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

Abstract

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

Description

Technical Field

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

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

[0003] For example, WO2016 / 040531 describes the production of complex fucosylated HMOs, showing that the α1,3-fucosyltransferase CafD can produce LNFP-III, and WO2019 / 008133 describes the α1,3-fucosyltransferase FucT109, which appears to fucosylate the glucose (Glc) and N-acetylglucosamine (GlcNAc) moieties of lacto-N-neotetraose (LNnT), potentially generating a mixture containing LNnT, LNFP-III, and LNFP-VI.

[0004] Dumon et al., 2004 (alpha-1,3-fucosyltransferase Biotechnol. Prog. 2004, 20, 412-419) further described an alpha 1,3-fucosyltransferase FutB, which is also able 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 a small amount, if any, of complex fucosylated HMOs and the amount of by-product formation is high.

[0006] In summary, the production of fucosylated HMOs, especially the more complex ones, can be challenging due to the lack of fucosyltransferases with the required substrate specificity and the low yield of the desired fucosylated HMOs compared to other HMO products present after fermentation (e.g., HMO precursor products, which may require tedious separation procedures). Summary of the invention

[0007] The need for highly substrate-specific α-1,3-fucosyltransferases and α-1,4-fucosyltransferases was addressed by identifying a bifunctional α-1,3(4)-fucosyltransferase Med1, which exhibits low or no substrate specificity for the glucose moiety in LNT and LNnT, but has high substrate specificity for the N-acetylglucosamine (GlcNAc) moiety in LNT and LNnT. This enables Med1 to produce high levels of very pure LNFP-III in the LNnT background strain without any other complex fucosylated HMOs. More importantly, the α-1,4-fucosyltransferase activity of Med1, which is very specific for the GlcNAc moiety in LNT, is rare, and this paper shows for the first time that an α-1,4-fucosyltransferase can produce the complex fucosylated HMOs LNFP-II and / or LNDFH-I. The HMO produced by the strain containing Med1α-1,3(4)-fucosyltransferase may be a mixture of HMOs comprising precursor HMOs (e.g., LNT, LNnT, or LNFP-I) and / or byproduct oligosaccharides or byproduct HMOs (e.g., 2'FL, 3FL, DFL, pLNH2, or pLNnH). However, the mixture is substantially free of alternative complex fucosylated HMOs LNFP-V, LNFP-VI, and / or LNDFH-II.

[0008] Thus, the α-1,3(4)-fucosyltransferases described herein can be used to produce pure or substantially pure LNFP-III and LNFP-II in a mixture of HMOs, from which they can be easily purified. In addition, the α-1,3(4)-fucosyltransferases identified herein are the first to be able to efficiently biosynthesize the difucosylated HMO LNDFH-I. Thus, provided herein are enzymes, mixtures, compositions, uses, genetically engineered cells and methods for producing LNFP-II, LNFP-III and / or LNDFH-I.

[0009] Thus, a first aspect described herein relates to a genetically engineered cell capable of producing one or more fucosylated HMOs, the genetically engineered cell comprising a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the glycosyltransferase is Med1 having an amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof having an amino acid sequence having at least 80% identity to SEQ ID NO: 1.

[0010] A second aspect of the present invention relates to a method for producing one or more fucosylated HMOs, preferably, wherein the HMO is selected from LNFP-II, LNFP-III and LNDFH-I, the method comprising culturing 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(4)-fucosyltransferase activity, wherein the glycosyltransferase is Med1 having an amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of the functional homologue having at least 80% identity to SEQ ID NO: 1.

[0011] A third aspect relates to the use of an enzyme having α-1,3(4)-fucosyltransferase activity for producing a fucosylated product (e.g., a fucosylated HMO, preferably selected from LNFP-II, LNFP-III and LNDFH-I), wherein the enzyme is Med1 having 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 to SEQ ID NO: 1.

[0012] A fourth aspect relates to an HMO mixture consisting essentially of:

[0013] a) LNFP-II, pLNH2 and LNT, or

[0014] b) LNFP-II, LNT and 3FL, or

[0015] c) LNFP-II, LNT and pLNH2, or

[0016] d) LNFP-II, LNT-II, LNT, 3FL and pLNH2, or

[0017] e) LNFP-III and LNnT, or

[0018] f) LNFP-III, LNnT and pLNnH, or

[0019] g) LNFP-III and 3FL, or

[0020] h) LNDFH-I, LNFP-I and 2'FL.

[0021] The fifth aspect involves

[0022] a) an HMO mixture consisting essentially of LNFP-II and LNT, and having a low 3FL or pLNnH content, for example less than 20% of the total HMO molar content in the mixture,

[0023] b) an HMO mixture consisting essentially of 5-35 mol % of LNFP-II,

[0024] 0-15 mol% LNT-II, 0-20 mol% 3FL and 50-85 mol% LNT, the total molar content is 100%,

[0025] c) an HMO mixture consisting essentially of LNFP-III and LNnT, and having a low content of 3FL or pLNnH, for example less than 10% of the total HMO molar content in the mixture,

[0026] d) an HMO mixture consisting essentially of 50-99 mol % of LNFP-III,

[0027] 0-45 mol% LNnT, 0-20% 3FL and 0-10% pLNnH, the total molar content is 100%,

[0028] e) an HMO mixture consisting essentially of LNDFH-I, LNFP-I and 2'FL, and

[0029] f) an HMO mixture consisting essentially of 7-15 mol % of LNDFH-I,

[0030] The mixture is composed of 65-90 mol% LNFP-I and 5-20 mol% 2'FL, and the total HMO molar content in the mixture adds up to 100 mol%.

[0031] A sixth aspect of the present invention relates to a composition comprising and / or consisting essentially of a mixture of a) to f), and its use in infant formula, dietary supplements and / or medical nutrition. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1is a description of the pathway used to make LNFP-II from a strain with an LNT background and potential byproducts that may be generated in the process. The acceptor for the α-1,3(4)-fucosyltransferase can be lactose, LNT, LNFP-V, and LNFP-II, depending on the specificity of the α-1,3(4)-fucosyltransferase. Fucosylated byproducts are, for example, 3FL, LNFP-V, and LNDFH-II. Neutral byproducts are, for example, LNT-II, LNnT, and pLNH2. Depending on the genetically modified cell, the starting substrate for the production of LNFP-II can be lactose, LNT-II, or even LNnT, as long as the strain can absorb the starting substrate. In the figure, β1,3-GlcNAcT is a β1,3-glucosaminyltransferase, β1,3-GalT is a β1,3-galactosyltransferase, Glcα1,3(4)-FucT is an α1,3(4)-fucosyltransferase active on the glucose part of the acceptor oligosaccharide, and GlcNAcα1,3(4)-FucT is an α1,3(4)-fucosyltransferase active on the GlcNAc part of the acceptor oligosaccharide; GDP is guanosine diphosphate, and UDP is uridine diphosphate.

[0033] Figure 2 The following is an illustration of the pathway for making LNDFH-I from a strain with an LNFP-I background and potential byproducts that may be produced during this process. Depending on the specificity of the α-1,3(4)-fucosyltransferase, the acceptor for the α-1,3(4)-fucosyltransferase can be lactose, LNT, LNFP-I and LNFP-II. Fucosylated byproducts are, for example, 2'FL, 3FL, LNFP-I and LNFP-II. Neutral byproducts are, for example, LNT-II, LNnT and pLNH2. Depending on the genetically modified cell, the starting substrate for the production of LNDFH-I can be lactose, LNT-II or even LNnT or LNFP-I, as long as the strain can absorb the starting substrate. In the figure, β1,3-GlcNAcT is a β1,3-glucosaminyltransferase, β1,3-GalT is a β1,3-galactosyltransferase, α1,2-FucT is an α1,2-fucosyltransferase, and α1,3(4)-FucT is an α1,3(4)-fucosyltransferase; GDP is guanosine diphosphate, and UDP is uridine diphosphate.

[0034] Figure 3The following is an illustration of the pathway for making LNFP-III from a strain with an LNT background and potential byproducts that may be produced during this 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, LNnT, and pLNnH. Depending on the genetically modified cell, the starting substrate for the production of LNFP-III can be lactose, LNT-II, or even LNnT, as long as the strain is able to absorb the starting substrate. In the figure, β1,3-GlcNAcT is a β1,3-glucosaminyltransferase, β1,3-GalT is a β1,3-galactosyltransferase, Glcα1,3-FucT is an α1,3-fucosyltransferase active at the glucose part of the acceptor oligosaccharide, and GlcNAcα1,3-FucT is an α1,3-fucosyltransferase active at the GlcNAc part of the acceptor oligosaccharide; GDP is guanosine diphosphate, and UDP is uridine diphosphate.

[0035] Figure 4 : Experimental setup showing the regeneration and viability assessment of freeze-dried probiotics under acidic conditions at pH 3.0.

[0036] Figure 5 : Show the regeneration and vigor of freeze-dried Lactobacillus casei (DSM 32382) incubated for 3 hours at pH 3.0. A) is the control without HMO, and the picture shows the dilution step 1:100 (E-2), 1:1000 (E-3) from left to right. B) is the combination of Lactobacillus casei (DSM 32382) and HMO mixture, and the HMO mixture contains 75% LNFP-I, 10% LNDFH-I and 15% 2'FL, and the picture shows the dilution step 1:100 (E-2), 1:1000 (E-3), 1:10.000 (E-4) and 1:100.000 (E-5) from left to right. C) shows the results expressed as mean (n=2) and standard deviation (SD) of colony forming units (CFU) per ml, which were calculated from the Lactobacillus casei colonies inoculated on agar plates according to dilution step E-3, ** indicates a statistically significant difference compared to the control, p<0.01. DETAILED DESCRIPTION

[0037] The present invention solves the biotechnological challenges of HMO production in vivo, in particular complex fucosylated HMOs comprising at least five monosaccharide units, wherein at least one monosaccharide unit is a fucosyl unit. Preferably, the complex fucosylated HMO is selected from the complex fucosylated HMOs LNFP-II, LNFP-III and LNDFH-I. The present invention provides a specific strain engineering solution to produce specific complex fucosylated HMOs, in particular LNFP-II, LNFP-III and / or LNDFH-I, by utilizing the substrate specificity of the α-1,3(4)-fucosyltransferase Med1 disclosed herein for the GlcNAc moiety in LNT and LNnT, and the dual activity of the α-1,3(4)-fucosyltransferase Med1 disclosed herein.

[0038] The genetically engineered cells of the present invention express genes encoding key enzymes for the biosynthesis of fucosylated HMOs. In addition, it is advantageous if the genetically engineered cells express genes required for the production of LNT, LNnT or LNFP-I, whether from lactose or LNT-II as a starting substrate. In some embodiments, the genetically engineered cells of the present invention also express the de novo GDP-fucose pathway genes responsible for the formation of GDP-fucose manA, manB, manC, gmd and wcaG. Overexpression of one or more of these genes and / or upregulation of the colanic acid gene cluster (CA), including genes gmd, wcaG, wcaH, wcaI, manC and manB from E. coli, by introduction of a nucleic acid construct encoding the colanic acid gene cluster (CA) as shown in SEQ ID NO: 3, may be advantageous, allowing the formation of GDP-fucose, which enables the cell to produce higher levels of fucosylated oligosaccharides from one or more oligosaccharide substrates (e.g., lactose or LNT-II, LNT, LNnT and / or LNFP-I). Depending on the intended use of the substrate, one or more additional glycosyltransferases and pathways for producing nucleotide-activated sugars, such as glucose-UDP-GlcNac, CMP-N-acetylneuraminic acid, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and / or CMP-N-acetylneuraminic acid, may also be present in the genetically engineered cells.

[0039] Production of LNFP-II, LNFP-III or LNDFH-I

[0040] The advantage of using the α-1,3(4)-fucosyltransferase of the present disclosure is that it can specifically recognize and fucosylate the GlcNAc moiety in LNT, LNnT and / or LNFP-I, thereby generating LNFP-II, LNFP-III and / or LNDFH-I, respectively (as shown in the reactions in Formulas 1, 2 and 3). In essence, the present disclosure describes for the first time an enzyme with dual α-1,3-fucosyltransferase and 1,4-fucosyltransferase activities (α-1,3(4)-fucosyltransferase) that is more active on the GlcNAc moiety in LNnT compared to, for example, pure α-1,3-fucosyltransferases FucT109 (WO2019 / 008133) and CafD (WO2016 / 040531) (which have previously been proposed to fucosylate GlcNAc and glucose (Glu) moieties at the α-1,3 positions on LNnT).

[0041] In addition, the α-1,3(4)-fucosyltransferase Med1 of the present invention is also able to fucosylate the GlcNAc portion of LNT with a high specificity at an α-1,4 bond, thereby producing LNFP-II, and substantially no LNFP-V. This is in contrast to FucT109, which has previously been proposed to also fucosylate the Glc portion of LNT, but only at an α-1,3 bond, thereby producing LNFP-V.

[0042] The α-1,3(4)-fucosyltransferase med1 described herein has little or no activity on the glucose and galactose moieties of oligosaccharides, particularly med1 has little or no activity on the glucose and galactose moieties of oligosaccharides having an LNT or LNnT backbone. Essentially, this means that the med1 enzyme is able to produce HMO mixtures in which only one complex fucosylated oligosaccharide is present in the mixture.

[0043] In addition, the α-1,3(4)-fucosyltransferases described herein have very low activity on lactose and the glucose moieties in LNT and LNnT. Therefore, if there is sufficient amount of LNT or LNnT in the genetically engineered cell, the α-1,3(4)-fucosyltransferases described in the present disclosure will produce very little, if any, 3FL.

[0044] Therefore, the properties of the α-1,3(4)-fucosyltransferase described herein are very suitable for high-level industrial production of LNFP-III without producing high levels of alternative fucosylated HMOs (byproducts), such as LNFP-VI and LNDFH-III, and other byproduct HMOs, such as LNnT and pLNnH. In addition, the α-1,3(4)-fucosyltransferase described herein is also very suitable for the production of LNFP-II and a mixture comprising LNFP-II and LNT and a small amount of LNT-II, 3FL and pLNH2. In addition, the α-1,3(4)-fucosyltransferase disclosed herein is also suitable for the production of LNDFH-I and a mixture comprising LNDFH-I and LNFP-I because it can further fucosylate LNFP-I to produce LNDFH-I.

[0045] The genetically engineered cells of the present disclosure express α-1,3(4)-fucosyltransferase Med1 having high substrate specificity for the GlcNAc moiety in LNnT, LNT and LNFP-I, and for the first time are capable of producing high titers of LNFP-III and HMO mixtures containing LNPF-II or LNDFH-I, from which it is possible to purify LNPF-II or LNDFH-I. In particular, the HMO mixture produced by the genetically engineered cells of the present disclosure expressing α-1,3(4)-fucosyltransferase Med1 produces only a single complex fucosylated HMO consisting of at least five monosaccharide units, which is a great advantage if complex HMOs, such as LNFP-II, LNFP-III or LNDFH-II, are to be purified from the mixture.

[0046] Therefore, the present disclosure enables more efficient production of LNFP-II, LNFP-III and / or LNDFH-I, which is very beneficial for the biotechnological production of more complex fucosylated HMOs (e.g., LNFP-II, LNFP-III and LNDFH-I).

[0047] In the following sections, the various elements of the present invention, in particular the various elements of the genetically engineered cells, will be described. It will be appreciated that these elements may be combined between the various parts.

[0048] Oligosaccharides

[0049] In this context, the term "oligosaccharide" refers to a sugar polymer containing at least three monosaccharide units, i.e., tri, tetra, penta, hexa or higher oligosaccharides. Oligosaccharides can have a linear or branched structure, which contains monosaccharide units interconnected by interglycosidic bonds. Specifically, oligosaccharides include the lactose residue of the reducing end and one or more naturally occurring monosaccharides of 5-9 carbon atoms, and the monosaccharide is selected from aldose (e.g., glucose, galactose, ribose, arabinose, xylose, etc.), ketose (e.g., fructose, sorbose, tagatose, etc.), deoxy sugar (e.g., rhamnose, fucose, etc.), deoxy amino sugar (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acid and ketouronic acid (e.g., N-acetylneuraminic acid). Preferably, oligosaccharides are HMOs.

[0050] Human Milk Oligosaccharides (HMO)

[0051] Preferred oligosaccharides of the present disclosure are human milk oligosaccharides (HMOs).

[0052] The term "human milk oligosaccharide" or "HMO" in this context refers to a complex carbohydrate found in human milk. HMOs have a core structure comprising a lactose unit at the reducing end, which may be extended by one or more β-N-acetyllactosamine units and / or one or more β-lacto-N-disaccharyl units, and which may be substituted by α-L-fucopyranyl and / or α-N-acetylneuraminic acid (fucosyl) moieties. The HMO structure is disclosed by, for example, Xi Chen in Chapter 4 of Advances in Carbohydrate Chemistry and Biochemistry 2015 vol 72.

[0053] The present invention focuses on fucosylated HMO. Examples of fucosylated HMO include 2'-fucosyllactose (2'FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-I), lacto-N-difucohexaose II (LNFP-II), lacto-N-difucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-I ... II), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-fucopentaose VI (LNFP-VI), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-p-lacto-N-hexaose I (FpLNH-I), fucosyl-p-lacto-N-neohexaose II (F-pLNNH-I), II), fucosyl-lacto-N-neohexaose (FLNnH), 3-fucosyl-3'-fucosyllactose (FSL), fucosyl-LST-a (FLST-a), fucosyl-LST b (FLST b), fucosyl-LST-c (FLST-c), fucosyl-LST d (FLST-d) and fucosyl-lacto-N-hexaose (SLNH).

[0054] 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 complex 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 the starting substrate lactose, for example, the formation of LNFP-II requires α-1,4-fucosyltransferase, β-1,3-N-acetylglucosaminyltransferase and β-1,3-galactosyltransferase, the formation of LNFP-III requires α-1,3-fucosyltransferase, β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase, and the formation of LNDFH-I requires α-1,2-fucosyltransferase, α-1,4-fucosyltransferase, β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase.

