at a site different from the native site and controlled by the PglpF promoter.

[0454] 4 FutA-PglpF - Gene encoding α-1,3(4)-fucosyltransferase (SEQ ID NO:5) controlled by the PglpF promoter (SEQ ID NO:27). One (1x), two (2x) or three (3x) copies inserted into the genome or expressed from plasmid pUC57, a high copy number (>300) plasmid with ColE1 / pMB1 / pBR322 / pUC replication origin. The antibiotic resistance marker on the pUC57 vector is ampicillin.

[0455] 5 FucT-III-PglpF – A single copy of the gene encoding α-1,4-fucosyltransferase (SEQ ID NO:39) controlled by the PglpF promoter (SEQ ID NO:27). et al. 2015 Bioorganic & Medicinal Chemistry 23: 6799–6806.

[0456] Deep well assay

[0457] The deep well test in this example was performed in the manner originally described by Lv et al. (Bioprocess Biosyst Eng 20 (2016) 39: 1737-1747) and optimized for the purposes of the present invention. More specifically, the strains disclosed in this example were screened in 96 deep well plates using a 4-day protocol. Within the first 24 hours, the preculture was grown to high density (OD600 up to 5) and then transferred to a medium that allowed induction of gene expression and product formation.

[0458] More specifically, on day 1, a fresh preculture was prepared using 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), KHzPO4 (7 g / L), NH4H4PO4 (7 g / L), citric acid (0.5 g / L), trace mineral solution (5 mL / L). The trace mineral stock solution contained: ZnSO4*7H4O 0.82 g / L, citric acid 20 g / L, Mn4*H4O 0.98 g / L, FeSO4*7H4O 3.925 g / L, CuSO4*5H4O 0.2 g / L. The pH of the basal minimal medium was adjusted to 7.0 with 5N NaOH and then sterilized by autoclave. The pre-culture was incubated at 34°C and 1000rpm shaking for 24 hours and then further transferred to 0.75mL new BMM (pH 7,5) to start the main culture. The new BMM was supplemented with magnesium sulfate (0.12g / L), thiamine (0.02g / L), glucose solution (0.1-0.15g / L) and lactose solution (5-20g / L). In addition, 20% sucrose stock solution (40-45g / L) or maltodextrin (19-20g / L) was provided as a carbon source, and specific hydrolases, sucrose hydrolases or glycosidases were added respectively at the same time 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 1000rpm shaking for 72 hours. To analyze the total culture medium, the 96-well plate was boiled at 100°C, then centrifuged, and the supernatant was finally analyzed by HPLC.

[0459] Fermentation

[0460] E. coli strains were cultured in 250 mL fermenters (Ambr250 HT bioreactor system, Sartorius) with a starting medium of 100 mL mineral medium consisting of 30 g / L glucose and mineral medium consisting of NH4H2PO4, KH2PO4, MgSO4×7H2O, KOH, NaOH, citric acid, trace element solution, defoamer and thiamine. The airflow was started at 700 rpm (up to 4500 rpm) and 1 VVM (up to 3 VVM) to maintain the dissolved oxygen level at 20% by a cascade of stirring followed by airflow. 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 pre-culture containing 10 g / L glucose, (NH4)2HPO4, KH2PO4, MgSO4×7H2O, KOH, NaOH, citric acid, trace element solution, defoamer 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 dropped to 30°C with a 3-hour linear ramp 12 hours after the start of the feed. Lactose was added once as a 25% lactose monohydrate solution 6 hours after the start of the feed, and then added every 19 hours to prevent lactose from becoming a rate-limiting factor. Cell growth, metabolic activity and metabolic state were tracked by online measurement of stirring, dissolved oxygen tension, reflectivity, NH4OH base addition, O2 absorption rate and CO2 release rate. Throughout the fermentation process, samples were collected to determine the concentration of HMO products, lactose and other minor byproducts using HPLC.

[0461] Example 1 - In vivo LNDFH-II synthesis

[0462] Genetically modified cells expressing individual α-1,3 / 4-fucosyltransferases were screened for their ability to produce the fucosylated HMO LNDFH-II and mixtures thereof.

