Novel fucosyltransferase for in vivo synthesis of LNFP-III

Abstract

The present disclosure relates to the production of fucosylated human milk oligosaccharides (HMOs). In particular, the present invention relates to alpha-1, 3-fucosyltransferases for the production of milk-N-fucopentasaccharide III (LNFP-III), as well as genetically engineered cells and their use in said production. Preferably, the alpha-1, 3-fucosyltransferase described herein does not perform fucosylation on the reducing terminal of the acceptor oligosaccharide that is used as a substrate for the alpha-1, 3-fucosyltransferase.

Description

Technical Field

[0001] The present disclosure relates to the production of fucosylated human milk oligosaccharides (HMOs). In particular, it relates to α-1,3-fucosyltransferases for producing lacto-N-fucopentaose III (LNFP-III), as well as genetically engineered cells, and their use in said production. Preferably, the α-1,3-fucosyltransferase described herein does not fucosylate the reducing end of the acceptor oligosaccharide used as a substrate for the α-1,3-fucosyltransferase. Background Art

[0002] Designing and constructing bacterial cell factories to produce fucosylated human milk oligosaccharides (HMOs), especially more complex fucosylated HMOs, is critical to providing innovative and scalable solutions for tomorrow's more complex products.

[0003] The production of complex fucosylated HMOs has been described, for example, in WO2016 / 040531, showing that the α-1,3-fucosyltransferase CafD can produce LNFP-III, and in WO2019 / 008133, the α-1,3-fucosyltransferase FucT109 was described, which appears to fucosylate the glucose (Glc) and N-acetylglucosamine (GlcNAc) moieties of lacto-N-neotetraose (LNnT), potentially generating a mixture containing all three of LNnT, LNFP-III and LNFP-VI.

[0004] Dumon et al., 2004 (Biotechnol. Prog. 2004, 20, 412-419) further described the α-1,3-fucosyltransferase FutB, which was also suggested to produce a mixture of LNnT, LNFP-III, LNFP-VI and LNDFH-III.

[0005] However, the fucosyltransferases disclosed in the prior art (CafD, FucT109 and FutB) produce only small amounts of complex fucosylated HMOs, if any, with high by-product formation.

[0006] 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 production of the desired fucosylated HMOs in low yields compared to other HMO products (e.g., HMO precursor products) present after fermentation, which may require tedious separation procedures. Summary of the invention

[0007] The need for highly substrate-specific α-1,3-fucosyltransferases was addressed by identifying a series of α-1,3-fucosyltransferases that exhibit low or no specificity for the glucose moiety in LNnT as a substrate for the fucosylation reaction, but have high substrate specificity for the N-acetylglucosamine (GlcNAc) moiety in LNnT, thereby producing almost exclusively complex fucosylated HMO LNFP-III or a mixture of HMOs containing a high content of LNFP-III. Therefore, the α-1,3-fucosyltransferases presented herein can be used to produce pure or nearly pure LNFP-III. Therefore, enzymes, mixtures, compositions, uses, genetically engineered cells and methods for producing LNFP-III are provided herein.

[0008] In a first aspect, the present invention relates to a genetically engineered cell capable of producing one or more fucosylated HMOs, preferably LNFP-III, and comprising a recombinant nucleic acid sequence encoding a glycosyltransferase having α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is selected from Camp1, Parm1 and Hpull1 having an amino acid sequence according to SEQ ID NO: 1, 2 and 3, or a functional homologue thereof having an amino acid sequence having at least 80% identity with SEQ ID NO: 1, 2 and 3 [Camp1, Parm1 and Hpull1 amino acids].

[0009] In a second aspect, the present invention relates to a method for producing one or more fucosylated HMOs, preferably LNFP-III, the method comprising culturing a genetically engineered cell capable of producing one or more fucosylated HMOs, the genetically engineered cell comprising a recombinant nucleic acid sequence encoding a glycosyltransferase having α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is selected from Camp1, Parm1, Hpull1 and Hacin2 having an amino acid sequence according to SEQ ID NO: 1, 2, 3 or 5, or a functional homologue thereof having an amino acid sequence having at least 80% identity with SEQ ID NO: 1, 2, 3 or 5 [Camp1, Parm1, Hpull1 and Hacin2 amino acids].

[0010] In a third aspect, the present invention relates to the use of an enzyme having α-1,3-fucosyltransferase activity for producing a fucosylated product (e.g. HMO, preferably LNFP-III), wherein the enzyme is selected from Camp1, Parm1, Hpull1 and Hacin2 having an amino acid sequence according to SEQ ID NO: 1, 2, 3 or 5, or a functional homolog thereof having an amino acid sequence having at least 80% identity with SEQ ID NO: 1, 2, 3 or 5 [Camp1, Parm1, Hpull1 and Hacin2 amino acids].

[0011] In a fourth aspect, the invention relates to a mixture of HMOs consisting essentially of LNFP-III and LNnT, and having small amounts of 3-FL, LNDFH-III and / or pLNnH, for example less than 10 mole % of the total molar HMO content in the mixture.

[0012] In some embodiments, the mixture of HMOs consists essentially of 30-99 mol% LNFP-III, 0-65 mol% LNnT, 0-5 mol% 3-FL, and 0-10% pLNnH, which together add up to 100% of the molar content of HMOs in the mixture.

[0013] A fifth aspect of the present invention provides

[0014] a) a mixture of HMOs consisting essentially of LNFP-III and LNnT, and minor amounts (e.g., less than 10% of the total molar HMO content of the composition) of 3-FL or pLNnH, or

[0015] b) A mixture of HMO consisting essentially of 30-99 mol % LNFP-III, 0-65 mol % LNnT, 0-20 mol % 3-FL, 0-5 mol % LNDFH-III and 0-10 mol % pLNnH, adding up to 100 mol % in total.

[0016] The invention also relates to a composition comprising a mixture of a) and b), and to the use of said composition in infant formula, dietary supplement or medical nutrition. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Data from the single copy strains in Table 5 are presented, clearly showing the difference in LNFP-III production from strains with the five novel enzymes compared to the prior art enzymes FutB, CafD and FucT109.

[0018] Figure 2is a schematic representation of the pathway for making LNFP-III and potential byproducts that may be produced in the process. Depending on the specificity of the α-1,3-fucosyltransferase, the acceptor for the α-1,3-fucosyltransferase can be lactose, LNnT, LNFP-VI, and LNFP-III. Fucosylated byproducts are, for example, 3FL, LNFP-VI, and LNDFH-III. Neutral byproducts are, for example, LNT-II and LNnT. Depending on the genetically modified cell, the initial substrate for LNFP-III production may be lactose, LNT-II, or even LNnT, as long as the strain is able to absorb the initial substrate. In the figure, β1,3-GlcNAcT is β1,3-glucosaminyltransferase, β1,3-GalT is β1,3-galactosyltransferase, Glcα1,3-FucT is α1,3-fucosyltransferase active on the glucose part of the acceptor oligosaccharide, GlcNAcα1,3-FucT is α1,3-fucosyltransferase active on the GlcNAc part of the acceptor oligosaccharide; GDP is guanosine diphosphate, and UDP is uridine diphosphate.

[0019] Figure 3 : Shows the experimental setup for regeneration and viability assessment of freeze-dried probiotics under acidic conditions at pH 3.0.

[0020] Figure 4 : Shows the regeneration and viability of freeze-dried Lactobacillus casei (DSM 32382), incubated for 3 hours at pH 3.0 and plated at two dilutions: 1:100 (E-2), 1:1000 (E-3), A) is a control without HMO, B) is Lactobacillus casei (DSM 32382) combined with a HMO mixture containing 50% LNFP-III and 50% LNnT (Mixture 1); C) is Lactobacillus casei (DSM 32382) combined with a HMO mixture containing 75% LNFP-III and 25% LNnT (Mixture 2); B) is Lactobacillus casei (DSM 32382) combined with a HMO mixture containing 40% LNFP-III, 50% LNnT and 5% pLNnH (Mixture 3).

[0021] Figure 5 :show Figure 4 The results of the medium plates are expressed as mean values ​​(n=2) and standard deviations (SD) of colony forming units (CFU) per ml, which were calculated from the Lactobacillus casei colonies inoculated onto agar plates at dilution E-3. Mixtures 1 and 2 showed statistically significant differences compared to the control at p<0.01, while mixture 3 showed statistically significant differences compared to the control at p<0.05. DETAILED DESCRIPTION

[0022] The present invention solves the biotechnological challenges of HMO production in vivo, in particular complex fucosylated HMOs comprising at least five monosaccharides, wherein at least one sugar moiety is a fucosyl moiety, such as the complex fucosylated HMO LNFP-III, which comprises a single fucosyl moiety. The present disclosure provides a specific strain engineering solution to produce specific complex fucosylated HMOs, in particular LNFP-III, by utilizing the substrate specificity and activity of the 1,3-fucosyltransferase of the present disclosure towards the GlcNAc moiety in LNnT.

[0023] The genetically engineered cells disclosed herein express genes encoding key enzymes for the biosynthesis of fucosylated HMOs. In addition, the genetically engineered cells may express additional genes required to produce LNnT from lactose or LNT-II as initial substrates. Alternatively, the cells may be engineered to absorb LNnT that only requires 1,3-fucosyltransferase activity in the cells (see, e.g., WO2023 / 099680). In some embodiments, the genetically engineered cells of the present specification further express one or more of the de novo GDP-fucose pathway genes, manA, manB, manC, gmd, and / or wcaG, responsible for the formation of GDP-fucose. It may be advantageous to overexpress one or more of these genes and / or upregulate CA, including the genes gmd, wcaG, wcaH, wcaI, manC and manB from E. coli, to form GDP-fucose by introducing a nucleic acid construct encoding the colanic acid gene cluster (CA) as shown in SEQ ID NO: 12, which enables the cell to produce higher levels of fucosylated oligosaccharides from one or more oligosaccharide substrates (e.g., lactose, LNT-II and / or LNnT). Depending on the intended use of the substrate, one or more additional glycosyltransferases and pathways for producing nucleotide activated sugars (e.g., glucose-UDP-GlcNac, CMP-N-acetylneuraminic acid, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and CMP-N-acetylneuraminic acid) may also be present in the genetically engineered cells.

[0024] Production of LNFP-III

[0025] The advantage of using any of the α-1,3-fucosyltransferases disclosed in this context is that they can specifically recognize and fucosylate the GlcNAc moiety in LNnT, thereby generating LNFP-III. In particular, the present disclosure describes an enzyme (α-1,3-fucosyltransferase) having α-1,3-fucosyltransferase activity, which is more active on the GlcNAc moiety of LNnT than the α-1,3-fucosyltransferases described in the prior art, such as FucT109 (see WO2019 / 008133) and FutB (see Dumon et al., 2004). In addition, the α-1,3-fucosyltransferases described herein have very low activity on the glucose moieties of lactose and LNnT. If LNnT is available in sufficient amounts in genetically engineered cells, very little (if any) 3-FL is produced by the α-1,3-fucosyltransferases described in the present disclosure. Therefore, the traits of the α-1,3-fucosyltransferase described herein are well suited for high-level industrial production of LNFP-III without producing high levels of alternative fucosylated HMOs (byproducts), such as 3FL, LNFP-VI, LNDFH-III and other byproduct HMOs or byproduct non-HMO oligosaccharides.

[0026] In an embodiment, the α-1,3-fucosyltransferase of the present disclosure mainly fucosylates the N-acetylglucosamine (GlcNAc) portion of the acceptor oligosaccharide (e.g., LNnT). The term mainly should be understood as less than 5%, such as less than 4%, such as less than 3%, such as less than 2% of the other parts in LNnT are fucosylated by the α-1,3-fucosyltransferase of the present disclosure.

[0027] In an embodiment, the α-1,3-fucosyltransferase of the present disclosure has low activity or no activity on the glucose moiety at the reducing end of the acceptor molecule. The acceptor molecule is a sugar or oligosaccharide, such as lactose or an HMO, such as LNT-II or LNnT, but also other HMOs. LNnT is a preferred acceptor molecule for the α-1,3-fucosyltransferase of the present disclosure. The term low activity or no activity is to be understood as less than 5%, such as less than 4%, such as less than 3%, such as less than 2% of the glucose moiety in the reducing end of the acceptor molecule is fucosylated by the α-1,3-fucosyltransferase of the present disclosure.

[0028] The genetically engineered cells of the present invention express an α-1,3-fucosyltransferase with high specificity for the GlcNAc moiety in LNnT, and are capable of producing high titers of LNFP-III. In particular, in the absence of other complex fucosylated HMOs (e.g., LNFP-VI and / or LNDFH-III). Thus, the present invention enables a more efficient production of LNFP-III, which is very beneficial in the biotechnological production of more complex fucosylated HMOs (e.g., LNFP-III), for example due to reduced purification requirements.

[0029] Thus, the mixture of HMOs produced by the cells and / or methods described herein contains a high percentage of LNFP-III in the total amount of HMOs produced, such as at least 30%, preferably at least 50%, such as at least 75%, such as at least 80%, such as at least 90% of the total amount of HMOs produced by the cells and / or methods.

[0030] In the following sections, individual elements of the invention, in particular genetically engineered cells, are described. It should be understood that these elements can be combined between the various sections.

[0031] Oligosaccharides

[0032] In this article, term " oligosaccharide " refers to the sugar polymer containing at least three monosaccharide units, i.e. three, four, five, six or higher oligosaccharides. Oligosaccharides can have the straight chain or branched structure containing the monosaccharide units connected to each other by glycosidic bonds. Especially, oligosaccharides are included in the lactose residue at the reducing end and the monosaccharide of one or more naturally occurring 5-9 carbon atoms, and it is selected from aldose (such as glucose, galactose, ribose, arabinose, xylose etc.), ketose (such as fructose, sorbose, tagatose etc.), deoxy sugar (such as rhamnose, fucose etc.), deoxy amino sugar (such as N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine etc.), uronic acid and ketouronic acid (such as N-acetylneuraminic acid). Preferably, oligosaccharide is HMO.

[0033] Human Milk Oligosaccharides (HMO)

[0034] Preferred oligosaccharides of the present disclosure are human milk oligosaccharides (HMOs).

[0035] The term "human milk oligosaccharide" or "HMO" refers herein to a complex carbohydrate found in human breast milk. HMOs have a core structure comprising a lactose unit at the reducing end, which may be extended by one or more β-N-acetyllactosamine and / or one or more β-lacto-N-disaccharide units, and which may be partially substituted by α-L-fucopyranosyl and / or α-N-acetylneuraminic acid (fucosyl) moieties. The HMO structure is disclosed, for example, by Xi Chen in Chapter 4 of Advances in Carbohydrate Chemistry and Biochemistry 2015 vol 72.

[0036] The present invention focuses on fucosylated HMOs. Examples of fucosylated HMOs include 2'-fucosyllactose (2'-FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-III), 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-pLNnHII), 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).

[0037] In the context of the present invention, 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 fucosylated HMOs with 6 monosaccharide units, such as but not limited to LNDFH-I, LNDFH-II and LNDFH-III. Preferably, a complex fucosylated HMO is an HMO that requires at least three different glycosyltransferase activities to be produced from lactose as an initial substrate, for example, the formation of LNFP-III requires α-1,3-fucosyltransferase, β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase.

[0038] In an embodiment of the invention, the fucosylated human milk oligosaccharide (HMO) produced by the cell is LNFP-III, such as only or substantially only LNFP-III. In another embodiment of the invention, at least 30%, such as at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the molar content of the total HMO produced by the cell is LNFP-III. Preferably, at least 75% of the molar content of the total HMO produced by the cell is LNFP-III.

[0039] In embodiments, the genetically engineered cells described herein produce at least 50% of the molar content of total HMOs produced by the cell as LNFP-III, and the remaining 50% of the total HMOs produced by the cell are byproduct HMOs selected from LNDFH-III, 3FL, LNnT and pLNnH.

