Novel fucosyltransferases for in vivo synthesis of complex fucosylated human milk oligosaccharide mixtures comprising LNFP-VI or LNFP-V

Abstract

The present invention relates to the production of complex fucosylated human milk oligosaccharides (HMOs), and in particular to the production of complex fucosylated HMO LNFP-VI or LNFP-V having five or more monosaccharide units, wherein the production method results in a product that is substantially free of LNFP-III and LNDFH-III, or LNFP-II and LNADH-II. The present disclosure also relates to genetically engineered cells and alpha-1,3-fucosyltransferases suitable for use in the production, as well as methods of producing the fucosylated HMOs.

Description

Technical Field

[0001] This disclosure relates to the production of complex fucosylated human lactose oligosaccharides (HMOs), and more particularly to the production of complex fucosylated HMOs LNFP-VI or LNFP-V having five or more monosaccharide units, wherein the products obtained from said production are substantially free of LNFP-III and LNDFH-III or LNFP-II and LNDFH-II. This disclosure also relates to genetically engineered cells and α-1,3-fucosyltransferase suitable for said production, and methods for producing said fucosylated HMOs. Background Technology

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

[0003] The production of complex fucosylated HMOs has been described, for example, in WO2019 / 008133, which reports that the α-1,3-fucosyltransferase FucT109 appears to be able to fucosylate both the glucose (Glc) and N-acetylglucosamine (GlcNAc) moieties in lact-N-neotetrasaccharide (LNnT), thereby potentially generating a mixture containing LNnT, LNFP-III, and LNFP-VI. At the same time, the enzyme can apparently also generate LNFP-V using LNT as a backbone.

[0004] Dumon et al. (2004) (Biotechnol. Prog. 2004, 20, 412−419) further described two α-1,3-fucosyltransferases, FutA and FutB, which were thought to produce a mixture of LNFP-VI, LNDFH-III and 3FL, or a mixture of LNnT, LNFP-III, LNFP-VI and LNDFH-III, respectively.

[0005] WO2023 / 110995 discloses a fucosyltransferase with α-1,3-fucosyltransferase activity that acts on N-acetylglucosamine (GlcNAc) and / or glucose (Glc) on various sugar structures, including lactose, LNT, and LNnT, to generate a mixture of fucosylated oligosaccharides, such as LNFP-III, LNFP-VI, and LNDFH-III or LNFP-II, LNFP-V, and LNDFH-II.

[0006] In addition, WO2016 / 040531 discloses a variety of α-1,3-fucosyltransferases, including CafC and CafF, which are capable of generating 3FL.

[0007] In summary, the production of fucosylated HMOs, especially certain complex fucosylated HMOs such as LNFP-VI and LNFP-V, is challenging and may require laborious separation procedures due to the lack of fucosylated transferases with the desired substrate specificity and the low productivity of the desired fucosylated HMOs compared to other HMO products present after fermentation, such as HMO precursors and complex fucosylated HMO byproducts. Summary of the Invention

[0008] The need for highly substrate-specific α-1,3-fucosyltransferases has been addressed by screening for a number of α-1,3-fucosyltransferases. These enzymes exhibit low or no specificity for the N-acetylglucosamine (GlcNAc) or galactose (Gal) moieties in LNnT or LNT, which are substrates for fucosylation reactions, but high substrate specificity for the glucose (Glc) moieties in LNnT or LNT. These α-1,3-fucosyltransferases can be used to produce large quantities of complex fucosylated HMOs LNFP-VI or LNFP-V, or mixtures of LNFP-VI or LNFP-V HMOs, while producing only very low levels of complex fucosylated HMO byproducts, such as LNFP-III and LNDFH-III or LNFP-II and LNDFH-II. Therefore, this article provides enzymes, mixtures, compositions, uses, genetically engineered cells, and methods for the production of LNFP-VI or LNFP-V.

[0009] The first aspect of this disclosure relates to a method for producing human milk oligosaccharides (HMOs) lacto-N-neofucopentose VI (LNFP-VI) or lacto-N-fucopentose V (LNFP-V), wherein the content of fucosylated byproduct oligosaccharides having 5 or 6 monosaccharide units in the total molar content of the HMO is less than 5%, the method comprising the steps of: a) providing a genetically engineered cell having a recombinant nucleic acid sequence encoding an α-1,3-fucosylation enzyme derived from Bacteroidales bacterium; b) culturing the genetically modified cell under conditions that allow for the formation of LNFP-VI or LNFP-V; and c) optionally purifying the LNFP-VI or LNFP-V by removing byproducts such as 3FL and / or LNnT or 3FL and / or LNT.

[0010] Preferably, α-1,3-fucosyltransferase has high specificity for the glucose (Glc) moiety in LNnT and / or LNT, while having low or no specificity for the N-acetylglucosamine (GlcNAc) or galactose (Gal) moiety in LNnT.

[0011] The second aspect is a genetically engineered cell capable of producing human lactooligosaccharides (HMOs) selected from lacto-N-neofucopentose VI (LNFP-VI) and lacto-N-fucopentose V (LNFP-V). This genetically engineered cell contains a recombinant nucleic acid sequence encoding α-1,3-fucosyltransferase Bacbac2 or a functional homolog thereof. Bacbac2 comprises or consists of an amino acid sequence as shown in SEQ ID NO: 2, and the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2.

[0012] In addition to LNFP-V, the cell may also produce one or more HMOs selected from 3FL, LNT-II, and LNT. Preferably, the cell does not substantially produce LNFP-II and / or LNDFH-II. The cell may contain other modifications, such as substrate input proteins selected from lactose input protein, lact-N-trisaccharide-II (LNT-II) input protein, and LNT input protein.

[0013] The third aspect is a genetically engineered cell capable of producing human lactose oligosaccharide (HMO) lact-N-neofucopentose VI (LNFP-VI), which contains a recombinant nucleic acid sequence encoding α-1,3-fucosyltransferase, which is selected from:

[0014] a) Bacbac1 or a functional homolog thereof comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 1; or

[0015] b) Bacbac2 or a functional homolog thereof comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 2, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2.

[0016] The genetically engineered cell also contains one or more recombinant nucleic acid sequences required to produce LNnT in the cell.

[0017] In addition to LNFP-VI, the cell may produce one or more HMOs selected from 3FL, LNT-II, and LNnT, and preferably, the cell substantially does not produce LNFP-III and / or LNDFH-III. The cell may contain other modifications, such as substrate input proteins selected from lactose input proteins, lact-N-trisaccharide-II (LNT-II) input proteins, and LNnT input proteins.

[0018] The fourth aspect relates to the use of α-1,3-fucosyltransferase in the production of LNFP-VI, wherein the α-1,3-fucosyltransferase is selected from Bacbac1 or Bacbac2, or their functional homologs, Bacbac1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1, Bacbac2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of the functional homolog having at least 80% identity with SEQ ID NO: 1 or 2.

[0019] The fifth aspect of this disclosure relates to mixtures of HMOs, preferably prepared using the methods of this disclosure, wherein the mixture of HMOs consists essentially of: a) LNFP-VI and 3-FL, or b) LNFP-VI, or c) LNnT, or LNFP-V, 3FL and LNnT, or d) LNFP-V, 3FL and LNT.

[0020] A sixth aspect of this disclosure relates to the use of mixtures or compositions of this disclosure in infant formula, dietary supplements, and / or medical nutrition products. Attached Figure Description

[0021] Figure 1 An overview of the synthesis of complex fucosylated HMOs with an LNnT backbone.

[0022] Figure 2 The data for each strain in Table 7 are presented, clearly showing the differences in LNFP-VI production between strains with different enzymes. LNFP-VI is represented by black bars, 3FL by gray bars, LNnT by diagonal stripes, LNDFH-III by square bars, LNFP-III by horizontal stripes, and pLNnH by dashed lines.

[0023] Figure 3 An overview of the synthesis of complex fucosylated HMOs with an LNT backbone.

[0024] Figure 4 This study demonstrates the regeneration and viability of lyophilized *Lactobacillus rhamnosus* (DSM 33156) after incubation at pH 3.0 for 3 hours, followed by plate inoculation at four dilutions: 1:1000 (E-3), 1:10,000 (E-4), 1:100,000 (E-5), and 1:1,000,000 (E-6). A) is the control group without HMO; B) is a combination of *Lactobacillus rhamnosus* (DSM 33156) and an HMO mixture containing 80% LNFP-VI, 10% 3FL, and 10% LNnT (mix 1); C) is a combination of *Lactobacillus rhamnosus* (DSM 33156) and an HMO mixture containing 60% LNFP-VI and 40% 3FL (mix 2). Detailed Implementation

[0025] This disclosure aims to address the biotechnological challenges of producing HMOs in vivo, particularly the production of complex fucosylated HMOs comprising at least five monosaccharide units, wherein at least one monosaccharide unit is a fucosylated unit, such as LNFP-VI and LNFP-V. This disclosure provides specific strain engineering solutions for producing specific complex fucosylated HMOs, particularly LNFP-VI or LNFP-V, by utilizing the substrate specificity of the identified α-1,3-fucosylation enzymes Bacbac1, Bacbac2, Para1, and CafF disclosed herein, especially their specificity for the glucose (Glc) moiety in LNnT (or LNT) rather than the N-acetylglucosamine (GlcNAc) or galactose (Gal) moiety.

[0026] The genetically engineered cells of this disclosure express genes encoding key enzymes for the biosynthesis of fucosylated HMOs. Furthermore, it is more advantageous if the genetically engineered cells express genes required for the production of LNnT or LNT using lactose or LNT-II as initial substrates. Alternatively, cells can be engineered to take up LNnT simply by possessing 1,3-fucosyltransferase activity within their cells (see, for example, WO2023 / 099680). In some embodiments, the genetically engineered cells of this disclosure also express one or more genes involved in the de novo GDP-fucose synthesis pathway, such as manA, manB, manC, gmd, and / or wcaG, which are responsible for GDP-fucose formation. By introducing nucleic acid constructs encoding CA (as shown in SEQ ID NO: 41), overexpressing one or more of these genes and / or upregulating the colanic acid gene cluster (CA) of *E. coli* (including gmd, wcaG, wcaH, wcaI, manC, and manB genes), it may be beneficial to the formation of GDP-fucose, thereby enabling cells to produce higher levels of fucosylated oligosaccharides from one or more oligosaccharide substrates (such as lactose, LNT-II, and / or LNnT). Depending on the intended use of the substrate, one or more additional glycosyltransferases and pathways may also be present in the genetically engineered cells for the production of 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.

[0027] LNFP-VI production

[0028] In the context of this disclosure, the advantage of using any of the α-1,3-fucosyltransferases of this disclosure lies in their ability to specifically recognize and fucosylate the Glc moiety in LNnT, thereby generating LNFP-VI. Specifically, this disclosure describes enzymes (α-1,3-fucosyltransferases) with α-1,3-fucosyltransferase activity that exhibit higher activity toward the Glc moiety of LNnT compared to α-1,3-fucosyltransferases described in the prior art (e.g., FutA and FutB (see Dumon et al., 2004), CafC (WO2016 / 040531), and FucT109 (WO2019 / 008133)). Furthermore, the α-1,3-fucosyltransferases described herein have extremely low activity toward the GlcNAc moiety of LNnT. If the intracellular LNnT content in genetically engineered cells is sufficient, the α-1,3-fucosyltransferases described herein will hardly produce LNFP-III and / or LNDFH-III. Therefore, the properties of the α-1,3-fucosyltransferases described herein are well-suited for the high-level industrial production of LNFP-VI without generating other complex fucosylated oligosaccharide byproducts, such as LNFP-III and LNDFH-III.

[0029] In embodiments, the α-1,3-fucosyltransferase of this disclosure primarily fucosylates the glucose (Glc) moiety of the acceptor oligosaccharide (e.g., LNnT). The term "primarily" should be understood to mean that the α-1,3-fucosyltransferase of this disclosure fucosylates other moieties of LNnT at a rate of less than 5%, for example less than 4%, for example less than 3%, or for example less than 2%.

[0030] In embodiments, the α-1,3-fucosyltransferase of this disclosure has low or no activity toward the N-acetylglucosamine (GlcNAc) moiety in the receptor molecule. The receptor molecule is an oligosaccharide, such as an HMO like LNT-II or LNnT, but may also be other oligosaccharides or HMOs. LNnT is a preferred receptor molecule for the α-1,3-fucosyltransferase of this disclosure. The terms “low activity” or “inactive” should be understood as: the proportion of the GlcNAc moiety in the receptor molecule that is fucosylated by the α-1,3-fucosyltransferase of this disclosure is less than 5%, for example less than 4%, for example less than 3%, or for example less than 2%. Therefore, in embodiments, the oligosaccharides produced by cells expressing the α-1,3-fucosyltransferase are substantially free of N-acetylglucosamine (GlcNAc) fucosylated oligosaccharides. In this embodiment, the oligosaccharides produced by cells expressing the α-1,3-fucosyltransferase are substantially free of N-acetylglucosamine (GlcNAc) fucosylated oligosaccharides LNFP-III and / or LNDFH-III.

[0031] This disclosure discloses a genetically engineered cell expression of an α-1,3-fucosyltransferase with high specificity for the Glc moiety in LNnT, enabling high-titer production of LNFP-VI. This is particularly true in the absence of other complex fucosylated oligosaccharide byproducts with 5 or 6 monosaccharide units (e.g., LNFP-III and LNDFH-III). Therefore, this disclosure enables more efficient production of LNFP-VI, which offers significant advantages for the biotechnological production of more complex fucosylated HMOs such as LNFP-VI.

[0032] Therefore, in the HMO mixture produced by the cells and / or methods described herein, LNFP-VI accounts for a high proportion of the total amount of HMOs produced, for example, at least 25% of the total amount of HMOs, preferably at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80% of the total amount of HMOs produced by the cells.

[0033] The ability to produce LNFP-VI via fermentation with very low or no fucosylated oligosaccharide byproducts of 5 or 6 monosaccharide units is advantageous for purification, as isolating fucosylated HMOs of similar length is very challenging. Therefore, the method and genetically engineered cells described herein have the advantage of yielding a final product with LNFP-VI purity of at least 60%, for example, at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 85%, for example, at least 90%, especially when one or more purification steps are applied after fermentation to remove unwanted byproducts (particularly HMO byproducts).

[0034] LNFP-V production

[0035] Furthermore, the genetically engineered cells of this disclosure can also be modified to produce fucosylated HMOs with an LNT backbone. These cells are further modified to express an α-1,3-fucosyltransferase with high specificity for the Glc moiety in LNTs, thereby enabling high-titer LNFP-V production, especially in the absence of other complex fucosylated HMO byproducts (e.g., LNFP-II and LNDFH-II). Therefore, this disclosure also enables more efficient production of LNFP-V, which is a significant advantage for the biotechnological production of more complex fucosylated HMOs such as LNFP-V.

[0036] Furthermore, the α-1,3-fucosyltransferase described herein exhibits extremely low activity towards the GlcNAc moiety of LNTs. If the intracellular LNT content in genetically engineered cells is sufficient, the α-1,3-fucosyltransferase described herein will hardly produce LNFP-II and / or LNDFH-II. Therefore, the characteristics of the α-1,3-fucosyltransferase described herein are highly suitable for the large-scale industrial production of LNFP-V without generating other complex fucosylated oligosaccharide byproducts, such as LNFP-II and LNDFH-II.

[0037] In embodiments, the α-1,3-fucosyltransferases of this disclosure primarily fucosylate the glucose (Glc) moiety of receptor oligosaccharides (e.g., LNTs). The term "primarily" should be understood to mean that the α-1,3-fucosyltransferases of this disclosure fucosylate other moieties of LNTs at a rate of less than 5%, for example less than 4%, for example less than 3%, for example less than 2%. Therefore, the α-1,3-fucosyltransferases of this disclosure that primarily fucosylate the glucose (Glc) moiety of receptor oligosaccharides can also be described as having high specificity for the glucose (Glc) moiety in the receptor oligosaccharides; thus, it should be understood that these terms are used interchangeably.

[0038] In some embodiments, the α-1,3-fucosyltransferase of this disclosure has very low or no activity toward the N-acetylglucosamine (GlcNAc) moiety of the receptor molecule. The receptor molecule is an oligosaccharide, such as an HMO, like LNT-II or LNT, but may also be other oligosaccharides or HMOs. LNT is a preferred receptor molecule for the α-1,3-fucosyltransferase of this disclosure. In particular, the α-1,3-fucosyltransferase of this disclosure has no or only very low α-1,4-fucosyltransferase activity, and therefore cannot fucosylate the GlcNAc moiety of LNT, because this moiety is linked to the terminal galactose via an α-1,3-linking bond, and therefore cannot be fucosylated by an enzyme with only α-1,3-fucosyltransferase activity. The terms "low activity" or "inactive" should be understood as meaning that the proportion of the GlcNAc portion of the receptor molecule (e.g., LNT) fucosylated by the α-1,3-fucosyltransferase of this disclosure is less than 2%, for example less than 1.5%, for example less than 1%, for example less than 0.5%, for example less than 0.1%. Therefore, in embodiments, the oligosaccharides produced by cells expressing the α-1,3-fucosyltransferase are substantially free of N-acetylglucosamine (GlcNAc)-fucosylated oligosaccharides, such as N-acetylglucosamine (GlcNAc)-fucosylated oligosaccharides having an LNT and / or LNnT backbone.

[0039] The genetically engineered cells of this disclosure express an α-1,3-fucosyltransferase with high specificity for the Glc moiety in LNTs, enabling the production of a mixture of LNFP-V containing trace amounts of other fucosylated oligosaccharides. In embodiments, these genetically engineered cells produce a mixture of LNFP-V and LNTs, wherein the content of other fucosylated HMOs is less than 5%, for example, less than 2%. Specifically, this LNFP-V mixture is free of other complex fucosylated HMOs, such as LNFP-II and LNDFH-II, which simplifies the purification of LNFP-V. Therefore, in embodiments, the oligosaccharides produced by cells expressing the α-1,3-fucosyltransferase are substantially free of the N-acetylglucosamine (GlcNAc) fucosylated oligosaccharides LNFP-II and / or LNDFH-II. Thus, this disclosure enables more efficient production of LNFP-V, which is highly advantageous for the biotechnological production of more complex fucosylated HMOs such as LNFP-V.