[0463] Four enzymes (Table 6) were compiled to test their ability to synthesize LNDFH-II when introduced into genetically modified cells producing LNT and GDP-fucose. These enzymes were compared with the known α-1,3-fucosyltransferase FutA.

[0464] Genetically modified strains expressing five individual α-1,3 / 4-fucosyltransferases (Table 4) were generated as described in the Methods section. Cells were screened in a deep well assay as described in the Methods section.

[0465] The genotypes of strains capable of producing LNDFH-II are listed in Table 5. The molar content of individual HMOs produced by the strains was determined by HPLC.

[0466] Table 6 shows the results for the LNDFH-II production cells, expressed as the fraction (percent, %) of the total HMO molar content produced by each strain.

[0467] Table 6: The percentage of individual HMO content produced by each strain to the total HMO molar (mM) content.

[0468]

[0469] No HMOs other than those shown in Table 6 were identified in the deep hole test.

[0470] As can be seen from the data shown in Table 6, a single copy of the two enzymes Bgall1 and Bbac1 can transfer a fucosyl unit to the Glc and GlcNAc moieties of LNT, linked with an α-1,3 bond on the Glc moiety and an α-1,4 bond on the GlcNAc moiety, to form LNDFH-II, which is more than 7% of the total HMO. FutA, which is known in the prior art as an α-1,3-fucosyltransferase (see, for example, Dumon et al., 2004 (Biotechnol. Prog. 2004, 20, 412-419), was surprisingly found to be able to fucosylate the GlcNAc moiety of LNT with an α-1,4 bond and produce LNDFH-II, respectively, accounting for 5% of the total HMO when expressed from a single genomic copy under the control of the PglpF promoter.

[0471] As can be seen from Table 6, increasing the copy number of Bgall1 to two genomic copies increased the relative yield of LNDFH-II from 16% to 24% of the total HMO content, while the relative yield of LNFP-V decreased from 54% to 37%, the relative yield of LNT slightly decreased from 9% to 3%, and the yield of 3FL increased from 21% to 38%. Further increasing the expression level of Bgall1 by introducing a high-copy plasmid encoding Bgall1 resulted in a decrease in the relative yield of LNDFH-II from 24% to 16% of the total HMO content, while the relative yield of LNFP-V further decreased from 37% to 23%, the relative yield of LNT slightly decreased from 3% to 2%, and the yield of 3FL further increased from 38% to 54%.

[0472] Increasing the copy number of Murba1 to two genomic copies resulted in an increase in the relative yield of LNDFH-II from 1% to 4% of the total HMO content, while the relative yield of LNFP-V decreased from 66% to 64%, the relative yield of LNT slightly decreased from 23% to 10%, and the yield of 3FL increased from 11% to 22%. Further increasing the expression level of Murba1 by introducing a high-copy plasmid encoding Murba1 resulted in a further increase in the relative yield of LNDFH-II from 4% to 7% of the total HMO content, while the relative yield of LNFP-V decreased from 64% to 40%, the relative yield of LNT decreased from 10% to 3%, and the yield of 3FL increased from 22% to 50%.

[0473] Increasing the copy number of Para1 to two genomic copies resulted in an increase in the relative production of LNDFH-II from 2% to 11% of the total HMO content, a slight increase in the relative production of LNFP-II from 4% to 5%, a slight increase in the relative production of LNFP-V from 4% to 5%, a decrease in the relative production of LNT from 51% to 34%, a decrease in the production of 3FL from 1% to 0%, and an increase in the production of LNT2 from 38% to 45%.

[0474] Increasing the copy number of FutA to two genomic copies resulted in an increase in the relative yield of LNDFH-II from 5% to 13% of the total HMO content, while the relative yield of LNFP-V increased from 75% to 79% and the relative yield of LNT decreased slightly from 20% to 8%, respectively. The expression level of FutA was further increased by introducing a high-copy plasmid encoding FutA, resulting in a further increase in the relative yield of LNDFH-II from 13% to 39% of the total HMO content, while the relative yield of LNFP-V decreased from 79% to 42% and the relative yield of LNT decreased slightly from 8% to 3%, respectively.