[0055] Specifically, the produced fucosylated HMO is selected from complex fucosylated HMOs comprising at least five monosaccharide units, wherein at least one monosaccharide unit is a fucosyl unit. In addition, in an embodiment, the fucosylated HMO is selected from complex fucosylated HMOs having a LNT and / or LNnT backbone structure, preferably, selected from LNFP-II, LNFP-III and LNDFH-I.

[0056] Examples of fucosylated HMOs with an LNT backbone structure are lacto-N-fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose I (LNDFH-I), lacto-N-difucohexaose II (LNDFH-II), sialyl-lacto-N-fucopentaose I (S-LNFP-I), sialyl-lacto-N-fucopentaose II (S-LNFP-II), monofucosyl-lacto-N-hexaose I (F-LNH-I), monofucosyl-lacto- -N-hexaose II (F-LNH-II), monofucosyl-lacto-N-hexaose III (F-LNH-III), difucosyl-lacto-N-hexaose I (DF-LNH-I), difucosyl-lacto-N-hexaose II (DF-LNH-II), difucosyl-lacto-N-hexaose III (DF-LNH-III), trifucosyl-lacto-N-hexaose (TF-LNH), fucosyl-sialo-lacto-N-hexaose I (FS-LNH) and disialyl-fucosyl-lacto-N-hexaose II (DS-F-LNH-II).

[0057] Examples of HMOs having an LNnT backbone structure are lacto-N-fucopentaose III (LNFP-III), lacto-N-fucopentaose VI (LNFP-VI), lacto-N-difucohexaose III (LNDFH-III), fucosyl-p-lacto-N-hexaose I (F-para-LNH-I), difucosyl-p-lacto-N-hexaose (DF-para-LNH), difucosyl-p-lacto-N-neohexose (DF-para-LNnH), and fucosyl-sialic acid-lacto-N-neohexose I (FS-LNnH-I).

[0058] In an embodiment of the present disclosure, the fucosyltransferase of the present invention primarily fucosylates the N-acetylglucosamine (GlcNAc) portion of LNT, LNnT or LNFP-I. In a further embodiment, the fucosyltransferase of the present invention only fucosylates the N-acetylglucosamine (GlcNAc) portion of LNT, LNnT or LNFP-I. In this regard, the one or more fucosylated HMOs are selected from LNFP-II, LNFP-III and LNDFH-I.

[0059] Receptor oligosaccharide

[0060] The genetically engineered cell according to the present invention comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-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 or an α-1,4 bond.

[0061] 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 a more complex HMO, which may also be referred to as a precursor molecule.

[0062] The acceptor oligosaccharide may be an intermediate product of the present fermentation process, the end product of a separate fermentation process using separate genetically engineered cells, or an enzymatically or chemically produced molecule.

[0063] In the present context, the acceptor oligosaccharide of the α-1,3(4)-fucosyltransferase is preferably an oligosaccharide having a lacto-N-tetraose (LNT) or lacto-N-neotetraose (LNnT) backbone. Preferably, the acceptor oligosaccharide of the α-1,3(4)-fucosyltransferase is lacto-N-tetraose (LNT) or lacto-N-neotetraose (LNnT). LNT and LNnT can be produced within genetic engineering from the precursor molecules lactose (e.g., the acceptor of β-1,3-N-acetylglucosaminyltransferase) and / or lacto-N-triose II (LNT-II) (e.g., the acceptor of β-1,4-galactosyltransferase or β-1,3-galactosyltransferase). In addition, the acceptor oligosaccharide of α-1,3(4)-fucosyltransferase may also be lacto-N-fucopentaose I (LNFP-I), which is produced from the precursor molecules lactose (e.g., the acceptor of β-1,3-N-acetylglucosaminyltransferase) and / or lacto-N-triose II (LNT-II) (e.g., the acceptor of β-1,3-galactosyltransferase) and / or LNT (e.g., the acceptor of α-1,2-fucosyltransferase). The precursor molecules are preferably fed to genetically engineered cells capable of producing LNT, LNnT or LNFP-I from the precursors.

[0064] Glycosyltransferase

[0065] The genetically engineered cells according to the 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.

[0066] The genetically engineered cells according to the present invention may comprise one or more other 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 cells to synthesize LNT, LNnT or LNFP-I from precursor molecules such as lactose or LNT-II or LNT (precursor of LNFP-I). In an embodiment, the genetically engineered cells of the present invention comprise one or more other recombinant nucleic acids encoding one or more heterologous glycosyltransferases.

[0067] The additional glycosyltransferase is preferably selected from the group consisting of galactosyltransferase, glucosaminyltransferase, fucosyltransferase and N-acetylglucosaminyltransferase.

[0068] In one aspect, the fucosyltransferase in the genetically engineered cell of the present invention is an α-1,3(4)-fucosyltransferase. Preferably, the α-1,3(4)-fucosyltransferase is capable of transferring a fucose unit to the GlcNAc portion of the LNT, LNnT or LNFP-I molecule.

[0069] In the present invention, the functional enzyme (α-1,3(4)-fucosyltransferase) capable of transferring a fucosyl moiety from a fucosyl donor to an acceptor oligosaccharide is Med1, whose amino acid sequence is according to SEQ ID NO: 1, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 1. This enzyme can, for example, be used to produce LNFP-II, LNFP-III and / or LNDFH-III.

[0070] Without being bound by theory, an α-1,3(4)-fucosyltransferase having a higher substrate specificity for the GlcNAc moiety in LNT, LNnT and LNFP-I than for the terminal galactose moiety in LNT, LNnT or LNFP-I is advantageous because such an α-1,3(4)-fucosyltransferase would theoretically produce less or no alternative complex fucosylated HMOs, such as LNFP-V and LNFP-VI, when the starting substrate is lactose and where the availability of LNnT is not limiting in this case. In this case, lower amounts of alternative complex-fucosylated HMOs will result in easier purification of the produced HMOs, since purification of LNFP-II, LNFP-III or LNDFH-I from a mixture of HMOs comprising mainly the desired product will be simpler, since it is easier to separate complex-fucosylated HMOs from smaller HMOs than to separate different fucosylated HMOs of the same or similar size from each other, e.g., LNFP-III from LNFP-VI or LNDFH-III, or LNFP-II from LNFP-V or LNDFH-II, or LNDFH-I from LNDFH-II. Therefore, it is believed that a lower starting amount of LNFP-VI and / or LNDFH-III in the production of LNFP-III is beneficial to the purification of LNFP-III, while a lower starting amount of LNFP-V and / or LNDFH-II in the production of LNFP-II is beneficial to the purification of LNFP-II, and a lower starting amount of LNDFH-II in the production of LNDFH-I is beneficial to the purification of LNDFH-I.

[0071] In a preferred embodiment, the use of an α-1,3(4)-fucosyltransferase according to the invention results in at least 80% of the total molar content of HMO produced by the cell according to the invention being LNFP-III. In other preferred embodiments, the use of an α-1,3(4)-fucosyltransferase according to the invention results in at least 15% of the total molar content of HMO produced by the cell according to the invention being LNFP-II. In other preferred embodiments, the use of an α-1,3(4)-fucosyltransferase according to the invention results in at least 8% of the total molar content of HMO produced by the cell according to the invention being LNDFH-I. In an embodiment, the α-1,3(4)-fucosyltransferase is Med1 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.

[0072] In an embodiment, the expression of the α-1,3(4)-fucosyltransferase according to the present invention in the genetically engineered cells is also combined with the expression of one or more other recombinant nucleic acids encoding one or more heterologous glycosyltransferases.

[0073] In an embodiment of the present invention, the cell further comprises one or more recombinant nucleic acid sequences encoding β-1,3-galactosyltransferase and / or β-1,4-galactosyltransferase.

[0074] In a preferred embodiment, the expression of the α-1,3(4)-fucosyltransferase of the invention in a genetically engineered cell is combined with the expression of a β-1,3-galactosyltransferase (e.g., galTK from Helicobacter pylori) or a β-1,4-galactosyltransferase (e.g., galT from Helicobacter pylori). In a further embodiment, a third enzyme is expressed, such as a β-1,3-N-acetylglucosaminyltransferase, such as LgtA from Neisseria meningitidis. In yet another embodiment, a fourth enzyme is expressed, such as an α-1,2-fucosyltransferase, such as Smob from Sulfuriflexus mobilis.

[0075] In addition to the α-1,3(4)-fucosyltransferase Med1, exemplary other glycosyltransferases are preferably selected from the glycosyltransferases described below (Tables 1, 2, and 3).

[0076] α-1,3(4)-fucosyltransferase

[0077] The term "α-1,3(4)-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of a fucosyl group from a donor substrate (e.g. GDP-fucose) to an acceptor molecule with an α-1,3-bond. Preferably, the α-1,3(4)-fucosyltransferase used in the present invention is not derived from a species of genetically engineered cells, i.e. the gene encoding the α-1,3(4)-fucosyltransferase is of heterologous origin and is Med1 from Mediterranea sp. An20, having an amino acid sequence according to SEQ ID NO: 1 and GenBank ID WP_087337236.1, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO: 1. In the context of the present invention, the acceptor molecule for the α-1,3(4)-fucosyltransferase is preferably the GlcNAc portion of an acceptor oligosaccharide of at least four monosaccharide units (e.g. LNT, LNnT or LNFP-I).

[0078] An exemplary reaction for producing LNFP-II from LNT using the α-1,3(4)-fucosyltransferase of the present invention is shown in Formula 1:

[0079]

[0080] An exemplary reaction for producing LNFP-III from LNnT using the α-1,3(4)-fucosyltransferase of the present invention is shown in Formula 2:

[0081]

[0082] An exemplary reaction for producing LNDFH-I from LNFP-I using the α-1,3(4)-fucosyltransferase of the present invention is shown in Formula 3:

[0083]

[0084] The α-1,3(4)-fucosyltransferase may be selected from functional homologues having 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, such as 100% identity to the amino acid sequence of any α-1,3(4)-fucosyltransferase of SEQ ID NO: 1. In a preferred embodiment, the functional homologue has a tryptophan (w) at the position corresponding to position 13 of SEQ ID NO: 1 when aligned according to the sequence identity assessment parameters and tools described herein.

[0085] Example 1 of the present application discloses the identification of fucosyltransferases Para1 (SEQ ID NO: 33) and Med1 (SEQ ID NO: 1), both of which are capable of generating LNFP-II. However, there are significant differences in the substrate specificity of Para1 and Med1 fucosyltransferases, as Med1 only fucosylates LNT at the GlcNAc moiety with an α-1,4 bond, thereby generating only a single fucosylated HMO with an LNT backbone, specifically LNFP-II, while Para1 fucosylates LNT at the glucose (Glc) moiety and the GlcNAc moiety, thereby generating three fucosylated HMOs with an LNT backbone, namely LNFP-II, LNFP-V, and LNDFH-II, respectively. Since a total of 50 fucosyltransferases were screened and only two were identified that were able to produce LNFP-II, and only Med1 was able to produce LNFP-II as the only complex-fucosylated HMO, LNTGlcNAc specificity appears to be rare among fucosyltransferases across bacterial species.

[0086] In addition, Example 1 of the present invention discloses that increasing the copy number of Med1 to two gene copies can increase the production of LNFP-II, while increasing the copy number of Para1 only leads to a slight increase in the formation of LNFP-II, while the amount of difucosylated HMOLNDFH-II increases to a greater extent, clearly indicating that Para1 has the same preference for fucosylation of the GlcNAc and Glc parts of LNT, while Med1 is only active on the GlcNAc part. Further increase in Med1 expression was studied by introducing a high copy plasmid pUC57-Med1-PglpF (Med1_3) containing the Med1 coding sequence, which further increased the level of LNFP-II. Similar effects were also observed for the production of LNFP-III (Example 2) and LNDFH-I (Examples 3 and 5).

[0087] If one wishes to purify LNFP-II, LNFP-III or LNDFH-I from a culture medium that does not contain other similarly sized fucosylated HMOs (e.g. LNFP-V, LNFP-VI or LNDFH-II), the absence of alternative fucosylated LNT or LNnT species is highly advantageous and preferred when using Med 1. Thus, when the goal is to produce large amounts of a single HMO, high levels of a specific HMO are beneficial.

[0088] In an embodiment of the present invention, the fucosyltransferase having α-1,3(4)-fucosyltransferase activity is Med1 from Mediterranean sp. An20, which comprises or consists of the amino acid sequence of SEQ ID NO: 1, or comprises or consists of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO: 1.

[0089] Specifically, the enzyme Med1 is introduced into a genetically engineered cell which also comprises a β-1,3-galactosyltransferase or a β-1,4-galactosyltransferase and preferably also a β-1,3-N-acetylglucosaminyltransferase.

[0090] β-1,3-N-acetylglucosaminyltransferase

[0091] β-1,3-N-acetylglucosaminyltransferase is any protein capable of transferring 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 β-1,3-N-acetylglucosaminyltransferase is a heterologous source. In the context of the present invention, the acceptor molecule is lactose or an oligosaccharide of at least four monosaccharide units, such as LNT, LNnT or a more complex HMO structure. Therefore, in an embodiment, the genetically engineered cell also comprises one or more recombinant nucleic acid sequences encoding β-1,3-N-acetylglucosaminyltransferase.

[0092] Non-limiting examples of β-1,3-N-acetylglucosaminyltransferases are given in Table 1. β-1,3-N-acetylglucosaminyltransferase variants may also be useful, preferably such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to the amino acid sequence of any β-1,3-N-acetylglucosaminyltransferase in Table 1.

[0093] Table 1. List of β-1,3-N-acetylglucosaminyltransferases

[0094]

[0095] In an embodiment, the genetically engineered cell comprises a recombinant nucleic acid sequence encoding a β-1,3-N-acetylglucosaminyltransferase. In one embodiment, the recombinant nucleic acid sequence encoding a β-1,3-N-acetylglucosaminyltransferase comprises or consists of the amino acid sequence of SEQ ID NO:4 (LgtA from Neisseria meningitidis), or a functional homolog thereof, the amino acid sequence of the functional homolog having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:4.

[0096] To produce LNnT or LNT from the substrate lactose, 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.

[0097] Some of the examples below use a heterologous β-1,3-N-acetylglucosaminyltransferase named LgtA from Neisseria meningitidis or a variant thereof.

[0098] β-1,3-galactosyltransferase

[0099] β-1,3-galactosyltransferase is any protein that can transfer galactose of UDP-galactose to N-acetylglucosamine moiety with β-1,3-bond and thus to acceptor molecule. Preferably, the β-1,3-galactosyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding β-1,3-galactosyltransferase is of heterologous origin.

[0100] Non-limiting examples of β-1,3-galactosyltransferases are given in Table 12. β-1,3-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 any of the β-1,3-galactosyltransferases in Table 12.

[0101] Table 12. List of β-1,3-glycosyltransferases

[0102]

[0103] In the context of the present invention, the acceptor molecule is an acceptor sugar, such as LNT-II, or a more complex HMO structure.

[0104] The following examples use a heterologous β-1,3-galactosyltransferase named GalTK or a variant thereof in combination with other glycosyltransferases to produce, for example, LNFP-II, LNDFH-I, or LNFP-I and LNDFH-I.

[0105] In an embodiment, the cell of the present invention further comprises a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosaminyltransferase. In one embodiment, the recombinant nucleic acid sequence encoding β-1,3-galactosyltransferase comprises or consists of the amino acid sequence of SEQ ID NO:31 (galTK from Helicobacter pylori), or a functional homolog thereof, the amino acid sequence of the functional homolog having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% identity with SEQ ID NO:31.

[0106] To produce LNT from the LNT-II precursor, a β-1,3-galactosyltransferase is required. In one embodiment, the genetically modified cell comprises a β-1,3-galactosyltransferase gene or a functional homologue or fragment thereof.

[0107] The following are examples of genetically modified strains according to the present invention having a specific combination of glycosyltransferases that will produce LNFP-II or LNDFH-I using lactose as a starting substrate.

[0108] In one example, LgtA from Neisseria meningitidis was used in combination with galTK from Helicobacter pylori and Med1 from Mediterranea An20 to initiate the production of LNFP-II using lactose as the starting substrate.

[0109] In another example, LgtA from Neisseria meningitidis was used in combination with galTK from Helicobacter pylori, Smob from Aspergillus motileus, and Med1 from Mediterranea An20 to initiate the production of LNDFH-I using lactose as the starting substrate.

[0110] In another example, galTK from Helicobacter pylori was used in combination with Med1 from Mediterranea sp. An20 to initiate the production of LNFP-II using LNT-II as the starting substrate.

[0111] In another example, galTK from Helicobacter pylori and Smob from Aspergillus motilus were used in combination with Med1 from Mediterranea sp. An20 to initiate the production of LNFP-II using LNT-II as the starting substrate.

[0112] β-1,4-galactosyltransferase

[0113] β-1,4-galactosyltransferase is any protein that can transfer the galactose of UDP-galactose to the N-acetylglucosaminyl part and thus to the acceptor molecule with a β-1,4-bond. 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.

[0114] The following example uses a heterologous β-1,4-galactosyltransferase named GalT or a variant thereof, in combination with other glycosyltransferases to produce, for example, LNFP-III. Thus, in an embodiment, the genetically engineered cell comprises one or more recombinant nucleic acid sequences encoding a β-1,4-galactosyltransferase.

[0115] Table 2 provides non-limiting examples of β-1,4-galactosyltransferases. 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 β-1,4-galactosyltransferase in Table 2.

[0116] Table 2. List of β-1,4-glycosyltransferases

[0117] Protein name GenBank ID source GalT WP_001262061.1 Helicobacter pylori LqCy AAF42257.1 Neisseria meningitidis MC58

[0118] In an embodiment of the present invention, the β-1,3-N-acetylglucosaminyltransferase is derived from Neisseria meningitidis, and the β-1,3-galactosyltransferase and / or β-1,4-galactosyltransferase are derived from Helicobacter pylori, respectively.

[0119] In one embodiment, the recombinant nucleic acid sequence encoding β-1,4-galactosyltransferase comprises or consists of the amino acid sequence of SEQ ID NO:5 (galT from Helicobacter pylori), or a functional homolog thereof, the amino acid sequence of which has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO:5.