[0040] In a further embodiment, at least 90% of the total molar content of HMOs produced by the cell is LNFP-III and LNnT. In other embodiments of the invention, less than 1%, such as 0.0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 0.99% of the total molar content of HMOs produced by the cell is LNFP-VI.

[0041] In a further embodiment of the invention, less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1%, such as 0.0%, 0.5%, 0.8%, 1.0%, 1.5%, 2.0%, 2.5% of the total molar content of HMOs produced by the cells is LNDFH-III.

[0042] In other embodiments, less than 5% of the molar content of total HMOs produced by the cell are alternative complex fucosylated HMOs.Alternative fucosylated HMOs are considered herein to be one or more fucosylated HMOs that are not LNFP-III.

[0043] In other embodiments, less than 20%, such as less than 15%, such as less than 10%, such as less than 5%, such as less than 1% of the total molar HMO content of the total HMO produced by the cell is non-complex fucosylated HMO, such as 3-FL. The alternative fucosylated HMO can be selected from 3-FL, DFL, LNFP-VI and LNDFH-III. The alternative complex fucosylated HMO can be selected from LNFP-VI and LNDFH-III.

[0044] The production of LNFP-III may require the presence of two or more glycosyltransferase activities, particularly if starting from lactose as the acceptor oligosaccharide.

[0045] Receptor oligosaccharide

[0046] The genetically engineered cell according to the present invention comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, which is capable of transferring fucose from an activated sugar to the GlcNAc portion of an acceptor oligosaccharide with an α-1,3 bond.

[0047] In the context of the present invention, 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 "Glycosyltransferase" section. Preferably, the acceptor oligosaccharide is a precursor for the preparation of more complex HMOs and may also be referred to as a precursor molecule.

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

[0049] In this context, the acceptor oligosaccharide for the α-1,3-fucosyltransferase is preferably lacto-N-neotetraose (LNnT), which is produced from the precursor molecules lactose (e.g., the acceptor for β-1,3-N-acetylglucosaminyltransferase) and / or lacto-N-triose II (LNT-II) (e.g., the acceptor for β-1,4-galactosyltransferase). The precursor molecules are preferably fed into genetically engineered cells capable of producing LNnT from the precursors.

[0050] Glycosyltransferase

[0051] The genetically engineered cells according to the present invention comprise at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase capable of transferring a fucosyl residue from a fucosyl donor to an acceptor oligosaccharide to synthesize one or more fucosylated human milk oligosaccharide products, i.e. a fucosyltransferase.

[0052] The genetically engineered cell according to the present invention may comprise at least one or more additional recombinant nucleic acids encoding one or more heterologous glycosyltransferases capable of transferring glycosyl residues from a glycosyl donor to an acceptor oligosaccharide. Preferably, the additional glycosyltransferases enable the genetically engineered cell to synthesize LNnT from a precursor molecule (e.g., lactose or LNT-II). In an embodiment, the genetically engineered cell of the present invention comprises one or more other recombinant nucleic acids encoding one or more heterologous glycosyltransferases.

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

[0054] In one aspect, the fucosyltransferase in the genetically engineered cell of the invention is an α-1,3-fucosyltransferase. Preferably, the α-1,3-fucosyltransferase is capable of transferring a fucose unit to the GlcNAc portion of the LNnT molecule.

[0055] In one embodiment, the α-1,3-fucosyltransferase in the genetically engineered cell primarily fucosylates the N-acetylglucosamine (GlcNAc) moiety of the acceptor oligosaccharide with an α-1,3 bond, such as the N-acetylglucosamine (GlcNAc) moiety LNnT. Advantageously, the fucosyltransferase has low or no activity on the glucose moiety at the reducing end of the acceptor molecule (e.g., lactose or HMO, such as LNT-II or LNnT, particularly LNnT), because the lack of such activity will result in fewer byproducts, such as LNFP-VI and / or 3-FL.

[0056] In the present invention, at least one functional enzyme (α-1,3-fucosyltransferase) capable of transferring a fucosyl moiety from a fucosyl donor to an acceptor oligosaccharide can be selected from Camp1, Parm1, Hpull1, Med1 and Hacin2 having an amino acid sequence according to SEQ ID NO: 1, 2, 3, 4 or 5, or a functional homologue thereof (Table 1), the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1, 2, 3, 4 or 5 [Camp1, Parm1, Hpull1, Med1 and Hacin2 amino acids]. These enzymes can, for example, be used to produce LNFP-III.

[0057] In a preferred embodiment, the α-1,3-fucosyltransferase is selected from Camp1, Parm1 and Hpull1 having an amino acid sequence according to SEQ ID NO: 1, 2 or 3, or a functional homologue thereof (Table 1) having an amino acid sequence at least 80% identical to SEQ ID NO: 1, 2 or 3 [Camp1, Parm1 and Hpull1 amino acids]. These enzymes can, for example, be used to produce LNFP-III.

[0058] Without being bound by theory, an α-1,3-fucosyltransferase having a higher substrate specificity for the GlcNAc moiety in LNnT than for the terminal glucose moiety in LNnT is advantageous because such an α-1,3-fucosyltransferase would theoretically produce less or no LNFP-VI and 3-FL when the initial substrate is lactose and where the availability of LNnT is not limited. Lower amounts of LNFP-VI allow for easier purification of LNFP-III because purification of LNFP-III from a mixture of HMOs containing primarily LNFP-III would be simpler because it is easier to separate LNFP-III from smaller HMOs than to separate different fucosylated HMOs of the same or similar size (e.g., LNFP-III from LNFP-VI or LNDFH-III) from each other. Therefore, lower initial amounts of LNFP-VI and / or LNDFH-III are believed to be beneficial for purification of LNFP-III.

[0059] In a preferred embodiment, the use of the α-1,3-fucosyltransferase according to the invention results in at least 90% of the molar content of the total HMO produced by the cell according to the invention being LNFP-III. In another preferred embodiment, the α-1,3-fucosyltransferase is camp1 having an amino acid sequence according to SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence at least 80% identical to SEQ ID NO: 1.

[0060] In an embodiment, the expression of the α-1,3-fucosyltransferase according to the present invention in a genetically engineered cell is further combined with the expression of one or more other recombinant nucleic acids encoding one or more heterologous glycosyltransferases. In a preferred embodiment, the expression of the α-1,3-fucosyltransferase of the present invention in a genetically engineered cell is combined with the expression of a β-1,4-galactosyltransferase such as galT from Helicobacter pylori. In another embodiment, a third enzyme is added, such as a β-1,3-N-acetylglucosaminyltransferase, such as LgtA from Neisseria meningitidis.

[0061] α-1,3-fucosyltransferase

[0062] The term "α-1,3-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 with an α-1,3-bond. Preferably, the α-1,3-fucosyltransferase used in the present invention is not a species derived from a genetically engineered cell, i.e., the gene encoding the α-1,3-fucosyltransferase is of heterologous origin and is selected from the α-1,3-fucosyltransferases shown in Table 1. In the context of the present invention, the acceptor molecule of the α-1,3-fucosyltransferase is preferably an acceptor oligosaccharide having at least four monosaccharide units of a GlcNAc moiety, such as LNnT. Heterologous α-1,3-fucosyltransferases capable of transferring a fucosyl moiety to LNnT are known in the art, in particular FutB having an amino acid sequence as provided in SEQ ID NO: 6 and encoded by the nucleic acid sequence of SEQ ID NO: 40 is known to produce a mixture of LNFP-III and LNFP-VI (Dumon et al. 2004 Biotechnol. Prog. 20: 412-419).

[0063] 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-fucosyltransferases listed in Table 1.

[0064] Table 1. List of α-1,3-fucosyltransferases capable of producing high levels of LNFP-III.

[0065]

[0066] 1 The GenBank ID reflects the full-length enzyme, and in the present invention, truncated or mutated versions may be used, which are represented by the sequences shown in SEQ ID NOs.

[0067] Example 1 of the present invention discloses the identification of heterologous α-1,3-fucosyltransferases Camp1, Parm1, Hpull1, Med1 and Hacin2 (SEQ ID NOs: 1, 2, 3, 4 and 5, respectively), which, when introduced into LNnT production cells, are capable of producing a mixture of HMOs having a higher LNFP-III content than the previously known α-1,3-fucosyltransferase FutB (SEQ ID NO: 6). Specifically, compared to the prior art enzyme FutB, which only produces 6% LNFP-III of the total HMO, the five new enzymes Camp1, Parm1, Hpull1, Med1 and Hacin2 can transfer a fucosyl unit to the GlcNAc portion of LNnT with an α-1,3 bond to form LNFP-III at a level of more than 30% of the total HMO, and in addition, none of the five new enzymes produces LNFP-VI.

[0068] Furthermore, the experiments performed in Example 1 showed that the enzymes Camp1, Hpull1, Med1 and Hacin2 also did not produce any LNDFH-III, indicating that these enzymes are highly substrate specific for the GlcNAc moiety in LNnT. In contrast, it was found that FutB, a single copy enzyme, produced a small amount of LNFP-VI and more 3-FL compared to the five new enzymes, indicating that FutB also has at least some activity on the terminal glucose (Glc) moiety of LNnT.

[0069] In an embodiment, the fucosyltransferase having α-1,3-fucosyltransferase activity is selected from: Camp1 having an amino acid sequence according to SEQ ID NO:1, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:1, Parm1 having an amino acid sequence according to SEQ ID NO:2, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:2, Hpull1 having an amino acid sequence according to SEQ ID NO:3, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:3, Med1 having an amino acid sequence according to SEQ ID NO:4, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:4, and Hacin2 having an amino acid sequence according to SEQ ID NO:5, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:5.

[0070] In an embodiment, the fucosyltransferase having α-1,3-fucosyltransferase activity is selected from: Camp1 having an amino acid sequence according to SEQ ID NO:1, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:1, Parm1 having an amino acid sequence according to SEQ ID NO:2, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:2, Hpull1 having an amino acid sequence according to SEQ ID NO:3, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO:3.

[0071] In one embodiment of the present invention, the fucosyltransferase having α-1,3-fucosyltransferase activity is Camp1 from Campylobacter sp., which comprises the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:1, or consists of the same.

[0072] In another embodiment of the present invention, the fucosyltransferase having α-1,3-fucosyltransferase activity is Parm1 from Parabacteroides faecalis, which comprises the amino acid sequence of SEQ ID NO:2, or 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, or consists of the same.

[0073] In another embodiment of the present invention, the fucosyltransferase having α-1,3-fucosyltransferase activity is Hpull1 from Helicobacter pullorum, which comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:3, or consists of the same.

[0074] In another embodiment of the present invention, the fucosyltransferase having α-1,3-fucosyltransferase activity is Med1 from Mediterranea An20, which comprises the amino acid sequence of SEQ ID NO:4, or 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, or consists of the same.

[0075] In another embodiment of the present invention, the fucosyltransferase having α-1,3-fucosyltransferase activity is Hacin2 from Helicobacter felis, which comprises the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:5, or consists of the same.

[0076] In one embodiment, the enzyme Camp1 is introduced into a genetically engineered cell which also comprises a β-1,4-galactosyltransferase, preferably a β-1,3-N-acetylglucosaminyltransferase.

[0077] β-1,3-N-acetylglucosaminyltransferase

[0078] β-1,3-N-acetylglucosaminyltransferase is any protein comprising the ability to transfer N-acetylglucosamine of UDP-N-acetylglucosamine to lactose or another acceptor molecule with a β-1,3-bond. 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 of the present invention, the acceptor molecule is lactose or an oligosaccharide of at least four monosaccharide units, such as LNnT or a more complex HMO structure.

[0079] Thus, in an embodiment, the genetically engineered cell further comprises one or more recombinant nucleic acid sequences encoding β-1,3-N-acetylglucosaminyltransferase.

[0080] 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 one of the β-1,3-N-acetylglucosaminyltransferases in Table 2.

[0081] Table 2. List of β-1,3-N-acetylglucosaminyltransferases

[0082]

[0083]

[0084] 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 the amino acid sequence of SEQ ID NO: 13 (LgtA, from Neisseria meningitidis) or a functional homolog thereof, or consists 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: 13.

[0085] To produce LNnT from lactose as a substrate, a β-1,3-N-acetylglucosaminyltransferase is used to form the LNT-II precursor. In one embodiment, the genetically engineered cell comprises a β-1,3-N-acetylglucosaminyltransferase gene or a functional homolog or fragment thereof to produce the intermediate LNT-II from lactose.

[0086] Some of the following examples use a heterologous β-1,3-N-acetylglucosaminyltransferase named LgtA from Neisseria meningitidis or a variant thereof.

[0087] β-1,4-galactosyltransferase

[0088] β-1,4-galactosyltransferase is any protein comprising the ability to transfer galactose of UDP-galactose to the N-acetylglucosamine moiety of an acceptor molecule with a β-1,4-linkage. Preferably, the β-1,4-galactosyltransferase used herein is not derived from a species of genetically engineered cells, i.e. the gene encoding the β-1,4-galactosyltransferase is of heterologous origin. In the context of the present invention, the acceptor molecule is an acceptor sugar, such as LNT-II or a more complex HMO structure.

[0089] The following examples use a heterologous β-1,4-galactosyltransferase designated GalT or a variant thereof to produce, for example, LNFP-III in combination with other glycosyltransferases. Thus, in an embodiment, the genetically engineered cells comprise one or more recombinant nucleic acid sequences encoding a β-1,4-galactosyltransferase.

[0090] Non-limiting examples of β-1,4-galactosyltransferases are provided in Table 3. β-1,4-galactosyltransferase variants may also be useful, preferably such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to the amino acid sequence of any one of the β-1,4-galactosyltransferases in Table 3.

[0091] Table 3. List of β-1,4-glycosyltransferases

[0092] Protein name GenBank ID source GalT WP_001262061.1 Helicobacter pylori LqCy AAF42257.1 Neisseria meningitidis MC58

[0093] In one embodiment, the recombinant nucleic acid sequence encoding β-1,4-galactosyltransferase comprises the amino acid sequence of SEQ ID NO:14 (GalT from Helicobacter pylori) or a functional homolog thereof, or consists of the same, and the amino acid sequence of the functional homolog has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:14.

[0094] In order to produce LNnT from LNT-II precursor, a β-1,4-galactosyltransferase is required. In one embodiment, the genetically engineered cell comprises a β-1,4-galactosyltransferase gene or a functional homologue or fragment thereof. In an embodiment, the β-1,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis and the β-1,4-galactosyltransferase is from Helicobacter pylori. In a further embodiment, the 1,3-N-acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO: 13 [LgtA amino acid] or a functional homologue thereof, the amino acid sequence of the functional homologue having at least 80% identity with SEQ ID NO: 13, and the β-1,4-galactosyltransferase has an amino acid sequence according to SEQ ID NO: 14 [GalT amino acid] or a functional homologue thereof, the amino acid sequence of the functional homologue having at least 80% identity with SEQ ID NO: 14.

[0095] The following are non-limiting examples of genetically modified strains according to the invention having a specific combination of glycosyltransferases that will result in the production of LST-c using lactose as initial substrate.

[0096] In one non-limiting example, LgtA from Neisseria meningitidis is used in combination with galT from Helicobacter pylori and Camp1 from Campylobacter to produce LNFP-III starting from lactose as the initial substrate.

[0097] In one non-limiting example, LgtA from Neisseria meningitidis is used in combination with galT from Helicobacter pylori and Parm1 from Parabacteroides faecalis to produce LNFP-III starting from lactose as the initial substrate.

[0098] In one non-limiting example, LgtA from Neisseria meningitidis is used in combination with galT from Helicobacter pylori and Hpull1 from Helicobacter gallinarum to produce LNFP-III starting from lactose as the initial substrate.

[0099] In one non-limiting example, LgtA from Neisseria meningitidis is used in combination with galT from Helicobacter pylori and Med1 from Mediterranea An20 to produce LNFP-III starting from lactose as the initial substrate.