[0475] Therefore, copy number variation of Bgall1, Para1 and FutA can be used to tailor a specific HMO mixture, in which case the mixture contains different ratios of LNDFH-II and LNT2, 3FL, LNT, LNFP-II and / or LNFP-V, depending on the requirements of a specific product.

[0476] It was found that the HMO mixture produced by Bbac1 consisted of 97% fucosylated HMOs, of which only 3% were LNTs, and the remaining 97% of HMOs produced were a mixture of 3FL, LNDFH-II, and LNFP-V. Therefore, Bbac1 is a good choice for a fucosylated HMO mixture, with a lower content of non-fucosylated HMOs (e.g., LNT-II and LNT).

[0477] Strains containing high copy number plasmids of Bgall1, Murba1 or FutA were also shown to be able to produce a mixture of fucosylated HMOs, with the content of fucosylated HMOs exceeding 90% of the total HMOs produced, and even up to 98% of the total HMOs produced by the Bgall1-3 strain were fucosylated HMOs.

[0478] Example 2 - Fermentation of Bgall1 or FutA strains to produce LNDFH-II

[0479] To confirm the HMO profile observed in the deep well test, especially the content of LNDFH-II in the total HMO, the Bgall1-2, Murba1-2, FutA-1 and FutA-2 strains in Example 1 (containing two Bgall1 genome copies or one or two FutA genome copies) were fermented as described in the "Methods" section above. The results are shown in Table 7.

[0480] Table 7: Percentage of individual HMO content in the total HMO content produced by the strain

[0481]

[0482] As can be seen from the data shown in Table 7, the LNDFH-II fractions of the FutA-1 and FutA-2 strains increased during the fermentation process compared to the deep well test results shown in Example 1. For the Bgall1-2 strain, the LNDFH-II content during the fermentation process increased slightly compared to the deep well test results shown in Example 1. The Murba1-2 strain produced almost double the level of LNDFH-II during the fermentation process compared to the deep well test results shown in Example 1, and although this level is lower than that of Bgall1 and FutA, it may be a strain of interest if high levels of LNFP-V and 3FL are required to be used in combination with some LNDFH-II.

[0483] Example 3 - Comparison of Bgall1, FutA and FucTIII strains for LNDFH-II production

[0484] Taking into account et al. 2015 (Bioorganic & Medicinal Chemistry 23: 6799–6806), using the DW assay described in the Methods section for comparative examples.

[0485] The genotypes of the strains used in the current example are listed in Table 5. The molar content of individual HMOs produced by the strains was determined by HPLC.

[0486] Table 8 shows the results for the LNDFH-II production cells, expressed as the fraction (percent, %) of the total HMO molar content produced by each strain.

[0487] Table 8: The percentage of individual HMO content produced by each strain to the total HMO molar (mM) content.

[0488]

[0489] From the results in Table 8, it can be seen that under the current conditions, Bgall1 is currently the best producer of LNDFH-II. However, FucTIII did not produce any LNDFH-II.

Claims

1. A genetically engineered cell capable of producing LNDFH-II, comprising a recombinant nucleic acid sequence encoding a fucosyltransferase, wherein the glycosyltransferase is selected from: a. Bgall1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1, b. Murba1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 2, c. Bbac1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 3, and d. Para1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 4, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO:

4.

2. The genetically engineered cell according to claim 1, wherein the cell further comprises one or more recombinant nucleic acid sequences encoding β-1,3-N-acetylglucosaminyltransferase and / or β-1,3-galactosyltransferase.

3. The genetically engineered cell of claim 1 or 2, wherein the cell further comprises a substrate importin selected from the group consisting of a lactose importin, a lacto-N-triose-II (LNT-II) importin or a LNT importin.

4. The genetically engineered cell according to any one of the preceding claims, wherein the cell also produces one or more HMOs selected from the group consisting of 3FL, LNT-II, LNT and LNFP-V.

5. A genetically engineered cell according to any one of the preceding claims, wherein the cell comprises two, three or more genomic copies and / or plasmid-borne copies of a recombinant nucleic acid sequence encoding the glycosyltransferase, the glycosyltransferase being selected from Bgall1, Murba1, Bbac1, Para1, comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, 2, 3 or 4, or a functional homologue thereof, the amino acid sequence of the functional homologue having at least 80% identity with SEQ ID NO: 1, 2, 3 or 4.