[0120] 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: 4, or a functional homologue thereof, the amino acid sequence of the functional homologue having at least 80% identity with SEQ ID NO: 4, and the β-1,4-galactosyltransferase has an amino acid sequence according to SEQ ID NO: 5, or a functional homologue thereof, the amino acid sequence of the functional homologue having at least 80% identity with SEQ ID NO: 5.

[0121] In order to produce LNT from LNT-II precursor, a β-1,3-galactosyltransferase is required. In one embodiment, the genetically engineered cell comprises a β-1,3-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-1,3-galactosyltransferase is from Helicobacter pylori. In a further embodiment, the 1,3-N-acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO: 4, or a functional homologue thereof, the amino acid sequence of the functional homologue having at least 80% identity with SEQ ID NO: 4, and the β-1,3-galactosyltransferase has an amino acid sequence according to SEQ ID NO: 31, or a functional homologue thereof, the amino acid sequence of the functional homologue having at least 80% identity with SEQ ID NO: 31.

[0122] The following are non-limiting examples of genetically modified strains according to the present invention having a specific combination of glycosyltransferases that will produce LNFP-II or LNFP-III using lactose as a starting substrate.

[0123] In a non-limiting example, LgtA from Neisseria meningitidis is used in combination with galT or galTK from Helicobacter pylori and Med1 from Mediterranea An20 to initiate the production of LNFP-III or LNFP-II, respectively, using lactose as a starting substrate.

[0124] In a non-limiting example, galT or galTK from Helicobacter pylori is used in combination with Med1 from Mediterranea an20 to initiate the production of LNFP-III or LNFP-II, respectively, using LNT-II as the starting substrate.

[0125] α-1,2-fucosyltransferase

[0126] α-1,2-fucosyltransferase is a protein that can catalyze the transfer of fucose from a donor substrate (e.g., GDP-fucose) to an acceptor molecule with an α-1,2-bond. Preferably, the α-1,2-fucosyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding the α-1,2-fucosyltransferase is of heterologous origin. Table 3 gives non-limiting examples of α-1,2-fucosyltransferases. α-1,2-fucosyltransferase 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 with one of the α-1,2-fucosyltransferases in Table 3.

[0127] Table 3. List of α-1,2-fucosyltransferases

[0128]

[0129]

[0130] In an embodiment, the cell of the present invention further comprises a recombinant nucleic acid sequence encoding an α-1,2-fucosyltransferase from Aspergillus motile, the α-1,2-fucosyltransferase having an amino acid sequence according to SEQ ID NO: 32, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO: 32.

[0131] The following are non-limiting examples of genetically modified strains according to the present invention having a specific combination of glycosyltransferases that will produce LNDFH-I using lactose as a starting substrate.

[0132] In a non-limiting example, LgtA from Neisseria meningitidis was used in combination with galTK from Helicobacter pylori, Smob from Aspergillus motileus, and Med1 from Mediterranea An20 to produce LNDFH-I starting from lactose as a starting substrate.

[0133] In one non-limiting example, galTK from Helicobacter pylori is used in combination with Smob from Aspergillus motileus and Med1 from Mediterranea sp. An20 to produce LNDFH-I starting from LNT-II as a starting substrate.

[0134] Glycosyl donor-nucleotide activated sugar pathway

[0135] When implementing the method of the present invention, preferably glycosylation reaction mediated by glycosyltransferase occurs, wherein activated sugar nucleotide is used as glycosyl donor. Activated sugar nucleotide usually has phosphorylated glycosyl residue attached to 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. Genetically engineered cells according to the present invention can include one or more pathways for producing nucleotide activated sugars, and nucleotide activated sugars are selected from glucose-UDP-GlcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and CMP-N-acetylneuraminic acid.

[0136] In one embodiment of the invention, the genetically engineered cells can produce one or more of the above-mentioned activated sugar nucleotides by de novo pathways. In this regard, activated sugar nucleotides are made by cells from simple carbon sources (such as glycerol, sucrose, fructose or glucose) in a stepwise reaction sequence under the action of enzymes participating in the de novo biosynthetic pathways of respective sugar nucleotides (for a review of monosaccharide metabolism, see, for example, HH Freeze and AD Elbein: Chapter 4: Glycosylation Precursors, in: Essentials of Glycobiology, 2nd edition (Eds. A. Varki et al.), Cold Spring Harbour Laboratory Press (2009)).

[0137] The enzymes involved in the de novo biosynthetic pathway of activated sugar nucleotides may be naturally present in the cell or introduced into the cell by means of genetic or recombinant DNA techniques, which are part of the general knowledge of the person skilled in the art.

[0138] In another embodiment, genetically engineered cells can utilize salvaged monosaccharides as sugar nucleotides. In the salvage pathway, monosaccharides from degraded oligosaccharides are phosphorylated by kinases and converted into nucleotide sugars by pyrophosphorylases. The enzymes involved in this process can be heterologous or native enzymes of the host cell.

[0139] colanic acid gene cluster

[0140] For the production of fucosylated HMOs, the de novo GDP-fucose pathway is very important to ensure the presence of sufficient GDP-fucose. The colanic acid gene cluster of E. coli encodes most of the 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 (e.g., wcaJ) are responsible for the production of the extracellular polysaccharide colanic acid, which is the major oligosaccharide of the bacterial cell wall. These genes can be deleted to prevent the conversion of GDP-fucose to colanic acid.

[0141] In order to ensure a sufficient amount of GDP-fucose, the promoter of the natural colanic acid gene cluster can be exchanged with a stronger promoter to generate a recombinant colanic acid gene cluster to drive additional GDP-fucose production. In addition, additional copies of the colanic acid gene cluster or selected genes thereof can be introduced into the genetically engineered cells described in the examples.

[0142] In an embodiment, the colanic acid gene cluster can be expressed from its native genomic site. Expression can be actively regulated. Expression can be regulated by exchanging a native promoter with a promoter of interest, and / or by expressing the gene cluster from another non-native genomic site or additionally expressing the colanic acid gene cluster or its specific gene to increase the copy number of the colanic acid gene encoding the protein.

[0143] With respect to the present disclosure, the term "natural genomic site" relates to the colanic acid gene cluster and to the original and natural location of the gene cluster in the genome of a genetically engineered cell.

[0144] The de novo GDP-fucose pathway genes responsible for GDP-fucose formation include or consist of the following genes:

[0145] i) manA, which encodes the protein mannose-6-phosphate isomerase (EC 5.3.1.8, UniProt accession number P00946), which promotes the interconversion of fructose 6-phosphate (F6P) and mannose-6-phosphate;

[0146] ii) manB, which encodes a protein phosphomannose mutase (EC 5.4.2.8, UniProt accession number P24175), which participates in the biosynthesis of GDP-mannose by catalyzing the conversion of mannose-6-phosphate to mannose-1-phosphate;

[0147] iii) manC, which encodes the protein mannose-1-phosphate guanylate transferase (EC: 2.7.7.13, UniProt accession number P24174), which is involved in the biosynthesis of GDP-mannose by synthesizing GDP-mannose from GTP and α-D-mannose-1-phosphate;

[0148] iv) gmd, which encodes the protein GDP-mannose-4,6-dehydratase (UniProt accession number P0AC88), which catalyzes the conversion of GDP-mannose to GDP-4-dehydro-6-deoxy-D-mannose;

[0149] v) wcaG (fcl), which encodes the protein GDP-L-fucose synthase (EC 1.1.1.271, UniProt accession number P32055), which catalyzes the two-step NADP-dependent conversion of GDP-4-dehydro-6-deoxy-D-mannose to GDP-fucose.

[0150] Therefore, when the genetically engineered cell produces one or more fucosylated heterologous products, it is preferred to overexpress the entire colanic acid gene cluster (e.g., as shown in SEQ ID NO: 3 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.

[0151] Lactose permease

[0152] Lactose permease is a membrane protein, a member of the major facilitator superfamily, and can be classified as a symporter, which utilizes the proton gradient toward the cell to transport β-galactosides (e.g., lactose) into the cell in the same direction. In oligosaccharide production, especially in the production of human milk oligosaccharides (HMOs), lactose is often the starting substrate that is modified to produce any HMO of interest in a biotransformation species that occurs inside the cell. Therefore, in the production of HMOs, it is desirable to be able to introduce lactose into the cell, for example by expressing and / or overexpressing a lactose permease, such as lacY of E. coli K.-12.

[0153] In an embodiment, the lactose permease is as shown in SEQ ID NO:6, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO:6, such as at least 85%, 90% or 95% identical.

[0154] In an embodiment, the expression of the lactose permease is regulated by a promoter according to the invention.

[0155] β-Galactosidase

[0156] Host cells suitable for HMO production, such as E. coli, may contain an endogenous β-galactosidase gene or an exogenous β-galactosidase gene, such as E. coli containing 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 so that they do not contain any β-galactosidase gene or contain an inactivated β-galactosidase gene. The gene may be inactivated by completely or partially deleting the corresponding nucleic acid sequence from the bacterial genome, or the gene sequence may be mutated and cannot be transcribed, or even if transcribed, the transcript will not be translated, or even if translated into a protein (i.e., β-galactosidase), the protein does not have the corresponding enzyme activity. In this way, the bacteria producing HMOs accumulate an increased intracellular lactose pool, which is beneficial for the production of HMOs.

[0157] Transporter

[0158] Oligosaccharide products (e.g., HMOs 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, i.e., it diffuses through the cell membrane to the outside. More complex HMO products may remain in the cell, which is likely to ultimately damage cell growth, thereby affecting the total yield of products that can be produced in a single fermentation. HMO transport can be facilitated by major facilitator superfamily transport proteins, which promote the outflow of sugar derivatives from cells to the supernatant. Major facilitator superfamily transport proteins can be exogenous or endogenous, and overexpressed under fermentation conditions to increase the output of oligosaccharide derivatives (HMOs) produced. Mutations by known recombinant DNA techniques can change the specificity of the sugar portion of the product to be secreted.

[0159] Thus, the genetically engineered cell according to the invention may further comprise a nucleic acid sequence encoding a major facilitator superfamily transporter protein capable of exporting a fucosylated human milk oligosaccharide product, such a transporter protein may be, for example, a member of the major facilitator superfamily transporter protein.

[0160] In recent years, several new and efficient major facilitator superfamily transporters have been identified, each specific for a different recombinantly produced HMO, and the development of recombinant cells expressing said proteins has facilitated large-scale industrial HMO manufacturing.

[0161] Therefore, in one or more exemplary embodiments, the genetically engineered cell according to the present invention further comprises a gene product used as a LNFP-II, LNFP-III or LNDFH-I transporter. The gene product used as a LNFP-II, LNFP-III or LNDFH-I transporter can be encoded by a recombinant nucleic acid sequence expressed in the genetically engineered cell. The recombinant nucleic acid sequence encoding LNFP-II, LNFP-III or LNDFH-I transporter can be integrated into the genome of the genetically engineered cell, or expressed using a plasmid.

[0162] In one embodiment, the genetically engineered cells of the present invention comprise a nucleic acid sequence encoding a major facilitator superfamily transporter capable of exporting fucosylated human milk oligosaccharide products into the extracellular medium, particularly preferably a transporter I that is specific for exporting complex fucosylated HMOs (e.g., LNFP-II, LNFP-III or LNDFH-).

[0163] Genetically engineered cells

[0164] In this context, the terms "genetically engineered cells" and "genetically engineered cells" are used interchangeably. As used herein, "genetically engineered cells" refer to host cells whose genetic material has been altered by human intervention using genetic engineering techniques, such as, but not limited to, transformation or transfection, such as using heterologous polynucleotide sequences, Crisper / Cas editing and / or random mutations. In one embodiment, the genetically engineered cells have been transformed or transfected by a recombinant nucleic acid sequence.

[0165] The genetic modification can, for example, be selected from the addition of glycosyltransferases, and / or metabolic pathway engineering and the addition of transporters, as described above, and the skilled person will know how to combine them into a genetically engineered cell capable of producing one or more fucosylated HMOs, preferably, the one or more fucosylated HMOs are selected from LNFP-II, LNFP-III and LNDFH-I.

[0166] In an embodiment of the present disclosure, the complex fucosylated human milk oligosaccharide (HMO) produced by the cell is selected from LNFP-II, LNFP-III and LNDFH-I. Generally speaking, LNFP-II, LNFP-III and LNDFH-I are not produced by the same cell, but require different genetic modifications in the background strain to provide LNT, LNnT or LNFP-I strains, respectively. Therefore, if starting from lactose as a starting substrate, the production of LNFP-II, LNFP-III or LNDFH-I requires the presence of three or more glycosyltransferase activities, or if starting from LNT-II as a starting substrate, the presence of two or more glycosyltransferase activities is required.

[0167] In this regard, the cell of the present invention expressing β-1,3-N-acetylglucosaminyltransferase and β-1,3-galactosyltransferase in combination with the α-1,3(4)-fucosyltransferase of the present invention is capable of producing complex fucosylated HMOLNFP-II.

[0168] The HMO produced by the strain containing the α-1,3(4)-fucosyltransferase described herein can be a mixture of HMOs comprising a precursor HMO (e.g., LNT, LNnT, or LNFP-I) and / or a byproduct oligosaccharide or byproduct HMO (e.g., 2'FL, 3FL, DFL, pLNH2, or pLNnH). Figure 1 As shown in Figure 2, pLNH2 is a potential oligosaccharide byproduct, but, although it is likely present in small amounts in human breast milk, it is not officially reported as an HMO.

[0169] However, in a preferred embodiment the mixture is substantially free of the alternative complex fucosylated HMOs LNFP-V, LNFP-VI and / or LNDFH-II.

[0170] In other embodiments, the cells of the invention produce a HMO mixture comprising LNFP-II, LNT and pLNH2 or LNFP-II, LNnT and 3FL or LNFP-II, LNT, 3FL and pLNH2 or LNFP-II, LNT, 3FL, LNT-II and pLNH2.

[0171] In other embodiments, the cells of the invention produce a HMO mixture comprising LNFP-III and LNnT, LNFP-III, LNnT and pLNnH, or LNFP-III and 3FL.

[0172] In additional embodiments, cells of the invention produce a HMO mixture comprising LNFP-I, 2'FL, and LNDFH-I.

[0173] In a preferred embodiment, the engineered cell is capable of producing one or more fucosylated HMOs according to any one of the preceding claims, wherein the cell produces a mixture of HMOs consisting essentially of:

[0174] a) LNFP-II, pLNH2 and LNT, or

[0175] b) LNFP-II, LNT and 3FL, or

[0176] c) LNFP-II, LNT, 3FL and pLNH2, or

[0177] d) LNT-II, LNFP-II, LNnT, 3FL, and pLNH2, or

[0178] e) LNFP-III and LNnT, or

[0179] f) LNFP-III, LNnT and pLNnH, or

[0180] g) LNFP-III and 3FL, or

[0181] h) LNFP-I, 2'FL and LNDFH-I.

[0182] In an embodiment, the total HMO produced by the cell is substantially free of LNFP-VI and / or LNDFH-II. In the present invention, "substantially free of LNFP-VI and / or LNDFH-III" is understood as the content of LNFP-VI and / or LNDFH-III in the total HMO produced by the cell is less than 1%. In the present invention, "substantially free of LNFP-V and / or LNDFH-II" is understood as the content of LNFP-V and / or LNDFH-II in the total HMO produced by the cell is less than 1%. In the present invention, "substantially free of 3FL" is understood as the content of 3FL in the total HMO produced by the cell is less than 1%.

[0183] In a preferred embodiment, a genetically engineered cell capable of producing one or more fucosylated HMOs, preferably one or more fucosylated HMOs selected from the group consisting of LNFP-II, LNFP-III and LNDFH-I, comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is Med1 having an amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof having an amino acid sequence having at least 80% identity to SEQ ID NO: 1.

[0184] Host cells

[0185] 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 function as host cells include bacterial cells, archaeal cells, algae cells, and fungal cells.

[0186] 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.

[0187] Regarding bacterial host cells, there are in principle no restrictions; they can be eubacteria (Gram-positive or Gram-negative) or archaea, as long as they allow genetic manipulation to insert the gene of interest and can be cultured on a manufacturing scale. Preferably, the host cell has properties that allow cultivation to high cell densities. Non-limiting examples of bacterial host cells suitable for 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, can also be used.Similarly, bacteria of the genera Lactobacillus and Lactococcus can be engineered using the methods of the invention, including, but not limited to, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also species of bacteria suitable for use in the invention described herein. Also included as part of the invention are strains engineered as described herein from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa). aeruginosa)).

[0188] Non-limiting examples of fungal host cells suitable for the recombinant industrial production of heterologous products are, for example, yeast cells, such as Komagataella, Kluyveromyces, Yarrowia, Pichia, Saccaromyces, Schizosaccharomyces or Hansenula, or filamentous fungi from the genera Aspargillus, Fusarium or Thricoderma.

[0189] In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of Escherichia coli, Bacillus, Lactobacillus, Corynebacterium sp., and Campylobacter.

[0190] 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.

[0191] In one or more exemplary embodiments, the genetically engineered cell is Bacillus subtilis.

[0192] In one or more exemplary embodiments, the genetically engineered cell is Saccharomyces cerevisiae or Pichia pastoris.

[0193] In one or more exemplary embodiments, the genetically engineered cell is Escherichia coli.

[0194] 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.

[0195] Cell-produced HMO mixtures

[0196] Genetically engineered cells described herein that contain more than one glycosyltransferase will typically produce a mixture of HMOs in a multi-step process to produce a final HMO product. In the production of LNFP-II using lactose as the starting substrate, LNT-II, LNT, LNFP-II, and pLNH2 may be present at the end of the culture, and 3FL (fucosylated lactose), LNFP-V, and LNDFH-II may also be present.

[0197] In the process of producing LNDFH-I using lactose as the starting substrate, 2'FL (fucosylated lactose), LNT-II, LNT, LNFP-I and LNDFH-I may be present at the end of the culture, and 3FL, LNFP-II, LNFP-V, LNDFH-II and pLNH2 may also be present.

[0198] In the process of producing LNFP-III using lactose as the starting substrate, LNT-II, LNnT, LNFP-III and pLNnH may be present at the end of the culture, and 3FL (fucosylated lactose), LNFP-VI and LNDFH-III may also be present.