[0100] In one non-limiting example, LgtA from Neisseria meningitidis is used in combination with galT from Helicobacter pylori and Hacin2 from Helicobacter felis to produce LNFP-III starting from lactose as the initial substrate.

[0101] In one non-limiting example, galT from Helicobacter pylori is used in combination with Camp1 from Campylobacter to produce LNFP-III starting from LNT-II as the initial substrate.

[0102] In one non-limiting example, galT from Helicobacter pylori is used in combination with Parm1 from Parabacteroides faecalis to produce LNFP-III starting from LNT-II as the initial substrate.

[0103] In one non-limiting example, galT from Helicobacter pylori is used in combination with Hpull1 from Helicobacter gallinarum to produce LNFP-III starting from LNT-II as the initial substrate.

[0104] In one non-limiting example, galT from Helicobacter pylori is used in combination with Med1 from Mediterranea An20 to produce LNFP-III starting from LNT-II as the initial substrate.

[0105] In one non-limiting example, galT from Helicobacter pylori is used in combination with Hacin2 from Helicobacter felis to produce LNFP-III starting from LNT-II as the initial substrate.

[0106] Sugar pathway for sugar-donor-nucleotide activation

[0107] When carrying out the method of the present invention, preferably glycosylation reaction mediated by glycosyltransferase occurs, wherein activated sugar nucleotide is used as sugar donor. Activated sugar nucleotide usually has phosphorylated glycosyl residue connected with nucleoside. Specific glycosyltransferase only accepts specific sugar nucleotide. Therefore, preferably, following activated sugar nucleotide participates in glycosyl transfer: glucose-UDP-GlcNAc, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine (GlcNAc) and CMP-N-acetylneuraminic acid.

[0108] The genetically engineered cells according to the present invention may comprise one or more pathways to produce a nucleotide activated sugar 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.

[0109] In one embodiment of the invention, the genetically engineered cells can produce one or more of the above-mentioned activated sugar nucleotides by a de novo approach. In this regard, activated sugar nucleotides are prepared by cells in a stepwise reaction sequence from simple carbon sources such as glycerol, sucrose, fructose or glucose under the action of enzymes participating in the de novo biosynthetic pathway of the corresponding 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)).

[0110] 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 genetic or recombinant DNA techniques, all of which is part of the general knowledge of the skilled person.

[0111] In another embodiment, the genetically engineered cells can use the recycled monosaccharides for sugar nucleotides. In the salvage pathway, monosaccharides derived from degraded oligosaccharides are phosphorylated by kinases and converted into nucleotide sugars by pyrophosphatases. The enzymes involved in this procedure can be host cell heterologous or native enzymes.

[0112] Colanic acid gene cluster

[0113] 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 involved in the de novo synthesis of GDP-fucose (gmd, wcaG, wcaH, wcal, manB, manC), while one or more genes downstream of GDP-L-fucose, such as wcaJ, which is responsible for the production of the extracellular polysaccharide colanic acid, the major oligosaccharide of the bacterial cell wall, can be deleted to prevent the conversion of GDP-fucose to colanic acid.

[0114] In order to ensure a sufficient amount of GDP-fucose, the promoter of the natural colanic acid gene cluster can be replaced with a stronger promoter to generate a recombinant colanic acid gene cluster to drive additional production of GDP-fucose. In addition, as described in the Examples, additional copies of the colanic acid gene cluster or its selected genes can be introduced into genetically engineered cells.

[0115] In an embodiment, the colanic acid gene cluster can be expressed from its natural genomic locus. Expression can be actively modulated. Expression can be modulated in the following ways: replacing the natural promoter with the target promoter and / or by expressing the gene cluster from another rather than the natural genomic locus, or free expressing the colanic acid gene cluster or its specific gene to increase the number of copies of the colanic acid gene encoding the protein.

[0116] With respect to the present disclosure, the term "native genomic locus", with respect to the colanic acid gene cluster, relates to the original and natural location of the gene cluster in the genome of a genetically engineered cell.

[0117] The de novo GDP-fucose pathway genes responsible for the formation of GDP-fucose comprise or consist of the following genes:

[0118] i) manA, which encodes the protein mannose-6-phosphate isomerase (EC 5.3.1.8, UniProt accession number nr. P00946), which promotes the interconversion of fructose 6-phosphate (F6P) and mannose-6-phosphate;

[0119] ii) manB, which encodes a protein phosphomannose mutase (EC 5.4.2.8, UniProt accession number nrP24175), which is involved in the biosynthesis of GDP-mannose by catalyzing the conversion of mannose-6-phosphate to mannose-1-phosphate;

[0120] iii) manC, which encodes the protein mannose-1-phosphate guanylyltransferase (EC: 2.7.7.13, UniProt accession number nr P24174), which is involved in the GDP-mannose biosynthesis process of GDP-mannose from GTP and α-D-mannose-1-phosphate;

[0121] iv) gmd, which encodes the protein GDP-mannose-4,6-dehydratase (UniProt accession number nr P0AC88), which catalyzes the conversion of GDP-mannose to GDP-4-dehydro-6-deoxy-D-mannose;

[0122] v) wcaG (fcl), which encodes the protein GDP-L-fucose synthase (EC 1.1.1.271, UniProt accession number nrP32055), which catalyzes the two-step NADP-dependent conversion of GDP-4-dehydro-6-deoxy-D-mannose to GDP-fucose.

[0123] Therefore, it is preferred that when producing one or more fucosylated heterologous products, the genetically engineered cells overexpress the entire colonic acid gene cluster (as shown in SEQ ID NO: 12, or a functional variant thereof) and / or one or more genes of the de novo GDP-fucose pathway selected from manA, manB, manC, gmd and wcaG.

[0124] Lactose permease

[0125] Lactose permease is a membrane protein that is a member of the major facilitator superfamily and can be classified as a symporter that uses the proton gradient toward the cell to transport β-galactosides such as lactose into the cell in the same direction. In oligosaccharide production, especially in the production of human milk oligosaccharides (HMOs), lactose is often the initial substrate for any HMO of interest that is modified to produce a biotransformation that occurs inside the cell. Therefore, when producing HMOs, it is desirable to be able to import lactose into the cell, for example by expressing / or overexpressing a lactose permease such as lacY of E. coli K.-12.

[0126] In an embodiment, the lactose permease is as shown in SEQ ID NO: 15, or a functional homologue thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO: 15, such as at least 85%, 90% or 95% identical.

[0127] In an embodiment, the expression of the lactose permease is regulated by a promoter according to the invention.

[0128] β-Galactosidase

[0129] 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 includes an endogenous lacZ gene (e.g., GenBank Accession No. V00296 (GI: 41901)). For the purposes of the present invention, when producing HMOs, the genetically engineered cells are genetically manipulated to not contain any β-galactosidase gene or to contain an inactivated β-galactosidase gene. The gene can be inactivated by completely or partially deleting the corresponding nucleic acid sequence from the bacterial genome, or the gene sequence is mutated in such a way that it is not transcribed, or if transcribed, the transcript is not translated or if translated into a protein (i.e., β-galactosidase), the protein does not have the corresponding enzymatic activity. In this way, the HMO-producing bacteria accumulate an increased intracellular lactose pool, which is beneficial for the production of HMOs.

[0130] Transporter

[0131] 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 to the outside of the cell membrane. More complex HMO products may remain in the cell, which may eventually damage cell growth, thereby affecting the possible total yield of the product from a single fermentation. HMO transport can be promoted by the main facilitator superfamily transporter, which promotes the outflow of sugar derivatives from cells to the supernatant. Transporters can be exogenous or endogenous, and overexpressed under fermentation conditions to enhance the output of the oligosaccharide derivatives (HMO) produced. The specificity of the sugar moiety of the product to be secreted can be changed by mutation by means of known recombinant DNA technology.

[0132] Thus, the genetically engineered cell according to the invention may further comprise a nucleic acid sequence encoding a transporter protein capable of exporting one or more fucosylated human milk oligosaccharide products, such as the transporter protein may for example be a member of the major facilitator superfamily of transporters.

[0133] In recent years, several new and potent major facilitator superfamily transporters have been identified, each specific for a different recombinantly produced HMO, and the development of recombinant cells expressing said proteins is advantageous for large-scale industrial HMO manufacturing, see, e.g., WO2010 / 142305, WO2017 / 042382, WO2021 / 148615, WO2021 / 148614, WO2021 / 148611 and WO2021 / 148620.

[0134] Therefore, in one or more exemplary embodiments, the genetically engineered cell according to the methods described herein also comprises a gene product that serves as the LNFP-III transporter. The gene product that serves as the LNFP-III transporter can be encoded by a recombinant nucleic acid sequence expressed in the genetically engineered cell. The recombinant nucleic acid sequence encoding the LNFP-III transporter can be integrated into the genome of the genetically engineered cell, or expressed using a plasmid.

[0135] Yj Y

[0136] In one embodiment, the genetically modified cell of the present disclosure comprises a nucleic acid sequence encoding an efflux transporter capable of exporting fucosylated oligosaccharide products, particularly LNFP-III, to the extracellular medium. In this article, the efflux transporter preferably encodes a putative MFS (major facilitator superfamily) transporter, such as a putative metabolite transporter YjhB, which is encoded by the gene yjhB of Escherichia coli. More specifically, the present disclosure relates to a genetically modified cell optimized to produce oligosaccharides, particularly LNFP-III, comprising a recombinant nucleic acid encoding a protein having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or 100% sequence identity to an amino acid sequence having the amino acid sequence of UniProt accession number P39352.

[0137] Yj Y

[0138] In one embodiment, the genetically modified cell of the present disclosure comprises a nucleic acid sequence encoding an efflux transporter capable of exporting fucosylated oligosaccharide products, particularly LNFP-III, to the extracellular medium. In this article, the efflux transporter preferably encodes a putative MFS (major facilitator superfamily) transporter, such as the uncharacterized MFS-type transporter YhhS encoded by the gene yhhS of Escherichia coli. More specifically, the present disclosure relates to a genetically modified cell optimized to produce oligosaccharides, particularly LNFP-III, comprising a recombinant nucleic acid encoding a protein having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or 100% sequence identity to an amino acid sequence having the amino acid sequence of UniProt accession number P37621.

[0139] YepQ

[0140] In one embodiment, the genetically modified cell of the present disclosure comprises a nucleic acid sequence encoding an efflux transporter capable of exporting a fucosylated oligosaccharide product, particularly LNFP-III, to the extracellular medium. In the present context, the efflux transporter preferably encodes a putative MFS (major facilitator superfamily) transporter, such as the uncharacterized transporter YebQ encoded by the gene yebQ from Escherichia coli. More specifically, the present disclosure relates to a genetically modified cell optimized to produce oligosaccharides, particularly LNFP-III, comprising a recombinant nucleic acid encoding a protein having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or 100% sequence identity with an amino acid sequence having the amino acid sequence of UniProt accession number P76269.

[0141] Genetically engineered cells

[0142] In this context, the terms "genetically engineered cells" and "genetically engineered cells" are used interchangeably. As used herein, a "genetically engineered cell" is a host cell whose genetic material has been altered by human intervention using genetic engineering techniques, such as, but not limited to, transformation or transfection with heterologous polynucleotide sequences, Crisper / Cas editing, and / or random mutagenesis. In one embodiment, the genetically engineered cell has been transformed or transfected with a recombinant nucleic acid sequence.

[0143] The genetic modification may for example be selected from the inclusion of glycosyltransferases as described in the above sections, and / or metabolic pathway engineering and inclusion of transporters, which the skilled person will know how to combine into a genetically engineered cell capable of producing one or more fucosylated HMOs.

[0144] In one aspect of the present invention, the genetically engineered cell comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, which is capable of producing at least 30% LNFP-III of the total molar HMO content produced by the cell. In a preferred embodiment, the one or more fucosylated HMOs are LNFP-III. In an embodiment, the total HMO produced by the cell is substantially free of LNFP-VI and / or LNDFH-III. In the present invention, substantially free of LNFP-VI and / or LNDFH-III should be understood as the content of LNFP-VI and / or LNDFH-III in the total HMO produced by the cell is less than 1%.

[0145] In additional embodiments, the cells of the invention produce a mixture of HMOs comprising LNFP-III, LNnT, LNDFH-III and / or pLNnH.

[0146] 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 alpha-1,3-fucosyltransferase activity, wherein the enzyme is selected from the group consisting of:

[0147] a) Camp1 having 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, [Camp1 amino acid],

[0148] b) Parm1 having 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, [Parm1 amino acid],

[0149] c) Hpull1 having 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, [Hpull1 amino acid],

[0150] d) Med1 having 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, [Med1 amino acid], and

[0151] e) Hacin2 having 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, [Hacin2 amino acid].

[0152] In a preferred 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-fucosyltransferase activity, wherein the enzyme is selected from the group consisting of:

[0153] a) Camp1 having 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, [Camp1 amino acid],

[0154] b) Parm1 having 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, [Parm1 amino acid],

[0155] c) Hpull1 having 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, [Hpull1 amino acid], and

[0156] d) Med1 having an amino acid sequence according to SEQ ID NO: 4, or a functional homologue thereof, said functional homologue having an amino acid sequence at least 80% identical to SEQ ID NO: 4, [Med1 amino acid].

[0157] In a further preferred 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-fucosyltransferase activity, wherein the enzyme is selected from the group consisting of:

[0158] a) Camp1 having 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, [Camp1 amino acid],

[0159] b) Parm1 having 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, [Parm1 amino acid], and

[0160] c) Hpull1 having 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, [Hpull1 amino acid].

[0161] In a further preferred 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-fucosyltransferase activity, wherein the enzyme is selected from the group consisting of:

[0162] a) Camp1 having 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, [Camp1 amino acid], and

[0163] b) Parm1 having an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof, said functional homologue having an amino acid sequence at least 80% identical to SEQ ID NO: 2, [Parm1 amino acid].

[0164] In a further preferred 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-fucosyltransferase activity, wherein the enzyme is selected from the group consisting of:

[0165] a) Camp1 having 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, [Camp1 amino acid],

[0166] b) Hpull1 having 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, [Hpull1 amino acid].

[0167] In a currently preferred embodiment, the genetically engineered cell capable of producing one or more fucosylated HMOs comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having alpha-1,3-fucosyltransferase activity, wherein the enzyme is Camp1 having an amino acid sequence according to SEQ ID NO: 1, or a functional homolog thereof having an amino acid sequence at least 80% identical to SEQ ID NO: 1, [Camp1 amino acid].

[0168] In a currently preferred embodiment, the genetically engineered cell capable of producing fucosylated HMO comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity as described herein, said cell being capable of producing LNFP-III in an amount of at least 30%, such as at least 50%, such as at least 80% of the total molar HMO content produced by the cell.

[0169] HMO mixture produced by cells

[0170] Genetically engineered cells containing more than two of the glycosyltransferases described herein typically produce a mixture of HMOs as a result of a multi-step process to the final HMO product. When producing LNFP-III from lactose as the initial substrate, it is expected that the cells will produce small amounts of 3-FL (fucosylated lactose), LNT-II, LNnT, LNFP-III and pLNnH, and potentially also LNFP-VI and LNDFH-.

[0171] In some embodiments, the genetically engineered cells of the invention express Camp1, which comprises or consists of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 1, and the molar % content of LNFP-III produced by the genetically engineered cells is higher than 90%, such as higher than 94%, such as higher than 96%, such as higher than 97% of the total HMOs.

[0172] In some embodiments, the genetically engineered cells of the invention express Parm1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or 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 thereto, and the molar % content of LNFP-III produced by the genetically engineered cells is higher than 70%, such as higher than 75%, such as higher than 80%, such as higher than 85% of the total HMOs.

[0173] In some embodiments, the genetically engineered cells of the invention express Hpull1 comprising or consisting of an amino acid sequence of SEQ ID NO:3, or 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 thereto, and the molar % content of LNFP-III produced by the genetically engineered cells is higher than 70%, such as higher than 75% of the total HMOs.