[0040] Therefore, in the HMO mixture produced by the cells and / or methods described herein, LNFP-V may account for a high proportion of the total amount of HMOs produced, for example, at least 40% of the total amount of HMOs, preferably at least 45%, for example, at least 50%, at least 55%, or at least 57% of the total amount of HMOs produced by the cells and / or methods.

[0041] The ability to produce LNFP-V via fermentation with very low or no fucosylated oligosaccharide byproducts of 5 or 6 monosaccharide units is advantageous for purification, as isolating fucosylated HMOs of similar length is very challenging. Therefore, the method and genetically engineered cells described herein have the advantage of yielding a final product with LNFP-V purity of at least 60%, for example, at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 85%, for example, at least 90%, especially when one or more purification steps are applied after fermentation to remove unwanted byproducts (particularly HMO byproducts).

[0042] The following sections will describe the various elements of this disclosure, particularly genetically engineered cells. It should be understood that these elements can be combined across different sections.

[0043] Oligosaccharides

[0044] In this context, the term "oligosaccharide" refers to a sugar polymer containing at least three monosaccharide units, i.e., trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or higher oligosaccharides. Oligosaccharides can have a linear or branched structure, containing monosaccharide units interconnected by glycosidic bonds. Preferably, the oligosaccharide comprises a lactose residue at its reducing end, and one or more naturally occurring monosaccharides of 5-9 carbon atoms, selected from aldoses (e.g., glucose, galactose, ribose, arabinose, xylose, etc.), ketoses (e.g., fructose, sorbose, tagatose, etc.), deoxysaccharides (e.g., rhamnose, fucose, etc.), deoxyaminosaccharides (e.g., N-acetylglucosamine, N-acetymannosamine, N-acetylgalactosamine, etc.), uronic acids, and ketouronic acids (e.g., N-acetylneuraminic acid). Preferably, the oligosaccharide is an HMO.

[0045] Human milk oligosaccharides (HMOs)

[0046] The preferred oligosaccharide disclosed herein is human milk oligosaccharide (HMO).

[0047] In this context, the term "human milk oligosaccharide" or "HMO" refers to a complex carbohydrate found in human breast milk. HMOs have a core structure comprising a lactose unit at a reducing end, which can be elongated by one or more β-N-acetyl-lactosamine and / or one or more β-lacto-N-disaccharide units, and this core structure can be partially substituted with α-L-fucopyranosyl and / or α-N-acetyl-ceramide (fucosyl) components. HMO structures are disclosed, for example, in Chapter 4 of Xi Chen's *Advances in Carbohydrate Chemistry and Biochemistry*, 2015, vol. 72.

[0048] This disclosure focuses on fucosylated HMOs. Examples of fucosylated HMOs include: 2'-fucosyllactose (2'FL), lacto-N-fucopentose I (LNFP-I), lacto-N-difucohexasose I (LNDFH-I), 3-fucosyllactose (3FL), difucosyllactose (DFL), lacto-N-fucopentose II (LNFP-II), lacto-N-fucopentose III (LNFP-III), lacto-N-difucohexasose III (LNDFH-III), fucosyl-lacto-N-hexasose II (FLNH-II), and lacto-N-fucopentose V (LN... FP-V), lacto-N-fucopentose VI (LNFP-VI), lacto-N-difucohexasose II (LNDFH-II), fucosyl-lacto-N-hexasose I (FLNH-I), fucosyl-p-lacto-N-hexasose I (FpLNH-I), fucosyl-p-lacto-N-neohexose II (F-pLNnHII), fucosyl-lacto-N-neohexose (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-hexasose (SLNH).

[0049] In the context described herein, a complex fucosylated HMO refers to a fucosylated HMO containing at least five monosaccharide units, wherein at least one monosaccharide unit is a fucosylated unit. Non-limiting examples of complex fucosylated HMOs include fucosylated HMOs consisting of five monosaccharide units, such as LNFP-I, LNFP-II, LNFP-III, LNFP-V, and LNFP-VI; and complex fucosylated HMOs having six monosaccharide units, such as, but not limited to, difucosylated HMOs, LNDFH-I, LNDFH-II, and LNDFH-III, or sialic acid-fucosylated-HMOs, FLST-a, FLST-b, FLST-c, and FLST-d. Preferably, a complex fucosylated HMO requires at least three different glycosyltransferase activities to be generated from lactose as an initial substrate. For example, the formation of LNFP-VI requires glucose-specific α-1,3-fucosyltransferase, β-1,3-N-acetylglucosamine transferase, and β-1,4-galactosyltransferase (see...). Figure 1 The formation of LNDFH-III requires at least one α-1,3-fucosyltransferase, β-1,3-N-acetylglucosamine transferase, and β-1,4-galactosyltransferase. The enzymes described herein are preferably active only on the Glc moiety of LNnT or LNT, thus enabling the generation of LNFP-VI from LNnT (see...).Figure 1 ), or generate LNFP-V from LNT ( Figure 3 It produces little or no complex fucosylation byproduct oligosaccharides, such as LNFP-III and / or LNDFH-III or LNFP-II and / or LNDFH-III.

[0050] In the context of this disclosure, a complex fucosylated HMO refers to a fucosylated HMO comprising at least five monosaccharide units, wherein at least one monosaccharide unit is a fucosylated unit; non-limiting examples of complex fucosylated HMOs include fucosylated HMOs composed of five monosaccharide units, such as LNFP-I, LNFP-II, LNFP-III, LNFP-V, and LNFP-VI, and fucosylated HMOs composed of six monosaccharide units, such as, but not limited to, LNDFH-I, LNDFH-II, and LNDFH-III. Preferably, a complex fucosylated HMO refers to an HMO that requires at least three different glycosyltransferases to form from lactose as an initial substrate; for example, the formation of LNFP-VI requires glucose-specific α-1,3-fucosylation enzyme, β-1,3-N-acetylglucosamine transferase, and β-1,4-galactosyltransferase.

[0051] In this disclosure, "complex fucosylation byproduct oligosaccharides" refers to complex fucosylated oligosaccharides that are not the desired product. This means that the aforementioned complex fucosylated HMOs, if not the desired component in the final product, can be considered byproducts (this term can also be used interchangeably with "HMO byproduct"), and in this disclosure, the final product is LNFP-VI or LNFP-V. Examples of fucosylation byproduct oligosaccharides having 5 or 6 monosaccharide units include LNFP-III, LNFP-II, LNDFH-III, and LNDFH-II.

[0052] In embodiments of this disclosure, the fucosylated human lactose oligosaccharide (HMO) produced by the cells is LNFP-VI, for example, LNFP-VI is the main component. In another embodiment of this disclosure, the content of LNFP-VI in the total molar content of the HMOs produced by the cells is at least 25%, for example, at least 30%, 50%, 55%, 60%, 70%, 75%, 80%, or 82%. Preferably, the content of LNFP-VI in the total molar content of the HMOs produced by the cells is at least 60%. In other embodiments, the content of LNFP-VI and LNnT in the total molar content of the HMOs produced by the cells is at least 80%. In other embodiments, the content of LNFP-VI and 3FL in the total molar content of the HMOs produced by the cells is at least 80%, for example, at least 85%, 90%, 95%, or at least 99% or 100%. In other embodiments of this disclosure, the LNFP-III content in the total molar content of HMOs produced by cells is less than 3%, for example, 0.0%, or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, or for example, less than 2.99%. In other embodiments of this disclosure, the LNDFH-III content in the total molar content of HMOs produced by cells is less than 3%, for example, 0.0%, or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, or for example, less than 2.99%. In other embodiments, the content of other complex fucosylated oligosaccharide byproducts in the total molar content of HMOs produced by cells is less than 5%. In this context, other fucosylated oligosaccharide byproducts refer to one or more non-LNFP-VI fucosylated oligosaccharides. Other fucosylated oligosaccharides, such as fucosylated HMOs, may be selected from 3-FL, DFL, LNFP-III, and LNDFH-III. Other complex fucosylated HMOs may be selected from LNFP-III and LNDFH-III.

[0053] Preferably, the cells do not produce LNFP-III or LNDFH-III.

[0054] The generation of LNFP-VI may require two or more glycosyltransferase activities, especially when lactose is used as the initial acceptor oligosaccharide.

[0055] In other embodiments of this disclosure, the fucosylated human lactose oligosaccharide (HMO) produced by the cells is LNFP-V, for example, primarily LNFP-V. In yet another embodiment of this disclosure, the total molar content of LNFP-V in the HMOs produced by the cells is at least 25%, for example, at least 30%, 50%, 55%, 56%, 57%, 58%, or 59%. Preferably, the total molar content of LNFP-V in the HMOs produced by the cells is at least 50%. In other embodiments, the total molar content of LNFP-V and LNT in the HMOs produced by the cells is at least 80%. In other alternative embodiments of this disclosure, the total molar content of LNFP-II in the HMOs produced by the cells is less than 2%, for example, 0.0%, or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 1.75%, or for example, less than 1.99%. In other alternative embodiments of this disclosure, the content of LNDFH-II in the total molar content of HMOs produced by cells is less than 2%, for example 0.0%, or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 1.75%, or for example less than 1.99%. In other alternative embodiments, the content of other complex fucosylated oligosaccharide byproducts in the total HMOs produced by cells is less than 5%. In this context, other fucosylated oligosaccharide byproducts (e.g., HMOs) besides LNFP-V are considered as one or more non-LNFP-V fucosylated oligosaccharides. Other fucosylated HMOs may be selected from 3-FL, DFL, LNFP-II, and LNDFH-II. Other complex fucosylated HMOs may be selected from LNFP-II and LNDFH-II.

[0056] Preferably, the cells do not produce LNFP-II or LNDFH-II.

[0057] The generation of LNFP-V may require the activity of two or more glycosyltransferases, especially when lactose is used as the initial oligosaccharide acceptor.

[0058] Receptor oligosaccharides

[0059] The genetically engineered cells according to this disclosure contain a recombinant nucleic acid sequence encoding a fucosyltransferase, which has α-1,3-fucosyltransferase activity and is capable of transferring fucose from the activated sugar to the glucose moiety of the acceptor oligosaccharide (preferably LNnT or LNT) via an α-1,3-linkage bond.

[0060] As described herein, the acceptor oligosaccharide is an oligosaccharide that can serve as a substrate for a glycosyltransferase, which transfers the glycosyl moiety from a glycosyl donor to the acceptor oligosaccharide. The glycosyl donor is preferably a nucleotide-activated sugar, as described in the section "Glycosyl Donor-Nucleotide-Activated Sugar Pathway". Preferably, the acceptor oligosaccharide is a precursor for the synthesis of more complex HMOs, and may also be referred to as a precursor molecule.

[0061] Receptor oligosaccharides can be intermediate products of this fermentation process, end products of an independent fermentation process using independently genetically engineered cells, or molecules prepared by enzymatic or chemical methods.

[0062] In this context, the receptor oligosaccharide for the α-1,3-fucosyltransferase is preferably lact-N-neotetrasaccharide (LNnT), which is generated from the precursor molecule lactose (e.g., the receptor for β-1,3-N-acetylglucosamine aminotransferase) and / or lact-N-trisaccharide II (LNT-II) (e.g., the receptor for β-1,4-galactosyltransferase). If lactose is provided during culture, it can also be considered as the initial substrate. If it is desired that cells do not produce 3FL, the precursor molecule (alternative initial substrate) provided to the culture is preferably LNT-II.

[0063] Alternatively, the acceptor oligosaccharide of the α-1,3-fucosyltransferase is preferably lacto-N-tetrasaccharide (LNT), which is generated from the precursor molecule lactose (e.g., the acceptor of β-1,3-N-acetylglucosamine transferase) and / or lacto-N-trisaccharide II (LNT-II) (e.g., the acceptor of β-1,3-galactosyltransferase). If lactose is provided during culture, it can also be considered as the initial substrate. If it is desired that the cells do not produce 3FL, it is preferable to provide LNT-II as the initial precursor molecule (alternative to the initial substrate) to the culture.

[0064] Preferably, the precursor molecule is provided to the genetically engineered cells at the start of culture or during culture via continuous feeding, pulse feeding, or a combination of both, so that the genetically engineered cells can use the initial precursor to produce LNnT or LNT.

[0065] Typically, the initial precursor is lactose, and genetically engineered cells are able to produce intermediate precursors (receptor oligosaccharides, such as LNT-II and LNnT or LNT) within the cell. However, if the cell is able to take up at least one of LNT-II, LNnT, or LNT, the initial precursor can also be LNT-II, LNnT, or LNT.

[0066] Glycosyltransferase

[0067] The genetically engineered cell according to this disclosure contains at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase, such as fucosyltransferase, which is capable of transferring fucosylation residues from a fucosylation donor to a recipient oligosaccharide to synthesize one or more fucosylated human milk oligosaccharide products, i.e., fucosyltransferase.

[0068] The genetically engineered cells according to this disclosure may also contain one or more additional recombinant nucleic acids encoding one or more recombinant and / or heteroglycosyltransferases capable of transferring glycosyl residues from a glycosyl donor to a recipient oligosaccharide. Preferably, the additional glycosyltransferase enables the genetically engineered cells to synthesize LNnT or LNT from a precursor molecule (e.g., lactose or LNT-II). In one embodiment, the genetically engineered cells described herein contain one or more recombinant nucleic acids encoding one or more recombinant and / or heteroglycosyltransferases.

[0069] The additional glycosyltransferase is preferably selected from galactosyltransferase, glucosyltransferase, fucosyltransferase, N-acetylglucosyltransferase, and sialic acid transferase.

[0070] α-1,3-fucosyltransferase

[0071] The term "α-1,3-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of fucosylation from a donor substrate (e.g., GDP-fucosylation) via an α-1,3-linkage bond to an acceptor molecule (preferably the Glc moiety in LNnT or LNT). Figure 1 Preferably, the α-1,3-fucosyltransferase used in this disclosure is not derived from a genetically engineered cell species, i.e., the gene encoding the α-1,3-fucosyltransferase is heterologous and selected from the α-1,3-fucosyltransferases listed in Table 1. In the context described herein, the acceptor molecule for the α-1,3-fucosyltransferase is preferably a acceptor oligosaccharide having a GlcNAc moiety consisting of at least four monosaccharide units, such as LNnT or LNT. Heterologous α-1,3-fucosyltransferases capable of transferring fucosyl groups to LNnT or LNT are known in the art; in particular, FutA has been shown to produce a mixture of LNFP-VI and LNDFH-III (Dumon et al. 2004 Biotechnol. Prog. 20:412-419).

[0072] In one aspect, the fucosyltransferase in the genetically engineered cells of this disclosure is an α-1,3-fucosyltransferase. Preferably, this α-1,3-fucosyltransferase is capable of transferring fucose units to the glucose (Glc) moiety of the LNnT or LNT molecule. More preferably, this α-1,3-fucosyltransferase is specific to the glucose (Glc) moiety of the LNnT or LNT molecule.

[0073] If the desired HMO product is LNFP-VI, then an α-1,3-fucosyltransferase with higher substrate specificity to the Glc moiety in LNnT is advantageous compared to the GlcNAc or Gal moiety in LNnT, because theoretically, such an α-1,3-fucosyltransferase would produce fewer or no byproduct oligosaccharides of 5 or 6 monosaccharide units, such as LNFP-III and LNDFH-III. Lower LNFP-III and LNDFH-III content is beneficial for LNFP-VI purification because it is easier to purify LNFP-VI from a mixture of HMOs that predominantly contain LNFP-VI. This is because separating LNFP-VI from smaller HMOs is easier than separating different fucosylated HMOs of the same or similar size (e.g., LNFP-VI from LNFP-III or LNDFH-III) from each other. Therefore, lower initial levels of LNFP-III and / or LNDFH-III, or the complete absence of LNFP-III and / or LNDFH-III, are considered beneficial for the purification of LNFP-VI.

[0074] In another embodiment, it is desirable for α-1,3-fucosyltransferase to have low activity toward the glucose fraction of lactose in order to reduce the formation of 3FL during fermentation with lactose as the initial substrate.

[0075] In a preferred embodiment, the use of the α-1,3-fucosyltransferase according to this disclosure results in the content of LNFP-VI being at least 14%, for example at least 25%, for example at least 50%, of the total molar content of HMOs produced by cells according to this disclosure. Specifically, the LNFP-III and / or LNDFH-III produced by the α-1,3-fucosyltransferase according to this disclosure is less than 5%, preferably, almost no LNFP-III and / or LNDFH-III is produced.

[0076] In this disclosure, the α-1,3-fucosyltransferases capable of transferring the fucosyl moiety from a fucosyl donor to the glucose (Glc) moiety in lacto-N-neotetrasaccharide (LNnT) are α-1,3-fucosyltransferases derived from Bacteroidalesbacterium. Examples of such α-1,3-fucosyltransferases are Bacbac1 or Bacbac2, having the amino acid sequence according to SEQ ID NO: 1 or 2, or functional homologs thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 or 2.

[0077] In a preferred embodiment, the α-1,3-fucosyltransferase is Bacbac2 having the amino acid sequence according to SEQ ID NO: 2, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO: 2. Bacbac2 can be used to produce LNFP-VI, which constitutes more than 50%, for example, more than 60%, of the total molar content of HMOs. Specifically, the α-1,3-fucosyltransferase Bacbac2 according to this disclosure produces LNFP-III and / or LNDFH-III in amounts less than 2%, for example, the total molar content of HMOs produced by cells or methods is almost entirely devoid of LNFP-III and LNDFH-III. Furthermore, Bacbac2 has a low affinity for lactose, thus producing 3FL in amounts less than 15%, for example, the 3FL content in the total molar content of HMOs produced by cells or methods is less than 10%. During fermentation, the conversion of substrate LNnT to LNFP-VI is highly efficient, with less than 15% of LNnT remaining at the end of fermentation. The high conversion rate of LNnT results in almost no production of pLNnH during fermentation. Furthermore, all LNT-II is converted to LNnT, as fermentation is essentially LNT-II-free.