[0199] In this regard, the HMO mixture can consist essentially of: a) LNFP-II, pLNH2 and LNT, or b) LNFP-II, LNT and 3FL, or c) LNFP-II, LNT, 3FL and pLNH2, or d) LNT-II, LNFP-II, LNT, 3FL and pLNH2, or e) LNFP-III and LNnT, or f) LNFP-III, LNnT and pLNnH, or g) LNFP-III and 3FL, or h) LNFP-I, 2'FL and LNDFH-I.

[0200] The HMO product produced by the methods disclosed herein can be described by its mole percentage (%) in the HMO mixture. The "mole percentage (%) of HMO" described herein is understood to be the mole percentage (%) of HMO in the total HMO produced.

[0201] In one embodiment of the invention, after culturing the genetically engineered cells described herein, the HMO mixture consists essentially of LNFP-II and LNT in combination with one or more of 3FL and / or LNT-II and / or pLNH2.

[0202] In one embodiment of the invention, after culturing the genetically engineered cells described herein, the HMO mixture consists essentially of 3-35% LNFP-II, 45-85% LNT, 0-20% 3FL, 0-15% LNT-II and 0-5% pLNH2, and the total HMO molar contents in the mixture add up to 100%.

[0203] In an embodiment of the invention, the fucosylated human milk oligosaccharide (HMO) produced by the cell is LNFP-II, for example, completely or substantially completely LNFP-II. In other embodiments of the invention, at least 5%, for example, at least 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25% or 30% of the total HMO molar content produced by the cell is LNFP-II. Preferably, at least 25% of the total HMO molar content produced by the cell is LNFP-II. In other embodiments, at least 75% of the total HMO molar content produced by the cell is LNFP-II and LNT. In other embodiments of the invention, less than 1% of the total HMO molar content produced by the cell, for example 0.0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 0.99% is LNFP-V. In other embodiments of the invention, less than 5% of the total molar content of HMO produced by the cell, 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% is LNDFH-II. In other embodiments, less than 5% of the total molar content of HMO produced by the cell is alternative complex fucosylated HMO. In other embodiments, less than 25%, such as less than 50%, such as less than 15%, such as less than 10%, such as less than 5%, such as less than 1% of the total molar content of HMO produced by the cell is non-complex fucosylated HMO, such as 3FL. In this context, it is considered that alternative fucosylated HMO is one or more fucosylated HMO that is not LNFP-II. Alternative fucosylated HMO can be selected from 3FL, DFL, LNFP-V and LNDFH-II. The alternative complex fucosylated HMO may be selected from LNFP-V and LNDFH-II. In one embodiment of the invention, after culturing the genetically engineered cells as described herein, the HMO mixture consists essentially of LNDFH-I, LNFP-I and 2'FL.

[0204] In one embodiment of the invention, after culturing the genetically engineered cells described herein, the HMO mixture consists essentially of 7-15% LNDFH-I, 65-90% LNFP-I and 5-20% 2'FL, the total HMO molar contents in the mixture adding up to 100%.

[0205] In other embodiments of the invention, at least 5%, such as at least 6%, 7%, 8%, 9%, 10% or 11% of the total molar content of HMO produced by the cell is LNDFH-I. In other embodiments, at least 80% of the total molar content of HMO produced by the cell is LNFP-I and LNDFH-I. In other embodiments of the invention, less than 1% of the total molar content of HMO produced by the cell is LNFP-V and / or LNDFH-II, 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%.

[0206] In other embodiments, less than 5% of the total molar content of HMO produced by the cell is alternative fucosylated HMO. In this context, when the predominantly produced HMOs are LNDFH-I and LNFP-I, alternative fucosylated HMOs are considered to be one or more fucosylated HMOs that are not LNDFH-I or LNFP-I. Thus, when the predominantly produced HMOs are LNDFH-I and LNFP-I, alternative fucosylated HMOs may be selected from 3FL, 2'FL, DFL, LNFP-V, and LNDFH-II.

[0207] In one embodiment of the invention, after culturing the genetically engineered cells described herein, the HMO mixture consists essentially of LNFP-III and LNnT, and the content of 3FL or pLNnH is relatively low, such as less than 10% of the total molar content of HMOs in the composition.

[0208] In one embodiment of the invention, after culturing the genetically engineered cells described herein, the HMO mixture consists essentially of 50-99 mole % LNFP-III, 0-45 mole % LNnT, 0-20% 3FL and 0-10% pLNnH, adding up to a total of 100 mole % content.

[0209] In an embodiment of the invention, the fucosylated human milk oligosaccharide (HMO) produced by the cell is LNFP-III, for example, completely or substantially completely LNFP-III. In other embodiments of the invention, at least 30%, for example at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the total molar content of HMO produced by the cell is LNFP-III. Preferably, at least 75% of the total molar content of HMO produced by the cell is LNFP-III.

[0210] In other embodiments, at least 90% of the total molar content of HMO produced by the cell is LNFP-III and LNnT. In other embodiments of the invention, less than 1% of the total molar content of HMO produced by the cell, 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% is LNFP-VI. In other embodiments of the invention, less than 5% of the total molar content of HMO produced by the cell, 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% is LNDFH-III. In another embodiment, less than 5% of the total molar content of HMO produced by the cell is alternative complex fucosylated HMO. In another embodiment, 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 HMO molar content produced by the cell is non-complex fucosylated HMO, such as FL. In this context, alternative fucosylated HMOs are considered to be one or more fucosylated HMOs that are not LNFP-III. Alternative fucosylated HMOs can be selected from 3FL, DFL, LNFP-VI and LNDFH-III. Alternative complex fucosylated HMOs can be selected from LNFP-VI and LNDFH-III.

[0211] In some embodiments, the genetically engineered cells of the invention express Med1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or comprising or consisting of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO: 1, and the 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% or such as higher than 85% of the total HMOs.

[0212] In some embodiments, the genetically engineered cells of the invention express Med1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or comprising or consisting of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 1, and the genetically engineered cells produce LNFP-II with a molar % content higher than 5% of total HMOs, such as higher than 10%, such as higher than 20%, such as higher than 30%.

[0213] In some embodiments, the genetically engineered cells of the invention express Med1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or comprising or consisting of an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 1, and the genetically engineered cells produce LNDFH-I with a molar % content higher than 5% of total HMOs, such as higher than 6%, such as higher than 8%, such as higher than 9%.

[0214] The supported mole % of the individual HMO components are supported by the experimental data of Examples 1-5, which show exemplary HMO mixture ranges for genetically engineered cells expressing the fucosyltransferase Med1.

[0215] Recombinant nucleic acid sequence

[0216] The present invention relates to a genetically engineered cell comprising a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, specifically α-1,3(4)-fucosyltransferase Med1, and wherein the cell produces human milk oligosaccharides (HMOs), preferably, the fucosylated HMOs are selected from fucosylated HMOs comprising at least five or six monosaccharide units, more preferably, one or more fucosylated HMOs are selected from LNFP-II, LNFP-III and LNDFH-I.

[0217] In this context, the terms "recombinant nucleic acid sequence", "recombinant gene / nucleic acid / nucleotide sequence / DNA code" or "coding nucleic acid sequence" are used interchangeably and refer to an artificial nucleic acid sequence (i.e., a sequence produced in vitro using standard experimental methods for preparing nucleic acid sequences) that contains a set of consecutive, non-overlapping triplets (codons) that are transcribed into mRNA and translated into protein when under the control of appropriate control sequences (i.e., promoter sequences).

[0218] The boundaries of the coding sequence are usually determined by the ribosome binding site, transcription start codon (AUG, GUG or UUG) and translation stop codon (UAA, UGA or UAG) located upstream of the open reading frame at the 5' end of the mRNA. The coding sequence may include, but is not limited to, genomic DNA, cDNA, synthetic and recombinant nucleic acid sequences.

[0219] The term "nucleic acid" includes RNA, DNA and cDNA molecules. It is understood that due to the degeneracy of the genetic code, a large number of nucleic acid sequences can be produced that encode a given protein.

[0220] The recombinant nucleic acid sequence can be a coding DNA sequence, such as a gene, or a non-coding DNA sequence, such as regulatory DNA, such as a promoter sequence or other non-coding regulatory sequences.

[0221] The recombinant nucleic acid sequence can also be heterologous. "Heterologous" as used herein refers to a polypeptide, amino acid sequence, nucleic acid sequence or nucleotide sequence that is foreign to a cell or organism, i.e., a polypeptide, amino acid sequence, nucleic acid molecule or nucleotide sequence that does not naturally occur in the cell or organism.

[0222] The present invention also relates to a nucleic acid construct comprising a coding nucleic acid sequence, i.e. a recombinant DNA sequence of a target gene (e.g. a fucosyltransferase gene), and a non-coding regulatory DNA sequence, such as a promoter DNA sequence, e.g. a recombinant promoter sequence derived from a promoter sequence of a lac operon or a glp operon, or a promoter sequence derived from another genomic promoter DNA sequence, or a synthetic promoter sequence, wherein the coding sequence and the promoter sequence are operably linked.

[0223] The term "operably linked" refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. It refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter sequence is operably linked to a coding sequence if it stimulates or regulates transcription of the coding sequence in a suitable host cell or other expression system.

[0224] Typically, promoter sequences that are operably linked to a transcribed sequence are physically adjacent to the transcribed sequence, ie, they are cis-acting.

[0225] 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.

[0226] Therefore, the term "nucleic acid construct" refers to an artificially constructed nucleic acid fragment, in particular a DNA fragment, which is intended to be inserted into a target cell, such as a bacterial cell, to modify the expression of a genomic gene or the expression of a gene / coding DNA sequence that may be contained in the construct. Therefore, in an embodiment, the present invention relates to a nucleic acid construct comprising a recombinant nucleic acid sequence encoding a fucosyltransferase, wherein the recombinant nucleic acid sequence is a nucleic acid sequence encoding Med1, such as a nucleic acid sequence according to SEQ ID NO: 2, or a functional variant thereof.

[0227] Thus, in one aspect of the invention, the genetically engineered cell comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity capable of producing at least 50%, preferably at least 80% of the total HMO molar content produced by the cell as LNFP-III.

[0228] In this regard, cells of the present invention expressing β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase in combination with the α-1,3(4)-fucosyltransferase of the present invention are capable of producing the complex fucosylated HMO, LNFP-III.

[0229] In one aspect of the invention, a genetically engineered cell comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having alpha-1,3(4)-fucosyltransferase activity capable of producing LNFP-II at least 5% of the total molar content of HMO produced by the cell.

[0230] In one aspect of the invention, a genetically engineered cell comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having alpha-1,3(4)-fucosyltransferase activity capable of producing LNDFH-I that is at least 8% of the total molar content of HMO produced by the cell.

[0231] Furthermore, cells expressing a combination of β-1,3-N-acetylglucosaminyltransferase, β-1,3-galactosyltransferase and α-1,2-fucosyltransferase and the α-1,3(4)-fucosyltransferase of the present invention are able to produce a combination of complex fucosylated HMO LNDFH-I and complex fucosylated HMO LNFP-I.

[0232] The genetically engineered cells according to the present invention can also comprise multiple copies of the recombinant nucleic acid sequence encoding fucosyltransferase. Examples 1 and 2 show that the copy number of fucosyltransferase Med1 is increased to change the ratio of HMO produced and to increase the level of LNFP-II and LNFP-III produced, respectively. Specifically, Example 2 discloses that increasing the copy number of Med1 to two genomic copies (strain Med1_2) increases the production of LNFP-III, and further increasing the expression by additionally expressing Med1 from a high copy plasmid, resulting in a slight decrease in LNFP-II levels, while a sudden increase in the production of 3FL is observed. In addition, for the high expression Med1 strain, it is observed that all LNnT produced by the cell are fucosylated.

[0233] Furthermore, Example 1 discloses that increasing the copy number of Med1 and fucosyltransferase Para1 to two genomic copies increased the production of LNFP-II by 10% from 5% to 15% for Med1, while Para1 only increased the formation of LNFP-II by 1%, and the amount of difucosylated LNDFH-II increased from 2% to 11% for Para1, clearly indicating that Para1 has an equal preference for fucosylation of the GlcNAc and Glc moieties of LNT, while Med1 is only active on the GlcNAc moiety. Further increasing the expression of Med1 increased the level of LNFP-II from 15% to 30%.

[0234] Furthermore, an increase in LNFP-II or LNFP-III was observed when the expression of Med1 was enhanced, which also resulted in a change in the amount of each HMO produced by the cells, as is apparent from Examples 1 to 3.

[0235] Thus, copy number variation can be used to produce customized HMO mixtures, in this case mixtures containing different ratios of LNFP-III, LNnT, and / or 3FL, mixtures containing different ratios of LNFP-II, LNT, and 3FL or pLNH2, or mixtures containing different ratios of LNDFH-I, LNFP-I, and 2'FL, depending on the requirements of a specific product.

[0236] 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 Med1 having an amino acid sequence according to SEQ ID NO: 1 or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 1.

[0237] In other embodiments, the recombinant nucleic acid sequence is encoded on a plasmid, which recombinant nucleic acid sequence encodes a glycosyltransferase Med1 having 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 to SEQ ID NO: 1. In a further embodiment, the plasmid is a high copy number plasmid, preferably a pUC57 plasmid.

[0238] In other embodiments, 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 Med1 having an amino acid sequence according to SEQ ID NO: 1 or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 1.

[0239] 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 Med1, which comprises or consists of a nucleic acid sequence of SEQ ID NO:2, or comprises or consists of a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:2.

[0240] Preferably, the fucosyltransferase coding sequence is under the control of a promoter sequence selected from the promoter sequences having the nucleic acid sequence as identified in Table 4.

[0241] Table 4 - Selected promoter sequences

[0242]

[0243] * Promoter activity was assessed in the LacZ assay described below, with the PglpF promoter as a positive reference in the same assay. To compare different assays, activity was calculated relative to the PglpF promoter, and ranges represent results from multiple assays.

[0244] The promoter may be heterologous, a promoter native to the genetically engineered cell, or it may be a recombinant promoter, a combination of heterologous and / or native elements.

[0245] One approach to increasing product yields may be to modulate the production of a desired enzyme activity for production of the product, such as a glycosyltransferase or an enzyme involved in the glycosyl donor biosynthetic pathway.

[0246] Increasing the strength of the promoter driving the desired enzyme expression may be a method to achieve this goal. The strength of the promoter can be evaluated using the lacZ enzyme test method, wherein the method for determining the activity of β-galactosidase is as described above (e.g., with reference to Miller JH Experiments in molecular genetics, Cold spring Harbor Laboratory Press, NY, 1972). In short, the cells are diluted in Z buffer and permeabilized with sodium dodecyl sulfate (0.1%) and chloroform. The LacZ test method is carried out at 30°C. Preheat the sample, start the test by adding 200μl of o-nitrophenyl-β-galactosidase (4mg / ml), and stop the test by adding 500μl 1M Na2CO3 when the sample turns slightly yellow. The release of o-nitrophenol is subsequently determined based on the change in optical density at 420nm. Specific activity is expressed in Miller units (MU) [A420 / (min*ml*A600)]. A regulatory element with an activity higher than 10,000MU is considered strong, a regulatory element with an activity lower than 3,000MU is considered weak, and those in between have medium strength. An example of a strong regulatory element is the PglpF promoter with an activity of about 14.000MU, and an example of a weak promoter is Plac, which has an activity of about 2300MU when induced with IPTG. In a preferred embodiment, the expression of the nucleic acid sequence is controlled by a strong promoter selected from SEQ ID NO 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18.

[0247] In an embodiment, the expression of the nucleic acid sequence of the present invention is controlled by the PglpF (SEQ ID NO: 19) or Plac (SEQ ID NO: 28) promoter or PmglB_UTR70 (SEQ ID NO: 16) or PglpA_70UTR (SEQ ID NO: 17) or PglpT_70UTR (SEQ ID NO: 18) or variants thereof (e.g., the promoters identified in Table 4), in particular the PglpF_SD4 variant of SEQ ID NO: 14 or the Plac_70UTR variant of SEQ ID NO: 10, or the PmglB_70UTR variants of SEQ ID NO: 7, 8, 9, 11, 12, 13 and 14. 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 for reference).

[0248] In a preferred embodiment, the recombinant nucleic acid sequence is individually controlled by one or more promoters selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 19, 28, 16, 17 and 18, respectively) and variants thereof.

[0249] The nucleic acid construct of interest contained in the construct (expression cassette) can be integrated into the bacterial genome by conventional methods, for example by using a linear fragment containing flanking sequences homologous to specific sites on the chromosome, such as described for the attTn7 site (Waddell CS and Craig NL, Genes Dev. (1988) February; 2(2): 137-49.); methods for genomic integration of recombinant nucleic acid sequences mediated by the Red recombinase function of bacteriophage λ or the RecE / RecT recombinase function of the Rac prophage (Murphy, J Bacteriol. (1998); 180(8): 2063-7; Zhang et al., Nature Genetics (1998) 20: 123-128; Muyrers et al., EMBO Rep. (2000) 1(3): 239-243); methods based on Red / ET recombination (Wenzel et al., Chem. Biol. (2003); 1(4): 237-243; Biol. (2005), 12(3):349-56.; Vetcher et al., Appl Environ Microbiol. (2005); 71(4):1829-35); or positive clones, i.e. clones carrying the expression cassette, can be selected by, for example, loss or gain of marker gene or gene function.

[0250] In one or more exemplary embodiments, the present disclosure relates to a recombinant nucleic acid sequence as shown in SEQ ID NO:2.

[0251] Specifically, the present disclosure relates to a recombinant nucleic acid sequence and / or a functional homolog thereof, the sequence of which is at least 70% identical to SEQ ID NO:2, such as at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical or 100% identical.

[0252] Sequence identity

[0253] The term "sequence identity" as used herein describes the association between two amino acid sequences or 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 invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo / . Biol. 48: 443-453), which is implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or higher (available at https: / / www.ebi.ac.uk / Tools / psa / emboss needle / ). The parameters used are gap open penalty (gap open penalty) 10, gap extension penalty (gap extension penalty) 0.5, and EBLOSUM62 (EMBOSS version of 30BLOSUM62) substitution matrix. The Needle output (obtained using the -nobrief option) labeled as "identity" is used as percentage identity. Usually, sequence identity can be calculated as follows: (same residue × 100) / (length of comparison region).