[0174] In some embodiments, the genetically engineered cells of the invention express Med1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, or 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 thereto, and the molar % content of LNFP-III produced by the genetically engineered cells is higher than 50%, such as higher than 60%, such as higher than 70%, such as higher than 75% of the total HMOs.

[0175] In some embodiments, the genetically engineered cells of the present invention express Hacin2, which comprises the amino acid sequence of SEQ ID NO:5, or 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, or consists of it, and the molar % content of LNFP-III produced by the genetically engineered cells is higher than 30% of the total HMOs.

[0176] The mole % of the individual HMO components, supported by experimental data from the Examples, show exemplary HMO composition ranges where the mixture of HMOs consists essentially of LNFP-III and LNnT, 3-FL, or pLNnH.

[0177] In this regard, the mixture of HMOs can consist essentially of i) LNFP-III and LNnT, ii) LNFP-III and 3-FL, iii) LNFP-III, LNnT, and LNDFH-III, or iv) LNFP-III, LNnT, and pLNnH.

[0178] In some embodiments, the genetically engineered cells of the invention express Camp1 comprising or consisting of the amino acid sequence of SEQ ID NO:1, or 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 resulting mixture consists essentially of LNFP-III and LNnT or LNFP-III and 3-FL, wherein 1-5% of the total molar HMO content in the mixture is LNnT, or 1-20% of the total molar HMO content in the mixture is 3-FL, and 95-99% of the total molar HMO content in the mixture is LNFP-III, totaling a total of 100% molar content.

[0179] In some embodiments, the genetically engineered cells of the invention express Parm1 comprising or consisting of the amino acid sequence of SEQ ID NO:2, or 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 resulting mixture consists essentially of LNFP-III, LNnT and LNDFH-III, wherein 3-25% of the total molar HMO content in the mixture is LNnT, 1-10% of the total molar HMO content in the mixture is LNDFH-III, and 70-90% of the total molar HMO content in the mixture is LNFP-III, totaling a total of 100% molar content.

[0180] In some embodiments, the genetically engineered cells of the invention express Hpull1 comprising or consisting of an amino acid sequence of SEQ ID NO:3, or 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 resulting mixture consists essentially of LNFP-III and LNnT, wherein 10-30% of the total molar HMO content in the mixture is LNnT, or 70-90% of the total molar HMO content in the mixture is LNFP-III, totaling a total of 100% molar content.

[0181] In some embodiments, the genetically engineered cells of the invention express Med1 comprising, or consisting of, the amino acid sequence of SEQ ID NO:4, or 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 resulting mixture consists essentially of LNFP-III and LNnT, or LNFP-III, LNnT and pLNnH, or LNFP-III and 3-FL, wherein 0-45% of the total molar HMO content in the mixture is LNnT, 0-8% of the total molar HMO content in the mixture is pLNnH, 0-20% of the total molar HMO content in the mixture is 3-FL, and 50-90% of the total molar HMO content in the mixture is LNFP-III, totaling 100% molar content.

[0182] In some embodiments, the genetically engineered cells of the present invention express Hacin2 comprising or consisting of the amino acid sequence of SEQ ID NO:5, or 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 resulting mixture consists essentially of LNFP-III, LNnT and pLNnH, wherein 40-70% of the total molar HMO content in the mixture is LNnT, 0-10% of the total molar HMO content in the mixture is pLNnH, and 20-60% of the total molar HMO content in the mixture is LNFP-III, totaling 100% molar content.

[0183] Host cells

[0184] 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.

[0185] 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.

[0186] Regarding bacterial host cells, there are in principle no restrictions; they may be fungal genus (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 cells have properties that allow cultivation to high cell densities. Non-limiting examples of bacterial host cells suitable for the recombinant industrial production of HMOs according to the invention may be 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, may also be used.Similarly, bacteria of the genera Lactobacillus and Lactococcus can be engineered using the methods described herein, including, but not limited to, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the invention described herein. Also included are strains engineered as described herein from Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa).

[0187] Non-limiting examples of fungal host cells suitable for the recombinant industrial production of heterologous products are e.g. yeast cells, e.g. of the genera Komagataella, Kluyveromyces, Yarrowia, Pichia, Saccaromyces, Schizosaccharomyces or Hansenula, or a filamentous fungus selected from the genera Aspargillus, Fusarium or Thricoderma.

[0188] In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of Escherichia sp., Bacillus sp., Lactobacillus sp., Corynebacterium sp., and Campylobacter sp.

[0189] 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.

[0190] In one or more exemplary embodiments, the genetically engineered cell is Bacillus subtilis.

[0191] In one or more exemplary embodiments, the genetically engineered cell is Saccharomyces cerevisiae or Pichia pastoris.

[0192] In one or more exemplary embodiments, the genetically engineered cell is Escherichia coli.

[0193] 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.

[0194] Recombinant nucleic acid sequence

[0195] The present invention relates to a genetically engineered cell comprising a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, such as an enzyme selected from Camp1, Parm1, Hpull1, Med1 and Hacin2, and wherein the cell produces human milk oligosaccharides (HMOs). In particular, fucosylated HMOs, and preferably having a molar % content of LNFP-III above, or at least 30% of the total HMOs produced.

[0196] In the present context, the terms "recombinant nucleic acid sequence", "recombinant gene / nucleic acid / nucleotide sequence / DNA encoding..." or "coding nucleic acid sequence" are used interchangeably and are intended to refer to an artificial nucleic acid sequence (i.e., produced in vitro using standard laboratory methods for making nucleic acid sequences) that comprises a set of 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).

[0197] 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) 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.

[0198] The term "nucleic acid" includes RNA, DNA and cDNA molecules. It will be appreciated that due to the degeneracy of the genetic code, a large number of nucleic acid sequences encoding a given protein can be generated.

[0199] 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, for example, a promoter sequence or other non-coding regulatory sequences.

[0200] In addition, the recombinant nucleic acid sequence can be heterologous. As used herein, "heterologous" refers to a polypeptide, amino acid sequence, nucleic acid sequence or nucleotide sequence that is foreign to a cell or organism, i.e., a polypeptide, amino acid sequence, nucleic acid molecule or nucleotide sequence that is not naturally present in the cell or organism.

[0201] The present invention also relates to a nucleic acid construct comprising a coding nucleic acid sequence, i.e. a recombinant DNA sequence of a gene of interest (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 the lac operon or the glp operon, or a promoter sequence derived from another genomic promoter DNA sequence, or a synthetic promoter sequence, wherein the coding and promoter sequences are operably linked.

[0202] 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 the promoter sequence stimulates or regulates transcription of the coding sequence in a suitable host cell or other expression system.

[0203] Typically, promoter sequences operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, ie, they are cis-acting.

[0204] 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.

[0205] 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 included 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 Camp1, Parm1, Hpull1, Med1 and Hacin2, such as the nucleic acid sequences according to SEQ ID NO: 7, 8, 9, 10 or 11, or functional variants thereof.

[0206] The genetically engineered cells according to the present invention may also comprise multiple copies of a recombinant nucleic acid sequence encoding a fucosyltransferase. Increasing the copy number of the fucosyltransferase is shown in Example 1 to change the ratio of the HMO produced. Specifically, it is shown that increasing the copy number of Camp1 by introducing a high copy number plasmid results in a slight increase in 3-FL while eliminating the production of LNnT from the strain. In addition, increasing the copy number of Parm1 to two genomic copies and also including a high copy plasmid (pUC57-Parm1-PglpF-amp) increases the relative amount of LNFP-III produced while reducing the amount of LNnT produced. The increase in the copy number of Parm1 also increases the formation of LNDFH-III, indicating that the increased activity also increases the fucosylation of the Glc portion in LNnT. In addition, increasing the copy number of Med1 to two genetic copies (strain Med1_2) increased LNFP-III production, while additional expression of Med1 from a high-copy plasmid further increased expression, resulting in a slight decrease in LNFP-II levels, while a sudden increase in 3-FL production was observed. In addition, it was observed that in the high-expressing Med1 strain, all LNnT produced by the cells was fucosylated.

[0207] Thus, copy number variation can be used to produce tailored specific HMO mixtures, in this case mixtures containing different ratios of LNFP-III, LNnT and / or 3-FL, depending on the need for a particular product.

[0208] Therefore, in an embodiment, the genetically engineered cell of the present invention comprises one, two, three or more genomic copies of a recombinant nucleic acid sequence encoding a glycosyltransferase, wherein the glycosyltransferase is selected from Camp1, Parm1, Hpull1, Med1 and Hacin2 having 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 is at least 80% identical to SEQ ID NO: 1, 2, 3 or 4 [Camp1, Parm1, Hpull1, Med1 and Hacin2 amino acids]. In other embodiments, the recombinant nucleic acid sequence encoding a glycosyltransferase is encoded on a plasmid, and the glycosyltransferase is selected from Camp1, Parm1, Hpull1, Med1 and Hacin2 having 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 or 4 [Camp1, Parm1, Hpull1, Med1 and Hacin2 amino acids]. In another embodiment, the plasmid is a high copy number plasmid, preferably a pUC57 plasmid.

[0209] In a further embodiment, the genetically engineered cell according to the present invention comprises one, two, three or more genomic copies and / or plasmid-borne copies of a recombinant nucleic acid sequence encoding a glycosyltransferase, wherein the glycosyltransferase is selected from Med1 and Hacin2 having 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 is at least 80% identical to SEQ ID NO: 1, 2, 3 or 4 [Camp1, Parm1, Hpull1, Med1 and Hacin2 amino acids].

[0210] One embodiment of 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: a) Camp1, which comprises a nucleic acid sequence of SEQ ID NO:7 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or for example at least 99% sequence identity with SEQ ID NO:7, or consists thereof; b) Parm1, which comprises a nucleic acid sequence of SEQ ID NO:8 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or for example at least 99% sequence identity with SEQ ID NO:8, or consists thereof; c) Hpull1, which comprises a nucleic acid sequence of SEQ ID NO:9 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or for example at least 99% sequence identity with SEQ ID NO:9, or consists thereof; d) Med1, which comprises a nucleic acid sequence of SEQ ID NO:10 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or for example at least 99% sequence identity with SEQ ID NO:11, or consists thereof; NO:10 has a nucleic acid sequence with 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, or consists of it; and / or e) Hacin2, which comprises the nucleic acid sequence of SEQ ID NO:11 or a nucleic acid sequence with 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:11, or consists of it.

[0211] Preferably, the fucosyltransferase coding sequence is under the control of a promoter sequence selected from the group consisting of those having the nucleic acid sequence shown in Table 4.

[0212] Table 4 - Selected promoter sequences

[0213]

[0214]

[0215] *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 relative to the PglpF promoter was calculated, and the range indicates results from multiple assays.

[0216] The promoter may be of heterologous origin, be native to the genetically modified cell, or may be a recombinant promoter combining heterologous and / or native elements.

[0217] One approach to increasing product yields may be to modulate the production of a desired enzyme activity for producing the product, such as a glycosyltransferase or an enzyme involved in a glycosyl donor biosynthetic pathway.

[0218] Increasing the promoter strength that drives the expression of the desired enzyme may be a way to achieve this goal. The strength of the promoter can be evaluated using the lacZ enzyme assay, in which the β-galactosidase activity is measured as described above (see Miller JH Experiments in molecular genetics, Cold spring Harbor Laboratory Press, NY, 1972). In short, cells are diluted with Z buffer and infiltrated with sodium dodecyl sulfate (0.1%) and chloroform. The LacZ assay is performed at 30°C. The sample is preheated, the assay is started by adding 200 μl of o-nitro-phenyl-β-galactosidase (4 mg / ml), and 500 μl of 1M Na2CO3 is added when the sample turns slightly yellow to stop the assay. The release of o-nitrophenol is subsequently determined as the change in optical density at 420 nm. The specific activity is reported in Miller units (MU) [A420 / (min*ml*A600)]. The regulatory elements with an activity higher than 10,000MU are considered strong, the regulatory elements with an activity lower than 3000MU are considered weak, and the regulatory elements between the two have medium strength. An example of a strong regulatory element is the PglpF promoter, which has 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 under the control of a strong promoter selected from SEQ ID NO 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27.

[0219] In an embodiment, the expression of the nucleic acid sequence of the present invention is controlled by: PglpF (SEQ ID NO:28) or Plac (SEQ ID NO:37) promoter or PmglB_UTR70 (SEQ ID NO:25) or PglpA_70UTR (SEQ ID NO:26) or PglpT_70UTR (SEQ ID NO:27), or variants thereof, such as the promoters shown in Table 4, in particular the PglpF_SD4 variant of SEQ ID NO:23 or the Plac_70UTR variant of SEQ ID NO:19 or the PmglB_70UTR variants of SEQ ID NO:16, 17, 18, 20, 21, 22, 24 and 25. Other 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 by reference).

[0220] In a preferred embodiment, the recombinant nucleic acid sequence is individually controlled by one or more promoters selected from the group consisting of PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 28, 37, 25, 26 and 27, respectively), and variants thereof.

[0221] Integration of the nucleic acid construct of interest contained in the construct (expression cassette) into the bacterial genome can be achieved by conventional methods, for example by using a linear cassette containing flanking sequences homologous to specific sites on the chromosome, as described for the attTn7 site (Waddell CS and Craig NL, Genes Dev. (1988) Feb; 2(2): 137-49.); methods for genomic integration of nucleic acid sequences, wherein recombination is mediated by the Red recombinase function of bacteriophage λ or the RecE / RecT recombinase function of the Rac prophage (Murphy, J Bacteriol. (1998); 180(8): 2063-7; Zhang et al., Nature Genetics (1998) 20: 123-128; Muyrers et al., EMBO Rep. (2000) 1(3): 239-243); methods based on Red / ET recombination (Wenzel et al., Chem. Biol. (2003); 1(4): 157-163; Muyrers et al., EMBO Rep ... 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, for example, by loss or gain of marker gene or gene function.

[0222] In one or more exemplary embodiments, the present disclosure relates to one or more recombinant nucleic acid sequences as shown in SEQ ID NOs: 7, 8, 9, 10 and 11 [nucleic acids encoding Camp1, Parm1, Hpull1, Med1 and Hacin1, respectively].

[0223] 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:7, 8, 9, 10 and 11 [nucleic acids encoding Camp1, Parm1, Hpull1, Med1 and Hacin1, respectively].

[0224] Sequence identity

[0225] The term "sequence identity" as used herein describes the correlation between two amino acid sequences or between two nucleotide sequences based on their paired comparison, i.e., a candidate sequence (e.g., a sequence of the present invention) and a reference sequence (e.g., a prior art sequence). For purposes of the present disclosure, 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 parameter used is gap open penalty (gap open penalty) 10, gap extension penalty (gap extension penalty) 0.5, and EBLOSUM62 (EMBOSS version of 30BLOSUM62) substitution matrix.The output (using-nobrief option to obtain) of Needle marked as "identity" is used as percentage identity.Usually sequence identity can be calculated as follows: (same residue x 100) / (length of comparison zone).

[0226] For the purpose of the present disclosure, use in EMBOSS bag (EMBOSS:The European Molecular BiologyOpen Software Suite, Rice et al., 2000, Trends Genet.16:276-277), 10 preferred versions 5.0.0 or higher versions of the Needle program implemented in the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, the same) determine the sequence identity between two nucleotide sequences.The parameter used is gap open penalty (gap open penalty) 10, gap extension penalty (gap extension penalty) 0.5, and DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.The output (using-nobrief option to obtain) of the Needle marked as "identity" is used as identity percentage.Usually sequence identity can be calculated as follows: (identical deoxyribonucleotide x 100) / (length of comparison zone).