[0078] In another embodiment, the α-1,3-fucosyltransferase Bacbac2 (SEQ ID NO: 2) disclosed herein, or a functional homolog thereof (with an amino acid sequence having at least 80% identity with SEQ ID NO: 2), can be used to produce LNFP-V comprising more than 50% of the total molar content of HMOs, for example, at least 55%. Specifically, the LNFP-II and / or LNDFH-II produced by the α-1,3-fucosyltransferase Bacbac2 according to this disclosure comprises less than 2%, for example, substantially no LNFP-II and LNDFH-II in the total molar content of HMOs produced by cells or methods. Furthermore, Bacbac2 has a low affinity for lactose and therefore produces less than 5% 3FL, for example, less than 2% of the total molar content of HMOs produced by cells or methods. In addition, all LNT-II is converted to LNT.

[0079] In another embodiment, the α-1,3-fucosyltransferase is Bacbac1 having the amino acid sequence according to SEQ ID NO: 1, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO: 1. Bacbac1 can be used to produce LNFP-VI comprising more than 15%, for example, more than 30%, of the total HMO molar content. Specifically, the α-1,3-fucosyltransferase Bacbac1 according to this disclosure produces less than 2% LNFP-III and / or LNDFH-III, for example, the total HMO molar content produced by the cell or method is substantially free of LNFP-III and LNDFH-III. During fermentation, the conversion of substrate LNnT to LNFP-VI is highly efficient, with less than 5%, for example, less than 2%, of residual LNnT at the end of fermentation. Furthermore, all LNT-II is converted to LNnT because the fermentation is substantially free of LNT-II.

[0080] In another embodiment, the α-1,3-fucosyltransferases capable of transferring the fucosylation moiety from the fucosylation donor to the acceptor oligosaccharide are selected from Bacbac1, Bacbac2, Para1, and CafF, having an amino acid sequence according to SEQ ID NO: 1, 2, 3, or 43, or selected from their functional homologs, the amino acid sequences of which have at least 80% identity with the amino acid sequences of SEQ ID NO: 1, 2, 3, or 43 (Table 1). These α-1,3-fucosyltransferases exhibit extremely high specificity for the glucose (Glc) moiety in lacto-N-neotetrasaccharide (LNnT), while showing low or no specificity for the N-acetylglucosamine (GlcNAc) or galactose (Gal) moiety in LNnT. These enzymes can be used to produce LNFP-VI, particularly in cases where the content of the fucosylation byproduct oligosaccharide having 5 or 6 monosaccharide units is less than 5% of the total molar content of HMOs.

[0081] In another embodiment, the α-1,3-fucosyltransferase is Para1 having the amino acid sequence according to SEQ ID NO: 3, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO: 3. Para1 can be used to produce LNFP-VI, which constitutes more than 25% of the total HMO molar content, wherein the content of LNFP-II and / or LNDFH-II is less than 2%, and for example, LNFP-III and LNDFH-III are substantially absent in the mixture.

[0082] In another embodiment, α-1,3-fucosyltransferase is CafF having the amino acid sequence according to SEQ ID NO: 43, or a functional homolog thereof having an amino acid sequence with at least 80% identity to SEQ ID NO: 43. CafF can be used to produce LNFP-VI in an amount greater than 14% of the total HMO molar content, with LNFP-III and LNDFH-III substantially absent in the mixture. When using CafF, it is likely to increase the yield of LNFP-VI if, for example, LNT-II is used as the starting substrate, because this will prevent the fucosylation of lactose, which CafF is known to efficiently perform (WO2019 / 008133).

[0083] For example, these enzymes can be used to produce LNFP-VI with little or no production of other complex fucosylation byproducts HMO, particularly with the content of fucosylation byproduct oligosaccharides (e.g., LNFP-III and / or LNDFH-III) having 5 or 6 monosaccharide units being less than 5% of the total molar content of HMO, for example, less than 2%.

[0084] The α-1,3-fucosyltransferase of this disclosure may be selected from an amino acid sequence having at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, or for example, at least 99% sequence identity with any of the α-1,3-fucosyltransferases listed in Table 1.

[0085] Table 1. List of α-1,3-fucosyltransferases in this disclosure that can produce LNFP-VI and produce little or no LNFP-III and / or LNDFH-III byproducts.

[0086]

[0087] 1 The GenBank ID reflects the full-length enzyme. Shortened, extended, or mutated versions may be used in this disclosure, which are represented by the sequence indicated by SEQ ID NO.

[0088] Example 1 of this disclosure discloses the identification of heterologous α-1,3-fucosyltransferases Bacbac1, Bacbac2, Para1, and CafF (SEQ ID NO: 1, 2, 3, and 43, respectively), which, when introduced into LNnT production cells, were able to produce a mixture of HMOs compared to previously known α-1,3-fucosyltransferases FutA, FutB, CafC, and FucT109 (SEQ ID NO: 5, 6, 47, and 45, respectively), wherein LNFP-VI was the major complex fucosylated HMO in the mixture (i.e., more than 10-fold, for example, 50-fold higher than LNFP-III and LNDFH-III). Specifically, three novel enzymes, Bacbac1, Bacbac2, and Para1 (which are entirely new in HMO production), along with the known 3FL-producing CafF enzyme, specifically transfer fucosylation units via α-1,3 linkages to the glucose moiety of LNnT, forming LNFP-VI at a concentration higher than 14%, for example, higher than 25% of total HMO, without producing any LNFP-III or LNDFH-III. In contrast, existing enzymes FutA, FutB, CafC, and FucT109 produce LNDFH-III and / or LNFP-III as byproducts, respectively.

[0089] The experimental results in Example 1 show that enzymes Bacbac1, Bacbac2, Para1, and CafF do not produce any LNFP-III or LNDFH-III, indicating that these enzymes have high substrate specificity for the glucose moiety in LNnT.

[0090] In embodiments, the α-1,3-fucosyltransferase expressed in genetically engineered cells according to the present disclosure is further combined with the expression of one or more recombinant nucleic acids encoding one or more heteroglycosyltransferases. In a preferred embodiment, the expression of the α-1,3-fucosyltransferase according to the present disclosure in genetically engineered cells is combined with the expression of a β-1,4-galactosyltransferase (e.g., galT from Helicobacter pylori) to enable LNT-II as an initial substrate to generate LNnT. In another embodiment, a third enzyme, such as β-1,3-N-acetylglucosamine transferase (e.g., LgtA from Neisseria meningitidis), is added to enable lactose as an initial substrate to generate LNnT.

[0091] In other embodiments, the expression of α-1,3-fucosyltransferase Bacbac2 (SEQ ID NO: 2) or a functional homolog thereof is combined with the expression of β-1,3-galactosyltransferase (e.g., galTK from Helicobacter pylori) to generate LNTs using LNT-II as the initial substrate. In another embodiment, a third enzyme, such as β-1,3-N-acetylglucosamine transferase (e.g., LgtA from Neisseria meningitidis), is added to generate LNTs using lactose as the initial substrate.

[0092] β-1,3-N-acetylglucosamine transferase

[0093] β-1,3-N-acetylglucosamine transferases are any proteins capable of transferring N-acetylglucosamine from UDP-N-acetylglucosamine via a β-1,3-linked bond to lactose or other receptor molecules (see [link to relevant documentation]). Figure 1 Preferably, the β-1,3-N-acetylglucosamine transferase used in this paper is not derived from a genetically engineered cell species, that is, the gene encoding this β-1,3-N-acetylglucosamine transferase is heterologous.

[0094] Therefore, in some embodiments, the genetically engineered cells also contain one or more recombinant nucleic acid sequences encoding β-1,3-N-acetylglucosamine transferase.

[0095] Table 2 lists non-limiting examples of β-1,3-N-acetylglucosamine transferases. Variants of β-1,3-N-acetylglucosamine transferases may also be useful, preferably having at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, or for example, 99% sequence identity with any of the β-1,3-N-acetylglucosamine transferases in Table 2.

[0096] Table 2. List of β-1,3-N-acetylglucosamine transferases

[0097]

[0098] In one embodiment, the genetically engineered cell comprises a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosamine transferase. In one embodiment, the recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosamine transferase comprises or consists of the amino acid sequence of SEQ ID NO: 14 (from LgtA of Neisseria meningitidis), or a functional homolog thereof, said functional homolog having at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or for example at least 99% sequence identity with SEQ ID NO: 14.

[0099] To produce LNnT using lactose as a substrate, a precursor of LNT-II is generated using β-1,3-N-acetylglucosamine transferase. In one embodiment, genetically engineered cells contain a β-1,3-N-acetylglucosamine transferase gene or a functional homolog or fragment thereof, which generates the intermediate LNT-II using lactose as an initial substrate.

[0100] Some of the following examples use a heterologous β-1,3-N-acetylglucosamine transferase or a variant thereof from Neisseria meningitidis, named LgtA.

[0101] β-1,3-galactosyltransferase

[0102] β-1,3-galactosyltransferase refers to any protein capable of transferring galactose from UDP-galactose via a β-1,3-linkage bond to the N-acetylglucosamine moiety of a receptor molecule. Preferably, the β-1,3-galactosyltransferase used herein is not derived from a genetically engineered cell species, i.e., the gene encoding this β-1,3-galactosyltransferase is heterologous.

[0103] Table 12 lists non-restrictive examples of β-1,3-galactosyltransferases. Variants of β-1,3-galactosyltransferases may also be used, preferably having at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, or for example, 99% sequence identity with one of the β-1,3-galactosyltransferases in Table 12.

[0104] Table 12. List of β-1,3-glycosyltransferases

[0105]

[0106] In this disclosure, the receptor molecule is a receptor sugar, such as LNT-II or a more complex HMO structure.

[0107] The following example uses a heterologous β-1,3-galactosyltransferase called GalTK or a variant thereof to produce, for example, LNFP-V.

[0108] In embodiments of this disclosure, the cell further comprises a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosyltransferase. 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: 42 (galTK from Helicobacter pylori), or a functional homolog thereof, said functional homolog having an amino acid sequence that is at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or for example at least 99% identity with SEQ ID NO: 42.

[0109] To generate LNTs from LNT-II precursors, β-1,3-galactosyltransferase is required. In one embodiment, the genetically modified cell contains the β-1,3-galactosyltransferase gene, or a functional homolog or fragment thereof.

[0110] Examples of genetically modified strains according to this disclosure are listed below, which have a specific combination of glycosyltransferases and are capable of producing LNFP-V using lactose as an initial substrate.

[0111] In one example, LgtA of Neisseria meningitidis was combined with galTK of Helicobacter pylori and Bacbac2 of Bacteroidetes to produce LNFP-V using lactose as the initial substrate.

[0112] In another example, galTK from Helicobacter pylori was combined with Bacbac2 from Bacteroidetes to produce LNFP-V using LNT-II as the initial substrate.

[0113] β-1,4-galactosyltransferase

[0114] β-1,4-galactosyltransferase is any protein capable of transferring galactose from UDP-galactose via a β-1,4-linkage bond to the N-acetylglucosamine moiety on the receptor molecule (see [link to relevant documentation]). Figure 1 Preferably, the β-1,4-galactosyltransferase used herein is not derived from a genetically engineered cell species; that is, the gene encoding this β-1,4-galactosyltransferase is heterologous. In the context described herein, the receptor molecule is a receptor sugar, such as LNT-II or a more complex HMO structure.

[0115] The following examples use heterologous β-1,4-galactosyltransferase GalT or a variant thereof to produce LNnT, for example, in combination with the α-1,3-fucosyltransferase described herein to produce LNFP-VI. Therefore, in some embodiments, genetically engineered cells contain one or more recombinant nucleic acid sequences encoding β-1,4-galactosyltransferases.

[0116] Table 2 provides non-limiting examples of β-1,4-galactosyltransferases. β-1,4-galactosyltransferase variants may also be useful, preferably having at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, or for example, at least 99% identity with the amino acid sequence of any of the β-1,4-galactosyltransferases in Table 3.

[0117] Table 3. List of β-1,4-glycosyltransferases

[0118]

[0119] In the embodiments described herein, β-1,3-N-acetylglucosamine transferase is derived from Neisseria meningitidis, and β-1,3-galactosyltransferase is derived from Helicobacter pylori.

[0120] 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: 15 (galT from Helicobacter pylori), or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or for example at least 99% sequence identity with SEQ ID NO: 15.

[0121] The generation of LNnT from the LNT-II precursor requires β-1,4-galactosyltransferase. In one embodiment, the genetically engineered cell contains a β-1,4-galactosyltransferase gene, or a functional homolog or fragment thereof. In some embodiments, β-1,3-N-acetylglucosamine transferase is derived from Neisseria meningitidis, while β-1,4-galactosyltransferase is derived from Helicobacter pylori. In other embodiments, the amino acid sequence of β-1,3-N-acetylglucosamine transferase is as shown in SEQ ID NO: 14, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 14; the amino acid sequence of β-1,4-galactosyltransferase is as shown in SEQ ID NO: 15, or a functional homolog thereof, the amino acid sequence of which has at least 80% identity with SEQ ID NO: 15.

[0122] Glycosyl-donor-nucleotide activation pathway

[0123] When performing the methods of this disclosure, a glycosyltransferase-mediated glycosylation reaction preferably occurs, wherein the activated glyconucleotide serves as a sugar donor. The activated glyconucleotide typically has phosphorylated glycosyl residues linked to a nucleoside. Specific glycosyltransferases accept only specific glyconucleotides. Therefore, preferably, the following activated glyconucleotides are involved in glycosyltransfer: glucose-UDP-GlcNAc, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine (GlcNAc), and CMP-N-acetylneuraminic acid.

[0124] The genetically engineered cells according to the present invention may contain one or more pathways to produce nucleotide-activated sugars selected from the following: glucose-UDP-GlcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine (GlcNAc), UDP-N-acetylglucosamine, and CMP-N-acetylneuraminic acid.

[0125] In one embodiment of this disclosure, genetically engineered cells are capable of producing one or more of the aforementioned activated glyconucleotides via a de novo pathway. In this regard, the activated glyconucleotides are prepared by the cells in a stepwise reaction sequence from simple carbon sources such as glycerol, sucrose, fructose, or glucose by enzymes involved in the de novo biosynthesis pathway of the respective glyconucleotide (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)).

[0126] Enzymes involved in the de novo biosynthesis of activated glyconucleotides can be naturally present in cells or introduced into cells through genetic technology or recombinant DNA technology, all of which are part of the general knowledge of technicians.

[0127] In another implementation, genetically engineered cells can use recycled monosaccharides for glyconucleotides. In the salvage pathway, monosaccharides derived from degraded oligosaccharides are phosphorylated by kinases and converted into nucleotide sugars by pyrophosphatases. The enzymes involved in this process can be heterologous to or native to the host cell.

[0128] Colanic acid gene cluster

[0129] For the production of fucosylated HMOs, the de novo GDP-fucose pathway is crucial to ensuring the availability of sufficient GDP-fucose. The E. coli capsular heteropolysaccharide gene cluster encodes selected enzymes (gmd, wcaG, wcaH, wcal, manB, manC) involved in the de novo synthesis of GDP-fucose, while one or more genes downstream of GDP-L-fucose, such as wcaJ (which is responsible for the production of the extracellular polysaccharide capsular heteropolysaccharide (the major oligosaccharide of the bacterial cell wall)), can be deleted to prevent the conversion of GDP-fucose to capsular heteropolysaccharide.

[0130] To ensure a sufficient quantity of GDP-fucose, the promoter of the natural capsular heteropolysaccharide gene cluster can be replaced with a stronger promoter to generate a recombinant capsular heteropolysaccharide gene cluster, thereby driving additional GDP-fucose production. Furthermore, as described in the examples, additional copies of the capsular heteropolysaccharide gene cluster or a selected gene thereof can be introduced into genetically engineered cells.

[0131] In this implementation, the capsular heteropolysaccharide gene cluster can be expressed from its natural genomic locus. Expression can be actively modulated. Modulation can be achieved by replacing the natural promoter with a target promoter and / or by expressing the gene cluster from another, not natural, genomic locus, or by expressing the capsular heteropolysaccharide gene cluster or a specific gene thereof in free form to increase the copy number of the capsular heteropolysaccharide gene encoding the protein.

[0132] In connection with this disclosure, the term "natural genomic locus" refers to the original and natural location of a gene cluster in the genome of a genetically engineered cell, in contrast to a capsular heteropolysaccharide gene cluster.

[0133] The de novo GDP-fucose pathway genes responsible for GDP-fucose formation include or are composed of the following genes:

[0134] i) manA, which encodes the protein mannose-6-phosphate isomerase (EC 5.3.1.8, UniProt accession number nr.P00946), promotes the interconversion of fructose-6-phosphate (F6P) and mannose-6-phosphate;

[0135] ii) manB, which encodes the protein phosphomannose mutase (EC 5.4.2.8, UniProt accession number nrP24175), participates in the biosynthesis of GDP-mannose by catalyzing the conversion of mannose-6-phosphate to mannose-1-phosphate;

[0136] iii) manC, which encodes the protein mannose-1-phosphate guanylate transferase (EC: 2.7.7.13, UniProt accession number nr P24174), is involved in the biosynthesis of GDP-mannose by synthesizing GDP-mannose from GTP and α-D-mannose-1-phosphate;

[0137] iv) gmd, which encodes the protein GDP-mannose-4,6-dehydratase (UniProt accession number nr P0AC88), which catalyzes the conversion of GDP-mannose to GDP-4-dehydro-6-deoxy-D-mannose;

[0138] v)wcaG(fcl) encodes the protein GDP-L-fucose synthase (EC 1.1.1.271, UniProt accession number nrP32055), which catalyzes a two-step NADP-dependent conversion of GDP-4-dehydro-6-deoxy-D-mannose to GDP-fucose.