[0254] For the purpose of the present invention, the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), which is implemented in the Needle program of the EMBOSS package (EMBOSS:The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277), 10 preferred versions 5.0.0 or higher. The parameters used are gap open penalty 10, gap extension penalty 0.5 and DNAFULL (EMBOSS version is NCBI NUC4.4) substitution matrix. The Needle output (obtained using the -nobrief option) labeled as "identity" is used as percentage identity. Typically, sequence identity can be calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment region).

[0255] Functional homologs

[0256] Functional homologues or functional variants of protein / nucleic acid sequences described herein are protein / nucleic acid sequences with altered genetic codes that retain their original functions. Functional homologues can be obtained by mutation, or can be natural variants from the same or other species. Compared with the function of the protein / nucleic acid sequence, functional homologues should have at least 50% of the remaining function, such as at least 60%, 70%, 80%, 90% or 100%.

[0257] Functional homologs of any of the disclosed amino acid or nucleic acid sequences may also have higher functionality. A functional homolog of the Med1 amino acid sequence having the amino acid sequence of SEQ ID NO: 1 or the encoding recombinant nucleic acid of SEQ ID NO: 2 should ideally be able to participate in the production of fucosylated HMOs in terms of increased HMO yield, improved purity / byproduct formation, reduced biomass formation, viability of genetically engineered cells, robustness of genetically engineered cells according to the present disclosure, or reduced consumables required for production.

[0258] Uses of genetically engineered cells or enzymes

[0259] 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).

[0260] Therefore, the present invention also relates to the use of a fucosyltransferase Med1 having α-1,3(4)-fucosyltransferase activity in the production of a fucosylated product, the fucosyltransferase having an amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof, the amino acid sequence of the functional homologue having at least 80% identity with SEQ ID NO: 1. In an embodiment, the fucosyltransferase of the present invention is also used to produce a fucosylated product, wherein the fucosylated product is one or more fucosylated oligosaccharides or one or more fucosylated polypeptides. Preferably, the one or more fucosylated oligosaccharides are one or more HMOs, preferably, the fucosylated HMOs are selected from fucosylated HMOs comprising at least five monosaccharide units, preferably selected from fucosylated HMOs having a backbone structure of LNT and / or LNnT, more preferably, the fucosyltransferase only fucosylates the N-acetylglucosamine (GlcNAc) portion of LNT or LNnT.

[0261] In an exemplary embodiment, the α-1,3(4)-fucosyltransferase, genetically engineered cells and / or nucleic acid constructs according to the present invention are used to produce one or more fucosylated HMOs, preferably LNFP-II, LNFP-III and LNDFH-I.

[0262] In an exemplary embodiment, the α-1,3(4)-fucosyltransferase, genetically engineered cells and / or nucleic acid constructs according to the present invention are used to produce HMOs. Preferably, the genetically engineered cells and / or nucleic acid constructs are used to produce HMOs LNFP-II, LNFP-III or LNDFH-I. In addition, the genetically engineered cells and / or nucleic acid constructs are preferably used to produce an HMO mixture, wherein at least 5% of the total molar content of HMOs produced by the cells is LNFP-II, wherein at least 50% of the total molar content of HMOs produced by the cells is LNFP-II, or wherein at least 8% of the total molar content of HMOs produced by the cells is LNDFH-I.

[0263] In an embodiment, a fucosyltransferase, a genetically engineered cell, and / or a nucleic acid construct is used to produce one or more fucosylated HMOs, wherein the cell produces a mixture of HMOs comprising

[0264] a) LNFP-II, pLNH2 and LNT, or

[0265] b) LNFP-II, LNT and 3FL, or

[0266] c) LNFP-II, LNT, 3FL and pLNH2, or

[0267] d) LNT-II, LNFP-II, LNT, 3FL, and pLNH2, or

[0268] e) LNFP-III and LNnT, or

[0269] f) LNFP-III, LNnT and pLNnH, or

[0270] g) LNFP-III and 3FL, or

[0271] h) LNDFH-I, LNFP-I, 2'FL.

[0272] When starting with lactose or LNT-II, production of these HMOs may require the presence of two or more glycosyltransferase activities.

[0273] A method for producing fucosylated human milk oligosaccharides (HMO)

[0274] 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.

[0275] One aspect is a method for producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising the steps of:

[0276] a) providing a genetically modified cell as described herein; and

[0277] b) culturing the cell according to (a) in a suitable cell culture medium to produce said one or more fucosylated HMOs, and

[0278] c) optionally, purifying said one or more fucosylated HMOs.

[0279] The present invention particularly relates to a method for producing human milk oligosaccharides (HMOs), wherein the genetically engineered cells produce LNFP-III in a molar percentage (%) content greater than 50% of the total amount of HMOs produced, such as greater than 75% of the total amount of HMOs produced, or such as greater than 85% of the total amount of HMOs produced. In an embodiment, the fucosylated HMO is LNFP-III.

[0280] The present invention particularly relates to a method for producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically engineered cell comprising a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is Med1 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, and wherein the cell produces a fucosylated HMO selected from a fucosylated HMO comprising at least five monosaccharide units.

[0281] In other embodiments of the method of the present invention, the fucosylated HMO produced is selected from fucosylated HMOs having a LNT and / or LNnT backbone structure. In another embodiment of the method of the present invention, the fucosyltransferase only fucosylates the N-acetylglucosamine (GlcNAc) portion of LNT or LNnT. In a preferred embodiment of the method of the present invention, one or more fucosylated HMOs are selected from LNFP-II, LNFP-III and LNDFH-I. In an embodiment, at least one HMO produced by the method of the present invention is LNFP-II, LNFP-III or LNDFH-I.

[0282] Other embodiments of the present invention relate to a method for producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically engineered cell comprising

[0283] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0284] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-galactosyltransferase or β-1,4-galactosyltransferase activity; and

[0285] c. A recombinant nucleic acid sequence encoding a fucosyltransferase Med1 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.

[0286] Further genetic modifications may be selected from, for example, the addition of additional glycosyltransferases and / or metabolic pathway engineering as described in the above section, and the addition of MFS transporters, which the skilled person will know how to combine into a genetically engineered cell capable of producing one or more fucosylated HMOs.

[0287] The method specifically comprises culturing a genetically engineered cell that produces fucosylated HMOs, wherein the LNFP-III content produced by the cell is at least 30% of the total amount of HMOs produced by the cell. The method specifically comprises culturing a genetically engineered cell that produces fucosylated HMOs, wherein the LNFP-II content produced by the cell is at least 5%, preferably at least 25%, of the total amount of HMOs produced by the cell. The method specifically comprises culturing a genetically engineered cell that produces fucosylated HMOs, wherein the LNFP-I content produced by the cell is at least 5%, preferably at least 8% of the total amount of HMOs produced by the cell.

[0288] The method comprises culturing a genetically engineered cell that produces a fucosylated HMO, and further comprises culturing the genetically engineered cell in the presence of a carbon source (energy source), such as a carbon source selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.

[0289] In an embodiment, the method of the invention produces a mixture of HMOs, wherein the cell produces a mixture of HMOs comprising

[0290] a) LNFP-II, pLNH2 and LNT, or

[0291] b) LNFP-II, LNT and 3FL, or

[0292] c) LNFP-II, LNnT, 3FL and pLNH2, or

[0293] d) LNT-II, LNFP-II, LNnT, 3FL, and pLNH2, or

[0294] e) LNFP-III and LNnT, or

[0295] f) LNFP-III, LNnT and pLNnH, or

[0296] g) LNFP-III and 3FL, or

[0297] h) LNFP-I, 2'FL and LNDFH-I.

[0298] In one aspect, the process according to the invention produces an HMO mixture wherein at least 50%, e.g. at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% of the total HMO molar content produced is LNFP-III.

[0299] In one aspect, the process according to the invention produces a HMO mixture wherein at least 5%, such as at least 10%, 15%, 20%, 25% or 30% of the total HMO molar content produced is LNFP-II.

[0300] In one aspect, the process according to the invention produces an HMO mixture wherein at least 5%, e.g., at least 6%, 7%, 8%, 9%, 10%, 11% or 12% of the total HMO molar content produced is LNDFH-I.

[0301] In one aspect, the method according to the invention produces a HMO mixture comprising LNFP-III, wherein the produced HMO mixture is substantially free of LNFP-VI and / or LNDFH-III.

[0302] In one aspect, the method according to the invention produces a HMO mixture comprising LNFP-II, wherein the produced HMO mixture is substantially free of LNFP-V and / or LNDFH-II.

[0303] In one aspect, the method according to the present invention produces an HMO mixture comprising LNDFH-I, wherein the produced HMO mixture is substantially free of 3FL.

[0304] In one aspect, the method according to the invention produces LNFP-III. In another aspect, the method according to the invention produces LNFP-II. In another aspect, the method according to the invention produces LNDFH-I.

[0305] 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 production of fucosyl nucleotides, such as GDP-fucose.

[0306] In a preferred embodiment of the method of the present invention, the genetically engineered cell comprises an upregulated biosynthetic pathway for producing a fuconucleotide. Preferably, in the method of the present invention, the fuconucleotide 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) of SEQ ID NO:3 from Escherichia coli. 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) of SEQ ID NO:3 from Escherichia coli into the genome of the host cell.

[0307] The methods of the present invention include providing a glycosyl donor either separately synthesized by one or more genetically engineered cells and / or added exogenously to the culture medium from an alternative source.

[0308] In one aspect, the method of the present invention further comprises providing an acceptor sugar as a substrate for HMO formation, the acceptor sugar comprising at least two monosaccharide units, the acceptor sugar being exogenously added to the culture medium and / or being produced by fermentation of a separate microorganism.

[0309] In one aspect, the method of the present invention comprises providing an acceptor sugar comprising at least two monosaccharide units, the acceptor sugar being exogenously added to the culture medium and / or produced by separate microbial fermentation, and being selected from lactose, LNT-II, LNT and LNnT. In a preferred embodiment, the substrate for HMO formation is lactose, which is fed to the culture during the fermentation of the genetically engineered cells.

[0310] Fucosylated human milk oligosaccharides (HMOs) are recovered from the culture or from the culture medium and / or genetically engineered cells.

[0311] 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

[0312] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0313] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,4-galactosyltransferase activity; and

[0314] c. A recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is Med1 having an amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, and

[0315] Wherein the fucosylated HMO is selected from fucosylated HMOs comprising at least five monosaccharide units, preferably, the one or more fucosylated HMOs are LNFP-III.

[0316] 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

[0317] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0318] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-galactosyltransferase activity; and

[0319] c. A recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is Med1, which has a sequence according to SEQ ID NO: 1

[0320] an amino acid sequence, or a functional homologue thereof, wherein the amino acid sequence of the functional homologue has at least 80% identity with SEQ ID NO: 1, and

[0321] Wherein the fucosylated HMO is selected from fucosylated HMOs comprising at least five monosaccharide units, preferably, the one or more fucosylated HMOs are LNFP-II.

[0322] 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

[0323] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0324] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-galactosyltransferase activity; and

[0325] c. A recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is Med1 having an amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, and

[0326] The fucosylated HMO is selected from fucosylated HMOs comprising at least five monosaccharide units, preferably, the one or more fucosylated HMOs are LNDFH-I.

[0327] 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

[0328] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetylglucosaminyltransferase activity; and

[0329] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-galactosyltransferase activity; and

[0330] c. a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,2-fucosyltransferase activity; and

[0331] d. A recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is Med1 having an amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, and

[0332] The fucosylated HMO is selected from fucosylated HMOs comprising at least five monosaccharide units, preferably, the one or more fucosylated HMOs are LNDFH-I.

[0333] Cultivation or fermentation (used interchangeably herein) in a controlled bioreactor generally includes (a) a first phase of exponential growth of cells in a medium secured 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 (e.g., lactose) to allow the formation of HMO products during this phase. Carbon (sugar) limitation refers to the phase in the fermentation process where the growth rate is kinetically controlled by the carbon source (sugar) concentration in the culture broth, which in turn is controlled by the rate of carbon addition (sugar feed rate) in the fermentor.

[0334] The terms "manufacturing" or "manufacturing scale" or "large-scale production" or "large-scale fermentation" are used interchangeably and in the meaning of the present invention define a fermentation with a minimum volume of 100 L, such as 1000 L, such as 10.000 L, such as 100.000 L, such as 200.000 L of culture broth. Typically, a "manufacturing scale" process is defined as one that is able to process large volumes of HMO products, e.g., in the case of therapeutic compounds or compositions, to meet the needs of toxicity testing, clinical trials and market supply. In addition to the large volumes, unlike simple laboratory scale methods such as shake flask cultures, manufacturing scale methods are characterized by technological systems using bioreactors (fermenters) equipped with stirring, aeration, nutrient feeding, monitoring and control devices for process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.). To a large extent, the behavior of the expression system in laboratory scale methods, such as shake flasks, benchtop bioreactors or the deep well format described in the examples of the present disclosure, does allow to predict the behavior of the system in the complex environment of the bioreactor.

[0335] There are no restrictions on suitable cell culture media used in the fermentation process. The culture medium can be semi-defined, i.e., containing complex culture media compounds (e.g., yeast extract, soy peptone, casamino acids, etc.), or can be chemically defined without any complex compounds. The carbon source can be selected from glucose, sucrose, fructose, xylose and glycerol. In one or more exemplary embodiments, the culture medium is supplemented with one or more energy and carbon sources selected from glycerol, sucrose and glucose. In other embodiments, lactose is added during the cultivation of genetically engineered cells as a substrate for HMO formation.

[0336] 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.

[0337] In one or more exemplary embodiments, the genetically engineered cell comprises a PTS-dependent sucrose utilization system further comprising the scrYA and scrBR operons described in WO2015 / 197082 (incorporated herein by reference).

[0338] 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.

[0339] The present invention also relates to a method for producing one or more fucosylated products, the method comprising:

[0340] a) providing a purified fucosyltransferase Med1 having 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 to SEQ ID NO: 1,

[0341] b) reacting the purified fucosyltransferase obtained in a) with

[0342] a. a substrate, such as an oligosaccharide or a glycosylated polypeptide, and

[0343] b. a fucose donor, such as GDP-fucose, and

[0344] c) recovering one or more fucosylation products from the reaction in b).

[0345] Recycling / Harvesting

[0346] Recovering fucosylated human milk oligosaccharides (HMO) from culture medium and / or genetically engineered cells. In the context, the term "recovery" is used interchangeably with the term "harvest". Both "recovery" and "harvesting" in the context refer to collecting the produced HMO from the culture / fermentation broth after the fermentation is terminated. In one or more exemplary embodiments, it may include collecting the HMO contained in the biomass (i.e., host cells) and the culture medium, i.e., before / without separation of the fermentation broth from the biomass. In other embodiments, the produced HMO may be collected separately from the biomass and the fermentation broth, i.e., after / subsequently separation of the biomass from the culture medium (i.e., the fermentation broth).

[0347] The separation of cells from the culture medium can be performed by any of the methods known to those skilled in the art, such as any suitable type of centrifugation or filtration. The separation of cells from the culture medium can be performed immediately after harvesting the fermentation broth, or can be performed at a later stage when the fermentation broth is stored under appropriate conditions. Recovering the produced HMO from the remaining biomass (or the total fermentation broth) includes extracting the HMO from the biomass (i.e., the production cells).

[0348] After recovery from fermentation, HMOs can be used for further processing and purification.

[0349] HMO can be purified according to methods known in the art, such as described in WO2017 / 182965. Purified HMO can be used as a nutraceutical, a pharmaceutical, or for any other purpose, such as for research.

[0350] At the end of the culture, oligosaccharides as products can be accumulated within cells and in the extracellular matrix.

[0351] The method according to the invention comprises culturing genetically engineered microbial cells in a culture medium that is intended to support the growth of the microorganism and that contains 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.

[0352] Manufactured products

[0353] The term "manufactured product" refers to one or more HMOs intended to be one or more product HMOs, depending on the use of the genetically engineered cell or nucleic acid construct. The various products are described above.

[0354] As can be seen from the data disclosed in Examples 1-5, Med1 fucosyltransferase is able to produce LNFP-II, LNFP-III, and LNDFH-I with little or no byproduct formation (e.g., LNFP-V, LNFP-VI, LNDFH-II, or LNDFH-III), highlighting the ability and suitability of Med1 for the production of specific fucosylated HMOs in large-scale manufacturing.

[0355] Advantageously, the methods disclosed herein both reduce the ratio of byproducts to products and increase the overall yield of products (and / or total HMOs). The low byproduct formation relative to product formation facilitates increased product yields and improves the efficiency of the production and product recovery processes, thereby providing a superior HMO manufacturing process.

[0356] The manufactured product may be a powder, composition, suspension or gel comprising one or more HMOs.

[0357] HMO mixtures and compositions thereof

[0358] Complex fucosylated HMOs, such as LNFP-II, LNFP-III and LNDFH-I and mixtures comprising LNFP-II, LNFP-III and / or LNDFH-I, are highly relevant as nutritional supplements or as therapeutic agents.

[0359] One aspect of the present disclosure is a manufactured product or composition comprising a HMO mixture consisting essentially of:

[0360] a) LNFP-II, pLNH2 and LNT, or

[0361] b) LNFP-II, LNT and 3FL, or

[0362] c) LNFP-II, LNT, 3FL and pLNH2, or

[0363] d) LNT-II, LNFP-II, LNT, 3FL, and pLNH2, or

[0364] e) LNFP-III and LNnT, or

[0365] f) LNFP-III, LNnT and pLNnH, or

[0366] g) LNFP-III and 3FL, or

[0367] h) LNDFH-I, LNFP-I and 2'FL.

[0368] As shown in Examples 1 and 2, copy number variation can be used in production to tailor specific HMO mixtures, such as those described above, depending on the demand for a particular product.

[0369] Thus, in an embodiment, the HMO mixture according to the invention consists essentially of 5-35% LNFP-II, 50-85% LNT, 0-15% LNT-II, 0-20% 3FL and 0-5% pLNH2, the total HMO molar contents in the mixture adding up to 100%.