[0227] Functional homologs

[0228] Functional homologues or functional variants of protein / nucleic acid sequences as described herein are protein / nucleic acid sequences with genetic code changes that retain their original functions. Functional homologues can be obtained by mutagenesis, or can be naturally occurring variants from the same or other species. Compared with the function of the protein / nucleic acid sequence, functional homologues should have at least 50%, for example at least 60%, 70%, 80%, 90% or 100% of the remaining function.

[0229] Functional homologs of any of the disclosed amino acid or nucleic acid sequences may also have higher functionality. Functional homologs of any of the amino acid sequences shown in Table 1 or recombinant nucleic acids encoding any of the sequences of SEQ ID NO: 7, 8, 9, 10 or 11 should ideally be able to participate in the production of fucosylated HMOs in terms of increased HMO yield, export of HMO products from cells or import of substrates for HMO production, such as acceptor oligosaccharides of at least three monosaccharide units, improved purity / byproduct formation, reduction in biomass formation, viability of genetically engineered cells, robustness of genetically engineered cells according to the present disclosure, or reduction in the number of consumables required for production.

[0230] Uses of genetically engineered cells or enzymes

[0231] 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).

[0232] Therefore, the present invention also relates to the use of a fucosyltransferase having α-1,3-fucosyltransferase activity in the production of a fucosylated product, wherein the fucosyltransferase is selected from Camp1, Parm1, Hpull1, Med1 and Hacin2 having 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 [Camp1, Parm1, Hpull1, Med1 and Hacin2 amino acids]. In a further embodiment, the fucosyltransferase used to produce the fucosylated product is selected from Camp1, Parm1, Hpull1 and Hacin2 having an amino acid sequence according to SEQ ID NO: 1, 2, 3 or 5, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO: 1, 2, 3 or 5 [Camp1, Parm1, Hpull1 and Hacin2 amino acids]. In an embodiment, the fucosyltransferase of the invention 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 LNFP-III, or one or more fucosylated polypeptides.

[0233] In an exemplary embodiment, the genetically engineered cells and / or nucleic acid constructs according to the invention are used to produce HMOs. Preferably, in the production of a mixture of HMOs, the molar % content of LNFP-III produced by the genetically engineered cells is higher than 30% of the total amount of HMOs produced. Preferably, in the production of HMOs in which pure LNFP-III is intended as the main product, the molar % content of LNFP-III produced by the genetically engineered cells is higher than 70%, such as higher than 75%, such as higher than 80%, such as higher than 90% of the total amount of HMOs produced.

[0234] In a further embodiment, the fucosyltransferase used for the production of the fucosylated product is selected from Camp1, Parm1, Hpull1 having an amino acid sequence according to SEQ ID NO: 1, 2 or 3, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO: 1, 2 or 3 [Camp1, Parm1 and Hpull1]. In an embodiment, the fucosyltransferase of the invention is also used for the production of a fucosylated product, wherein the fucosylated product is one or more fucosylated oligosaccharides, such as one or more HMOs, preferably LNFP-III, or one or more fucosylated polypeptides, wherein the mole % content of LNFP-III produced by the genetically engineered cell is higher than 75%.

[0235] In one exemplary embodiment, the genetically engineered cells and / or nucleic acid constructs according to the present invention are used to produce one or more fucosylated HMOs, preferably LNFP-III.

[0236] The production of these HMOs may require the presence of two or more glycosyltransferase activities.

[0237] Method for producing fucosylated human milk oligosaccharides (HMO)

[0238] The present invention also relates to a method for producing one or more fucosylated human milk oligosaccharides (HMOs), said method comprising culturing a genetically engineered cell according to the present invention.

[0239] One aspect is a method of producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising the steps of:

[0240] a) providing a genetically modified cell as described herein; and

[0241] b) culturing the cell according to (a) in a suitable cell culture medium to produce said one or more fucosylated HMOs, and

[0242] c) optionally, purifying the one or more fucosylated HMOs.

[0243] In one embodiment of the invention, after culturing the genetically engineered cells as described herein, the mixture of HMOs consists essentially of LNFP-III and LNnT, wherein the amount of 3-FL, LNDFH-III and / or pLNnH is low, for example, less than 10% of the total molar HMO content in the composition.

[0244] In one embodiment of the invention, after culturing the genetically engineered cells as described herein, the mixture of HMOs consists essentially of 30-55 mol% LNFP-III, 40-65 mol% LNnT and 3-8% pLNnH, totaling 100 mol% in total.

[0245] In one embodiment of the invention, after culturing the genetically engineered cells as described herein, the mixture of HMOs consists essentially of 30-99 mol% LNFP-III, 0-65 mol% LNnT, 0-20% 3-FL, 0-10% LNDFH-III and 0-8% pLNnH, totaling 100% molar content in total.

[0246] In one embodiment of the invention, after culturing the genetically engineered cells as described herein, the mixture of HMOs consists essentially of 70-99% LNFP-III and 1-50% LNnT, totaling 100% molar content in total.

[0247] In one embodiment of the invention, after culturing the genetically engineered cells as described herein, the mixture of HMOs consists essentially of 80-99% LNFP-III, 0-5% LNnT and 0-10% LNDFH-III, totaling 100% molar content.

[0248] In one embodiment of the invention, after culturing the genetically engineered cells as described herein, the mixture of HMOs consists essentially of 80-99 mole % LNFP-III and 0-20 mole % 3-FL.

[0249] The present invention particularly relates to a method for producing human milk oligosaccharides (HMOs), wherein the mole % content of LNFP-III produced by the genetically engineered cells is higher than 30% of the total amount of HMOs produced, such as higher than 50% or such as higher than 80% of the total amount of HMOs. In an embodiment, the fucosylated HMO is LNFP-III.

[0250] Thus, the present invention relates to a method for producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically engineered cell comprising:

[0251] A recombinant nucleic acid sequence encoding a fucosyltransferase having alpha-1,3-fucosyltransferase activity, wherein the enzyme is selected from the group consisting of:

[0252] a. Camp1 having 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, [Camp1 amino acid];

[0253] b. Parm1 having 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, [Parm1 amino acid];

[0254] c. Hpull1 having 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, [Hpull1 amino acid];

[0255] d. Med1 having 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, [Med1 amino acid];

[0256] e. Hacin2 having 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, [Hacin2 amino acid]; and

[0257] Wherein the cells produce a fucosylated HMO, preferably LNFP-III.

[0258] Another embodiment of the present invention is a method of producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically engineered cell comprising:

[0259] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0260] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,4-galactosyltransferase activity; and

[0261] c. A recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, wherein the enzyme is selected from:

[0262] i. Camp1, which has 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, [Camp1 amino acid];

[0263] ii. Parm1 having 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, [Parm1 amino acid];

[0264] iii. Hpull1 having 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, [Hpull1 amino acid], and

[0265] iv. Med1 having 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, [Med1 amino acid];

[0266] v. Hacin2, which has 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, [Hacin2 amino acid]; and

[0267] Wherein at least one of the fucosylated HMOs is LNFP-III.

[0268] Another embodiment of the present invention relates to a method of producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically engineered cell comprising:

[0269] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0270] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,4-galactosyltransferase activity; and

[0271] c. A recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, wherein the enzyme is selected from:

[0272] i. Camp1, which has 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, [Camp1 amino acid];

[0273] ii. Parm1 having 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, [Parm1 amino acid]; and

[0274] iii. Hpull1 having 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, [Hpull1 amino acid]. iii.

[0275] In an embodiment, the fucosylated HMO is LNFP-III.

[0276] The method particularly comprises culturing a genetically engineered cell producing a fucosylated HMO, wherein the LNFP-III content produced by the cell is at least 30% of the total HMO content produced by the cell. In addition, the method comprises culturing a genetically engineered cell producing a fucosylated HMO.

[0277] 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 an energy source (carbon source) selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.

[0278] In one aspect, the process according to the invention produces a mixture of HMOs, wherein at least 50%, such as at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% of the molar content of the total amount of HMOs produced is LNFP-III.

[0279] In one aspect, the process according to the invention produces a mixture of HMOs, wherein the mixture of HMOs produced is substantially free of LNFP-VI and / or LNDFH-III.

[0280] In one aspect, a mixture of HMOs is produced according to the method of the invention, wherein the cell produces a mixture of HMOs comprising LNFP-III, LNnT, 3-FL and / or pLNnH.

[0281] In one aspect, LNFP-III is produced according to the methods of the invention.

[0282] In order to be able to produce fucosylated HMOs in the method according to the invention, the genetically engineered cells comprise a biosynthetic pathway for the preparation of a fucose sugar nucleotide, such as GDP-fucose.

[0283] In a preferred embodiment of the method of the present invention, the genetically engineered cell comprises an upregulated biosynthetic pathway for preparing a fucose sugar nucleotide. Preferably, in the method of the present invention, the fucose sugar nucleotide is GDP-fucose. Therefore, in the method of the present invention, the sugar nucleotide pathway is expressed and / or upregulated in the genetically engineered cell, wherein the GDP-fucose pathway is encoded by the colanic acid gene cluster (CA) from the Escherichia coli of SEQ ID NO: 12. In the method of the present invention, the upregulation of the GDP-fucose pathway is obtained by integrating one or more copies of the colanic acid gene cluster (CA) from the Escherichia coli of SEQ ID NO: 12 into the genome of the host cell.

[0284] The methods of the invention include providing a glycosyl donor that is synthesized separately by one or more genetically engineered cells and / or added exogenously to the culture medium from an alternative source.

[0285] In one aspect, the method of the present invention further comprises providing an acceptor sugar as a substrate for HMO formation, wherein the acceptor sugar comprises at least two monosaccharide units, which is exogenously added to the culture medium and / or produced by a separate microbial fermentation.

[0286] In one aspect, the method of the invention comprises providing an acceptor sugar comprising at least two monosaccharide units, which is exogenously added to the culture medium and / or produced by separate microbial fermentation, and is selected from lactose, LNT-II and LNnT. In a preferred embodiment, the substrate for HMO formation is lactose, which is fed into the culture during fermentation of the genetically engineered cells.

[0287] Fucosylated human milk oligosaccharides (HMOs) are recovered from the culture medium and / or genetically engineered cells.

[0288] Another embodiment of the present invention is a method of producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically engineered cell comprising:

[0289] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0290] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,4-galactosyltransferase activity; and

[0291] c. A recombinant nucleic acid sequence encoding a fucosyltransferase having alpha-1,3-fucosyltransferase activity, wherein the enzyme is Camp1 having an amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof having an amino acid sequence at least 80% identical to SEQ ID NO: 1, [Camp1 amino acid], and

[0292] Wherein at least one of the fucosylated HMOs is LNFP-III.

[0293] Another embodiment of the present invention is a method of producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically engineered cell comprising:

[0294] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0295] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,4-galactosyltransferase activity; and

[0296] c. A recombinant nucleic acid sequence encoding a fucosyltransferase having alpha-1,3-fucosyltransferase activity, wherein the enzyme is Parm1 having an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof having an amino acid sequence at least 80% identical to SEQ ID NO: 2, [Parm1 amino acid], and

[0297] Wherein at least one of the fucosylated HMOs is LNFP-III.

[0298] Another embodiment of the present invention is a method of producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically engineered cell comprising:

[0299] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0300] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,4-galactosyltransferase activity; and

[0301] c. A recombinant nucleic acid sequence encoding a fucosyltransferase having alpha-1,3-fucosyltransferase activity, wherein the enzyme is Hpull1 having an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof having an amino acid sequence at least 80% identical to SEQ ID NO: 3, [Hpull1 amino acid], and

[0302] Wherein at least one of the fucosylated HMOs is LNFP-III.

[0303] Another embodiment of the present invention is a method of producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically engineered cell comprising:

[0304] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0305] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,4-galactosyltransferase activity; and

[0306] c. A recombinant nucleic acid sequence encoding a fucosyltransferase having alpha-1,3-fucosyltransferase activity, wherein the enzyme is Med1 having an amino acid sequence according to SEQ ID NO: 4, or a functional homologue thereof having an amino acid sequence at least 80% identical to SEQ ID NO: 4, [Med1 amino acid], and

[0307] Wherein at least one of the fucosylated HMOs is LNFP-III.

[0308] Another embodiment of the present invention is a method of producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically engineered cell comprising:

[0309] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0310] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,4-galactosyltransferase activity; and

[0311] c. A recombinant nucleic acid sequence encoding a fucosyltransferase having alpha-1,3-fucosyltransferase activity, wherein the enzyme is Hacin2 having an amino acid sequence according to SEQ ID NO:5, or a functional homologue thereof having an amino acid sequence at least 80% identical to SEQ ID NO:5, [Hacin2 amino acid], and

[0312] Wherein at least one of the fucosylated HMOs is LNFP-III.

[0313] Cultivation or fermentation (used interchangeably herein) in a controlled bioreactor generally includes (a) a first phase of exponential cell growth 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 along with an acceptor oligosaccharide (such as lactose) to allow the formation of HMO products during this phase. Carbon (sugar) limitation refers to the phase in the fermentation where the growth rate is controlled by the concentration kinetics of the carbon source (sugar) in the culture broth, which in turn is determined by the rate of carbon addition (sugar feed rate) to the fermentor.

[0314] 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, for example 1,000 L, such as 10,000 L, 100,000 L, 200,000 L of culture broth. In general, a "manufacturing scale" process is defined as one capable of processing large volumes of the HMO product of interest, 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 means for stirring, aeration, nutrient feeding, monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.). To a large extent, the behavior of the expression system in laboratory scale methods, such as shake flasks, benchtop bioreactors or the deep well format described in the examples of the present disclosure, does allow prediction of the behavior of the system in the complex environment of the bioreactor.

[0315] 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 it can be chemically defined without any complex compounds. The carbon source can be selected from glucose, sucrose, fructose, xylose and glycerol. In one or more exemplary embodiments, the culture medium is supplemented with one or more energy sources and carbon sources selected from glycerol, sucrose and glucose. In other embodiments, lactose is added during the cultivation of the genetically engineered cells as a substrate for HMO formation.

[0316] 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.

[0317] In one or more exemplary embodiments, the genetically engineered cell comprises a PTS-dependent sucrose utilization system further comprising scrYA and scrBR operons as described in WO2015 / 197082 (incorporated herein by reference).

[0318] 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.

[0319] Recycling / Harvesting

[0320] Recovering fucosylated human milk oligosaccharides (HMOs) from culture medium and / or genetically engineered cells. In the present context, the term "recovering" is used interchangeably with the term "harvesting". Both "recovering" and "harvesting" in the context refer to collecting the produced HMOs from the culture / culture broth after the fermentation is terminated. In one or more exemplary embodiments, it may include collecting the HMOs included in both the biomass (i.e., host cells) and the culture medium, i.e., before / without separating the fermentation broth from the biomass. In other embodiments, the produced HMOs may be collected separately from the biomass and the fermentation broth, i.e., after / after separating the biomass from the culture medium (i.e., fermentation broth).

[0321] Separation of the cells from the culture medium may be performed by any method known to those skilled in the art, such as any suitable type of centrifugation or filtration. Separation of the cells from the culture medium may be performed immediately after harvesting the fermentation broth, or at a later stage after storage of the fermentation broth under appropriate conditions. Recovering the produced HMO from the remaining biomass (or total fermentation broth) comprises extracting the HMO from the biomass (i.e., the production cells).

[0322] After recovery from the fermentation, the HMOs can be used for further processing and purification.

[0323] HMOs can be purified according to procedures 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.

[0324] At the end of the culture, oligosaccharides can accumulate as products within cells and in the extracellular matrix.

[0325] The method according to the invention comprises culturing genetically engineered microbial cells in a culture medium, the culture medium being designed to support the growth of the microorganism and containing one or more carbohydrate sources or only carbon sources, for example 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.

[0326] Manufactured products

[0327] Depending on the use of the genetically engineered cell or nucleic acid construct, the term "manufactured product" refers to one or more HMOs that are intended to be the product HMO or HMOs. Various products are described above.