[0139] Therefore, it is preferred that when one or more fucosylated heterologous products are produced, the genetically engineered cells overexpress the entire capsular heteropolysaccharide gene cluster (as shown in SEQ ID NO: 41, or its functional variants) and / or one or more genes selected from the de novo GDP-fucosylation pathway of manA, manB, manC, gmd, and wcaG.

[0140] Lactose permease

[0141] Lactose permease is a membrane protein, a member of the major promoting factor superfamily, and can be classified as a symporter. It uses a proton gradient towards the cell to transport β-galactosides such as lactose into the cell in the same direction. In oligosaccharide production, particularly in the production of human lactose oligosaccharides (HMOs), lactose is often modified to produce the initial substrate for any desired HMO undergoing biotransformation within the cell. Therefore, in HMO production, it is desirable to introduce lactose into cells, for example, by expressing / or overexpressing lactose permeases such as lacY in *E. coli*.

[0142] In the embodiments, lactose permease is as shown in SEQ ID NO: 16, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 16, for example at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0143] In this embodiment, the expression of lactose permease is regulated by a promoter according to the present disclosure.

[0144] β-galactosidase

[0145] Suitable host cells for HMO production, such as *Escherichia coli*, may contain endogenous or exogenous β-galactosidase genes; for example, *E. coli* includes the endogenous *lacZ* gene (e.g., GenBank accession number V00296 (GI: 41901)). For the purposes of this invention, when producing HMOs, it is preferable that the genetically engineered cells do not express functional β-galactosidase to avoid lactose degradation when using lactose as the initial substrate for producing complex fucosylated HMOs. In embodiments, the *lacZ* gene can be inactivated by completely or partially deleting the corresponding nucleic acid sequence from the bacterial genome, or by mutating the gene sequence in a manner that prevents transcription, or if transcribed, the transcript is not translated, or if translated into a protein (i.e., β-galactosidase), the protein does not have the corresponding enzymatic activity. In this way, the bacteria producing HMOs accumulate an increased intracellular lactose pool, which is beneficial for HMO production.

[0146] Input protein

[0147] The most common HMO-producing cells are genetically engineered to use lactose as a starting substrate because, as mentioned above, lactose is readily absorbed by lactose permease. However, using a starting substrate may necessitate reducing the amount of intracellular glycosyltransferases, as this alleviates the stress on the cell to produce multiple enzymes and reduces byproduct generation. For example, without using lactose as a starting substrate, cells containing fucosyltransferases will not produce 3FL as a byproduct, thus making fucose available for production, for example, more LNFP-VI.

[0148] Therefore, in some embodiments, the cell may also contain a substrate importer selected from lactose importers, lacto-N-trisaccharide-II (LNT-II) importers, and LNnT importers. WO2022 / 242860 discloses a method for identifying LNT-II importers. WO2023 / 099680 also discloses several potential LNT and LNT-II importers.

[0149] Examples of suitable LNT-II input proteins include:

[0150] - Lactose permease (LacY) mutants, such as LacY mutant Y236H or LacY mutant A177V+S306T, wherein the mutation is equivalent to the corresponding position in the sequence SEQ ID NO: 16;

[0151] - ABC transporter complexes, such as ABC transporters from *B. pseudocatenulatum* JCM1200 BBPC_1775, 1776, and 1777 (NCBI accession numbers BAR04453.1, BAR04454.1, and BAR04455.1, respectively) or ABC transporters from *B. breve* UCC2003 BBR_0527 / lntP1, BBR_0528 / lntP2, BBR_0530 / lntS, and BBR_0531 (NCBI accession numbers ABE95224.1, ABE95225.1, ABE95226.1, and ABE95228.1, respectively), and / or

[0152] - MFS transporter proteins, such as, but not limited to, Blon_0962 (NCBI accession number ACJ52061.1).

[0153] In other embodiments, nucleic acids or clusters of nucleic acids encoding one of these transport proteins can be introduced into genetically modified cells as described herein. Expression of such transport proteins enables the production of complex fucosylated oligosaccharides using LNT-II as an initial substrate.

[0154] Export protein

[0155] Oligosaccharide products, such as cell-produced HMOs, can accumulate in both the intracellular and extracellular matrix. These products can be passively transported into the supernatant, i.e., they diffuse beyond the cell membrane. More complex HMO products may remain within the cell, which can ultimately impair cell growth and thus affect the potential overall yield of products from a single fermentation. HMO transport can be facilitated by major facilitator superfamily transporter proteins, which promote the efflux of sugar derivatives from the cell into the supernatant. Exporting proteins can be present exogenously or endogenously and can be overexpressed under fermentation conditions to enhance the export of produced oligosaccharide derivatives (HMOs). Specificity for the oligosaccharide products to be secreted can be altered through mutation using known recombinant DNA techniques.

[0156] Therefore, the genetically engineered cells according to this disclosure may further include a nucleic acid sequence encoding an export protein, said transporter being capable of exporting one or more fucosylated human lactose oligosaccharide products, for example, the transporter may be a member of the major promoter superfamily of transporters.

[0157] In recent years, several novel and effective major promoting factor superfamily transporters have been identified as export proteins for HMOs. Each protein is specific for different recombinant-derived HMOs, and the development of recombinant cells expressing these proteins is advantageous for large-scale industrial HMO manufacturing (see, for example, WO2010 / 142305, WO2017 / 042382, WO2021 / 148615, WO2021 / 148614, WO2021 / 148611 and WO2021 / 148620).

[0158] Therefore, in one or more exemplary embodiments, the genetically engineered cell according to the method described herein further comprises a gene product that acts as an LNFP-VI and / or LNFP-V transporter. The gene product acting as an LNFP-VI or LNFP-V transporter may be encoded by a recombinant nucleic acid sequence expressed in the genetically engineered cell. The recombinant nucleic acid sequence encoding the LNFP-VI or LNFP-V transporter may be integrated into the genome of the genetically engineered cell or expressed using a plasmid.

[0159] Genetically engineered cells

[0160] In this context, the terms "genetically engineered cell" and "genetically modified cell" are used interchangeably. As used herein, a "genetically engineered cell" is a host cell whose genetic material has been altered through human intervention using genetic engineering techniques, such techniques being, but not limited to, transformation or transfection with heterologous and / or recombinant polynucleotide sequences, Crisper / Cas editing, and / or random mutagenesis. In one embodiment, the genetically engineered cell has been transformed or transfected with a recombinant nucleic acid sequence.

[0161] Genetic modifications may be selected, for example, from glycosyltransferases described in the foregoing sections, and / or metabolic pathway engineering repressors or the absence of unwanted enzymes and the inclusion of transport proteins, which will be known to those skilled in the art to be incorporated into genetically engineered cells capable of producing one or more fucosylated HMOs.

[0162] In one aspect of this disclosure, genetically engineered cells comprise a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, as described in the "α-1,3-fucosyltransferase" section above. Such genetically modified cells are capable of producing at least 14%, for example, at least 25%, of the total molar HMO content produced by the cells. In some embodiments, the total HMO produced by the cells is substantially free of LNFP-III and / or LNDFH-III, or the content of each of LNFP-III and LNDFH-III is less than 5%, for example, less than 4%, 3%, or 2%. In some embodiments, the total HMO produced by the cells is substantially free of LNFP-III and / or LNDFH-III. In this disclosure, "substantially free of LNFP-III and / or LNDFH-III" means that the content of LNFP-III and / or LNDFH-III in the total HMOs produced by the cells is less than 1% of the total molar content of the total HMOs produced by the cells, for example, less than 0.5%, less than 0.2%, or less than 0.1%. In other embodiments, the cells produced by this disclosure contain a mixture of HMOs comprising LNFP-VI, LNnT, 3FL, and / or pLNnH.

[0163] Preferably, the fucosyltransferase has α-1,3-fucosyltransferase activity, capable of fucosylation of the oligosaccharide at position 3 of the Glc moiety in LNnT or LNT, while simultaneously performing limited or no fucosylation at positions 2 or 3 of the Gal or GlcNAc moiety (see...). Figure 1 and Figure 3 Preferably, only the reducing end Glc portion is fucoidylated; more preferably, only the reducing end Glc portion of the oligosaccharide is fucoidylated by LNnT or LNT.

[0164] In one embodiment, the genetically engineered cells of this disclosure are capable of producing LNFP-VI or LNFP-V, wherein the cells contain a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase that has high specificity for the glucose (Glc) moiety in lacto-N-neotetrasaccharide (LNnT) and / or lacto-N-tetrasaccharide (LNT), and low or no specificity for the N-acetylglucosamine (GlcNAc) or galactose (Gal) moiety in LNnT or LNT, and wherein the cells produce:

[0165] a) Of the total molar content of HMOs produced by the cells, the content of LNDFH-III and / or LNFP-III is less than 5%, for example less than 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, or less than 0.1%; or

[0166] b) Of the total molar content of HMOs produced by the cells, the content of LNDFH-II and / or LNFP-II is less than 2%, for example less than 1%, 0.5%, 0.3%, 0.2% or less than 0.1%.

[0167] In other embodiments, the genetically engineered cells of this disclosure produce:

[0168] a) Of the total molar content of HMOs produced by the cells, the content of LNFP-VI is greater than 14%, for example greater than 20%, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, or for example greater than 90%; or

[0169] b) Of the total molar content of HMOs produced by the cells, the content of LNFP-V is greater than 50%, for example greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%, or for example greater than 90%.

[0170] In a preferred embodiment, the genetically engineered cells of this disclosure are capable of producing human milk oligosaccharide (HMO) lact-N-neofucopentose VI (LNFP-VI), wherein the cells contain a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase that has high specificity for the glucose (Glc) moiety in lact-N-neofetrasaccharide (LNnT) but low or no specificity for the N-acetylglucosamine (GlcNAc) or galactose (Gal) moiety in LNnT, and wherein the cells produce:

[0171] a) Of the total molar content of HMOs produced by the cells, the content of LNDFH-III and / or LNFP-III is less than 5%, for example less than 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, or less than 0.1%, and

[0172] b) Of the total molar content of HMOs produced by the cells, the content of LNFP-VI is greater than 14%, for example greater than 20%, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, or for example greater than 90%.

[0173] In other embodiments, the genetically engineered cells of this disclosure also produce one or more HMOs selected from 3FL, LNT-II, LNnT and pLNnH.

[0174] In a preferred embodiment, the cells substantially do not produce LNFP-III and / or LNDFH-III.

[0175] In a preferred embodiment, the recombinant nucleic acid encodes an α-1,3-fucosyltransferase derived from Bacteroidetes bacteria.

[0176] In other embodiments, the recombinant nucleic acid encoding α-1,3-fucosyltransferase is selected from:

[0177] a) Bacbac1, comprising or consisting of an amino acid sequence as shown in 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;

[0178] b) Bacbac2, which comprises or consists of an amino acid sequence as shown in SEQ ID NO: 2, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2;

[0179] c) Para1, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 3; and

[0180] d) CafF, which comprises or consists of an amino acid sequence as shown in SEQ ID NO: 43, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 43.

[0181] In a preferred embodiment of the invention, the genetically engineered cells capable of producing LNFP-VI comprise a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, wherein the fucosyltransferase is Bacbac1, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, or for example, at least 99% sequence identity with SEQ ID NO: 1. In these embodiments, the genetically engineered cells produce

[0182] i. LNFP-VI, whose molar content accounts for at least 30%, for example at least 35%, for example at least 40%, for example at least 45%, for example at least 50%, or for example at least 55% of the total HMOs produced by the cells, and / or

[0183] ii. LNFP-VI and 3FL, wherein the molar content of LNFP-VI and 3FL constitutes at least 90%, for example at least 95%, for example at least 99%, or for example 100% of the total HMOs produced by the cells, and / or

[0184] iii. 25-70 mol% LNFP-VI, 30-70 mol% 3FL, and 0-5 mol% LNnT, totaling 100% molar content, and

[0185] Wherein, the total molar content of HMOs produced by the cells contains less than 5% of LNnT, LNFP-III and LNDFH-III, for example less than 4%, 3%, 2%, 1%, 0.5%, 0.3% or less than 0.1%.

[0186] In a preferred embodiment of the invention, the genetically engineered cells capable of producing LNFP-VI or LNFP-V comprise a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, wherein the fucosyltransferase is Bacbac2, comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 2, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, or for example, at least 99% sequence identity with SEQ ID NO: 2. In an embodiment, the genetically engineered cells produce

[0187] i. LNFP-VI, whose molar content accounts for at least 60%, for example at least 65%, for example at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, or for example at least 90% of the total HMOs produced by the cells, and / or

[0188] ii. The LNFP-VI and LNnT produced by the cells are, for example, 85%, at least 90%, or at least 95%, or at least 99%, or at least 100% of the total HMOs produced by the cells, and / or

[0189] iii. 55-90 mol% LNFP-VI, 0-15 mol% 3FL, 0-35 mol% LNnT, and 0-10 mol% pLNnH, totaling 100% molar content, and

[0190] Wherein, the total molar content of HMOs produced by the cells contains less than 5% LNFP-III and LNDFH-III, for example less than 4%, 3%, 2%, 1%, 0.5%, 0.3%, or for example less than 0.1%; or

[0191] iv. The LNFP-V and LNT produced by the cells account for at least 90%, for example at least 95%, for example at least 99%, or for example 100% of the total HMOs produced by the cells; and / or

[0192] v. 50-70 mol% LNFP-V, 0-5 mol% 3FL, 30-50 mol% LNT, totaling 100% molar content.

[0193] Wherein, the total molar content of HMOs produced by the cells contains less than 2% LNFP-II and LNDFH-II, for example less than 1.5%, 1%, 0.5%, 0.3%, 0.2%, or for example less than 0.1%.

[0194] In a preferred embodiment of the invention, the genetically engineered cells capable of producing LNFP-VI comprise a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, wherein the fucosyltransferase is Para1, comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 3, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, or for example, at least 99% sequence identity with SEQ ID NO: 3. In these embodiments, the genetically engineered cells produce

[0195] i. The molar content of LNFP-VI produced by the cells accounts for at least 25%, for example at least 30%, for example at least 40%, for example at least 50%, for example at least 55%, for example at least 60%, or for example at least 65% of the total HMOs produced by the cells, and / or

[0196] ii. The molar amounts of LNFP-VI and LNnT produced by the cells account for at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, or for example at least 90% of the total HMOs produced by the cells, and / or

[0197] iii. 25-70 mol% LNFP-VI, 0-25 mol% 3FL, 15-65 mol% LNnT, and 0-15 mol% PLNnH, totaling 100% molar content, and

[0198] Wherein, the total molar content of HMOs produced by the cells contains less than 5% LNFP-III and LNDFH-III, for example less than 4%, 3%, 2%, 1%, 0.5%, 0.3%, or for example less than 0.1%.

[0199] In a preferred embodiment of the invention, the genetically engineered cell capable of producing LNFP-VI comprises a recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, wherein the fucosyltransferase is a CafF comprising or composed of the amino acid sequence according to SEQ ID NO:43, comprising or composed of the amino acid sequence shown in SEQ ID NO:43, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or for example at least 99% sequence identity with SEQ ID NO:43. Preferably, the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding a β-1,4-galactosyltransferase and a recombinant nucleic acid sequence encoding an LNT-II input protein. In one embodiment, the genetically engineered cell produces:

[0200] a) The molar content of LNFP-VI produced by the cells is at least 15%, for example at least 20%, for example at least 25%, for example at least 30%, for example at least 35%, for example at least 40%, or for example at least 45% of the total HMOs produced by the cells; and / or

[0201] b) The molar amounts of LNFP-VI, 3FL, and LNnT produced by the cells account for at least 80%, for example at least 85%, or for example at least 90% of the total HMOs produced by the cells; and / or

[0202] c) 10-30 mol% LNFP-VI, 35-60 mol% 3FL, 25-40 mol% LNnT, and 0-10 mol% pLNnH, totaling 100% molar content; and

[0203] Wherein, the total molar content of HMOs produced by the cells contains less than 5% LNFP-III and LNDFH-III, for example less than 4%, 3%, 2%, 1%, 0.5%, 0.3%, or for example less than 0.1%.

[0204] Another aspect of this disclosure relates to a genetically engineered cell capable of producing human lactose oligosaccharide (HMO) lact-N-fucopentose V (LNFP-V), comprising a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase that exhibits high specificity for the glucose (Glc) moiety of lact-N-tetrasaccharide (LNT) and low or no specificity for N-acetylglucosamine (GlcNAc) or galactose (Gal), wherein the total molar content of HMO produced by the cell contains less than 2%, for example less than 1%, 0.5%, 0.3%, 0.2%, or for example less than 0.1%.

[0205] Preferably, the cells also produce LNFP-V at a total molar content of more than 50% of the total HMO content.

[0206] In an embodiment, cells capable of producing LNFP-V contain one or two copies or more copies (preferably genomically integrated) of a nucleic acid sequence encoding Bacbac2 or a functional homolog thereof, wherein Bacbac2 comprises or consists of an amino acid sequence such as SEQ ID NO:2, and the amino acid sequence of the functional homolog has at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or for example at least 99% identity with SEQ ID NO:2.

[0207] The genetically engineered cells described in this article preferably express genes encoding key enzymes for the biosynthesis of fucosylated HMOs. Furthermore, if the genetically engineered cells express genes required for the production of LNnT or LNT using lactose or LNT-II as initial substrates (see [link to article]), they may express these genes. Figure 1 and Figure 3 ), and / or alternatively, it is more advantageous for cells to express the input proteins LNT-II, LNnT, or LNT.

[0208] In some embodiments, the genetically engineered cells contain one or more additional glycosyltransferases. These one or more additional glycosyltransferases are preferably selected from: galactosyltransferases, glucosyltransferases, fucosyltransferases, and N-acetylglucosyltransferases.