[0370] In other embodiments, the HMO mixture according to the present invention consists essentially of 50-90% LNFP-III, 0-40% LNnT, 0-20% 3FL, 0-8% pLNnH, and the total HMO molar content in the mixture adds up to 100%.

[0371] In a further embodiment, the HMO mixture according to the invention consists essentially of 65-75% LNFP-I, 7-15% LNDFH-I and 5-20% 2'FL, the total HMO molar contents in the mixture adding up to 100%.

[0372] The HMO mixture may also form part of a composition comprising additional moieties, such as active pharmaceutical ingredients, food supplements, excipients, surfactants and the like.

[0373] Therefore, the present invention also relates to compositions comprising HMOs having different amounts of a specific HMO.

[0374] In this regard, in one embodiment, the composition of the present invention relates to a composition comprising a mixture of HMOs, wherein the composition comprises 5-35% of LNFP-II, 50-85% of LNT, 0-15% of LNT-II, 0-20% of 3FL and 0-5% of pLNH2, totaling 100% molar content, based on the total HMO molar content in the composition. A preferred embodiment is that the mixture consists essentially of 15-20% of LNFP-II and 80-85% of LNnT, totaling 100% molar content, based on the total HMO molar content in the composition. Another preferred embodiment is that the mixture consists essentially of 25-35% of LNFP-II, 50-60% of LNnT and 15-25% of 3FL, totaling 100% molar content, based on the total HMO molar content in the composition.

[0375] In another embodiment, the composition of the present invention is a composition comprising a HMO mixture, wherein the composition comprises 50-90% LNFP-III, 0-40% LNnT, 0-20% 3FL, 0-8% pLNnH, totaling 100% molar content, based on the total HMO molar content in the composition.

[0376] In another embodiment, the composition of the present invention is a composition comprising a mixture of HMOs, wherein the composition comprises 65-75% of LNFP-I, 7-15% of LNDFH-I and 5-20% of 2'FL, which add up to a total of 100% molar content of the total HMO molar content in the composition. A preferred embodiment is a mixture consisting essentially of, for example, 75% of LNFP-I, 10% of LNDFH-I and 15% of 2'FL, which add up to a total of 100% molar content.

[0377] Therefore, the present invention also relates to the use of a composition comprising a mixture of HMOs, wherein the composition comprises LNFP-II, pLNH2 and LNT, LNFP-II, LNT and 3FL, LNFP-II, LNnT, 3FL and pLNH2, or LNT-II, LNFP-II, LNnT, 3FL and pLNH2, and wherein the composition is substantially free of LNFP-V and contains less than 5% LNDFH-II in an infant formula, dietary supplement or medical nutritional product.

[0378] Furthermore, the present invention relates to the use of a composition comprising a mixture of HMOs, wherein the composition comprises or consists essentially of a) LNFP-II and LNnT, LNFP-III and 3FL, b) LNFP-III, LNnT and pLNnH or c) LNFP-III, LNnT and 3FL, and wherein the composition is substantially free of LNFP-VI and contains less than 5% LNDFH-III in an infant formula, dietary supplement or medical nutritional product.

[0379] Furthermore, the present invention relates to the use of a composition comprising a mixture of HMOs, wherein the composition comprises or consists essentially of LNFP-I, 2'FL and LNDFH-I, and wherein the composition is substantially free of LNFP-V and contains less than 5% LNDFH-II in an infant formula, dietary supplement or medical nutritional product.

[0380] In an embodiment, the composition comprising the HMO mixture is a pharmaceutical composition.

[0381] Uses of HMO mixtures and compositions

[0382] Clinical data from infants suggests that human milk oligosaccharide supplementation may help develop a desired microbiota by serving as a food source for beneficial bacteria in the gut. HMOs are naturally present in human milk and have evolved over thousands of years, and existing research (clinical and preclinical) on HMOs suggests that supplementation with appropriate levels of specific HMOs may provide us with unique health benefits. In particular, human milk oligosaccharide supplementation may help support immunity and gut health, including supporting a balanced microbiota, with a potential role in cognitive development, which may open the door to future innovation opportunities.

[0383] Therefore, in an embodiment, the present invention relates to the use of a mixture or a composition according to the invention in infant nutrition.

[0384] The invention also relates to the use of the mixture or composition according to the invention as a dietary supplement or medical nutrition.

[0385] The HMO mixture or composition produced according to the methods described herein can be used to enhance the beneficial bacteria in the intestinal microbiota. Beneficial bacteria are, for example, bacteria of the genus Bifidobacterium, Lactobacillus or Barnesiella sp. The enhancement of beneficial bacteria in turn may lead to increased production of short-chain fatty acids (SCFAs), such as acetate, propionate and butyrate, which have been shown to have many benefits for infants and young children. These benefits are, 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).

[0386] The HMO mixtures or compositions produced according to the methods described herein can be used to reduce the number of harmful viruses and bacteria in the intestinal microbiota. Examples of pathogenic bacteria and viruses that can be reduced by the HMO mixtures described herein include Candida albicans, Clostridium difficile, Enterococcus faecium, Escherichia coli, Helicobacter pylori, Streptococcus agalactiae, Shigella dysenteriae, Staphylococcus aureus, nora virus, and rotavirus. Each mixture or composition described herein can also be used to treat and / or reduce the risk of infection of humans with a variety of bacteria.

[0387] The HMO mixture or composition produced according to the method described herein can be used to improve the regeneration and vitality of freeze-dried probiotics (including probiotics of the genus Bifidobacterium and / or Lactobacillus), especially in acidic environments (such as the stomach or acidic foods). Improving regeneration and vitality as well as extending the shelf life are advantages of using the HMO mixture described herein. Examples of Bifidobacterium that may have increased regeneration and vitality are Bifidobacterium animals lactis BB12 DSM 32269, Bifidobacterium animals lactis BIF6, Bifidobacterium longum DSM 32946, Bifidobacterium longum BB536, Bifidobacterium bifidum DSMZ 32403, Bifidobacterium infantis, Bifidobacterium breve DSM 33789, Bifidobacterium infantis SP37 DSM 32687, Bifidobacterium adolescentis DSM 34065 and / or Bifidobacterium animalis subsp. animalis DSM 16284. Examples of Lactobacillus species that may have increased regeneration and viability are Lactobacillus rhamnosus GG DSM 32550, Lactobacillus rhamnosus 19070-2 DSM 26357, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus LBrGG, Lactobacillus reuteri DSM 12246, Lactobacillus plantarum TIFN101, Lactobacillus gasseri Lg-36200B 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 Lactobacillus reuteri S12 DSM 33752.

[0388] In the context of this application, "regeneration" refers to the process of regaining / restoring the vitality of dried bacteria (i.e. "reviving" bacterial cells by rehydration, where "rehydration" means restoring liquid). This process is sometimes also called "reconstitution".

[0389] In the context of this application, "viability" is the ability of bacterial cells to survive and function as living cells. One method of determining bacterial cell viability is to plate them on agar plates with a suitable growth medium and count the number of colonies formed after incubation for a predetermined time (plate count). Alternatively, FACS analysis can be used.

[0390] In the context of the present application, "improved regeneration" of Bifidobacterium and / or Lactobacillus bacteria means an increase in the number (number) of successfully regenerated / resuscitated Bifidobacterium and / or Lactobacillus bacteria compared to a corresponding control (i.e. the number / number of Bifidobacterium and / or Lactobacillus bacteria without the addition of HMO).

[0391] In the context of the present application, "improved viability" of Bifidobacterium and / or Lactobacillus bacteria means an increase in the number (number) of Bifidobacterium and / or Lactobacillus bacteria compared to a corresponding control (i.e. the number / number of Bifidobacterium and / or Lactobacillus bacteria without addition of HMO).

[0392] In the context of this application, "acidic" means a pH value below 7.0 (e.g., a pH value ≤ 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 value measured in the stomach is in the range of about 1.5-3.5. The pH value measured in a healthy vagina is in the range of about 3.8-5.0. The pH value of fruit juice is in the range of about 2.0-4.5.

[0393] One embodiment of the invention is a composition comprising a HMO mixture as described herein (particularly in the "HMO mixture" section) and one or more probiotics. Preferably, the probiotics are of the genus Bifidobacterium and / or Lactobacillus, such as any of the specific species mentioned above.

[0394] The HMO mixture or composition produced according to the methods described herein can be used to extend the shelf life of probiotics (eg, Bifidobacterium and / or Lactobacillus).

[0395] The HMO mixtures or compositions produced according to the methods described herein can be used to improve the flow properties of powders or to reduce the viscosity of liquids.

[0396] The HMO mixture or composition produced according to the methods described herein is used in nutritional compositions. The nutritional composition is, for example, infant formula, rehydration, or dietary maintenance, medical nutrition or supplements for the elderly or immunocompromised population. Such anti-infective compositions may also include macronutrients such as edible fats, carbohydrates and proteins. Edible fats include, for example, coconut oil, soybean oil, and mono- and diglycerides. Carbohydrates include, for example, glucose, edible lactose and hydrolyzed corn starch. Proteins include, for example, soy protein, whey and skim milk. Such anti-infective compositions may also include vitamins and minerals (e.g., calcium, phosphorus, potassium, sodium, chlorine, magnesium, manganese, iron, copper, zinc, selenium, iodine and vitamins A, E, D, C and B complex).

[0397] sequence

[0398] This application contains a Sequence Listing in both text and electronic formats, which is incorporated herein by reference.

[0399] An overview of the SEQ ID NOs used in this application can be found in Table 4 (Promoter sequences). Additional sequences described in this application are the amino acid sequence of α-1,3-fucosyltransferase Med1 (SEQ ID NO: 1) and the DNA sequence encoding α-1,3-fucosyltransferase Med1 (SEQ ID NO: 2), the DNA sequence encoding the colanic acid gene cluster from Escherichia coli (SEQ ID NO: 3), the amino acid sequence of the lactose permease LacY from Escherichia coli (SEQ ID NO: 6), the amino acid sequence of the β-1,3-N-acetylglucosaminyltransferase LgtA from Neisseria meningitidis (SEQ ID NO: 4), the amino acid sequence of the β-1,4-galactosyltransferase galT from Helicobacter pylori (SEQ ID NO: 5), the amino acid sequence of the β-1,3-galactosyltransferase galTK from Helicobacter pylori (SEQ ID NO: 6). NO:31), the amino acid sequence of α-1,2-fucosyltransferase Smob from Aspergillus motileus (SEQ ID NO:32), the amino acid sequence of α-1,3-fucosyltransferase Para1 from Parabacteroides AM08-6 (SEQ ID NO:33) and the DNA sequence encoding α-1,3-fucosyltransferase Para1 from Parabacteroides AM08-6 (SEQ ID NO:34), the amino acid sequence of α-1,3-fucosyltransferase FutB from Helicobacter pylori (SEQ ID NO:35), the nucleic acid sequence encoding α-1,3-fucosyltransferase FutB from Helicobacter pylori (SEQ ID NO:36), the amino acid sequence of α-1,3-fucosyltransferase CafD from Helicobacter pylori ATCC 51449 (SEQ ID NO:37), the amino acid sequence of α-1,3-fucosyltransferase CafD from Bacteroides fragilis NCTC The amino acid sequence of the α-1,3-fucosyltransferase FucT109 from Helicobacter pylori ATCC51449 (SEQ ID NO:38), the nucleic acid sequence encoding the α-1,3-fucosyltransferase CafD from Helicobacter pylori ATCC51449 (SEQ ID NO:39), and the nucleic acid sequence encoding the α-1,3-fucosyltransferase FucT109 from Bacteroides fragilis NCTC 9343 (SEQ ID NO:40).

[0400] project

[0401] 1. A genetically engineered cell capable of producing one or more fucosylated HMOs, comprising a recombinant nucleic acid sequence encoding an enzyme having α-1,3(4)-fucosyltransferase activity, wherein the enzyme is Med1 having an amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof having an amino acid sequence having at least 80% identity to SEQ ID NO: 1.

[0402] 2. The genetically engineered cell according to item 1, wherein the fucosylated HMO is selected from complex fucosylated HMOs comprising at least five monosaccharide units.

[0403] 3. The genetically engineered cell according to claim 1 or 2, wherein the fucosylated HMO is selected from fucosylated HMOs having a LNT and / or LNnT backbone structure.

[0404] 4. The genetically engineered cell according to item 1 or 2, wherein the fucosylated HMO is LNFP-III.

[0405] 5. The genetically engineered cell according to item 1 or 2, wherein the fucosylated HMO is LNFP-II and / or LNDFH-I.

[0406] 6. The genetic engineering according to items 1 to 5, wherein the α-1,3(4)-fucosyltransferase fucosylate only the N-acetylglucosamine (GlcNAc) moiety in the acceptor oligosaccharide.

[0407] 7. The genetic engineering according to item 6, wherein the acceptor oligosaccharide of the α-1,3(4)-fucosyltransferase has a LNT or LNnT backbone.

[0408] 8. The genetic engineering according to item 6 or 7, wherein the acceptor oligosaccharide of the α-1,3(4)-fucosyltransferase is LNT or LNnT.

[0409] 9. The genetically engineered cell according to any one of the preceding items, wherein the α-1,3(4)-fucosyltransferase does not fucosylate glucose and galactose moieties in acceptor oligosaccharides, particularly in acceptor oligosaccharides having a LNT or LNnT backbone.

[0410] 10. The genetically engineered cell according to any one of the preceding items, wherein the genetically engineered cell comprises one or more recombinant nucleic acids encoding one or more heterologous glycosyltransferases.

[0411] 11. The genetically engineered cell according to any one of the preceding items, wherein the cell further comprises a recombinant nucleic acid sequence encoding β-1,3-galactosyltransferase and / or β-1,4-galactosyltransferase.

[0412] 12. The genetically engineered cell according to item 10 or 11, wherein the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosaminyltransferase.

[0413] 13. The genetically engineered cell according to item 11 or 12, wherein the β-1,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis and the β-1,3-galactosyltransferase is from Helicobacter pylori.

[0414] 14. The genetically engineered cell according to items 10 to 13, wherein the 1,3-N-acetylglucosaminyltransferase has 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 the β-1,3-galactosyltransferase has an amino acid sequence according to SEQ ID NO: 31, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 31.

[0415] 15. The genetically engineered cell according to any one of items 11 to 13, wherein the 1,3-N-acetylglucosaminyltransferase has 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 the β-1,4-galactosyltransferase 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.

[0416] 16. The genetically engineered cell according to any one of the preceding items, wherein the cell further comprises a recombinant nucleic acid sequence encoding an α-1,2-fucosyltransferase.

[0417] 17. The genetically engineered cell according to item 16, wherein the α-1,2-fucosyltransferase is derived from Aspergillus motileus.

[0418] 18. The genetically engineered cell according to item 15 or 10, wherein the α-1,2-fucosyltransferase has an amino acid sequence according to SEQ ID NO: 32, or a functional homologue thereof, the amino acid sequence of which has at least 80% identity to SEQ ID NO: 32.

[0419] 19. A genetically engineered cell according to any one of the preceding items, wherein the recombinant nucleic acid sequence is individually controlled by a promoter selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 19, 28, 16, 17 and 18, respectively) and variants thereof.

[0420] 20. The genetically engineered cell according to item 19, wherein the promoter is a strong promoter selected from SEQ ID NO 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19.

[0421] 21. 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 Med1 or a functional homologue thereof, wherein the glycosyltransferase has an amino acid sequence according to SEQ ID NO: 1, and the amino acid sequence of the functional homologue has at least 80% identity with SEQ ID NO: 1.

[0422] 22. The genetically engineered cell according to any of the preceding items, wherein the recombinant nucleic acid sequence encodes a glycosyltransferase Med1 having 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 to SEQ ID NO: 1, and the recombinant nucleic acid sequence is encoded on a plasmid.

[0423] 23. The genetically engineered cell according to item 22, wherein the plasmid is a high copy number plasmid.

[0424] 24. The genetically engineered cell according to item 22 or item 23, wherein the plasmid is a pUC57 plasmid.

[0425] 25. 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 Med1 or a functional homologue thereof, wherein the glycosyltransferase has an amino acid sequence according to SEQ ID NO: 1, and the amino acid sequence of the functional homologue has at least 80% identity with SEQ ID NO: 1.

[0426] 26. The genetically engineered cell according to any of the preceding items, wherein the cell overexpresses at least one enzyme in the de novo GDP-fucose pathway that is responsible for GDP-fucose formation.

[0427] 27. The genetically engineered cell according to item 26, wherein the one or more enzymes are selected from mannose-6-phosphate isomerase (manA), phosphomannose mutase (manB), mannose-1-phosphate guanylate transferase (manC), GDP-mannose-4,6-dehydratase (gmd) and GDP-L-fucose synthase (wcaG).

[0428] 28. The genetically engineered cell according to any of the preceding items, wherein the cell comprises a recombinant nucleic acid sequence according to SEQ ID NO: 3 encoding the colanic acid (CA) gene cluster.

[0429] 29. The genetically engineered cell according to any one of the preceding items, wherein the engineered cell is a microorganism.

[0430] 30. The genetically engineered cell according to any one of the preceding items, wherein the engineered cell is a bacterium or a fungus.

[0431] 31. The genetically engineered cell according to item 30, wherein the fungus is selected from yeast cells of the genera Komagata, Kluyveromyces, Yarrowia, Pichia, Saccharomyces, Schizosaccharomyces or Hansenula, or a filamentous fungus selected from the genera Aspergillus, Fusarium or Trichoderma.

[0432] 32. The genetically engineered cell according to item 30, wherein the bacterium is selected from the genera Escherichia coli, Bacillus, Lactobacillus, Corynebacterium and Campylobacter.

[0433] 33. The genetically engineered cell according to any one of items 29 to 32, wherein the engineered cell is selected from Escherichia coli, Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris and Saccharomyces cerevisiae.

[0434] 34. The genetically engineered cell according to item 32 or 33, wherein the engineered cell is an Escherichia coli microorganism.

[0435] 35. The genetically engineered cell according to any of the preceding items, wherein at least 5% of the total molar content of HMO produced by the cell is complexly fucosylated HMO.