[0328] From the data presented in Example 2, it can be seen that LNFP-III produced by Camp1 fucosyltransferase has very high purity, demonstrating the ability and suitability of Camp1 to produce high purity LNFP-III in large-scale manufacturing.

[0329] Advantageously, the methods disclosed herein provide a reduced byproduct to product ratio and an increased overall yield of product (and / or total HMO). This less byproduct formation associated with product formation promotes increased product production and improves the efficiency of the production and product recovery processes, providing an excellent HMO manufacturing process.

[0330] The manufactured product may be a powder, composition, suspension or gel comprising one or more HMOs.

[0331] Mixture of HMOs

[0332] LNFP-III is a very well-studied HMO that has been associated with many different effects, including hepatic steatosis and insulin resistance, as well as autoimmunity and transplantation in murine models of type 1 diabetes (T1D) (Triantis et al., Immunological Effects of Human Milk Oligosaccharides, Front Pediatr. 2018 Jul2;6:190e Collection 2018.).

[0333] Therefore, LNFP-III and mixtures thereof are highly relevant as nutritional supplements or as therapeutic agents.

[0334] Thus, the product of manufacture can be a mixture of HMOs consisting essentially of i) LNFPIII and LNnT, ii) LNFP-III and 3-FL, iii) LNFP-III LNnT and pLNnH, or iv) LNFP-III LNnT and LNDFH-III, and wherein the composition is further substantially free of LNFP-VI.

[0335] Furthermore, the product of manufacture may be a mixture of HMO consisting essentially of i) LNFPIII and LNnT, ii) LNFP-III and 3-FL and iii) LNFP-III LNnT and pLNnH, and wherein the composition is further substantially free of LNFP-VI and LNDFH-III.

[0336] In this respect, in an embodiment, the mixture of HMOs according to the invention consists essentially of 30-55 mol % LNFP-III, 40-65 mol % LNnT and 3-8 mol % pLNnH, adding up to a total of 100 mol % molar content.

[0337] In a further embodiment, the mixture of HMOs according to the invention consists essentially of 30-99 mol % LNFP-III, 0-65 mol % LNnT, 0-20 mol % 3-FL, 0-10 mol % LNDFH-III and 0-8 mol % pLNnH, totaling 100 mol % in total.

[0338] In other embodiments, the mixture of HMOs according to the present invention consists essentially of 70-99 mol% LNFP-III and 1-50 mol% LNnT, which together add up to 100 mol%. In other embodiments, the mixture of HMOs according to the present invention consists essentially of 80-99 mol% LNFP-III, 0-5 mol% LNnT and 0-10 mol% LNDFH-III, which together add up to 100 mol%.

[0339] In other embodiments, the mixture of HMOs according to the present invention consists essentially of 80-99 mol % LNFP-III and 0-20 mol % 3-FL, totaling 100 mol % in total.

[0340] In an embodiment, the present invention also relates to a composition comprising a mixture of HMOs, wherein the composition comprises 30-55 mol% of LNFP-III, 40-65 mol% of LNnT and 3-8 mol% of pLNnH of the total molar HMO content in the composition, totaling 100% molar content. A preferred embodiment is a mixture consisting essentially of 45-55 mol% of LNFP-III and 45-55 mol% of LNnT of the total molar HMO content in the composition, totaling 100% molar content. In another embodiment, a mixture consisting essentially of 35-45 mol% of LNFP-III and 50-60 mol% of LNnT and 4-6 mol% of pLNnH of the total molar HMO content in the composition, totaling 100% molar content.

[0341] In another embodiment, the present invention also relates to a composition comprising a mixture of HMOs, wherein the composition comprises 30-99 mol% of LNFP-III, 0-65 mol% of LNnT, 0-20 mol% of 3-FL, 0-10 mol% of LNDFH-III and 0-8 mol% of pLNnH of the total molar HMO content in the composition, totaling 100% molar content. A preferred embodiment is a mixture consisting essentially of 50-85 mol% of LNFP-III and 15-50 mol% of LNnT of the total molar HMO content in the composition, totaling 100% molar content. Another preferred embodiment is a mixture consisting essentially of 60-85 mol% of LNFP-III and 15-40 mol% of LNnT of the total molar HMO content in the composition, totaling 100% molar content.

[0342] In other embodiments, the mixture of HMOs according to the present invention consists essentially of 50 mol % LNFP-III and 50 mol % LNnT.

[0343] In other embodiments, the mixture of HMOs according to the present invention consists essentially of 75 mol % LNFP-III and 25 mol % LNnT.

[0344] In other embodiments, the mixture of HMOs according to the invention consists essentially of 40 mol % LNFP-III and 55 mol % LNnT and 5 mol % pLNnH.

[0345] In other embodiments, the present invention is also directed to a composition comprising a mixture of HMOs, wherein the composition comprises 70-99 mole % LNFP-III and 1-50 mole % LNnT, totaling 100 mole % total, based on the total mole HMO content of the composition.

[0346] In another embodiment, the present invention is also directed to a composition comprising a mixture of HMOs, wherein the composition comprises 80-99 mole % LNFP-III, 0-5 mole % LNnT and 0-10 mole % LNDFH-III, totaling 100 mole % total, based on the total mole HMO content of the composition.

[0347] In other embodiments, the present invention is also directed to a composition comprising a mixture of HMOs, wherein the composition comprises 80-99 mole % LNFP-III and 0-20 mole % 3-FL, totaling 100 mole % based on the total mole HMO content of the composition.

[0348] Clinical data in infants suggest that human milk oligosaccharide supplementation may help develop a desired microbiota by serving as a food source for good bacteria in the gut. The HMOs that occur naturally in breast milk have evolved over thousands of years, with HMO research (clinical and preclinical) now suggesting that specific HMOs at the right supplementation levels may provide us with unique health benefits. In particular, human milk oligosaccharide supplementation may help support immune and gut health, with a potential role in cognitive development, which could open up future opportunities for innovation.

[0349] The mixture of HMOs may also form part of a composition comprising additional moieties, such as active pharmaceutical ingredients, food supplements, excipients, surfactants and the like.

[0350] Therefore, the present invention also relates to the use of a composition comprising a mixture of HMOs in an infant formula, a dietary supplement or a medical nutritional agent, wherein the composition comprises LNFPIII and LNnT, LNFP-III and 3-FL, LNFP-III LNnT and pLNnH or LNFP-III, LNnT and 3-FL, and wherein the composition is substantially free of LNFP-VI and has less than 5% LNDFH-III.

[0351] In an embodiment, the composition comprising the mixture of HMOs is a pharmaceutical composition.

[0352] Uses of HMO mixtures and compositions

[0353] Clinical data in infants suggest that human milk oligosaccharide supplementation may help develop a desired microbiota by serving as a food source for good bacteria in the gut. HMOs naturally present in breast milk have evolved over thousands of years, with HMO research (clinical and preclinical) now suggesting that specific HMOs at the right supplementation levels may provide us with unique health benefits. In particular, human milk oligosaccharide supplementation may help support immune and gut health, including supporting a balanced microbiome, with a potential role in cognitive development, which could open up future opportunities for innovation.

[0354] Thus, in an embodiment, the invention relates to the use of a mixture or a composition according to the invention in infant nutrition.

[0355] The invention also relates to the use of the mixture or composition according to the invention as a dietary supplement or medical nutrition.

[0356] A mixture or composition of HMOs produced according to the methods described herein can be used to enhance the beneficial bacteria in the intestinal microbiome. Beneficial bacteria are, for example, Bifidobacterium sp., Lactobacillus sp., or Barnesiella sp. The enhancement of beneficial bacteria can 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 in infants and young children. Its benefits include, for example, inhibition of pathogens, prevention of infection and diarrhea, and reduction of the risk of allergies and metabolic disorders (see, for example, WO2006 / 130205, WO2017 / 129644, WO2017 / 129649).

[0357] The mixture or composition of HMO produced according to the methods described herein can be used to reduce the abundance of undesirable viruses and bacteria in the intestinal microbiome. Examples of pathogenic bacteria and viruses that can be reduced by the HMO mixture described herein include Candida albicans, Clostridium difficile, Enterococcus faecium, Escherichia coli, Helicobacter pylori, Streptococcus agalactiae, Shigella dysenteriae, Staphylococcus aureus, nora virus and rota virus. Each mixture or composition described herein can also be used to treat and / or reduce the risk of widespread bacterial infections in humans.

[0358] A mixture or composition of HMOs produced according to the methods described herein can be used to increase the regeneration and viability of freeze-dried probiotics (including probiotics of the genera Bifidobacterium and / or Lactobacillus), in particular increasing the regeneration and / or viability and / or shelf life in an acidic environment (such as the stomach or acidic foods) is an advantage of using the HMO mixture described herein. Examples of Bifidobacteria that may have increased regeneration and vitality are Bifidobacterium animals lactis BB12 DSM 32269, Bifidobacterium animal 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 ssp. 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 paracasei ssp. Paracasei and / or Lactobacillus Limosilactobacillus reuteri S12 DSM 33752.

[0359] In the context of this application, "regeneration" means the process of regaining / restoring the vitality of dried bacteria (i.e., "regenerating" the bacterial cells by rehydration, where "rehydration" means restoring fluid). This process is sometimes also referred to as "reconstitution".

[0360] In the context of the present application, "viability" is the ability of bacterial cells to survive and function as living cells. One way to determine the viability of bacterial cells is by spreading them on agar plates with a suitable growth medium and counting the number of colonies formed after incubation for a predetermined time (plate count). Alternatively, FACS analysis can be used.

[0361] In the context of the present application, "improving the regeneration of Bifidobacterium and / or Lactobacillus bacteria" means that the amount (number) of Bifidobacterium and / or Lactobacillus bacteria is increased and the bacteria are successfully regenerated / recovered compared to a corresponding control (i.e. the amount / number of Bifidobacterium and / or Lactobacillus bacteria without addition of HMO).

[0362] In the context of the present application, "improving the viability of Bifidobacterium and / or Lactobacillus bacteria" means increasing the amount (number) of viable Bifidobacterium and / or Lactobacillus bacteria compared to a corresponding control (i.e. the amount / number of Bifidobacterium and / or Lactobacillus bacteria without the addition of HMO).

[0363] In the context of this application, "acidic" means a pH below 7.0 (e.g., pH ≤ 6.0, or ≤ 5.0, or ≤ 4.0, or ≤ 3.0, or in the range of 1.0-6.0, such as 2.0 to 5.0). The pH measured in the stomach is in the range of about 1.5-3.5. The pH measured in a healthy vagina is in the range of about 3.8-5.0. The pH of fruit juice is in the range of about 2.0-4.5.

[0364] A mixture or composition of HMOs produced according to the methods described herein can be used to extend the shelf life of probiotics, such as Bifidobacterium and / or Lactobacillus.

[0365] One embodiment of the invention is a composition comprising a mixture of HMOs as described herein, in particular in the "Mixture of HMOs" section, and one or more probiotics. Preferably, the probiotics are of the genus Bifidobacterium and / or Lactobacillus. For example any of the specific species mentioned above.

[0366] Mixtures or compositions of HMOs produced according to the methods described herein can be used to improve the flow properties of powders or to reduce the viscosity of liquids.

[0367] Mixtures or compositions of HMO produced according to the methods described herein are used in nutritional compositions. The nutritional compositions are, for example, infant formula powders, rehydration solutions or dietary maintenance, medical nutrients, or supplements for the elderly or immunocompromised individuals. Macronutrients such as edible fats, carbohydrates, and proteins may also be included in such anti-infective compositions. 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. 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) may also be included in such anti-infective compositions.

[0368] The invention also relates to the use of the mixture or composition according to the invention as a dietary supplement and / or medical nutrition.

[0369] In an embodiment, the invention relates to the use of a mixture or a composition according to the invention in infant nutrition.

[0370] sequence

[0371] This application contains a Sequence Listing in both text and electronic formats, which is incorporated herein by reference.

[0372] An overview of the SEQ ID NOs used in this application can be found in Table 1 (α-1,3-fucosyltransferase protein sequences) and Table 4 (promoter sequences). Additional sequences described in this application are the colanic acid gene cluster (gmd-wcaG-wcaH-wcaI-manC-manB SEQ ID NO: 12), the amino acid sequences of the fucosyltransferases FutB (SSEQ ID NO: 6, GenBank ID WP_000487430.1), and CafD (SEQ ID NO: 41) and FucT109 (SEQ ID NO: 42) from Helicobacter pylori, DNA sequences encoding α-1,3-fucosyltransferases (SEQ ID NOs: 7 to 11), including FutB (SEQ ID NO: 40), CafD (SEQ ID NO: 44) and FucT109 (SEQ ID NO: 43), the DNA sequence encoding the colanic acid gene cluster from Escherichia coli (SEQ ID NO: 1 NO: 12) and β-1,3-N-acetylglucosaminyltransferase LgtA from Neisseria meningitidis (SEQ ID NO: 13), β-1,4-galactosyltransferase galT from Helicobacter pylori (SEQ ID NO: 14).

[0373] project

[0374] 1. A genetically engineered cell capable of producing one or more fucosylated HMOs, comprising a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is selected from:

[0375] a. Camp1, which has 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;

[0376] b. Parm1 having 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;

[0377] c. Hpull1, which has 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;

[0378] d. Med1 having 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; and

[0379] e. Hacin2 having 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.

[0380] 2. The genetically engineered cell according to item 1, wherein the fucosyltransferase mainly fucosylates the N-acetylglucosamine (GlcNAc) portion of the acceptor oligosaccharide with an α-1,3 linkage, such as the N-acetylglucosamine (GlcNAc) portion LNnT.

[0381] 3. The genetically engineered cell according to item 1 or 2, wherein the fucosyltransferase has low or no activity on the glucose moiety in the reducing end of an acceptor molecule, such as lactose or an HMO, such as LNT-II or LNnT, in particular LNnT.

[0382] 4. The genetically engineered cell according to any one of items 1 to 3, wherein the one or more fucosylated HMO is LNFP-III.

[0383] 5. A genetically engineered cell according to any of the preceding clauses, wherein at least 30% of the molar content of total HMOs produced by the cell is LNFP-III.

[0384] 6. Genetically engineered cell according to any of the preceding items, wherein less than 5%, preferably less than 3%, of the molar content of total HMOs produced by the cell is LNFP-VI and / or LNDFH-III.

[0385] 7. The genetically engineered cell according to any one of the preceding items, wherein the HMO produced by the cell is substantially free of LNFP-VI.

[0386] 8. 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.

[0387] 9. The genetically engineered cell according to item 8, wherein the genetically engineered cell comprises a recombinant nucleic acid sequence encoding β-1,4-galactosyltransferase.

[0388] 10. The genetically engineered cell according to item 8 or 9, wherein the genetically engineered cell comprises a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosaminyltransferase.

[0389] 11. The genetically engineered cell according to any one of item 9 or 10, wherein the β-1,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis and the β-1,4-galactosyltransferase is from Helicobacter pylori.

[0390] 12. A genetically engineered cell according to any one of item 9 or 12, wherein the 1,3-N-acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO: 13, or a functional homologue thereof having an amino acid sequence at least 80% identical to SEQ ID NO: 13, and the β-1,4-galactosyltransferase has an amino acid sequence according to SEQ ID NO: 14, or a functional homologue thereof having an amino acid sequence at least 80% identical to SEQ ID NO: 14.

[0391] 13. A genetically engineered cell according to any one of the preceding items, wherein the recombinant nucleic acid sequences are each under the control of a promoter selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 28, 37, 25, 26 and 27, respectively) and variants thereof.

[0392] 14. The genetically engineered cell according to item 13, wherein the promoter is a strong promoter selected from SEQ ID NOs 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27.