[0209] In one embodiment, the genetically engineered cell comprises one or more recombinant nucleic acid sequences encoding β-1,4-galactosyltransferase, and optionally a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosyltransferase. In some embodiments, the β-1,3-N-acetylglucosyltransferase is derived from Neisseria meningitidis, while the β-1,4-galactosyltransferase is derived from Helicobacter pylori.

[0210] In some embodiments, the genetically engineered cells comprise one or more recombinant nucleic acid sequences encoding β-1,3-galactosyltransferase, and optionally a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosyltransferase. In some embodiments, the β-1,3-N-acetylglucosyltransferase is derived from Neisseria meningitidis, while the β-1,3-galactosyltransferase is derived from Helicobacter pylori.

[0211] In one embodiment, the genetically engineered cells disclosed herein comprise one or more pathways for producing nucleotide-activated sugars selected from UDP-GlcNAc, GDP-fucose, UDP-galactose, and UDP-glucose. Preferably, the cells comprise all the pathways required to produce UDP-GlcNAc, GDP-fucose, UDP-galactose, and UDP-glucose. Alternatively, one or more activated nucleotides may be added to the culture medium.

[0212] In some embodiments, the genetically engineered cells described herein express the genes manA, manB, manC, gmd, and wcaG responsible for the de novo GDP-fucose synthesis pathway. Overexpression of one or more of these genes and / or upregulation of the capsular heteropolysaccharide (CA) gene cluster (including gmd, wcaG, wcaH, wcaI, manC, and manB genes) in *E. coli* by introducing nucleic acid constructs encoding CA (as shown in SEQ ID NO: 41 or equivalents) may favor GDP-fucose formation, thereby enabling cells to produce higher levels of fucosylated oligosaccharides from one or more intermediate oligosaccharide substrates (such as lactose or LNnT and / or LNT). Depending on the intended use of the substrate, genetically engineered cells may contain one or more additional glycosyltransferases and pathways for generating sugars that activate nucleotides, 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.

[0213] It is also understood that the genetically engineered cells described herein may also contain any of the modifications described above, such as additional glycosyltransferases, suitable input proteins (e.g., overexpression of lactose permease, LNT-II, or LNT input proteins), β-galactosidase inactivation (especially when lactose is used as an initial substrate), and suitable output proteins for the production of complex fucosylated HMOs by the cells.

[0214] host cells

[0215] In this embodiment, the engineered cells are microorganisms. Genetically engineered cells are preferably microbial cells, such as prokaryotic or eukaryotic cells. Suitable microbial cells that can be used as host cells include bacterial cells, archaea cells, algal cells, and fungal cells.

[0216] Genetically engineered cells can be, for example, bacterial or yeast cells. In a preferred embodiment, the genetically engineered cell is a bacterial cell.

[0217] There are no limitations on the bacterial host cells; they can be fungi (Gram-positive or Gram-negative) or archaea, as long as they allow genetic manipulation to insert the target gene and can be cultured on a manufacturing scale. Preferably, the host cells have properties that allow culture to high cell densities. Non-limiting examples of bacterial host cells suitable for the industrial production of recombinant HMOs according to this disclosure can be members of the order Enterobacterales, preferably of the genus Escherichia, and more preferably of the species E. coli. Other examples of suitable host cells are Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Campylobacter sp., Corynebacterium sp., Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillus can also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans.Similarly, the methods disclosed herein can be used to engineer bacteria of the genera *Lactobacillus* and *Lactococcus*, including but not limited to *Lactobacillus acidophilus*, *Lactobacillus salivarius*, *Lactobacillus plantarum*, *Lactobacillus helveticus*, *Lactobacillus delbrueckii*, *Lactobacillus rhamnosus*, *Lactobacillus bulgaricus*, *Lactobacillus crispatus*, *Lactobacillus gasseri*, *Lactobacillus casei*, *Lactobacillus reuteri*, *Lactobacillus jensenii*, and *Lactococcus lactis*. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species. Also included are engineered strains as described herein, derived from the genera *Enterococcus* (e.g., *Enterococcus faecium* and *Enterococcus thermophiles*), *Bifidobacterium* (e.g., *Bifidobacterium longum*, *Bifidobacterium infantis*, and *Bifidobacterium bifidum*), *Sporolactobacillus* spp., *Micromomosporas* pp., *Micrococcus* spp., *Rhodococcus* spp., and *Pseudomonas* (e.g., *Pseudomonas fluorescens* and *Pseudomonas aeruginosa*).

[0218] Non-limiting examples of fungal host cells suitable for recombinant industrial production of heterologous products are, for example, yeast cells, such as those from the genera *Komagataella*, *Kluyveromyces*, *Yarrowia*, *Pichia*, *Saccaromyces*, *Schizosaccharomyces*, or *Hansenula*, or filamentous fungi selected from the genera *Aspargillus*, *Fusarium*, or *Thricoderma*.

[0219] In one or more exemplary embodiments, the genetically engineered cells are selected from the genera *Escherichiasp.*, *Bacillus* sp., *Lactobacillus* sp., *Corynebacterium* sp., and *Campylobacter* sp.

[0220] In one or more exemplary embodiments, the genetically engineered cells are selected from Escherichia coli, Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

[0221] In one or more exemplary embodiments, the genetically engineered cell is Bacillus subtilis.

[0222] In one or more exemplary embodiments, the genetically engineered cell is Saccharomyces cerevisiae or Pichia pastoris.

[0223] In one or more exemplary embodiments, the genetically engineered cell is Escherichia coli.

[0224] In one or more exemplary embodiments, this disclosure relates to genetically engineered cells, wherein the cells are derived from Escherichia coli K-12 strain or Escherichia coli DE3 strain.

[0225] Recombinant nucleic acid sequence

[0226] This disclosure relates to genetically engineered cells comprising a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase having Glc-specific α-1,3-fucosyltransferase activity, said fucosyltransferase being, for example, an enzyme selected from Bacbac1, Bacbac2, and Para1, and said cells producing human lactose oligosaccharides (HMOs). In particular, at least one fucosylated HMO, and preferably, wherein LNFP-VI accounts for more than 25% of the total HMOs produced, for example, more than 50%.

[0227] In this context, the terms “recombinant nucleic acid sequence,” “recombinant gene / nucleic acid / nucleotide sequence / DNA encoding…” or “encoding nucleic acid sequence” are used interchangeably and are intended to refer to an artificial nucleic acid sequence (i.e., produced in vitro using standard laboratory methods for manufacturing nucleic acid sequences) that comprises a set of consecutive, non-overlapping triplets (codons) that, when controlled by appropriate control sequences (i.e. promoter sequences), are transcribed into mRNA and translated into protein.

[0228] The boundaries of a coding sequence are typically defined by the ribosome binding site upstream of the open reading frame at the 5' end of the mRNA, the transcription start codon (AUG, GUG, or UUG), and the translation stop codon (UAA, UGA, or UAG). Coding sequences can include, but are not limited to, genomic DNA, cDNA, synthetic and recombinant nucleic acid sequences.

[0229] The term "nucleic acid" includes RNA, DNA, and cDNA molecules. It should be understood that due to the degeneracy of the genetic code, a large number of nucleic acid sequences encoding a given protein can be generated.

[0230] Recombinant nucleic acid sequences can be coding DNA sequences, such as genes, or non-coding DNA sequences, such as regulatory DNA, such as promoter sequences or other non-coding regulatory sequences.

[0231] Furthermore, the recombinant nucleic acid sequence can be heterologous. As used herein, "heterologous" means a polypeptide, amino acid sequence, nucleic acid sequence, or nucleotide sequence that is foreign to the cell or organism, i.e., a polypeptide, amino acid sequence, nucleic acid molecule, or nucleotide sequence that is not naturally present in the cell or organism.

[0232] This disclosure also relates to nucleic acid constructs comprising a recombinant DNA sequence encoding a nucleic acid sequence, i.e., a target gene (such as the α-1,3-fucosyltransferase gene described herein), and a non-coding regulatory DNA sequence, such as a promoter DNA sequence, such as a recombinant promoter sequence derived from a promoter sequence of the lac operon or glp operon, or a promoter sequence derived from a promoter DNA sequence of another genome, or a synthetic promoter sequence, wherein the encoding and promoter sequences are operatively linked.

[0233] The term "operably linked" refers to a functional relationship between two or more segments of nucleic acids (such as DNA). It refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, if a promoter sequence stimulates or regulates the transcription of a coding sequence in a suitable host cell or other expression system, then the promoter sequence and coding sequence are operably linked.

[0234] Typically, promoter sequences that are operatively linked to the transcription sequence are physically continuous with the transcription sequence, meaning they act in cis.

[0235] In one exemplary embodiment, the nucleic acid construct of this disclosure may be part of a vector DNA; in another embodiment, the construct is an expression cassette / box integrated into the host cell genome.

[0236] Therefore, the term "nucleic acid construct" refers to an artificially constructed nucleic acid fragment, particularly a DNA fragment, designed to be inserted into target cells, such as bacterial cells, to modify the expression of genomic genes or the expression of gene / coding DNA sequences that may be included in the construct. Thus, in embodiments, this disclosure relates to nucleic acid constructs comprising a recombinant nucleic acid sequence encoding α-1,3-fucosyltransferase, wherein the recombinant nucleic acid sequence is selected from nucleic acid sequences encoding Bacbac1, Bacbac2, Para1, or CafF, such as the nucleic acid sequences according to SEQ ID NO: 7, 8, 9, or 44, or functional variants thereof.

[0237] The genetically engineered cells according to this disclosure may also contain multiple copies of recombinant nucleic acid sequences encoding α-1,3-fucosyltransferase. Example 1 shows that increasing the copy number of fucosyltransferase can alter the proportion of HMOs produced. Specifically, studies have shown that increasing the copy number of Para1 by introducing a high copy number plasmid (pBB-B9-Para1-PglpF) can increase the yield of LNFP-VI while decreasing the yields of LNnT and pLNnH in the strain. Furthermore, in Example 2, increasing the copy number of Bacbac2 to two genomic copies slightly increased the relative yield of LNFP-VI while decreasing the yield of LNnT.

[0238] Therefore, changes in the copy number of glycosyltransferases can be used to optimize HMO production, in this case, to optimize the yield of LNFP-VI.

[0239] Therefore, in embodiments, the genetically engineered cells of this disclosure contain one, two, three, or more copies of the genome encoding glycosyltransferases selected from the following:

[0240] a) Bacbac1, comprising or consisting of an amino acid sequence as shown in 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;

[0241] b) Bacbac2, which comprises or consists of an amino acid sequence as shown in SEQ ID NO: 2, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2;

[0242] c) Para1, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 3; and

[0243] d) CafF, which comprises or consists of an amino acid sequence as shown in SEQ ID NO: 43, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 43.

[0244] In other embodiments, the recombinant nucleic acid sequence encoding a glycosyltransferase is selected from those encoding on a plasmid:

[0245] a) Bacbac1, comprising or consisting of an amino acid sequence as shown in 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;

[0246] b) Bacbac2, which comprises or consists of an amino acid sequence as shown in SEQ ID NO: 2, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2;

[0247] c) Para1, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 3; and

[0248] d) CafF, which comprises or consists of an amino acid sequence as shown in SEQ ID NO: 43, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 43.

[0249] In other embodiments, the plasmid is a high copy number plasmid, preferably pUC57 or pBB-B9 plasmid.

[0250] One embodiment of this disclosure relates to a nucleic acid construct comprising a recombinant nucleic acid sequence encoding α-1,3-fucosylation enzyme, wherein the recombinant nucleic acid sequence is selected from:

[0251] a) A nucleic acid encoding Bacbac1, comprising or consisting of a nucleic acid sequence as shown in SEQ ID NO: 7, or a functional homolog thereof, said functional homolog having at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, or for example at least 99% sequence identity with SEQ ID NO: 7;

[0252] b) A nucleic acid encoding Bacbac2, comprising or consisting of a nucleic acid sequence as shown in SEQ ID NO: 7, or a functional homolog thereof, said functional homolog having at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, or for example at least 99% sequence identity with SEQ ID NO: 8; and

[0253] c) A nucleic acid encoding Para1, comprising or consisting of a nucleic acid sequence as shown in SEQ ID NO: 9, or a functional homolog thereof, said functional homolog having at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, or for example at least 99% sequence identity with SEQ ID NO: 9.

[0254] Preferably, the sequence encoding α-1,3-fucosyltransferase is controlled by a promoter sequence selected from promoter sequences having the nucleic acid sequences identified in Table 4. Preferably, the nucleic acid construct is suitable for genome integration in the desired host cell.

[0255] Table 4 – Selected Promoter Sequences

[0256]

[0257] * Promoter activity was evaluated in the LacZ assay described below, with the PglpF promoter serving as a positive reference in the same assay. To compare different assays, activity relative to the PglpF promoter was calculated, with ranges indicating results from multiple assays.

[0258] Promoters can be of heterologous origin, be natural promoters of gene-modified cells, or be recombinant promoters that combine heterologous and / or natural elements.

[0259] One way to increase product yield is to regulate the production of the desired enzyme activity used to produce the product, such as glycosyltransferases or enzymes involved in the biosynthesis pathway of glycosyl donors.

[0260] Increasing the promoter strength that drives the expression of the desired enzyme may be one way to achieve this goal. Promoter strength can be assessed using the lacZ enzyme assay, where β-galactosidase activity is measured as previously described (see Miller JH Experiments in molecular genetics, Cold Spring Harbor Laboratory Press, NY, 1972). Briefly, cells are diluted with Z buffer and permeated with sodium dodecyl sulfate (0.1%) and chloroform. The LacZ assay is performed at 30°C. Samples are preheated, and the assay is initiated by adding 200 μl of o-nitrophenyl-β-galactosidase (4 mg / ml), and stopped by adding 500 μl of 1 M Na₂CO₃ when the sample turns slightly yellow. The release of o-nitrophenol is then determined as a change in optical density at 420 nm. Specific activities are reported in Miller units (MU) [A420 / (min*ml*A600)]. Regulatory elements with an activity higher than 10,000 MU are considered strong, those with an activity lower than 3,000 MU are considered weak, and those in between are considered moderately strong. An example of a strong regulatory element is the PglpF promoter, with an activity of approximately 14,000 MU, while an example of a weak promoter is Plac, which has an activity of approximately 2,300 MU when induced with IPTG.

[0261] In a preferred embodiment, the expression of the nucleic acid sequence is controlled by a strong promoter selected from SEQ ID NO 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28.

[0262] The expression of the nucleic acid sequences described herein is controlled by the following: the PglpF (SEQ ID NO: 29) or Plac (SEQ ID NO: 38) promoter or PmglB_UTR70 (SEQ ID NO: 26) or PglpA_70UTR (SEQ ID NO: 27) or PglpT_70UTR (SEQ ID NO: 28), or variants thereof, such as the promoters shown in Table 4, particularly the PglpF_SD4 variant of SEQ ID NO: 24 or the Plac_70UTR variant of SEQ ID NO: 20 or the PmglB_70UTR variants of SEQ ID NO: 17, 18, 19, 21, 22, 23, 25 and 26. Other suitable variants of the PglpF, PglpA_70UTR, PglpT_70UTR and PmglB_70UTR promoter sequences are described in WO2019 / 123324 and WO2020 / 255054, respectively (incorporated herein by reference).

[0263] In a preferred embodiment, the recombinant nucleic acid sequence is individually controlled by one or more promoters selected from the group consisting of PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NO: 29, 38, 26, 27 and 28, respectively), and variants thereof.

[0264] Integrating a target nucleic acid construct contained in an expression cassette into the bacterial genome can be achieved through conventional methods, such as using linear cassettes containing flanking sequences homologous to specific sites on the chromosome, as described for the attTn7 site (Waddell CS and Craig NL, Genes Dev. (1988) Feb;2(2):137-49.); methods for nucleic acid sequence genome integration, where recombination is mediated by the Red recombinase function of phage λ or the RecE / RecT recombinase function of 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); and Red / ET recombination-based methods (Wenzel et al., Chem Biol. (2005). 12(3):349-56; Vetcher et al., Appl Environ Microbiol. (2005);71(4): 1829-35); or positive clones, i.e. clones carrying expression cassettes, which can be selected, for example, by marker genes or loss or gain of gene function.

[0265] In one or more exemplary embodiments, this disclosure relates to one or more recombinant nucleic acid sequences as shown in SEQ ID NO: 7, 8 and 9 [nucleic acid sequences encoding Bacbac1, Bacbac2 and Para1, respectively].

[0266] In particular, this disclosure relates to one or more recombinant nucleic acid sequences and / or their functional homologs, the sequences of which have at least 70% identity with SEQ ID NO: 7, 8 or 9 [nucleic acid sequences encoding Bacbac1, Bacbac2 and Para1 respectively], for example at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity or 100% identity.

[0267] Sequence identity

[0268] As used herein, the term "sequence identity" describes the correlation between two amino acid sequences or two nucleotide sequences based on their pairwise comparison, i.e., candidate sequences (e.g., sequences of this disclosure) and reference sequences (e.g., prior art sequences). For the purposes of this disclosure, sequence identity between two amino acid sequences was determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mo / .Biol. 48:443-453), 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 later (available at https: / / www.ebi.ac.uk / Tools / psa / emboss needle / ). The parameters used are gap open penalty 10, gap extension penalty 0.5, endopen 10.0, endextend 0.5, and an EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. The Needle output labeled "Identity" (obtained using the -nobrief option) is used as the percentage identity. Sequence identity is typically calculated as: (identical residues x 100) / (aligned region).

[0269] For the purposes of this publication, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) implemented in the Needle program, version 5.0.0 or higher of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), was used to determine sequence identity between two nucleotide sequences. The parameters used were a gap open penalty of 10, a gap extension penalty of 0.5, an endopen of 10.0, an endextend of 0.5, and a DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled “Identity” (obtained using the -nobrief option) was used as the identity percentage. Sequence identity can generally be calculated as: (identical nucleotide residues x 100) / (aligned region).