[0436] 36. The genetically engineered cell of any one of items 1 to 15 or 19 to 35, wherein at least 5% of the total molar content of HMOs produced by the cell is LNFP-II.

[0437] 37. The genetically engineered cell of any one of items 1 to 15 or 19 to 35, wherein at least 50% of the total molar content of HMOs produced by the cell is LNFP-III.

[0438] 38. A genetically engineered cell according to any one of items 1 to 35, wherein at least 9% of the total HMO molar content is LNDFH-I.

[0439] 39. The genetically engineered cell according to any of the preceding items, wherein the cell produces a HMO mixture comprising LNFP-II and LNT or LNFP-III and LNnT or LNFP-I and LNDFH-I.

[0440] 40. The genetically engineered cell according to any of the preceding items, wherein the cell produces a HMO mixture consisting of:

[0441] a) LNFP-II and LNT, or

[0442] b) LNFP-II and 3FL, or

[0443] c) LNFP-II, LNT and 3FL, or

[0444] d) LNFP-II, LNT, 3FL and pLNH2, or

[0445] e) LNFP-III and LNnT, or

[0446] f) LNFP-III, LNnT and pLNnH, or

[0447] g) LNFP-III and 3FL, or

[0448] h) LNDFH-I, LNFP-I and 2'FL.

[0449] 41. A method of producing one or more fucosylated HMOs, the method comprising culturing the genetically engineered cell according to any one of items to 40.

[0450] 42. The method according to item 41, wherein the method comprises culturing the genetically engineered cells in the presence of an energy source selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.

[0451] 43. The method according to any one of items 41 to 42, wherein lactose is added as a substrate for HMO formation during the cultivation of the genetically engineered cells.

[0452] 44. The method according to items 41 to 43, wherein the fucosylated HMO is selected from LNFP-II, LNFP-III and LNDFH-I.

[0453] 45. The process according to items 41 to 44, wherein the process produces a HMO mixture, and wherein the mixture is optionally purified to a purity of at least 75%.

[0454] 46. ​​The method according to items 41 to 44, wherein the fucosylated HMO produced by the method is purified to a purity of at least 75%.

[0455] 47. The method according to items 41 to 46, wherein one of the fucosylated HMOs is selected from LNFP-II, LNFP-III and LNDFH-I and is purified to a purity of at least 75%.

[0456] 48. Use of an enzyme having α-1,3(4)-fucosyltransferase activity in producing a fucosylated product, wherein the enzyme is Med1 having an amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.

[0457] 49. Use of the enzyme according to item 48, wherein the fucosylation product is one or more fucosylated oligosaccharides.

[0458] 50. Use of an enzyme according to any one of items 48 or 49, wherein the fucosylation product is one or more fucosylated HMOs.

[0459] 51. Use of the enzyme according to item 50, wherein the fucosylation product is LNFP-II or LNFP-III or LNDFH-I.

[0460] 52. Use of the enzyme according to item 48, wherein the fucosylation product is one or more fucosylated polypeptides.

[0461] 53. An HMO mixture consisting essentially of

[0462] a) LNFP-II, pLNH2 and LNT, or

[0463] b) LNFP-II, LNT, 3FL and pLNH2, or

[0464] c) LNT-II, LNFP-II, LNT, 3FL, and pLNH2, or

[0465] d) LNFP-III and LNnT, or

[0466] e) LNFP-III, LNnT and pLNnH, or

[0467] f) LNFP-III and 3FL or

[0468] g) LNDFH-I, LNFP-I and 2'FL.

[0469] 54. The HMO mixture according to claim 53, which consists essentially of 5-35% LNFP-II, 50-85% LNT, 0-15% LNT-II, 0-20% 3FL and 0-5% pLNH2, the total HMO molar content in the mixture adding up to 100%.

[0470] 55. The HMO mixture according to item 53, consisting essentially of 50-90% LNFP-III, 0-40% LNnT, 0-20% 3FL and 0-8% pLNnH, the total HMO molar contents in the mixture adding up to 100%.

[0471] 56. The HMO mixture according to item 53, consisting essentially of 65-75% LNFP-I, 7-15% LNDFH-I and 5-20% 2'FL, the total HMO molar contents in the mixture adding up to 100%.

[0472] 57. A composition comprising a mixture of HMOs, wherein the composition comprises 5-35% LNFP-II, 50-85% LNT, 0-15% LNT-II, 0-20% 3FL and 0-5% pLNH2, based on the total HMO molar content in the composition.

[0473] 58. A composition comprising a mixture of HMOs, wherein the composition comprises 50-90% LNFP-III, 0-40% LNnT, 0-20% 3FL and 0-8% pLNnH, based on the total HMO molar content in the composition.

[0474] 59. A composition comprising a mixture of HMOs, wherein the composition comprises 65-75% LNFP-I and 7-15% LNDFH-I and 5-20% 2'FL based on the total HMO molar content in the composition.

[0475] 60. Use of the mixture or composition according to any one of items 53 to 59 as a dietary supplement or medical nutrition.

[0476] 61. Use of a mixture or composition according to any one of items 53 to 59 in infant nutrition.

[0477] 62. A method for producing a fucosylated product, the method comprising,

[0478] a. providing a purified fucosyltransferase Med1 having an amino acid sequence according to SEQ ID NO: 1, or a functional homolog thereof, the amino acid sequence of the functional homolog having at least 80% identity with SEQ ID NO: 1,

[0479] b. The purified fucosyltransferase of a) is reacted with the following

[0480] i. a substrate, such as an oligosaccharide or a glycosylated polypeptide, and

[0481] ii. a fucose donor, such as GDP-fucose, and

[0482] c. Recovering one or more fucosylated products from the reaction in b), and optionally purifying said products.

[0483] 63. The method according to item 62, wherein the fucosylated product is purified to a purity of at least 75%.

[0484] Example

[0485] method

[0486] Unless otherwise indicated, nucleic acid manipulation, conversion and expression are performed using standard techniques, vectors, control sequence elements and other expression system elements known in the field of molecular biology. Such standard techniques, vectors and elements can be found in the following literature: Ausubel et al. (write), Current Protocols in Molecular Biology (1995) (John Wiley & Sons); Sambrook, Fritsch, & Maniatis (write), Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, NY); Berger & Kimmel, Methods in Enzymology 152: Guide to Molecular Cloning Techniques (1987) (Academic Press); Bukhari et al. (write), DNA Insertion Elements, Plasmids and Episomes (1977) (Cold Spring Harbor Laboratory Press, NY); Miller, JH Experiments in molecular genetics (1972.) (Cold Spring Harbor Laboratory Press, NY).

[0487] The embodiments described below are chosen to illustrate the present invention and are not intended to limit the present invention in any way.

[0488] Enzymes:

[0489] Screening of 50 enzymes with fucosyltransferase activity resulted in a single enzyme, Med1, which was able to fucosylate only the GlcNAc moiety in LNT and LNnT, meaning that it had very specific α-1,4-fucosyltransferase activity and α-1,3-fucosyltransferase activity (also written as α-1,3(4)-fucosyltransferase activity). Another enzyme, para1, was identified that had α-1,4-fucosyltransferase activity on the GlcNAc moiety in LNT. However, the Para1 enzyme also had α-1,3-fucosyltransferase activity on the glucose moiety of LNT. Therefore, both enzymes have α-1,3(4)-fucosyltransferase activity and are able to produce the complex HMO LNFP-II when introduced into a strain with an LNT background. The GenBank IDs and sources of two fucosyltransferases, Med1 and Para1, are listed in Table 5 along with three prior art enzymes, and an additional 45 enzymes tested are not shown because they were unable to fucosylate the GlcNAc moiety of LNT.

[0490] Table 5. List of enzymes showing results within the framework of the present invention

[0491] Enzyme name GenBank ID* SEQ ID NO source Med1 WP_087337236.1 1 Mediterranean bacteria An20 Para1 WP_147381213.1 33 Parabacteroides AM08-6 FutB** WP_000487430.1 35 Helicobacter pylori CafD*** AAP76669.1 37 Helicobacter hepatica ATCC 51449 FucT109 CAH09151.1 38 Bacteroides fragilis ATCC 25285

[0492] *Compared to the GenBank sequences, the sequences used in this application may be truncated at the N-terminus or C-terminus, and these sequences are indicated by SEQ ID NO.

[0493] **FutB is known to produce a mixture of LNFP-III and LNFP-VI (Dumon et al. 2004 Biotechnol. Prog. 20:412-419).

[0494] ***The use of CafD for the production of LNFP-III was suggested in WO2016 / 040531.

[0495] ^The use of FucT109 for the production of LNFP-III was suggested in WO2019 / 008133.

[0496] strain

[0497] The strain (genetically engineered cell) constructed in this application is based on Escherichia coli K-12DH1, and its genotype is: Fˉ, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. The E. coli K-12DH1 strain was additionally modified to generate the MDO strain with the following modifications: lacZ: 1.5 kbp deletion, lacA: 0.5 kbp deletion, nanKETA: 3.3 kbp deletion, melA: 0.9 kbp deletion, wcaJ: 0.5 kbp deletion, mdoH: 0.5 kbp deletion, and the Plac promoter was inserted upstream of the gmd gene. Methods for inserting a gene of interest into the E. coli genome are well known to those skilled in the art. Gene gorging can be used to insert the gene cassette into the E. coli chromosome using specific selection marker genes and screening methods (see, for example, Herring and Blattner 2004 J. Bacteriol. 186:2673-81 and Warming et al. 2005 Nucleic Acids Res. 33(4):e36).

[0498] To obtain an LNT-producing strain, the MDO strain was further modified by integrating β-1,3-GlcNAc transferase (LgtA from Neisseria meningitidis, homologous to NCBI Accession No. WP_033911473.1, and as shown in SEQ ID NO:4) and β-1,3-galactosyltransferase (GalTK from Helicobacter pylori, homologous to GenBank Accession No. BD182026.1, and as shown in SEQ ID NO:31) both under the control of the PglpF promoter (SEQ ID NO:19) on the chromosome, and the strain was named LNT strain.

[0499] To obtain the LNnT producing strain, the MDO strain was further modified by integrating β-1,3-GlcNAc transferase (LgtA from Neisseria meningitidis, homologous to NCBI Accession No. WP_033911473.1 and shown in SEQ ID NO:4) and β-1,4-galactosyltransferase (GalT from Helicobacter pylori, homologous to GenBank ID WP_001262061.1 and shown in SEQ ID NO:5) both controlled by the PglpF promoter (SEQ ID NO:19) on the chromosome, and the strain was named LNnT strain.

[0500] Codon-optimized DNA sequences encoding med1 or para1 were genomically integrated into the LNT strain. In addition, med1 was integrated into the LNnT strain and the LNFP-I strain.

[0501] Table 6 provides the genotypes of the background strain (MDO), LNT strain, LNnT strain, and α-1,3(4)-fucosyltransferase expressing strain capable of producing LNFP-II and / or LNDFH-I.

[0502] Table 6. Genotypes of strains capable of producing LNFP-II, LNFP-III and / or LNDFH-I used in this example.

[0503]

[0504] *1,3(4)FT is the abbreviation for α-1,3(4)-fucosyltransferase.

[0505] 1 lgtA-PglpF - two genomically inserted copies of a gene encoding β-1,3-N-acetylglucosaminyltransferase (SEQ ID NO:4) under the control of the PglpF promoter (SEQ ID NO:19).

[0506] 2 galTK-PglpF - a genomic insertion of a gene encoding β-1,3-galactosyltransferase (SEQ ID NO:31) under the control of the PglpF promoter (SEQ ID NO:19).

[0507] 3 galT-PglpF - a genomic insertion of a gene encoding β-1,4-galactosyltransferase (SEQ ID NO:5) under the control of the PglpF promoter (SEQ ID NO:19).

[0508] 4 CA = additional colanic acid gene cluster (gmd-wcaG-wcaH-wcaI-manC-manB, SEQ ID NO: 3) located at a site different from the native site and controlled by the PglpF promoter (SEQ ID NO: 19).

[0509] 5 Para1-PglpF - a genomic inserted gene encoding α-1,3(4)-fucosyltransferase (SEQ ID NO:33) under the control of the PglpF promoter (SEQ ID NO:19).

[0510] 6med1-PglpF – Gene encoding α-1,3(4)-fucosyltransferase (SEQ ID NO:2) controlled by the PglpF promoter (SEQ ID NO:19). One (1x), two (2x), or three (3x) copies inserted into the genome or expressed from plasmid pUC57, a high copy number (>300) plasmid with the ColE1 / pMB1 / pBR322 / pUC origin of replication. The antibiotic resistance marker on the pUC57 vector is ampicillin.

[0511] 7 Smob-PglpF is a gene encoding α-1,2-fucosyltransferase (SEQ ID NO: 32) inserted into the genome and controlled by the PglpF promoter.

[0512] Deep well assay

[0513] The deep well test in this example was performed in the manner originally described by Lv et al. (Bioprocess Biosyst Eng 20 (2016) 39: 1737-1747) and optimized for the purposes of the present invention. More specifically, the strains disclosed in this example were screened in 96 deep well plates using a 4-day protocol. Within the first 24 hours, the preculture was grown to high density (OD600 up to 5) and then transferred to a medium that allowed induction of gene expression and product formation.

[0514] More specifically, on day 1, a fresh preculture was prepared using basal minimal medium (BMM) (pH 7.0) supplemented with magnesium sulfate (0.12 g / L), thiamine (0.004 g / L) and glucose (5.5 g / L). The basal minimal medium had the following composition: NaOH (1 g / L), KOH (2.5 g / L), KHzPO4 (7 g / L), NH4H4PO4 (7 g / L), citric acid (0.5 g / L), trace mineral solution (5 mL / L). The trace mineral stock solution contained: ZnSO4*7H4O 0.82 g / L, citric acid 20 g / L, Mn4*H4O 0.98 g / L, FeSO4*7H4O 3.925 g / L, CuSO4*5H4O 0.2 g / L. The pH of the basal minimal medium was adjusted to 7.0 with 5N NaOH and then sterilized by autoclave. The pre-culture was incubated at 34°C and 1000 rpm shaking for 24 hours and then further transferred to 0.75 mL of new BMM (pH 7,5) to start the main culture. The new BMM was supplemented with magnesium sulfate (0.12 g / L), thiamine (0.02 g / L), glucose solution (0.1-0.15 g / L) and lactose solution (5-20 g / L). In addition, 20% sucrose stock solution (40-45 g / L) or maltodextrin (19-20 g / L) was provided as a carbon source, and specific hydrolases, sucrose hydrolases or glycosidases were added at the same time to release glucose at a rate suitable for carbon-limited growth, and similar to a typical fed-batch fermentation process. The main culture was incubated at 28°C and 1000 rpm shaking for 72 hours. To analyze the total culture medium, the 96-well plate was boiled at 100°C, then centrifuged, and finally the supernatant was analyzed by HPLC to identify the HMO produced by the cells. pLNH2 is not officially reported as an HMO, although it is likely present in human breast milk in small amounts. In this application, it is included in the total HMO calculation.

[0515] Fermentation-Ambr

[0516] E. coli strains were cultured in 250 mL fermenters (Ambr250 HT bioreactor system, Sartorius) with a starting medium of 100 mL mineral medium consisting of 30 g / L glucose and mineral medium consisting of NH4H2PO4, KH2PO4, MgSO4×7H2O, NaOH, citric acid, trace element solution, defoamer and thiamine. The airflow was started at 700 rpm (up to 4500 rpm) and 1 VVM (up to 3 VVM) to maintain the dissolved oxygen level at 20% by a cascade of stirring followed by airflow. The pH was maintained at 6.8 by titration with 8.5% NH4OH solution. The culture was started with a 2% (v / v) inoculum from a pre-culture containing 10 g / L glucose, (NH4)2HPO4, KH2PO4, MgSO4×7H2O, KOH, NaOH, citric acid, trace element solution, defoamer and thiamine. After the glucose contained in the basal minimal medium is exhausted, a feed solution containing glucose is continuously added to the fermenter at a rate to maintain carbon limiting conditions. The temperature was initially 33°C, but dropped to 30°C after 3 hours of feeding. Lactose was added once as a 25% lactose monohydrate solution 36 hours after the start of feeding, and then added once every 19 hours to prevent lactose from becoming a rate-limiting factor. The growth, metabolic activity and metabolic state of the cells were tracked by online measurement of reflectivity and CO2 release rate. Throughout the fermentation process, samples were collected to determine the concentration of HMO products, lactose and other minor by-products using HPLC.

[0517] Fermentation-Dasbox

[0518] E. coli strains were cultured in 100 mL fermenters (Dasbox, Eppendorf) with 100 mL of mineral medium consisting of 30 g / L glucose and mineral medium containing NH4H2PO4, KH2PO4, MgSO4×7H2O, KOH, NaOH, citric acid, trace element solution, defoamer and thiamine. The airflow was started at 700 rpm (up to 2000 rpm) and 1 VVM (up to 3 VVM) to maintain the dissolved oxygen level at 20% by a cascade of stirring followed by airflow. The pH was maintained at 6.8 by titration with 8.5% NH4OH solution. The culture was started with 2% (v / v) inoculum from a pre-culture containing 10 g / L glucose, (NH4)2HPO4, KH2PO4, MgSO4×7H2O, KOH, NaOH, citric acid, trace element solution, defoamer and thiamine. After the glucose contained in the basal minimal medium is exhausted, a feed solution containing glucose, MgSO4×7H2O, H3PO4 and trace mineral solution is continuously added to the fermenter at a rate to maintain carbon limiting conditions. The temperature was initially 33°C, but dropped to 25°C 15 minutes after the start of the feed. Lactose was added once as a 25% lactose monohydrate solution at the beginning of the feed and 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 stirring, dissolved oxygen tension, reflectivity, NH4OH base addition, O2 absorption rate and CO2 release rate. Throughout the fermentation process, samples were collected to determine the concentration of HMO products, lactose and other small by-products using HPLC.

[0519] Example 1 - In vivo LNFP-II synthesis

[0520] In this example, genetically modified cells expressing a single fucosyltransferase were screened for their ability to produce the fucosylated HMO LNFP-II in an LNT strain with an additional colanic acid gene cluster to ensure sufficient GDP-fucose as donor material for the fucosyltransferase.