[0393] 15. A genetically engineered cell according to any of the preceding items, wherein the cell comprises one, two, three or more genomic copies of a recombinant nucleic acid sequence encoding a glycosyltransferase, wherein the glycosyltransferase is selected from Camp1, Parm1, Hpull1, Med1 and Hacin2 having 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 is at least 80% identical to SEQ ID NO: 1, 2, 3, 4 or 5.

[0394] 16. A genetically engineered cell according to any of the preceding items, wherein a recombinant nucleic acid sequence encoding a glycosyltransferase is encoded on a plasmid, wherein the glycosyltransferase is selected from: Camp1, Parm1, Hpull1, Med1 and Hacin2 having 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.

[0395] 17. The genetically engineered cell according to item 16, wherein the plasmid is a high copy number plasmid.

[0396] 18. The genetically engineered cell according to item 16 or 17, wherein the plasmid is a pUC57 plasmid.

[0397] 19. A genetically engineered cell according to any of the preceding items, wherein the cell comprises one, two, three or more genomic copies and plasmid-borne copies of a recombinant nucleic acid sequence encoding a glycosyltransferase, wherein the glycosyltransferase is selected from: Camp1, Parm1, Hpull1, Med1 and Hacin2 having 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 is at least 80% identical to SEQ ID NO: 1, 2, 3, 4 or 5.

[0398] 20. The genetically engineered cell according to any of the preceding items, wherein the cell overexpresses at least one enzyme of the de novo GDP-fucose pathway responsible for GDP-fucose formation.

[0399] 21. The genetically engineered cell according to item 20, wherein the one or more enzymes are selected from mannose-6-phosphate isomerase (manA), phosphomannomutase (manB), mannose-1-phosphate guanylyltransferase guanylate transferase (manC), GDP-mannose-4,6-dehydratase (gmd) and GDP-L-fucose synthase (wcaG).

[0400] 22. The genetically engineered cell according to any one of the preceding items, wherein the cell further comprises a recombinant nucleic acid sequence according to SEQ ID NO: 12 encoding the colanic acid (CA) gene cluster.

[0401] 23. The genetically engineered cell according to any of the preceding items, wherein the cell further comprises a nucleic acid sequence encoding a MFS transporter capable of exporting one or more fucosylated HMOs into the extracellular medium, in particular LNFP-III.

[0402] 24. A genetically engineered cell according to item 23, wherein the MFS transporter is a yjhB, YhhS or YebQ protein or a functional variant thereof, such as a protein having at least 80% identity with a protein having UniProt accession number P39352 or P37621 or P76269.

[0403] 25. A genetically engineered cell according to any one of the preceding items, wherein the engineered cell is a microorganism.

[0404] 26. A genetically engineered cell according to any one of the preceding items, wherein the engineered cell is a bacterium or a fungus.

[0405] 27. The genetically engineered cell of item 26, wherein the fungus is selected from yeast cells of the genera Komagataella, Kluyveromyces, Yarrowia, Pichia, Saccharomyces, Schizosaccharomyces, or Hansenula, or a filamentous fungus selected from the genera Mold, Fusarium, or Trichoderma.

[0406] 28. The genetically engineered cell according to item 26, wherein the bacterium is selected from the genera Escherichia, Bacillus, Lactobacillus, Corynebacterium and Campylobacter.

[0407] 29. The genetically engineered cell according to any one of items 25 to 28, wherein the engineered cell is selected from Escherichia coli, Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris and Saccharomyces cerevisiae.

[0408] 30. The genetically engineered cell according to item 29, wherein the engineered cell is a microorganism, which is Escherichia coli.

[0409] 31. A genetically engineered cell according to any of the preceding items, wherein the glycosyltransferase is selected from Camp1, Parm1, Hpull1 and Med1 having 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 [Camp1, Parm1, Hpull1 and Med1 amino acids], and wherein at least 75% of the molar content of the total HMO produced by the cell is LNFP-III.

[0410] 32. The genetically engineered cell according to any of the preceding items, wherein the glycosyltransferase is selected from Camp1, Hpull1 and Med1 having an amino acid sequence according to SEQ ID NO: 1, 3 or 4, or a functional homologue thereof having an amino acid sequence at least 80% identical to SEQ ID NO: 1, 3 or 4 [Camp1, Hpull1 and Med1 amino acids], and wherein the total HMO produced by the cell is substantially free of LNFP-VI.

[0411] 33. The genetically engineered cell according to any of the preceding items, wherein the glycosyltransferase is selected from Camp1, Hpull1 and Med1 having an amino acid sequence according to SEQ ID NO: 1, 3 or 4, or a functional homologue thereof having an amino acid sequence at least 80% identical to SEQ ID NO: 1, 3 or 4 [Camp1, Hpull1 and Med1 amino acids], and wherein the total HMO produced by the cell is substantially free of LNDFH-III.

[0412] 34. The genetically engineered cell according to any of the preceding items, wherein the cell produces a mixture of HMOs comprising LNFP-III and LNnT.

[0413] 35. A genetically engineered cell according to any of the preceding clauses, wherein the cell produces a mixture of HMOs consisting essentially of 30-99% LNFP-III, 0-65% LNnT, 0-20% 3-FL and 0-10% pLNnH, totaling 100% molar content.

[0414] 36. A genetically engineered cell according to any of the preceding items, wherein the cell produces LNFP-III of at least 50% of the molar content of total HMOs produced by the cell, and the remaining 50% of the total HMOs produced by the cell are byproduct HMOs selected from LNDFH-III, 3FL, LNnT and pLNnH.

[0415] 37. A method for producing one or more fucosylated HMOs, the method comprising culturing the genetically engineered cell according to any one of items 1 to 35.

[0416] 38. The method of any one of clauses 37, 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.

[0417] 39. The method according to any one of item 37 or 38, wherein lactose is added as a substrate for HMO formation during the cultivation of the genetically engineered cells.

[0418] 40. The method according to item 37 to item 39, wherein the fucosylated HMO is LNFP-III.

[0419] 41. The method according to items 37 to 40, wherein the method produces a mixture of HMOs, and wherein the mixture is optionally purified to a purity of at least 85%.

[0420] 42. The method according to items 37 to 40, wherein the LNFP-III produced by the method is purified to at least 90% purity.

[0421] 43. Use of an enzyme having α-1,3-fucosyltransferase activity in the production of a fucosylated product, wherein the enzyme is selected from Camp1, Parm1, Hpull1, Med1 and Hacin2 having 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 has at least 80% identity with SEQ ID NO: 1, 2, 3, 4 or 5.

[0422] 44. Use of the enzyme according to item 43, wherein the fucosylation product is one or more fucosylated oligosaccharides.

[0423] 45. Use of an enzyme according to any one of item 43 or 44, wherein the fucosylation product is one or more HMOs.

[0424] 46. ​​Use of the enzyme according to item 45, wherein the fucosylation product is LNFP-III.

[0425] 47. Use of the enzyme according to item 46, wherein LNFP-III is substantially the only fucosylated HMO produced when LNnT is used as an acceptor substrate for α-1,3-fucosyltransferase.

[0426] 48. Use of the enzyme according to item 43, wherein the fucosylation product is one or more fucosylated polypeptides.

[0427] 49. A mixture of HMOs consisting essentially of LNFP-III and LNnT and less than 20% of 3FL, LNDFH-III and / or pLNnH.

[0428] 50. A mixture of HMOs according to item 49, consisting essentially of 30-99% LNFP-III, 0-65% LNnT, 0-20% 3-FL, 0-10% LNDFH-III and 0-10% pLNnH, totaling 100% molar content in total.

[0429] 51. A mixture of HMOs according to item 49, consisting essentially of 75-99% LNFP-III and 1-25% LNnT, adding up to 100% molar content.

[0430] 52. A mixture of HMOs according to item 49, consisting essentially of 55-85% LNFP-III and 15-45% LNnT, adding up to 100% molar content.

[0431] 53. A mixture of HMOs according to item 49, consisting essentially of 65-85% LNFP-III and 15-35% LNnT, adding up to 100% molar content.

[0432] 54. A mixture of HMOs according to item 49, consisting essentially of 25-60% LNFP-III, 35-65% LNnT and 2-8% pLNnH, adding up to 100% molar content in total.

[0433] 55. A mixture of HMOs according to item 49, consisting essentially of 85-95% LNFP-III, 0-5% LNnT and 2-15% LNDFH-III, adding up to 100% molar content in total.

[0434] 56. A mixture of HMOs according to item 49, consisting essentially of 75-85% LNFP-III and 15-25% 3FL, adding up to 100% molar content.

[0435] 57. The mixture of HMOs according to any one of items 49 to 55, wherein the mixture is substantially free of LNFP-VI.

[0436] 58. A composition comprising a mixture of the HMOs of items 49 to 57.

[0437] 59. The composition according to item 58, wherein the mixture further comprises one or more probiotics.

[0438] 60. The composition according to item 59, wherein the probiotic is of the genus Bifidobacterium and / or Lactobacillus.

[0439] 61. The composition according to item 59 or 60, wherein the probiotic is selected from Bifidobacterium animalis BB12 DSM 32269, Bifidobacterium animalis BIF6, Bifidobacterium longum DSM 32946, Bifidobacterium longum BB536, Bifidobacterium bifidum DSMZ 32403, Bifidobacterium infantis, Bifidobacterium breve DSM 33789, Bifidobacterium infantis SP37 DSM 32687, Bifidobacterium adolescentis DSM 34065, Bifidobacterium animalis subsp. animalis DSM 16284, Lactobacillus rhamnosus GG DSM 32550, Lactobacillus rhamnosus 19070-2 DSM 26357, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus LBrGG, Lactobacillus reuteri DSM12246, Lactobacillus plantarum TIFN101, Lactobacillus gasseri Lg-36 200B FloraFit Danisco, Lactobacillus casei DSMZ32382, Lactobacillus paracasei, Lactobacillus plantarum PS128, Lactobacillus plantarum (Sacco) DSM 32383, Lactococcus lactis PAREVE, Lactobacillus paracasei subsp. paracasei and Lactobacillus Lactobacillus reuteri S12 DSM33752.

[0440] 62. The composition according to item 59 or item 60, wherein the probiotic is Lactobacillus casei DSMZ 32382.

[0441] 63. Use of the mixture or composition according to any one of items 49 to 62 as a dietary supplement and / or medical nutrition.

[0442] 64. Use of the mixture or composition according to any one of items 49 to 62 in infant nutrition.

[0443] Example

[0444] method

[0445] Unless otherwise indicated, standard techniques, vectors, control sequence elements and other expression system elements known in the field of molecular biology are used for nucleic acid manipulation, conversion and expression. Such standard techniques, vectors and elements can be found in the following documents: Ausubel et al. (editor), Current Protocols in Molecular Biology (1995) (John Wiley & Sons); Sambrook, Fritsch, & Maniatis (editor), 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. (editor), DNA Insertion Elements, Plasmids and Episomes (1977) (Cold Spring Harbor Laboratory Press, NY); Miller, JH Experiments in molecular genetics (1972.) (Coldspring Harbor Laboratory Press, NY).

[0446] The embodiments described below are chosen to illustrate the present invention and are not intended to limit the present invention in any way.

[0447] Enzymes:

[0448] Screening of 50 enzymes with fucosyltransferase activity provided 6 enzymes with α-1,3-fucosyltransferase activity that were also able to produce the complex HMO LNFP-III. The GenBank IDs and sources of the 6 fucosyltransferases are provided in Table 5.

[0449] Table 5. List of enzymes tested in the framework of the present invention

[0450]

[0451] *Compared to the GenBank sequence represented by SEQ ID NO, the sequence used in this application may be truncated at the N- or C-terminus.

[0452] **FutB is known to produce a mixture of LNFP-III and LNFP-VI (Dumon et al. 2004 Biotechnol. Prog. 20:412-419).

[0453] ***CafD has been suggested in WO2016 / 040531 to produce LNFP-III

[0454] ^FucT109 has been suggested in WO2019 / 008133 to produce LNFP-III

[0455] strain

[0456] The strain (genetically engineered cell) constructed in this application is based on Escherichia coli K-12DH1 with the following genotype: 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.

[0457] Methods for inserting a target gene into the E. coli genome are well known to those skilled in the art. Specific selectable marker genes and screening methods can be used to insert a gene cassette into the E. coli chromosome using gene gorging (see, e.g., Herring and Blattner 2004 J. Bacteriol. 186: 2673-81 and Warming et al. 2005 Nucleic Acids Res. 33 (4): e36).

[0458] To obtain a strain producing LNnT, the MDO strain was further engineered by chromosomal integration of β-1,3-GlcNAc transferase (LgtA, from Neisseria meningitidis, homologous to NCBI Accession No. WP_033911473.1 and shown as SEQ ID NO: 13) and β-1,4-galactosyltransferase (GalT from Helicobacter pylori, homologous to GenBank ID WP_001262061.1 and shown as SEQ ID NO: 14), both under the control of the PglpF promoter (SEQ ID NO: 28), and this strain was designated as the LNnT strain.

[0459] A codon-optimized DNA sequence encoding a single α-1,3-fucosyltransferase was genomically integrated into the LNnT strain.

[0460] The genotypes of the background strain (MDO), the LNnT strain, and the α-1,3-fucosyltransferase expressing strain capable of producing LNFP-III are provided in Table 6.

[0461] Table 6. Genotypes of strains capable of producing LNFP-III used in this example.

[0462]

[0463]

[0464] *1,3FT is the abbreviation of α-1,3-fucosyltransferase, and the sequence is inserted into the genome of the host strain.

[0465] 1 lgtA-PglpF - Two genomic inserted copies of the gene encoding β-1,3-N-acetylglucosaminyltransferase (SEQ ID NO: 13) under the control of the PglpF promoter.

[0466] 2 galT-PglpF - A genomic insert encoding β-1,4-galactosyltransferase (SEQ ID NO: 14) under the control of the PglpF promoter.

[0467] 3 CA = additional colanic acid gene cluster under the control of the PglpF promoter at a locus different from the native locus (gmd-wcaG-wcaH-wcaI-manC-manB, SEQ ID NO: 12).

[0468] 4 pUC57 is a high copy number (>300) plasmid with ColE1 / pMB1 / pBR322 / pUC replication origin. The antibiotic resistance marker on the pUC57 vector is ampicillin. The a1,3FT shown is expressed from the plasmid.

[0469] **FutB is known to produce a mixture of LNFP-III and LNFP-VI (Dumon et al. 2004 Biotechnol. Prog. 20:412-419)

[0470] ***CafD has been suggested in WO2016 / 040531 to produce LNFP-III

[0471] Deep well assay

[0472] The deep well assay in this example was performed as 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. During 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.

[0473] More specifically, during the first day, 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), NH&HzPO4 (7 g / L), citric acid (0.5 g / l), trace mineral solution (5 mL / L). The trace mineral stock solution contained: ZnSO~*7H~O 0.82 g / L, citric acid 20 g / L, Mn$04*H~O 0.98 g / L, FeSO4*7H~O 3.925 g / L, CuSO4*5H~O 0.2 g / L. The pH of the basal minimal medium was adjusted to 7.0 with 5N NaOH and autoclaved. The pre-culture was incubated at 34°C and 1000rpm shaking for 24 hours, 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), a glucose solution (0.1-0.15g / L) and a lactose solution (5-20g / L). In addition, 20% sucrose stock solution (40-45g / L) or maltodextrin (19-20g / L) was used as a carbon source, and specific hydrolases, sucrose hydrolases or glucoamylases were added respectively, so that glucose was released at a rate suitable for carbon-restricted growth, and similar to a typical fed-batch fermentation process. The main culture was incubated at 28°C and 1000rpm shaking for 72 hours. In order to analyze the total fermentation broth, 96-well plates were boiled at 100°C, then centrifuged, and the supernatant was analyzed by HPLC at last.