[0270] Functional homologs

[0271] Functional homologs or variants of protein / nucleic acid sequences as described herein are protein / nucleic acid sequences with altered genetic code that retain their original function. Functional homologs can be obtained through mutagenesis or can be naturally occurring variants from the same or other species. Functional homologs should retain at least 50%, for example, at least 60%, 70%, 80%, 90%, or 100% of the function of the protein / nucleic acid sequence.

[0272] Functional homologs of any disclosed amino acid or nucleic acid sequence may also possess higher functionality. Ideally, functional homologs of any of the α-1,3-fucosyltransferase amino acid sequences shown in Table 1, or recombinant nucleic acids encoding any of SEQ ID NO: 7, 8, 9, or 44, should be able to participate in the production of fucosylated HMOs in terms of: increased HMO yield, export of HMO products from cells or input of substrates for HMO production, such as acceptor oligosaccharides with at least three monosaccharide units, improved purity / byproduct formation, reduced biomass formation, viability of genetically engineered cells, robustness of genetically engineered cells according to this disclosure, or reduction in the amount of consumer products required for production. Specifically, any of the functional homologs of α-1,3-fucosyltransferase disclosed herein, when expressed in suitable genetically engineered cells as described herein, can produce lact-N-neofucopentose VI (LNFP-VI), wherein the proportion of fucosylated byproduct oligosaccharides having 5 or 6 monosaccharide units to the total molar content of HMO is less than 5%, for example less than 2%, and for example essentially free of LNFP-III and LNDFH-I.

[0273] Uses of genetically engineered cells or enzymes

[0274] This 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, methods for producing one or more fucosylated human lactose oligosaccharides (HMOs), preferably LNFP-VI.

[0275] Therefore, this disclosure also relates to the use of α-1,3-fucosyltransferase in the production of one or more fucosylated human lactose oligosaccharides (HMOs), wherein the α-1,3-fucosyltransferase is selected from Bacbac1, Bacbac2, Para1, and CafF, comprising or consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, or 43, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 1, 2, 3, or 43.

[0276] In some embodiments, the α-1,3-fucosyltransferase of this disclosure can also be used to produce fucosylated products, wherein the fucosylated products comprise one or more oligosaccharides, such as one or more HMOs, and wherein the products comprise LNFP-VI or LNFP-V.

[0277] In one exemplary embodiment, the genetically engineered cells and / or nucleic acid constructs described in this disclosure are used for the manufacture of HMOs. Preferably, in the manufacture of HMO mixtures, the molar percentage of LNFP-VI produced by the genetically engineered cells is higher than 14%, for example, higher than 25%, of the total amount of HMOs produced. Preferably, in the manufacture of HMOs with pure LNFP-VI as the main product, the molar percentage of LNFP-VI produced by the genetically engineered cells accounts for a proportion of higher than 70%, for example, higher than 75%, for example, higher than 80%, for example, higher than 90% of the total amount of HMOs produced.

[0278] In other embodiments, the genetically engineered cells and / or nucleic acid constructs according to this disclosure are used to manufacture HMO mixtures, wherein the molar percentage content of LNFP-VI produced by the genetically engineered cells is higher than 40% of the total amount of HMOs produced. Preferably, in the production of HMOs with pure LNFP-V as the main product, the molar percentage content of LNFP-V produced by the genetically engineered cells is higher than 50% of the total amount of HMOs produced, for example, higher than 55%, higher than 57%, or higher than 58%.

[0279] In other embodiments, the α-1,3-fucosyltransferase used to produce LNFP-VI is selected from Bacbac1, Bacbac2, Para1, and CafF, with amino acid sequences as shown in SEQ ID NO: 1, 2, 3, or 43, or functional homologs thereof, the amino acid sequences of which have at least 80% identity with SEQ ID NO: 1, 2, 3, or 43. This enzyme can be used in vivo (as described herein) and in in vitro cell-free processes.

[0280] In one exemplary embodiment, the genetically engineered cells and / or nucleic acid constructs according to this disclosure are used to manufacture LNFP-VI or LNFP-V.

[0281] The production of these HMOs may require two or more glycosyltransferase activities.

[0282] A method for producing fucoidylated human milk oligosaccharides (HMOs)

[0283] This disclosure also relates to a method for producing LNFP-VI or LNFP-V, the method comprising culturing genetically engineered cells according to this disclosure under conditions suitable for producing HMOs.

[0284] Therefore, this disclosure also relates to a method for producing human milk oligosaccharide (HMO) lact-N-neofucopentose VI (LNFP-VI), wherein the total molar content of the HMO contains less than 5% fucosylated byproducts having 5 or 6 monosaccharide units, the method comprising the following steps:

[0285] a) Provide genetically engineered cells having a recombinant nucleic acid sequence encoding α-1,3-fucosyltransferase, which has high specificity for the glucose (Glc) moiety of lact-N-neotetrasaccharide (LNnT) and low or no specificity for the N-acetylglucosamine (GlcNAc) or galactose (Gal) moiety of LNnT;

[0286] b) Culture the gene-modified cells under conditions that allow for the formation of LNFP-VI; and

[0287] c) Optionally, the LNFP-VI is purified to remove byproducts such as 3FL and / or LNnT.

[0288] Preferably, the method produces LNFP-VI with a total HMO molar content of more than 14%, for example, more than 25%.

[0289] This disclosure also relates to a method for producing human milk oligosaccharide (HMO) lact-N-neofucopentose VI (LNFP-VI), wherein the content of the fucoidylated byproduct oligosaccharide having 5 or 6 monosaccharide units in the total molar content of the HMO is less than 5%, the method comprising the following steps:

[0290] a) Provide genetically engineered cells having a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase derived from Bacteroidetes bacteria; and

[0291] b) Culture the gene-modified cells under conditions that allow for the formation of LNFP-VI; and

[0292] c) Optionally, the LNFP-VI is purified to remove byproducts.

[0293] Preferably, the method produces LNFP-VI with a total HMO molar content of more than 25%, for example, more than 30%.

[0294] Therefore, this disclosure also relates to a method for producing HMO LNFP-VI, the method comprising providing and culturing genetically engineered cells containing a recombinant nucleic acid sequence encoding α-1,3-fucosyltransferase, wherein:

[0295] a) The total molar content of LNDFH-III in the HMO produced by the method is less than 2%, for example less than 1%, for example less than 0.5%, for example less than 0.2%;

[0296] b) The total molar content of LNFP-III in the HMO produced by the method is less than 2%, for example less than 1%, for example less than 0.5%, for example less than 0.2%; and

[0297] c) The total molar content of LNFP-VI in the HMO produced by the method is greater than 25%.

[0298] In an embodiment of the method, the recombinant nucleic acid encodes α-1,3-fucosyltransferase, wherein the α-1,3-fucosyltransferase is selected from:

[0299] a) Bacbac1, comprising or consisting of an amino acid sequence as shown in 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;

[0300] b) Bacbac2, which comprises or consists of an amino acid sequence as shown in SEQ ID NO: 2, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2;

[0301] c) Para1, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 3; and

[0302] d) CafF, which comprises or consists of an amino acid sequence as shown in SEQ ID NO: 43, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 43.

[0303] Another embodiment of this disclosure is a method for producing LNFP-VI, the method comprising culturing genetically engineered cells, the genetically engineered cells comprising:

[0304] a) A recombinant nucleic acid sequence encoding an enzyme with β-1,3-N-acetylglucosamine transferase activity; and

[0305] b) A recombinant nucleic acid sequence encoding an enzyme with β-1,4-galactosyltransferase activity; and

[0306] c) A recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, wherein the enzyme is selected from:

[0307] i. Bacbac1, which comprises or consists of an amino acid sequence as shown in 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;

[0308] ii. Bacbac2, which comprises or consists of an amino acid sequence as shown in SEQ ID NO: 2, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2;

[0309] iii. Para1, comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 3, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 3; and

[0310] Lactose was used as the initial substrate for the culture.

[0311] Another embodiment of this disclosure is a method for producing LNFP-VI, the method comprising culturing genetically engineered cells, the genetically engineered cells comprising:

[0312] a) A recombinant nucleic acid sequence encoding an enzyme with β-1,4-galactosyltransferase activity; and

[0313] b) A recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, wherein the enzyme is selected from:

[0314] i. Bacbac1, which comprises or consists of an amino acid sequence as shown in 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;

[0315] ii. Bacbac2, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2; and

[0316] iii. Para1, comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 3, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 3; and

[0317] LNT-II was used as the initial substrate for culture.

[0318] In some embodiments, the total molar content of HMOs produced by the method includes less than 5% LNDFH-III and / or LNFP-III. Preferably, LNDFH-III is less than 2% and LNFP-III is less than 2%, for example, less than 1% and less than 1%, or LNDFH-III and / or LNFP-III are between 0-5%. Therefore, in embodiments, the total molar content of HMOs produced in the cultivation step includes less than 5% LNFP-III and less than 5% LNDFH-III. In some embodiments, LNFP-III and / or LNDFH-III are substantially not produced in the cultivation step.

[0319] The method particularly includes culturing genetically engineered cells that produce fucosylated HMOs, wherein the content of LNFP-VI produced in the method accounts for at least 25% of the total HMO content produced by the method, and wherein LNDFH-III and / or LNFP-III account for less than 5% of the total molar content of the produced HMOs.

[0320] In some embodiments, this disclosure also relates to a method for producing human milk oligosaccharide (HMO) lacto-N-fucopentose V (LNFP-V), wherein the content of fucosylated byproducts having 5 or 6 monosaccharide units in the total molar content of the HMO is less than 5%, the method comprising the following steps:

[0321] a) Provide genetically engineered cells having a recombinant nucleic acid sequence encoding α-1,3-fucosyltransferase, which exhibits high specificity for the glucose (Glc) moiety of lacto-N-tetrasaccharide (LNT) and low or no specificity for the N-acetylglucosamine (GlcNAc) or galactose (Gal) moiety of LNT; and

[0322] b) Culture the genetically modified cells under conditions that allow for the formation of LNFP-VI; and

[0323] c) Optionally, the LNFP-VI is purified to remove byproducts such as 3FL and / or LNT.

[0324] Preferably, the content of LNDFH-II and / or LNFP-II in the total molar content of HMO produced by the method is less than 2%. More preferably, the content of LNFP-V in the total molar content of HMO produced by the method is greater than 50%.

[0325] In other embodiments, a method for producing LNFP-V includes culturing genetically engineered cells comprising:

[0326] a) A recombinant nucleic acid sequence encoding an enzyme with β-1,3-galactosyltransferase activity; and

[0327] b) A recombinant nucleic acid sequence encoding a fucosyltransferase having α-1,3-fucosyltransferase activity, wherein the fucosyltransferase is Bacbac2, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2; and

[0328] LNT-II was used as the initial substrate for culture.

[0329] Another embodiment of the method for producing LNFP-V includes: culturing genetically engineered cells, the cells comprising:

[0330] a) A recombinant nucleic acid sequence encoding an enzyme with β-1,3-N-acetylglucosamine transferase activity; and

[0331] b) A recombinant nucleic acid sequence encoding an enzyme with β-1,3-galactosyltransferase activity; and

[0332] c) At least one copy (e.g., 1, 2, 3, 4, 5, 10, 20, or 50 copies) or more than 50 copies of a recombinant nucleic acid sequence encoding fucosyltransferase Bacbac2 or a functional homolog thereof, wherein Bacbac2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:2, and the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO:2, and

[0333] Lactose was used as the initial substrate for the culture.

[0334] Culture or fermentation in a controlled bioreactor (these terms are used interchangeably herein) typically comprises (a) a first-stage exponential cell growth in a carbon-source-secured medium, and (b) a second-stage cell growth in a carbon-limited medium, wherein the carbon source is added sequentially along with an acceptor oligosaccharide (such as lactose), thereby allowing the formation of HMO products at this stage. Carbon (sugar) limitation refers to a stage in fermentation where the growth rate is controlled by the concentration kinetics of the carbon source (sugar) in the culture medium, and the concentration of the carbon source (sugar) is determined by the rate of carbon addition to the fermenter (sugar feed rate).

[0335] The method described herein also includes providing a receptor sugar as an initial substrate for HMO formation, the receptor sugar comprising at least two monosaccharide units, the receptor sugar being exogenously added to the culture medium and / or produced by individual microbial fermentation. In addition to adding an initial substrate for HMO production to the fermentation medium, cells can be further genetically modified to produce the initial substrate intracellularly (see, for example, WO2015 / 150328).

[0336] In some embodiments, the genetically engineered cells are cultured in the presence of an initial receptor substrate selected from lactose, LNT-II, and LNnT.

[0337] Initial acceptor substrates (e.g., lactose, LNT-II, and LNnT) may be added before and / or during the culture of genetically modified cells. In a preferred embodiment, the initial substrate for HMO formation is lactose, which is added to the culture medium during fermentation of genetically engineered cells.

[0338] Furthermore, the method disclosed herein includes providing a glycosyl donor for glycosylation of a receptor substrate. Preferably, the glycosyl donor is generated by an endogenous or recombinant de novo synthetic pathway in genetically engineered cells. In a preferred embodiment of the method of the invention, the genetically engineered cells contain an upregulated biosynthetic pathway for the synthesis of fuconucleotides (e.g., GDP-fucose). Alternatively, the glycosyl donor may be synthesized individually by one or more genetically engineered cells, and / or may be exogenously added to the culture medium from other sources.

[0339] The terms “manufacturing,” “manufacturing-scale,” “large-scale production,” or “large-scale fermentation” are used interchangeably and, in the sense of this disclosure, define a fermentation with a minimum volume of 100 L, such as 1,000 L, or culture media such as 10,000 L, 100,000 L, or 200,000 L. Generally, a “manufacturing-scale” process is defined as capable of processing large volumes of desired HMO products, for example, in the case of therapeutic compounds or compositions, to meet the needs of toxicity testing, clinical trials, and market supply. Beyond the large volume, unlike simple laboratory-scale methods such as shake-flask culture, manufacturing-scale methods are characterized by a technical system using a bioreactor (fermenter) equipped with devices for agitation, aeration, nutrient feeding, and monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.). To a large extent, the behavior of the system expressed in laboratory-scale methods, such as shake flasks, benchtop bioreactors, or the deep-well format described in the examples of this disclosure, does allow for prediction of the system's behavior in the complex environment of a bioreactor.

[0340] Existing fucosyltransferases, such as FutA or FutB reported by Dumon et al., 2004, may not be suitable for large-scale production of complex fucosylated HMOs, such as LNFP-VI. This is because cells expressing such fucosyltransferases yield lower yields than those obtained by the strains disclosed herein, or because these enzymes are more heterogeneous in their specificity, i.e., they produce one or more other byproducts, such as LNFP-III and / or LNDFH-III, which complicates the purification of LNFP-VI.

[0341] Therefore, in this disclosure, a suitable fucosyltransferase refers to an enzyme capable of large-scale production of LNFP-VI or LNFP-V. Thus, the fucosyltransferases disclosed herein, namely Bacbac1, Bacbac2, and Para1, are particularly suitable for HMO production after introduction into suitable production strains.

[0342] In some embodiments, the genetically engineered cells of this disclosure are suitable for large-scale production of HMOs.

[0343] In some implementations, the methods of this disclosure are suitable for large-scale manufacturing.

[0344] There are no restrictions on the suitable cell culture medium used in the fermentation process. The medium can be semi-defined, i.e., containing complex culture medium compounds (e.g., yeast extract, soybean peptone, casein amino acids, etc.), or it can be chemically defined, without any complex compounds. The carbon source can be selected from glucose, sucrose, fructose, xylose, and glycerol. In one or more exemplary embodiments, the culture medium is supplemented with one or more energy and carbon sources selected from glycerol, sucrose, and glucose. In another embodiment, lactose is added as a substrate for HMO formation during the culture of genetically engineered cells.

[0345] The method includes culturing genetically engineered cells that produce LNFP-VI or LNFP-V, and culturing the genetically engineered cells in a medium containing an energy source (carbon source) selected from glucose, sucrose, fructose, xylose and glycerol.

[0346] In one or more exemplary embodiments, the culture medium contains sucrose as the sole source of carbon and energy. In one or more exemplary embodiments, the genetically engineered cells comprise one or more heterologous nucleic acid sequences encoding one or more heterologous polypeptides capable of utilizing sucrose as the sole source of carbon and energy for the genetically engineered cells.

[0347] In one or more exemplary embodiments, the genetically engineered cell comprises a PTS-dependent sucrose utilization system, which also comprises the scrYA and scrBR operons as described in WO2015 / 197082 (incorporated herein by reference).

[0348] Following the methods disclosed herein, LNFP-VI or LNFP-V can be collected from cell cultures or fermentation broths using conventional methods. LNFP-VI or LNFP-V can be recovered from cultures, whether from culture media and / or genetically engineered cells.

[0349] Recycling / Harvesting

[0350] Recovery of fucosylated human lactose oligosaccharides (HMOs) from culture medium and / or genetically engineered cells. In this context, the terms "recovery" and "harvest" are used interchangeably. Both "recovery" and "harvest" in this context refer to the collection of HMOs generated from the culture / culture medium after fermentation has ceased. In one or more exemplary embodiments, this may include collecting HMOs contained in both the biomass (i.e., host cells) and the culture medium, i.e., before / without separating the fermentation broth from the biomass. In other embodiments, the generated HMOs may be collected separately from the biomass and fermentation broth, i.e., after / after separating the biomass from the culture medium (i.e., fermentation broth).

[0351] Cell isolation from the culture medium can be performed using any method well known to those skilled in the art, such as any suitable type of centrifugation or filtration. Cell isolation from the culture medium can be performed immediately after harvesting the fermentation broth or at a later stage after the fermentation broth has been stored under appropriate conditions. Recovery of the resulting HMOs from the remaining biomass (or total fermentation broth) includes the extraction of HMOs from the biomass (i.e., the producing cells).