[0521] Using the deep well assay described in the Methods section, only two tested enzymes, med1 and para1, had α-1,4-fucosyltransferase activity towards the GlcNAc moiety in LNT, an activity required for the production of LNFP-II (Table 5).

[0522] The initial screening of fucosyltransferases was done using a single genomic copy. The genotypes of these strains are shown in Table 6, with strain reference numbers Para1_1 and Med1_1. Based on the results of the initial screening, additional strains with additional copy numbers of the corresponding fucosyltransferases were generated. Table 6 lists the genotypes of strains capable of producing LNFP-II.

[0523] The molar content of individual HMOs produced by the strains was determined by HPLC. The results for the LNFP-II producing cells and three prior art strains are shown in Table 7 as the fraction (percent, %) of the total HMO content produced by each strain.

[0524] Table 7: Percentage (%) of individual HMO content produced by each strain to total HMO content (mM).

[0525]

[0526] *FT = fucosyltransferase.

[0527] No HMOs other than those shown in Table 7 were identified in the deep hole test.

[0528] From the data shown in Table 7 , it can be seen that strains with one copy of Para1 and Med1 are both able to produce a small amount of LNFP-II, while the FutB and FucT109 strains only produce LNFP-V, and the CafD strain does not fucosylate LNT at all.

[0529] There are clear differences in the specificity of Para1 and Med1 fucosyltransferases, where Med1 fucosylates LNT only at the GlcNAc moiety with an α-1,4 bond, thus producing only a single fucosylated HMO with an LNT backbone, specifically LNFP-II, whereas Para1 fucosylates LNT at both the glucose (Glc) moiety and the GlcNAc moiety, thus producing three fucosylated HMOs with an LNT backbone, namely LNFP-II, LNFP-V, and LNDFH-II, respectively.

[0530] Furthermore, increasing the copy number of Med1 and Para1 to two gene copies increased the production of LNFP-II by 10% for Med1, from 5% to 15%, while Para1 only increased the amount of LNFP-II formed by 1%, and the amount of difucosylated LNDFH-II increased from 2% to 11%, which clearly indicated that Para1 had the same preference for fucosylation of the GlcNAc and Glc parts of LNT, while Med1 was only active on the GlcNAc part. The ability to increase LNFP-II formation by further increasing Med1 expression was investigated by introducing a high copy plasmid pUC57-Med1-PglpF (Med1_3) containing the Med1 coding sequence. The results in Table 7 show that an increase in Med1 expression increased LNFP-II levels from 15% to 30%. However, when Med1 was highly overexpressed, it appeared to be able to fucosylate the Glc part on lactose to form 3FL.

[0531] Therefore, copy number variation can be used in production to tailor a specific HMO mixture containing a specific amount of a single HMO, depending on the needs of a specific product. Increasing the copy number of Med1 does not result in the production of any additional fucosylated HMO species with an LNT backbone.

[0532] The absence of alternative fucosylated LNT species is highly advantageous and preferred when using Medl if one wishes to purify LNFP-II from a culture medium that does not contain similarly sized (e.g. LNFP-V) or slightly larger fucosylated HMOs (e.g. LNDFH-II). Thus, high levels of a specific HMO are advantageous when the goal is to produce large amounts of a single HMO.

[0533] Example 2 In vivo LNFP-III synthesis

[0534] Considering the high specificity of Med1 for GlcNAc moieties in Example 1, it was investigated whether this was also applicable with LNnT as the backbone, which requires α-1,3-fucosyltransferase activity (in Example 1, this activity was also observed in high copy numbers with lactose as the substrate, where Med1 produced some 3FL).

[0535] Genetically modified cells expressing Med1 were screened for their ability to produce the fucosylated HMO LNFP-III. The genotype of the Med1 strain generated in this example is shown in Table 6. The strains were screened in a deep well test apparatus as described in the "Methods" section. The molar content of the individual HMOs produced by the strains was determined by HPLC.

[0536] Table 8 shows the results for LNFP-III producing cells, ie the fraction (percent, %) of the total HMO content produced by each strain.

[0537] Table 8: Percentage (%) of the content of individual HMOs produced by each strain to the total HMO (mM) content.

[0538] <![CDATA[FT ＊ Number of copies]]> LNFP-III LqCy HkDJ 3FL LNDFH-III LNFP-VI Cafd_LNnT 1 0 82 18 0 0 0 FutB_LNnT 1 6 79 13 1 0 1 FucT109_LNnT 1 26 29 1 2 24 18 Med1_6 1 52 42 6 0 0 0 Med1_7 2 90 10 0 0 0 0 Med1_8 >50 82 0 0 18 0 0

[0539] *FT = fucosyltransferase.

[0540] No HMOs other than those shown in Table 8 were identified in the deep hole test.

[0541] From the data shown in Table 8, it can be seen that Med1 can effectively transfer the fucosyl unit to the GlcNAc part of LNnT with an α-1,3 bond, thereby producing a high level of LNFP-III. It can also be seen that Med1 does not produce any complex fucosylated HMO (LNFP-VI and LNDFH-III) with fucosyl on the glc part, while FucT109 produces 18% of LNFP-VI, which clearly shows that FucT109 has similar specificity for the GlcNAc and Glc parts of LNnT. Similarly, it was found that FutB with a single copy enzyme produced a small amount of LNFP-VI and more 3-FL, indicating that FutB also has some activity on the terminal glucose (Glc) part of LNnT.

[0542] Prior art enzymes FutB and FucT109 produced only 6% and 26% of total HMOs, respectively, of LNFP-III. CafD, which was claimed to produce LNFP-III in WO2016 / 040531, did not appear to be able to fucosylate LNnT or lactose in this assay, as it did not produce any fucosylated HMOs.

[0543] Furthermore, increasing the copy number of Med1 to two gene copies (strain Med1_7) increased the production of LNFP-III from 52% to 90%. After the introduction of a high-copy plasmid (pUC57-Med1-PglpF-amp) encoding Med1 (strain Med1_8), a slight decrease in LNFP-III levels to 82% was observed, instead of producing 18% 3FL. Given the higher copy number of the Med1 gene (strain Med1_8), all LNnT produced by the cells was fucosylated, and therefore the formation of LNnT by the cells became the rate-limiting step for the production of LNFP-III, which could result in Med1 fucosylating lactose instead of LNnT, thereby further reducing LNnT formation by consuming lactose, leading to a reduction in LNFP-III.

[0544] Therefore, copy number variation can be used in production to tailor specific HMO mixtures, in which case the mixture contains different ratios of LNFP-III, LNnT, and / or 3FL, depending on the needs of a specific product. The data also suggest that if the level of LNFP-III is to be further increased, then the copy number of the enzyme that forms LNnT should be increased along with the Med1 enzyme.

[0545] If production of pure LNFP-III is desired, the absence of alternative similarly sized fucosylated LNnT species (eg LNFP-VI or LNDFH-III) is highly advantageous and preferred when using Med1.

[0546] Example 3 In vivo LNDFH-I synthesis

[0547] In view of the results in Example 1, in which Med1 showed activity only on the GlcNAc portion of LNT, it was investigated whether Med1 was able to produce the complex difucosylated hexasaccharide HMO LNDFH-I using an LNFP-I background strain with an α-1,2-fucosyltransferase Smob and two copies of the Med1 α-1,3(4) fucosyltransferase (Med1_4, Table 6).

[0548] The strains were screened in a deep well test set-up as described in the Methods section. The molar content of individual HMOs produced by the strains was determined by HPLC.

[0549] The results of LNDFH-I production cells are shown in Table 9, i.e., the fraction (percent, %) of the total HMO content produced by the Med1_4 strain.

[0550] Table 9: Percentage (%) of the content of individual HMOs produced by each strain to the total HMO (mM) content.

[0551] FT* copy number 2'FL 3FL LNFP-I LNDFH-I Med1_4 2×Med1, 17 0 70 12

[0552] *FT = fucosyltransferase

[0553] No HMOs other than those shown in Table 9 were identified in the deep hole test.

[0554] From the data shown in Table 9, it can be seen that Med1 can transfer the fucosyl unit to the GlcNAc moiety of LNFP-I with an α-1,4 linkage to form LNDFH-I. The production of difucosylated HMOs is complex and requires the presence of two fucosyltransferases with different activities, in this case α-1,2- and α1,4-fucosyltransferase activities, because it requires highly specific fucosyltransferases to produce the correct product without a large amount of fucosylated byproducts. Steric hindrance can also be a problem associated with the synthesis of complex HMOs. In the present case, the presence of the fucosyl moiety on the terminal galactose (Gal) unit of LNT can easily cause steric hindrance to the Med1 α1,4-fucosyltransferase, or the fucosyl moiety on the GlcNAc can cause steric hindrance to the Smob α1,2-fucosyltransferase. The 2'FL produced in the current strain is produced by the α-1,2-fucosyltransferase activity of the Smob enzyme, which is also able to fucosylate lactose, while the Med1 α-1,3-fucosyltransferase activity in this example obviously does not have this ability, because 3FL was not detected, which confirms the observations made in Example 1 for the Med1 strain with two copies, in which 3FL was not produced (Med1_2 Table 7).

[0555] Example 4 - Fermentation using Med1α-1,3(4)-fucosyltransferase strain to produce LNFP-II

[0556] To confirm the ability to produce LNFP-II without any complex fucosylated HMO byproducts, as observed in the deep well assay in Example 1, a high copy number strain (Med1_3 strain) was fermented using the ambr fermentation method described in the "Methods" section above. The results are shown in Table 10.

[0557] Table 10: The percentage (%) of individual HMO content produced by the strains to the total HMO content.

[0558] Strain number FT copy number 3FL LNFP-II LNT pLNH2 Med1_3 >50 4 15 78 2

[0559] As can be seen from the data shown in Table 10, the fraction of LNFP-II was higher for the Med1_3 strain when cultured in the deep well assay shown in Example 1 compared to the fermentation, despite this, the Med1 expressing cells showed the ability to produce only a single complex fucosylated HMO with an LNT backbone, at a level of 15% of the total HMO produced by the strain. Interestingly, the level of 3FL in the fermentation was also lower than that observed in the deep well assay, and a small amount of pLNH2 was also observed to be produced.

[0560] Example 5 - Fermentation using Med1α-1,3(4)-fucosyltransferase strain to produce LNDFH-I

[0561] To confirm the ability to produce LNDFH-I observed in the deep well test of Example 3, a strain containing 3× genomic copies of Med1-1,3(4)-fucosyltransferase and a single genomic copy of Smobα-1,2-fucosyltransferase was generated (strain Med1_5, Table 6). The strain was fermented using the ambr fermentation method described in the "Methods" section above. The results are shown in Table 11.

[0562] Table 11: The percentage (%) of individual HMO content produced by the strains to the total HMO content.

[0563] Strain number 2'-FL LNFP-I LNDFH-I Med1_5 6 86 9

[0564] As can be seen from the data shown in Table 11, the production of LNDFH-I in the fermentation device can be confirmed using the Med1_5 strain, although the level of LNDFH-I is slightly lower than that in the deep well test shown in Example 3.

[0565] Example 6 - Fermentation using Med1α-1,3(4)-fucosyltransferase strain to produce LNFP-III

[0566] To confirm the ability to produce LNFP-III observed in the deep well assay of Example 2, a strain containing 1× genomic copy of Med1-1,3(4)-fucosyltransferase was generated (strain Med1_6, Table 6). The strain was fermented using the dashbox fermentation method described in the "Methods" section above. The results are shown in Table 12.

[0567] Table 12: Percentage (%) of individual HMO content produced by strains to total HMO content.

[0568] Strain number 3-FL LNT-II LqCy LNFP-III HkDJ LNDFH-IIII Med1_6 0 3 61 24 12 0

[0569] As can be seen from the data shown in Table 12, when using the Med1_6 strain in the fermentation unit, the only complexly fucosylated HMO produced was LNFP-III. Compared to the deep well experiment shown in Example 2, the conversion rate from LNnT to LNFP-III was slightly lower, which can be optimized by, for example, using a strain with multiple copies of Med1 on the genome.

[0570] Example 7 - Regeneration and viability of freeze-dried Lactobacillus bacteria

[0571] Probiotics can be consumed as live bacteria or dried (e.g., freeze-dried) products. Regardless of the drying method, rehydration is an important step in the recovery of dehydrated bacteria; an insufficient rehydration / regeneration step may result in poor cell viability and ultimately low survival. Therefore, rehydration is a key step in the recovery of freeze-dried cultures. For both live and rehydrated bacteria, bacterial survival under acidic conditions is critical because they need to pass through the acidic environment of the stomach and may also face storage (shelf life) in acidic foods.

[0572] In this example, it was tested whether the HMO mixture produced by the LNDFH-I production strains in Examples 3 and 5 could provide benefits for the rehydration (regeneration) and vitality of probiotics. The test was conducted under acidic conditions to simulate the conditions that the bacteria must survive when passing through the stomach or when added to an acidic beverage.

[0573] Freeze-dried probiotic Lactobacillus casei (DSM 32382) (0.4 mg / ml) alone (control) or in combination with a HMO mixture consisting of 75% LNFP-I, 10% LNDFH-I and 15% 2'FL at a 5% w / v dose was dissolved in sterile phosphate buffered saline (PBS, pH=3), heated to 37°C and mixed vigorously for about 30 seconds until no visible clumps remained. The test tubes were incubated at 37°C for 3 hours. The samples were further diluted and 100 μl samples were plated in duplicate on MRS agar plates, which were incubated in an anaerobic chamber at 37°C for 48 hours. The regeneration and viability of the probiotics were determined by counting colonies after 48 hours of incubation on the plates. If the colonies grew slowly, the incubation time was extended to 72 hours. See the experimental setup for details. Figure 4 The results are expressed as standard deviation of the mean of colony forming units (CFU) per milliliter calculated from colonies formed on agar plates.

[0574] As assessed by colony counts, after 3 hours of incubation at 37°C pH 3.0 under acidic conditions, freeze-dried Lactobacillus casei containing the HMO mixture according to the present invention showed a strongly and significantly enhanced (P<0.01) regeneration and survival capacity compared to the control without the HMO mixture. Figure 5 C shows cfu / ml. The plates forming the basis for the counts are Figure 5 A (control) and 5B (HMO mix). These data clearly show that the regeneration and survival of Lactobacillus casei after exposure to low pH conditions (such as in the stomach or in acidic beverages) can be improved in the presence of the HMO mix.

[0575] To our knowledge, a mixture of LNFP-I, LNnT and 2'FL has not been previously shown to have the benefit of improving the ability of probiotics to reproduce and survive 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(4)-fucosyltransferase activity, wherein the fucosyltransferase is Med1 having an amino acid sequence according to SEQ ID NO: 1, or a functional homologue thereof having an amino acid sequence having at least 80% identity to SEQ ID NO:

1.

2. The genetically engineered cell according to claim 1, wherein the fucosylated HMO is selected from fucosylated HMOs comprising at least five monosaccharide units.

3. The genetically engineered cell according to claim 1 or 2, wherein the fucosyltransferase fucosylate only the N-acetylglucosamine (GlcNAc) portion of LNT, LNnT or LNFP-I.

4. The genetically engineered cell according to any one of claims 1 to 3, wherein the one or more fucosylated HMOs are selected from LNFP-II, LNFP-III and LNDFH-I.

5. The genetically engineered cell according to any one of the preceding claims, wherein the cell further comprises a recombinant nucleic acid sequence encoding a β-1,3-galactosyltransferase and / or a β-1,4-galactosyltransferase.

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

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

8. The genetically engineered cell of any one of the preceding claims, wherein the cell further comprises a recombinant nucleic acid sequence encoding an alpha-1,2-fucosyltransferase from Sulfuriflexus mobilis.

9. A genetically engineered cell according to any one of the preceding claims, wherein the cell comprises two, three or more genomic copies and / or plasmid-borne copies of a recombinant nucleic acid sequence encoding glycosyltransferase Med1 or a functional homolog thereof, the amino acid sequence of which is as shown in SEQ ID NO: 1, and the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO:

1.

10. The genetically engineered cell of any one of the preceding claims, wherein the engineered cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

11. A genetically engineered cell according to any one of the preceding claims, wherein the cell produces a HMO mixture consisting essentially of a. LNFP-II, pLNH2 and LNT, or b. LNFP-II, LNT and 3FL, or c.LNFP-II, LNT, 3FL, and pLNH2, or d.LNT-II, LNFP-II, LNT, 3FL, and pLNH2, or e.LNFP-III and LNnT, or f. LNFP-III, LNnT and pLNnH, or g.LNFP-III and 3FL, or h. LNDFH-I, LNFP-I and 2'FL.

12. A method for producing one or more fucosylated HMOs, the method comprising the steps of a. providing a genetically modified cell according to any one of claims 1 to 11; and b. culturing the cell according to (a) in a suitable cell culture medium to produce said one or more fucosylated HMOs, and c. Optionally, purifying the one or more fucosylated HMOs.

13. The method according to claim 12, wherein a. at least 5% of the total molar content of HMO produced by the cell is LNFP-II, or b. at least 50% of the total molar content of HMO produced by the cell is LNFP-III, or c. At least 9% of the total molar content of HMOs produced by the cell is LNDFH-I.

14. Use of an enzyme having α-1,3(4)-fucosyltransferase activity in producing fucosylated HMO, wherein the enzyme is Med1 or a functional homologue thereof, the amino acid sequence of Med1 being as shown in SEQ ID NO: 1, and the amino acid sequence of the functional homologue being at least 80% identical to SEQ ID NO:

1.

15. Use of the enzyme according to claim 14, wherein the fucosylated HMO is LNFP-II, LNFP-III or LNDFH-I.

16. An HMO mixture consisting essentially of a. LNFP-II, pLNH2 and LNT, or b. LNFP-II, LNT, 3FL, and pLNH2, or c.LNT-II, LNFP-II, LNT, 3FL, and pLNH2, or d. LNFP-III and LNnT, or e.LNFP-III, LNnT and pLNnH, or f. LNFP-III and 3FL, or g. LNDFH-I, LNFP-I and 2'FL.

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

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