[0474] Fermentation-Amber

[0475] E. coli strains were cultured in 250 mL fermenters (Ambr250 HT Bioreactor system, Sartorius) from 100 mL of a mineral medium consisting of 30 g / L glucose and NH4H2PO4, KH2PO4, MgSO4 x 7H2O, KOH, NaOH, citric acid, trace element solution, defoamer and thiamine. The dissolved oxygen level was maintained at 20% by first stirring and then starting aeration at 700 rpm (up to 4500 rpm) and 1 VVM (up to 3 VVM). The pH was maintained at 6.8 by titration with 8.5% NH4OH solution. The culture was started with 2% (v / v) inoculum from a pre-culture comprising 10 g / L glucose, (NH4)2HPO4, KH2PO4, MgSO4 x 7H2O, KOH, NaOH, citric acid, trace element solution, defoamer and thiamine. After the glucose contained in the basic minimal medium is exhausted, the feed solution containing glucose, MgSO4 x 7H2O, H3PO4 and trace mineral solution is continuously added to the fermentor at a rate to maintain carbon-limited conditions. The temperature was initially 33°C but dropped to 30°C with a linear ramp of 3 hours 12 hours after the start of feeding. 6 hours after the start of feeding, lactose was added in the form of a bolus addition of 25% lactose monohydrate solution to prevent lactose from becoming a rate-limiting factor, then every 19 hours. The growth, metabolic activity and metabolic state of the cells are tracked by online measurement of stirring, dissolved oxygen tension, reflectivity, NH4OH base addition, O2 absorption rate and CO2 release rate. Throughout the fermentation process, HPLC sampling was used to determine the concentration of HMO products, lactose and other minor byproducts.

[0476] Fermentation-Dasbox

[0477] E. coli strains were cultured in 100 mL fermenters (Dasbox, Eppendorf) from 100 mL of a mineral medium consisting of 30 g / L glucose and NH4H2PO4, KH2PO4, MgSO4 x7H2O, KOH, NaOH, citric acid, trace element solution, defoamer and thiamine. The dissolved oxygen level was maintained at 20% by first stirring and then starting aeration at 700 rpm (up to 2000 rpm) and 1 VVM (up to 3 VVM). The pH was maintained at 6.8 by titration with 8.5% NH4OH solution. The culture was started from a 2% (v / v) pre-culture inoculum consisting of 10 g / L glucose, (NH4)2HPO4, KH2PO4, MgSO4 x7H2O, 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 x7H2O, H3PO4 and trace mineral solution was continuously added to the fermentor at a rate to maintain carbon limiting conditions. The temperature was initially 33°C, but began to drop to 25°C 15 minutes after the start of feeding. Lactose was added as a bolus of 25% lactose monohydrate solution at the beginning of feeding and was added together with the glucose feed to prevent lactose from becoming the rate limiting factor. The growth, metabolic activity and metabolic state of the cells were tracked by online measurement of agitation, dissolved oxygen tension, reflectivity, NH4OH base addition, O2 absorption rate and CO2 evolution rate. Throughout the fermentation, samples were taken to determine the concentration of HMO products, lactose and other minor by-products using HPLC.

[0478] Example 1 - In vivo LNFP-III synthesis

[0479] Genetically modified cells expressing individual α-1,3-fucosyltransferases were screened for their ability to produce fucosylated HMOLNFP-III.

[0480] Five enzymes (Table 7) were compiled for testing their ability to synthesize LNFP-III when introduced into genetically modified cells producing LNnT and GDP-fucose.

[0481] Genetically modified strains expressing six individual α-1,3-fucosyltransferases were generated as described in the Methods section (Table 5). Cells were screened in a deep well assay as described in the Methods section.

[0482] The genotypes of strains capable of producing LNFP-III are listed in Table 6. The molar content of individual HMOs produced by the strains was measured by HPLC.

[0483] The results for LNFP-III producing cells are shown in Table 7 as the fraction (in percent, %) of the total molar HMO content produced by each strain.

[0484] Table 7: The content of each HMO is expressed as % of the total HMO molar (mM) content produced by each strain.

[0485]

[0486]

[0487] No HMOs other than those shown in Table 7 were identified in the deep well assays.

[0488] From the data presented in Table 7, it can be seen that the five new enzymes Camp1, Parm1, Hpull1, Med1 and Hacin2 can transfer the fucosyl unit to the GlcNAc part of LNnT with an α-1,3 bond to form LNFP-III at a level higher than 30% of the total HMO, compared to the prior art enzymes FutB and FucT109, which only produced 6% and 26% of the total HMO of LNFP-III, respectively. CafD, which is said to produce LNFP-III in WO2016 / 040531, does not seem to be able to fucosylate LNnT or lactose in this assay, as it does not produce any fucosylated HMO.

[0489] None of the five novel enzymes produced any LNFP-VI, while FucT109 produced 18% LNFP-VI, clearly indicating that FucT109 has similar specificity for both the GlcNAc and Glc moieties of LNnT. Similarly, FutB, a single copy enzyme, was found to produce a small amount of LNFP-VI as well as more 3-FL compared to the five novel enzymes, indicating that FutB also has some activity on the terminal glucose (Glc) moiety of LNnT.

[0490] Furthermore, the enzymes Camp1, Hpull1, Med1 and Hacin2 did not produce any LNDFH-III, indicating that these enzymes are highly specific for the GlcNAc moiety in LNnT, compared to FutT109 which produced 24% LNDFH-III.

[0491] Figure 1 The data from the single copy strains in Table 7 are illustrated, clearly illustrating the difference in LNFP-III production from the strains with the five novel enzymes compared to the prior art enzymes FutB, CafD and FucT109.

[0492] As can be seen in Table 7, increasing the copy number of Camp1 expressed from a plasmid to a high copy number (Camp1_2) resulted in slight 3-FL production, while abolishing LNnT production by this strain, indicating that the formation of LNnT in this strain is the limiting factor when the Camp1 copy number is high.

[0493] Increasing the copy number of Parm1 to two genomic copies, and further including a high copy plasmid (pUC57-Parm1-PglpF), increased the relative amount of LNFP-III produced from 76% to 81% to 86% of the total HMO content, respectively, while decreasing the amount of LNnT produced from 21% to 14% to 5%. Increasing the copy number of Parm1 also increased the formation of LNDFH-III, indicating that the increased activity also increased the fucosylation of the Glc moiety in LNnT.

[0494] Increasing the copy number of Med1 to two genetic copies (strain Med1_2) increased LNFP-III production from 52% to 90%. By introducing a high-copy plasmid encoding Med1 (pUC57-Med1-PglpF-amp) (strain Med1_3), a slight decrease in LNFP-III levels to 82% was observed, and 18% 3-FL was produced instead. Given the high copy number of the Med1 gene (strain Med1_8), all LNnT produced by the cells is fucosylated, so the formation of LNnT by the cells becomes the rate-limiting step in LNFP-III production, which may cause Med1 to fucosylate lactose instead of LNnT, thereby further reducing LNnT formation by consuming lactose, resulting in a reduction in LNFP-III.

[0495] Therefore, depending on the need for a particular product, copy number variants can be used to tailor a specific HMO mixture, in this case a mixture containing different ratios of LNFP-III, LNnT and / or 3-FL. Increasing the copy number of Camp1, Hpull1, Hacin2 and Med1 did not result in the production of any additional fucosylated HMO species with the LNnT backbone, and none of the strains with increased copy number produced 3-FL, except for Med1 with the high copy number plasmid, most likely due to the formation of LNnT becoming the rate-limiting step in the production process.

[0496] If it is desired to purify the produced LNFP-III, the absence of alternative fucosylated species in the produced mixture is highly advantageous and preferred. In addition, if it is desired to obtain pure LNFP-III, low levels of LNnT achieved by expressing Camp1 and Med1 genes (high copy number) are beneficial. If med! is applied in high copy number, the data suggest that the enzyme forming LNnT should potentially be increased along with the Med1 enzyme to ensure sufficient formation of LNnT.

[0497] Example 2 - Fermentation for LNFP-III production using Camp1_1α-1,3-fucosyltransferase strain

[0498] To confirm the HMO profile observed in the deep well assay, in particular the content of LNFP-III in the total HMO produced, the Camp1_1 strain of Example 1 containing a single genomic copy of Camp1 was fermented using ambr fermentation as described above in the "Methods" section. The results are shown in Table 8.

[0499] Table 8: The content of each HMO is % of the total HMO content produced by the strain

[0500] Strain reference number 3-FL LNT-II LNFP-III LNFP-VI LNDFH-III LqCy Camp1_1 0 0 98 0 0 2

[0501] As can be seen from the data presented in Table 8, the fraction of LNFP-III of the Camp1_1 strain was the same in the fermentation as the results presented in Example 1, further demonstrating the ability and applicability of Camp1 to produce high-purity LNFP-III in fermentation.

[0502] Example 3 - Fermentation for LNFP-III production using Med1_1α-1,3-fucosyltransferase strain

[0503] To confirm the HMO distribution characteristics observed in the deep well assay, in particular the content of LNFP-III in the total HMO produced, the Med1_1 strain of Example 1 containing a single genomic copy of Med1 was fermented using dashbox fermentation as described above in the "Methods" section. The results are shown in Table 9. The Med1_1 strain was run in parallel

[0504] Table 9: Content of individual HMOs as % of the total HMO content produced by the strains

[0505] Strain reference number 3-FL LNT-II LNFP-III LNFP-VI LNDFH-IIII LqCy HkDJ Med1_1 0 3 24 0 0 61 12 Camp1_1 0 2 95 0 0 2 0

[0506] As can be seen from the data shown in Table 9, using the Med1_1 strain in the fermentation setup, the only complexly fucosylated HMO produced was LNFP-III. Compared to the deep well assay presented in Example 2, the conversion rate from LNnT to LNFP-III was slightly lower, which could be optimized, for example, by using a strain with multiple copies of Med1 on the genome.

[0507] Camp1_1 was almost identical to the fermentation performed in Example 2 and showed high stability in different fermentation settings.

[0508] Example 4 - Regeneration and viability of freeze-dried Lactobacillus species

[0509] Probiotics can be consumed as live bacteria or as dried (e.g., freeze-dried) products. Independent of the drying method, rehydration involves an important step to restore dehydrated bacteria; an insufficient rehydration / regeneration step may result in poor cell viability and low final survival. Therefore, rehydration is a very critical step in the reactivation of freeze-dried cultures. For 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.

[0510] In this example, it was tested whether a mixture of HMOs produced by the LNFP-III producing strains in Example 1 could provide benefits in terms of rehydration (regeneration) and vitality of probiotics. The test was conducted under acidic conditions to resemble the conditions that the bacteria must survive when passing through the stomach or when administered in an acidic beverage.

[0511] The lyophilized probiotic Lactobacillus casei (DSM 32382) (0.4 mg / ml) (control) as shown in Table 10 was dissolved in sterile phosphate buffered saline (PBS, pH=3), warmed to 37°C and mixed vigorously for about 30 seconds until no visible clumps remained. The tubes were incubated at 37°C for 3 hours. The samples were further diluted and 100 μl were spread in duplicate on MRS agar plates, which were incubated at 37°C in an anaerobic chamber for 48 hours. The regeneration and viability of the probiotics were determined by counting the colonies on the plates after 48 h of incubation. If the colonies grew slowly, the incubation was extended to 72 h or even 96 h. For experimental setup, see Figure 3 The results were expressed as the mean plus standard deviation of colony forming units (CFU) per milliliter calculated from the colonies formed on the agar plates.

[0512] Table 10: HMO compositions tested in this example

[0513] HMO C Mix1 Mix2 Mix3 LNFP-III 0 50 75 40 LqCy 0 50 25 55 HkDJ 0 0 0 5

[0514] Figure 4 A plate with Lactobacillus casei colonies formed after 3 h acid treatment and 72 h incubation is shown. E-3 dilution plates were used for counting and the results are shown in Figure 5 middle.

[0515] Compared with the control without HMO mixture, the freeze-dried Lactobacillus casei dissolved with HMO mixture as described herein showed significant (between P<0.01 and P<0.05) enhanced regeneration and vitality. These data clearly show that in the presence of any HMO mixture, after being exposed to low pH conditions, such as in the stomach or in acidic beverages, the regeneration and vitality of Lactobacillus casei can be improved. It can also be seen that the amount of LNFP-III should preferably constitute at least 50% of the HMO mixture, and even more preferably constitute at least 75% of the HMO mixture.

[0516] To our knowledge, a mixture of LNFP-III and LNnT has not previously been shown to provide benefits that improve the regeneration and viability of probiotics in an acidic environment.

Claims

1. A genetically engineered cell capable of producing one or more fucosylated HMOs, comprising a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is selected from: a. Camp1, which has 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. Parm1 having 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; and c. Hpull1 having 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.

2. The genetically engineered cell of claim 1, wherein the fucosyltransferase has low or no activity towards a glucose moiety in the reducing end of an acceptor molecule for producing one or more fucosylated HMOs.

3. The genetically engineered cell according to claim 1 or 2, wherein the one or more fucosylated HMO produced by the cell is LNFP-III.

4. The genetically engineered cell according to any one of the preceding claims, wherein the genetically engineered cell comprises a recombinant nucleic acid sequence encoding β-1,4-galactosyltransferase.

5. The genetically engineered cell according to any one of the preceding claims, wherein the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosaminyltransferase.

6. The genetically engineered cell according to claim 5, wherein the β-1,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis and the β-1,4-galactosyltransferase is from Helicobacter pylori.

7. A genetically engineered cell according to any of the preceding claims, wherein the cell comprises two, three or more genomic and / or plasmid-borne copies of a recombinant nucleic acid sequence encoding a glycosyltransferase selected from Camp1, Parm1 and Hpull1 having an amino acid sequence according to SEQ ID NO: 1, 2 or 3, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1, 2 or 3.

8. A genetically engineered cell capable of producing one or more fucosylated HMOs according to 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.

9. A method for producing one or more fucosylated HMOs, the method comprising the steps of: a. Providing a genetically engineered cell having a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, wherein the glycosyltransferase is selected from: i. Camp1, which has 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; ii. Parm1 having 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; iii. Hpull1 having 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, and iv. Hacin2 having 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, b. culturing the cell according to (a) in a suitable cell culture medium to produce one or more fucosylated HMOs, and c. optionally, purifying one or more fucosylated HMOs, and Wherein the fucosylated HMO is LNFP-III.

10. The method according to claim 9, wherein a. at least 30% of the molar content of the total HMO produced by the method is LNFP-III; and b. less than 5% of the molar content of the total HMO produced by the method is another complex fucosylated HMO; and / or c. Less than 3% of the molar content of the total HMO produced by the process is LNFP-VI and / or LNDFH-III.

11. The method according to claim 9 or 10, wherein at least 90% of the molar content of total HMOs produced by the cells is LNFP-III and LNnT.

12. The method according to claims 9 to 11, wherein the LNFP-III produced by said method is at least 90% pure.

13. Use of a fucosyltransferase having α-1,3-fucosyltransferase activity in producing one or more fucosylated HMOs, wherein the enzyme is selected from Camp1, Parm1 and Hpull1 having an amino acid sequence according to SEQ ID NO: 1, 2, 3 or 5, or a functional homologue thereof having an amino acid sequence at least 80% identical to SEQ ID NO: 1, 2, 3 or 5, and wherein the fucosylated product is LNFP-III.

14. An HMO mixture, wherein the mixture is selected from: a. 30-55 mol% LNFP-III, 40-65 mol% LNnT and 2-8 mol% pLNnH, totaling 100% molar content; b. 30-99 mol% LNFP-III, 0-65 mol% LNnT, 0-20 mol% 3-FL, 0-10 mol% LNDFH-III and 0-8 mol% pLNnH, totaling 100% molar content; c. 70-99 mol% LNFP-III and 1-50 mol% LNnT, totaling 100 mol%; d. 80-99 mol% LNFP-III, 0-5 mol% LNnT and 0-10 mol% LNDFH-III, totaling 100 mol%; and e. 80-99 mol% of LNFP-III and 0-20 mol% of 3-FL, totaling 100 mol% content.

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

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