[0352] For example, after fermentation recovery, HMO can be used for further processing and purification. Purification may require multiple filtration steps; for instance, separating the byproduct LNDFH-III from LNFP-VI requires a size-dependent purification step, which is very cumbersome because LNFP-III and LNFP-VI have the same charge and mass. Therefore, it is best to avoid generating LNFP-III or LNDFH-III during fermentation processes that produce LNFP-VI.

[0353] HMOs can be purified according to procedures known in the art, such as those described in WO2017 / 152918, WO2017 / 182965, or WO2015 / 188834, the latter of which describes the purification of fucosylated HMOs. Purified HMOs can be used as nutritional products, pharmaceuticals, or for any other purpose, such as research.

[0354] At the end of the culture, oligosaccharides can accumulate as products in the intracellular and extracellular matrix.

[0355] The method according to this disclosure includes culturing genetically engineered microbial cells in a culture medium designed to support microbial growth and containing one or more carbohydrate sources or only carbon sources, such as those 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.

[0356] manufactured products

[0357] The term "manufactured product" refers to a composition of one or more HMOs intended as the product HMO, or a mixture of HMOs. Preferably, the product HMO or composition is produced using the genetically engineered cells described herein by the methods described herein.

[0358] The data from Example 2 show that Bacbac2 fucosyltransferase can produce high-purity LNFP-VI, indicating that Bacbac2 has the ability and applicability to produce high-purity LNFP-VI on a large scale.

[0359] Advantageously, the method disclosed herein provides a reduced by-product to product ratio and an increased overall yield of product (and / or total HMO). This reduced by-product formation associated with product formation promotes improved product production and enhances the efficiency of the production and product recovery processes, providing an excellent HMO manufacturing procedure.

[0360] One embodiment relates to a composition containing HMOs, for example, a nutrient product containing LNFP-VI, wherein the LNFP-VI is produced by the method of this disclosure.

[0361] The manufactured product can be a powder, composition, suspension or gel, containing one or more HMOs.

[0362] HMO mixture

[0363] The genetically engineered cells described herein, capable of producing one or more HMOs (preferably LNFP-VI), typically generate a mixture of HMOs during the multi-step production process of the final HMO product. When producing LNFP-VI using lactose as the initial substrate, cells are expected to produce 3-FL (fucosylated lactose), LNT-II, LNnT, and pLNnH, and possibly LNFP-III and LNDFH-III (see...). Figure 1 The method described herein can produce any of the following mixtures.

[0364] The molar percentages of each HMO component are supported by experimental data from the examples, and exemplary HMO compositions are shown, wherein the HMO mixture is essentially composed of LNFP-VI and LNnT, 3-FL and / or pLNnH.

[0365] In this regard, the HMO mixture may consist primarily of the following components: a) LNFP-VI and 3-FL; or b) LNFP-VI and LNnT; or c) LNFP-VI, 3-FL, and LNnT; or d) LNFP-VI, 3-FL, LNnT, and pLNnH. In embodiments, the HMO mixture is substantially composed of HMOs in the following concentration ranges: 25-90 mol% LNFP-VI, 0-70 mol% 3-FL, 0-65 mol% LNnT, 0-15 mol% pLNnH, less than 1% LNFP-III, and less than 1% LNDFH-III, totaling 100% molar content.

[0366] In this regard, one embodiment of the present disclosure relates to an HMO mixture that is substantially composed of 25-70 mol% LNFP-VI, 30-70 mol% 3FL and 0-5% LNnT, totaling 100 mol% in content.

[0367] Another embodiment of this disclosure relates to an HMO mixture, which is substantially composed of 55-90 mol% LNFP-VI, 0-15 mol% 3FL, 0-35% LNnT and 0-10 mol% pLNnH, totaling 100% mol content.

[0368] Another embodiment of this disclosure relates to an HMO mixture, which is substantially composed of 25-70 mol% LNFP-VI, 0-25 mol% 3FL, 15-65% LNnT and 0-15 mol% totaling 100 mol%.

[0369] Another embodiment of this disclosure relates to an HMO mixture that is substantially composed of 80 mol% LNFP-VI, 10 mol% 3FL and 10% LNnT.

[0370] Another embodiment of this disclosure relates to an HMO mixture that is substantially composed of 60 mol% LNFP-VI and 40 mol% LNFP-VI.

[0371] The genetically engineered cells described in this article, capable of producing LNFP-V, typically generate a mixture of HMOs during the multi-step production process of the final HMO product. When producing LNFP-V using lactose as the initial substrate, cells are expected to produce 3-FL (fucosylated lactose), LNT-II, LNT, and pLNnH, and possibly LNFP-II and LNDFH-II (see [link to article]). Figure 3 Any of the following mixtures can be produced by the methods described herein.

[0372] Example 3 illustrates an exemplary HMO composition, wherein the HMO mixture is essentially composed of LNFP-V, 3FL, and LNT.

[0373] One embodiment of this disclosure relates to an HMO mixture comprising essentially 50-70 mol% LNFP-V, 0-5 mol% 3FL, and 30-50 mol% LNT, totaling 100% molar content. Specifically, the content of byproducts LNFP-II and LNDFH-II is less than 1 mol%. Clinical data from infants suggest that human milk oligosaccharide supplements can help develop a desired microbiome by serving as a food source for beneficial bacteria in the gut. HMOs naturally present in breast milk have been studied for millennia, with HMO research (clinical and preclinical) now indicating that specific HMOs at the correct supplementation levels can provide unique health benefits. In particular, human milk oligosaccharide supplements can help support immune and gut health and have a potential role in cognitive development, which could open up future innovation opportunities.

[0374] The HMO mixtures described herein may also form part of a composition that includes other ingredients, such as active pharmaceutical ingredients, food supplements, probiotics, excipients, surfactants, etc.

[0375] Uses of HMO compositions or mixtures

[0376] HMOs are naturally present in breast milk. Through thousands of years of evolution, current HMO research (including clinical and preclinical studies) indicates that specific HMOs, when supplemented at appropriate levels, can provide unique health benefits. Since fucosylated HMOs account for more than 60% of the total HMOs in human milk, blends rich in fucosylated HMOs are particularly desirable.

[0377] Therefore, LNFP-VI and mixtures of HMOs containing LNFP-VI are highly relevant as nutritional supplements or therapeutic agents.

[0378] Clinical data from infants suggest that human milk oligosaccharide supplements can help develop a desired gut microbiota by serving as a food source for beneficial bacteria. In particular, human milk oligosaccharide supplements contribute to enhanced immunity and gut health and may play a role in cognitive development, potentially opening up opportunities for future innovations.

[0379] Mixtures or compositions of HMOs can be used to enhance beneficial bacteria in the gut microbiome. Beneficial bacteria are, for example, *Bifidobacterium* sp., *Lactobacillus* sp., or *Barnesiella* sp. Enhancement of beneficial bacteria can lead to increased production of short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate, which have been shown to have numerous benefits in infants and young children, such as inhibiting pathogens, preventing infections and diarrhea, and reducing the risk of allergies and metabolic disorders (see, for example, WO2006 / 130205, WO2017 / 129644, WO2017 / 129649).

[0380] Mixtures or compositions of HMOs produced according to the methods described herein can be used to reduce the abundance of undesirable viruses and bacteria in the gut microbiome. 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, noravirus, and rotavirus. Each of the HMOs or compositions described herein can also be used to treat and / or reduce the risk of widespread bacterial infections in humans.

[0381] The mixtures or compositions of HMOs produced according to the methods described herein can be used to increase the regeneration and viability of freeze-dried probiotics (including probiotics of the genus Bifidobacterium and Lactobacillus), and in particular, increased regeneration and / or viability in acidic environments (such as the stomach or acidic foods) and / or shelf life are advantages of using the HMO mixtures described herein. Examples of Bifidobacteria species that may have increased regeneration and viability include *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 exhibit increased regeneration and viability include *Lactobacillus rhamnosus* GG DSM 32550, *Lactobacillus rhamnosus* 19070-2 DSM 26357, *Lactobacillus rhamnosus* GG, *Lactobacillus rhamnosus* LBrGG, *Lactobacillus reuteri* DSM 12246, *Lactobacillus plantarum* TIFN101, *Lactobacillus gasseri* Lg-36 200B FloraFit Danisco, *Lactobacillus casei* DSM 32382, *Lactobacillus paracasei*, *Lactobacillus plantarum* PS 128, *Lactobacillus plantarum* (Sacco) DSM 32383, *Lactococcus lactis* PAREVE, *Lactobacillus paracasei* ssp. Paracasei and / or *Lactobacillus Probio-Tec® LGG®*, and *Lactobacillus reuteri* S12 DSM. 33752 (Limosilactobacillusreuteri S12 DSM 33752).

[0382] In the context of this application, "regeneration" means the process of regaining / restoring the vitality of dried bacteria (i.e., "regenerating" bacterial cells through rehydration, where "rehydration" means restoring fluid). This process is sometimes also referred to as "reconstruction".

[0383] In the context of this application, "viability" refers to the ability of bacterial cells to survive and function as living cells. One way to determine the viability of bacterial cells is by spreading them on an agar plate with a suitable growth medium and counting the number of colonies that form after a predetermined incubation time (plate counting). Alternatively, FACS analysis can be used.

[0384] In the context of this application, “improving the regeneration of Bifidobacterium and / or Lactobacillus” means that by increasing the amount (quantity) of Bifidobacterium and / or Lactobacillus bacteria compared to the corresponding control (i.e., the amount / quantity of Bifidobacterium and / or Lactobacillus bacteria without HMO), the bacteria are successfully regenerated / restored.

[0385] In the context of this application, “improving the viability of Bifidobacterium and / or Lactobacillus” means increasing the amount (number) of viable Bifidobacterium and / or Lactobacillus bacteria compared to the corresponding control (i.e., the amount / quantity of Bifidobacterium and / or Lactobacillus bacteria without HMO).

[0386] In the context of this application, "acidic" means a pH below 7.0 (e.g., pH ≤ 6.0, or ≤ 5.0, or ≤ 4.0, or ≤ 3.0, or in the range of 1.0–6.0, such as 2.0–5.0). pH measured in the stomach is in the range of about 1.5–3.5. pH measured in a healthy vagina is in the range of about 3.8–5.0. The pH of fruit juice is in the range of about 2.0–4.5.

[0387] Mixtures or compositions of HMOs produced according to the methods described herein can be used to extend the shelf life of probiotics such as Bifidobacterium and / or Lactobacillus.

[0388] One embodiment of the invention comprises a composition of a mixture of HMOs as described herein, particularly in the "Mixtures of HMOs" section, and one or more probiotics. Preferably, the probiotics are Bifidobacterium and / or Lactobacillus. For example, any of the specific species described above.

[0389] The mixtures or compositions of HMOs produced according to the methods described herein can be used to improve the flowability of powders or reduce the viscosity of liquids.

[0390] The HMO compositions and mixtures described in the “Manufactured Products” and “HMO Mixtures” sections may also form part of compositions that include other components, such as active pharmaceutical ingredients, food supplements, probiotics, excipients, carriers, etc.

[0391] The mixtures or compositions of HMOs described herein are used in nutritional compositions. Nutritional compositions are, for example, infant formula, rehydration solutions or dietary maintenance, medical nutrition, or supplements for the elderly or immunocompromised individuals. Macronutrients such as edible fats, carbohydrates, and proteins may also be included in such anti-infective compositions. Edible fats include, for example, coconut oil, soybean oil, and monoglycerides and diglycerides. Carbohydrates include, for example, glucose, edible lactose, and hydrolyzed corn starch. Proteins include, for example, soy protein, whey, and skim milk. Vitamins and minerals (e.g., calcium, phosphorus, potassium, sodium, chlorine, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E, D, C, and B complex) may also be included in such anti-infective compositions.

[0392] Therefore, the embodiments described herein relate to the use of HMO mixtures or compositions containing HMO mixtures (e.g., HMO mixtures produced according to this disclosure) in infant formula, dietary supplements, or medical nutrition products. In other embodiments, the compositions for use in infant formula, dietary supplements, or medical nutrition products comprise: a) LNFP-VI and 3-FL; b) LNFP-VI and LNnT; or c) LNFP-V, 3-FL, and LNnT. Such infant formula, dietary supplements, or medical nutrition products can be obtained using the methods disclosed herein.

[0393] In some embodiments, a composition comprising the HMO mixture described herein (e.g., a composition produced according to this disclosure) is a pharmaceutical composition.

[0394] This disclosure also relates to the use of mixtures or compositions according to this disclosure as dietary supplements and / or medical nutrition.

[0395] In embodiments, this disclosure relates to the use of mixtures or compositions according to the invention in infant nutrition.

[0396] sequence

[0397] This application contains a list of sequences in both text and electronic formats, which is incorporated herein by reference.

[0398] The following table summarizes the sequences described in this application. In addition, Table 3 lists the promoter sequences used in this application (promoter sequences SEQ ID NO: 17-40).

[0399] sequence

[0400]

[0401] project

[0402] 1. A method for producing human milk oligosaccharide (HMO) lacto-N-neofucopentose VI (LNFP-VI), comprising the following steps:

[0403] a) Provide genetically engineered cells having a recombinant nucleic acid sequence encoding α-1,3-fucosyltransferase, which has high specificity for the glucose (Glc) moiety of lact-N-neotetrasaccharide (LNnT) and low or no specificity for the N-acetylglucosamine (GlcNAc) or galactose (Gal) moiety of LNnT;

[0404] b) Culture the gene-modified cells under conditions that allow for the formation of LNFP-VI; and

[0405] c) Optionally, the LNFP-VI is purified to remove byproducts.

[0406] 2. The method according to Project 1, wherein the α-1,3-fucosyltransferase is derived from Bacteroidetes bacteria.

[0407] 3. The method according to Project 1, wherein the α-1,3-fucosyltransferase is selected from:

[0408] a) Bacbac2, which comprises or consists of an amino acid sequence as shown in SEQ ID NO: 2, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2;

[0409] b) Bacbac1, which comprises or consists of an amino acid sequence as shown in 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;

[0410] c) Para1, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 3; and

[0411] d) CafF, which comprises or consists of an amino acid sequence as shown in SEQ ID NO: 43, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 43.

[0412] 4. The method according to items 1 to 3, wherein the LNFP-VI produced by cells expressing the α-1,3-fucosyltransferase is substantially free of any N-acetylglucosamine (GlcNAc) fucosylated oligosaccharides.

[0413] 5. The method according to any one of the preceding items, wherein the content of fucosylated byproducts having 5 or 6 monosaccharide units in the total molar content of HMO produced by the method is less than 5%, for example less than 2.5%, for example less than 1%, for example less than 0.2%.

[0414] 6. The method according to item 4 or 5, wherein the N-acetylglucosamine (GlcNAc) fucoidylated oligosaccharide or fucoidylated byproduct having 5 or 6 monosaccharide units is LNDFH-III and / or LNFP-III.

[0415] 7. The method according to any one of the preceding items, wherein the content of LNFP-III in the total HMO molar content produced is less than 5%, for example less than 4%, 3%, 2%, 1% or for example less than 0.1%, and the molar content of LNDFH-III in the total HMO molar content produced is less than 5%, for example less than 4%, 3%, 2%, 1% or for example less than 0.1%.

[0416] 8. The method according to item 6 or 7, wherein fucoidylation byproducts having 5 or 6 monosaccharide units, such as LNFP-III and / or LNDFH-III, are substantially not generated in the culture step (b).

[0417] 9. The method according to any one of the preceding items, wherein the cells further produce one or more HMOs selected from 3FL, LNT-II and LNnT.

[0418] 10. The method according to any one of the preceding items, wherein the byproduct may be one or more HMO byproducts selected from 3FL, LNT-II, LNnT, LNFP-III, LNDFH-III and pLNnH.

[0419] 11. The method according to Item 10, wherein the only HMO byproducts are 3FL and / or LNnT, and possibly less than 5% pLNnH and / or LNT-II.

[0420] 12. The method according to any one of the preceding items, wherein the content of LNFP-VI in the total molar content of HMO produced in the cultivation step (b) is greater than 25%, for example greater than 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, or greater than 83%.

[0421] 13. The method according to any one of the preceding items, wherein the genetically engineered cells are cultured in a medium containing a receptor substrate selected from lactose, LNT-II and LNnT.

[0422] 14. The method according to any one of the preceding items, wherein the cell further comprises a substrate input protein selected from lactose input protein, lact-N-trisaccharide-II (LNT-II) input protein or LNnT input protein.

[0423] 15. The method according to any one of the preceding items, wherein the cells contain lactose input protein, and lactose is added as a substrate to the culture medium during the culture step.

[0424] 16. The method according to any one of the preceding items, wherein the conditions allowing the formation of LNFP-VI include a culture medium containing energy, preferably selected from glucose, sucrose, fructose, xylose and glycerol.

[0425] 17. The method according to any one of the preceding items, wherein the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding β-1,4-galactosyltransferase.

[0426] 18. The method according to any one of the preceding items, wherein the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosamine transferase.

[0427] 19. The method according to any one of items 13 to 18, wherein the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosamine transferase and a recombinant nucleic acid sequence encoding β-1,4-galactosyltransferase, and lactose is added as an acceptor substrate for HMO formation during the culture of the genetically engineered cell.

[0428] 20. The method according to any one of the preceding items, wherein the genetically engineered cell comprises one or more pathways for producing nucleotide-activated sugars selected from UDP-GlcNAc, GDP-fucose, UDP-galactose and UDP-glucose.

[0429] 21. The method according to Project 20, wherein the cell overexpresses at least one enzyme responsible for forming GDP-fucose in the de novo GDP-fucose synthesis pathway.

[0430] 22. The method according to item 21, wherein the one or more enzymes are selected from mannose-6-phosphate isomerase (manA), mannose phosphate mutase (manB), mannose-1-phosphate guanylate transferase (manC), GDP-mannose-4,6-dehydratase (gmd) and GDP-L-fucose synthase (wcaG).