6. The genetically engineered cell of any one of the preceding claims, wherein the engineered cell is selected from the group consisting of 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, the method comprising the steps of: a. Provide a genetically modified cell comprising a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the fucosyltransferase is selected from: i. Bgall1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1, ii. Murba1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 2, iii. Bbac1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 3, iv. Para1 comprising or consisting of the amino acid sequence according to SEQ ID NO: 4, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 4, and v. FutA comprising or consisting of the amino acid sequence according to SEQ ID NO: 5, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 5, and b. culturing the cell 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-II, and c. Optionally, purifying the one or more fucosylated HMOs.

8. The method of claim 7, wherein at least 5% of the total molar content of HMOs produced by the genetically engineered cells cultured in the method is LNDFH-II.

9. The method according to claim 7 or 8, wherein at least 65%, such as at least 70%, 80%, 90%, 95% or at least 97% of the total molar content of HMO produced by the method is fucosylated HMO selected from 3FL, LNFP-V and LNDFH-II.

10. The method according to any one of claims 7 to 9, wherein the genetically engineered cell is a cell according to any one of claims 1 to 6.

11. The method according to any one of claims 7 to 10, wherein the genetically engineered cells are cultured in the presence of an acceptor substrate selected from lactose, LNT-II or LNT.

12. Use of a fucosyltransferase having α-1,3 / 4-fucosyltransferase activity in the production of one or more fucosylated HMOs, wherein the enzyme is selected from the group consisting of Bgall1, Murba1, Bbac1, Para1 and FutA or a functional homologue thereof, wherein the Bgall1, Murba1, Bbac1, Para1 and FutA comprises or consists of an amino acid sequence according to SEQ ID NO: 1, 2, 3, 4 or 5, the amino acid sequence of the functional homologue having at least 80% identity with SEQ ID NO: 1, 2, 3, 4 or 5, wherein one of the HMOs produced is LNDFH-II.

13. An HMO mixture produced according to the process of any one of claims 7 to 11, consisting essentially of: a. LNDFH-II, LNFP-V and LNT, or b. LNDFH-II, LNFP-V and 3FL, or c.LNDFH-II, LNFP-V, LNT and 3FL, or d.LNDFH-II, LNT-II, LNFP-V, LNFP-II and LNT.

14. An HMO composition consisting essentially of 1-40 mol% LNDFH-II, 2-85 mol% LNFP-V, 0-10 mol% LNFP-II, 0-65 mol% 3FL, 0-55 mol% LNT and 0-50 mol% LNT-II, which add up to a total of 100 mol%.

15. The composition according to claim 14, wherein the mixture is selected from: a. A mixture consisting essentially of 15-40 mol % LNDFH-II, 20-60 mol % LNFP-V, 0-15 mol % LNT and 15-60 mol % 3FL, the total molar content being added to 100%; b. A mixture consisting essentially of 15-40 mol % LNDFH-II, 15-40 mol % LNFP-V, 0-5 mol % LNT and 25-60 mol % 3FL, the total molar content being added to 100%; c. A mixture consisting essentially of 1-15 mol % LNDFH-II, 35-70 mol % LNFP-V, 0-25 mol % LNT and 5-55 mol % 3FL, the total molar content being added to 100%; d. A mixture consisting essentially of 2-15 mol % LNDFH-II, 60-75 mol % LNFP-V, 0-10 mol % LNT and 15-30 mol % 3FL, adding up to a total of 100 mol %; and e. A mixture consisting essentially of 2-15 mol % LNDFH-II, 0-10 mol % LNFP-V, 0-10 mol % LNFP-II, 30-50 mol % LNT-II, 30-60 mol % LNT and 0-5 mol % 3FL, the total molar content being 100%, and f. A mixture consisting essentially of 2-40 mol % LNDFH-II, 35-85 mol % LNFP-V, 0-25 mol % LNT and 0-20 mol % 3FL, adding up to a total of 100 mol % content.

16. Use of the HMO mixture according to claim 13 or the HMO composition according to claim 14 or 15 in infant formula, dietary supplements and / or medical nutrition.

Images