[0431] 23. The method according to any one of the preceding items, wherein the LNFP-VI produced by the method has a purity of at least 90% in the final product.

[0432] 24. The method according to Item 23, wherein the content of other HMOs in the purified LNFP-VI product is less than 5%.

[0433] 25. A genetically engineered cell capable of producing human lactooligosaccharides (HMOs) selected from lacto-N-neofucopentose VI (LNFP-VI) or lacto-N-fucopentose V (LNFP-V), comprising a recombinant nucleic acid sequence encoding α-1,3-fucosyltransferase Bacbac2 or a functional homolog thereof, wherein Bacbac2 comprises or consists of an amino acid sequence as shown in SEQ ID NO: 2, and the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2.

[0434] 26. The genetically engineered cell according to item 25, wherein the genetically engineered cell comprises one or more recombinant nucleic acids encoding one or more heteroglycosyltransferases.

[0435] 27. The genetically engineered cell according to item 25 or 26, wherein the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosamine transferase and β-1,4-galactosyltransferase or β-1,3-galactosyltransferase.

[0436] 28. Genetically engineered cells according to any one of items 25 to 27, wherein the total molar content of HMOs produced by the cells contains less than 2%, for example less than 1.5%, for example less than 1%, for example less than 0.1%, of the following amounts: fucosylated byproducts having 5 or 6 monosaccharide units, such as LNDFH-III and / or LNFP-III or LNDFH-II and / or LNFP-II.

[0437] 29. Genetically engineered cells according to any one of items 25 to 28, wherein the total molar content of HMOs produced by the cells contains more than 25% LNFP-VI or LNFP-V, for example, more than 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, or more than 83%.

[0438] 30. The genetically engineered cell according to any one of items 25 to 29, wherein the cell further produces one or more HMOs selected from 3FL, LNT-II and LNT.

[0439] 31. The genetically engineered cell according to item 30, wherein the cell also produces LNT, and the content of 3FL and / or LNT-II in the total molar content of HMO produced by the cell is less than 2%.

[0440] 32. The genetically engineered cell according to any one of items 25 to 29, wherein the cell further produces one or more HMOs selected from 3FL, LNT-II, LNnT and pLNnH.

[0441] 33. The genetically engineered cell according to Item 32, wherein the cell produces LNnT and the content of 3FL and / or pLNnH in the total molar content of HMO produced by the cell is less than 15%.

[0442] 34. A genetically engineered cell capable of producing human milk oligosaccharide (HMO) lact-N-neofucopentose VI (LNFP-VI), comprising a recombinant nucleic acid sequence encoding α-1,3-fucosyltransferase, wherein the content of fucosylated byproducts having 5 or 6 monosaccharide units in the total molar content of HMO produced by the cell is less than 5%.

[0443] 35. The genetically engineered cells according to Item 34, wherein the total molar content of HMOs produced by said cells contains more than 25% LNFP-VI, for example, more than 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% or more than 83%.

[0444] 36. Genetically engineered cells according to any one of items 34 and 35, wherein the recombinant nucleic acid encodes α-1,3-fucosyltransferase, said α-1,3-fucosyltransferase being selected from:

[0445] a) Bacbac1, comprising or consisting of an amino acid sequence as shown in 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;

[0446] b) Bacbac2, which comprises or consists of an amino acid sequence as shown in SEQ ID NO: 2, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2;

[0447] c) Para1, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 3; and

[0448] d) CafF, which comprises or consists of an amino acid sequence as shown in SEQ ID NO: 43, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 43.

[0449] 37. The genetically engineered cell according to item 36, wherein the genetically engineered cell further comprises recombinant nucleic acid sequences encoding β-1,3-N-acetylglucosamine transferase and β-1,4-galactosyltransferase.

[0450] 38. The genetically engineered cell according to any one of items 37, wherein the β-1,3-N-acetylglucosamine transferase is derived from Neisseria meningitidis, for example, the amino acid sequence shown in SEQ ID NO: 14 or a functional homolog thereof, and the β-1,4-galactosyltransferase is derived from Helicobacter pylori, for example, the amino acid sequence shown in SEQ ID NO: 15 or a functional homolog thereof.

[0451] 39. The genetically engineered cell according to any one of items 34 to 38, wherein the fucosylation byproducts LNDFH-III and / or LNFP-III produced by the genetically engineered cell account for less than 5% of the total molar content of HMO, for example less than 2.5% or less than 1%.

[0452] 40. Genetically engineered cells according to Item 39, wherein the cells substantially do not produce LNFP-III and / or LNDFH-III.

[0453] 41. Genetically engineered cells according to any one of items 34 or 40, wherein the cells further produce one or more HMOs selected from 3FL, LNT-II, LNnT and pLNnH.

[0454] 42. The genetically engineered cell according to item 41, wherein the cell produces 3FL and / or LNnT, and less than 12% pLNnH and LNT-II, preferably less than 5% pLNnH and does not produce LNT-II.

[0455] 43. The genetically engineered cell according to any one of items 25 to 41, wherein the cell further comprises a substrate input protein selected from lactose input protein, lact-N-trisaccharide-II (LNT-II) input protein, LNT, and LNnT input protein.

[0456] 44. A genetically engineered cell according to any one of items 25 to 43, wherein the cell comprises a pathway for producing a nucleotide-activated sugar selected from UDP-GlcNAc, GDP-fucose, UDP-galactose, and UDP-glucose.

[0457] 45. Genetically engineered cells according to any one of Items 25 to 43, wherein the cells overexpress at least one enzyme responsible for GDP-fucose formation in the de novo GDP-fucose synthesis pathway.

[0458] 46. ​​The genetically engineered cell according to Item 44, wherein one or more enzymes are selected from mannose-6-phosphate isomerase (manA), mannose phosphate mutase (manB), mannose-1-phosphate guanylate transferase (manC), GDP-mannose-4,6-dehydratase (gmd) and GDP-L-fucose synthase (wcaG).

[0459] 47. The genetically engineered cell according to any one of items 25 to 46, wherein the cell further comprises a recombinant nucleic acid sequence encoding a capsular heteropolysaccharide (CA) gene cluster, as shown in SEQ ID NO: 41.

[0460] 48. A genetically engineered cell according to any one of items 25 to 47, wherein the cell further comprises a nucleic acid sequence encoding an MFS transporter protein capable of exporting LNFP-VI or LNFP-V into an extracellular medium.

[0461] 49. Genetically engineered cells according to any one of items 25 to 48, wherein the recombinant nucleic acid sequence is controlled by one or more promoters selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NO: 29, 38, 26, 27 and 28) and variants thereof.

[0462] 50. The genetically engineered cell according to any one of items 25 to 49, wherein the genetically engineered cell is a microorganism.

[0463] 51. The genetically engineered cell according to any one of items 25 to 50, wherein the genetically engineered cell is a bacterium or fungus.

[0464] 52. The genetically engineered cell according to Item 51, wherein the fungus is selected from yeast cells of the genera *Komagataella*, *Kluyveromyces* sp., *Yarrowia* sp., *Pichia* sp., *Saccaromyces* sp., *Schizosaccharomyces* sp., or *Hansenula* sp., or filamentous fungi selected from the genera *Aspargillus* sp., *Fusarium* sp., or *Thricoderma* sp.

[0465] 53. The genetically engineered cell according to Item 51, wherein the bacteria are selected from the genera Escherichia sp., Bacillus sp., lactobacillus sp., Corynebacterium sp. and Campylobacter sp.

[0466] 54. Genetically engineered cells according to any one of items 25 to 53, wherein the engineered cells are selected from Escherichia coli, Bacillus subtilis, lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

[0467] 55. Use of α-1,3-fucosyltransferase Bacbac2 or a functional homolog thereof comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 2 for the production of one or more fucosylated HMOs, wherein the amino acid sequence of the functional homolog has at least 80% identity with the amino acid sequence of SEQ ID NO: 2.

[0468] 56. According to the use described in item 55, wherein the fucosylated HMO is selected from LNFP-V and LNFP-VI.

[0469] 57. As described in item 55 or 56, wherein the total molar content of HMO produced contains less than 15% 3FL and substantially no LNFP-II, LNFP-III, LNDFH-II and / or LNDFH-III are produced.

[0470] 58. Use of α-1,3-fucosyltransferase in the production of LNFP-VI, wherein the α-1,3-fucosyltransferase is selected from Bacbac1, Bacbac2, Para1, and CafF, or their functional homologs, wherein Bacbac1, Bacbac2, Para1, and CafF respectively comprise or consist of the amino acid sequence shown in SEQ ID NO: 1, 2, 3, or 43, and the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 1, 2, 3, or 43.

[0471] 59. An HMO mixture, basically composed of the following:

[0472] a) LNFP-VI and 3-FL, or

[0473] b) LNFP-VI and LNnT, or

[0474] c) LNFP-VI, 3-FL and LNnT.

[0475] 60. The HMO mixture according to item 59 consists essentially of the following:

[0476] a) 25-70 mol% LNFP-VI, 35-70 mol% 3FL, 0-5% LNnT; or

[0477] b) 55-90 mol% LNFP-VI, 0-15 mol% 3FL, 0-35% LNnT, and 0-10 mol% pLNnH; or

[0478] c) 25-70 mol% LNFP-VI, 0-25 mol% 3FL, 15-65% LNnT and 0-15 mol% pLNnH,

[0479] Total content: 100% molar content.

[0480] 61. The HMO mixture according to item 59 or 60, wherein the mixture is produced by any one of items 1 to 24, or by genetically engineered cells according to any one of items 34 to 54.

[0481] 62. A composition comprising an HMO mixture according to any one of items 59 to 61.

[0482] 63. The composition according to item 62, wherein the mixture further comprises one or more probiotics.

[0483] 64. The composition according to item 63, wherein the probiotic is Bifidobacterium sp. and / or Lactobacillus sp.

[0484] 65. Use of the mixture according to any one of items 59 to 61 or the HMO composition according to any one of items 62 to 64 in infant formula, dietary supplements and / or medical nutrition products.

[0485] 66. A method for producing human milk oligosaccharide (HMO) lacto-N-fucopentose V (LNFP-V), wherein the total molar content of HMOs in the fucosylation byproduct having 5 or 6 monosaccharide units is less than 2%, the method comprising the following steps:

[0486] a) Provide genetically engineered cells having a recombinant nucleic acid sequence encoding α-1,3-fucosyltransferase, which has high specificity for the glucose (Glc) moiety of lact-N-neotetrasaccharide (LNnT) and low or no specificity for the N-acetylglucosamine (GlcNAc) or galactose (Gal) moiety of LNnT;

[0487] b) Culture the genetically modified cells under conditions that allow for LNFP-V formation; and

[0488] c) Optionally, the LNFP-V is purified to remove byproducts.

[0489] 67. The method according to item 66, wherein the genetically engineered cell is a cell according to any one of items 25 to 31, and wherein the total molar content of HMO produced by the method contains less than 2%, for example less than 1%.

[0490] 68. The method according to any one of items 66 or 67, wherein the total molar content of HMO produced by the method comprises at least 50% LNFP-V, for example at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or for example at least 59%.

[0491] 69. The method according to any one of items 66 to 68, wherein the content of LNDFH-II in the total molar content of HMO produced by the culture is less than 4%.

[0492] 70. The method according to any one of items 66 to 69, wherein LNFP-II and / or LNDFH-II are substantially not generated during the culture step.

[0493] 71. The method according to any one of items 66 to 70, wherein genetically engineered cells are cultured in a medium containing a receptor substrate selected from lactose, LNT-II and LNT.

[0494] 72. The method according to items 66 to 71, wherein the conditions allowing LNFP-V formation include a culture medium containing an energy source preferably selected from glucose, sucrose, fructose, xylose and glycerol.

[0495] 73. An HMO mixture, which is basically composed of LNFP-V, 3-FL and LNT.

[0496] 74. The HMO mixture according to item 73 is basically composed of 50-70 mol% LNFP-V, 0-5 mol% 3FL and 30-50 mol% LNT, totaling 100 mol% content.

[0497] 75. An HMO mixture according to item 73 or 74, wherein the mixture is produced by a method according to any one of items 66 to 72, or by genetically engineered cells according to any one of items 25 to 31.

[0498] 76. A composition comprising an HMO mixture according to any one of items 59 to 61 or according to any one of items 73 to 75.

[0499] 77. The composition according to item 76, wherein the mixture further comprises one or more probiotics.

[0500] 78. The composition according to item 77, wherein the probiotic is Bifidobacterium and / or Lactobacillus.

[0501] 79. Using a mixture or composition according to any one of items 73 to 76 to enhance the viability and / or regeneration capacity of Bifidobacterium and / or Lactobacillus bacteria for probiotic use in humans or animals.

[0502] 80. Use of the mixture according to any one of items 73 to 74 or the HMO composition according to any one of items 76 to 78 in infant formula, dietary supplements and / or medical nutrition products.

[0503] Example

[0504] method

[0505] Unless otherwise stated, standard techniques, vectors, control sequence elements, and other expression system elements known in the field of molecular biology are used for nucleic acid manipulation, transformation, and expression. Such standard techniques, vectors, and elements can be found in the following literature: Ausubel et al. (eds.), Current Protocols in Molecular Biology (1995) (John Wiley & Sons); Sambrook, Fritsch, & Maniatis (eds.), Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, NY); Berger & Kimmel, Methods in Enzymology 152: Guide to Molecular Cloning Techniques (1987) (AcademicPress); Bukhari et al. (eds.), 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).

[0506] The invention is described below using embodiments which are not intended to limit the invention in any way.

[0507] Enzymes:

[0508] Fifty enzymes with fucosyltransferase activity were screened, and four previously unreported enzymes capable of glucose-specific α-1,3-fucosyltransferase activity in treating LNnT were obtained. These enzymes can generate complex fucosylated HMO LNFP-VI without producing other complex fucosylation byproduct oligosaccharides during culture. Table 5 lists the GenBank IDs and sources of these four glucose-specific α-1,3-fucosyltransferases (Bacbac1, Bacbac2, Para1, and CafF), a multispecific α-1,3-fucosyltransferase (Prev1), and existing α-1,3-fucosyltransferases FutA, FutB, and FutT109 / CafA.

[0509] Table 5. List of enzymes tested within the framework of this disclosure

[0510]

[0511] ^The sequences used in this application may be truncated at their N-terminus or C-terminus relative to the GenBank sequences, and these sequences are represented by SEQ ID NO.

[0512] ^Dumon et al., 2004 (α-1,3-fucosyltransferase, Biotechnol. Prog. 2004, 20, 412−419) showed that FutA can produce a mixture of LNFP-VI, LNDFH-III and 3FL, and FutB can produce a mixture of LNFP-VI, LNFP-III, LNDFH-III and 3FL.

[0513] *WO2016 / 040531 indicates that CafC and CafF can produce 3FL.

[0514] **WO2019 / 000133 indicates that FucT109 can produce LNFP-V and LNFP-VI.

[0515] strain

[0516] The strains (genetically engineered cells) constructed in this application are based on *Escherichia coli* K-12 DH1 with the following genotypes: Fˉ, λˉ, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. The *Escherichia coli* K-12 DH1 strain underwent additional modifications to generate MDO strains 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 a Plac promoter was inserted upstream of the gmd gene.

[0517] The method of inserting a target gene into the genome of Escherichia coli is well known to those skilled in the art. Gene cassettes can be inserted into the E. coli chromosome using specific selectable 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).

[0518] To obtain an LNnT-producing strain, the MDO strain was further modified by integrating β-1,3-GlcNAc transferase (LgtA from Neisseria meningitidis, homologous to NCBI accession number WP_033911473.1, shown as SEQ ID NO: 14) and β-1,4-galactosyltransferase (GalT from Helicobacter pylori, homologous to GenBank ID WP_001262061.1, shown as SEQ ID NO: 15) onto the chromosome. Both enzymes are under the control of the PglpF promoter (SEQ ID NO: 29). This strain was named the LNnT strain.

[0519] 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 number WP_033911473.1, shown as SEQ ID NO: 14) and β-1,3-galactosyltransferase (GalTK, from Helicobacter pylori, homologous to GenBank accession number BD182026.1, shown as SEQ ID NO: 42) onto the chromosome. Both enzymes are controlled by the PglpF promoter (SEQ ID NO: 29). This strain was named the LNT strain.

[0520] Codon-optimized DNA sequences encoding a single α-1,3-fucosyltransferase were integrated into the genome of LNnT or LNT strains.

[0521] Table 6 lists the genotypes of the background strain (MDO), the LNnT strain, and the α-1,3-fucosyltransferase expression strain capable of producing LNFP-VI.

[0522] Table 6. Genotypes of the strains used in this example that produce LNDFH-III.

[0523]

[0524] *1,3FT is an abbreviation for α-1,3-fucosyltransferase, which inserts DNA sequences into the host strain's genome or integrates them via plasmids.

[0525] 1 lgtA-PglpF - Two genomic insert copies of the gene encoding β-1,3-N-acetylglucosamine transferase (SEQ ID NO: 14) under the control of the PglpF promoter.

[0526] 2galT-PglpF - A genomic insert gene encoding β-1,4-galactosyltransferase (SEQ ID NO:15) under the control of the PglpF promoter.

[0527] 3 CA = an additional capsular heteropolysaccharide gene cluster (gmd-wcaG-wcaH-wcaI-manC-manB, SEQ ID NO: 41) located at a locus different from the natural locus and under the control of the PglpF promoter.

[0528] 4 pUC57 is a high copy number (>300) plasmid with the pUC origin of replication. The antibiotic resistance marker on the pBB vector is ampicillin. The α-1,3-galactosyltransferase shown is expressed by this plasmid.

[0529] 4 galTK-PglpF – a genomic insertion gene encoding β-1,3-galactosyltransferase (SEQ ID NO: 42), controlled by the PglpF promoter.

[0530] Deep well assay

[0531] The deep-well assays in this embodiment were performed as originally described by Lv et al. (Bioprocess Biosyst Eng 20 (2016) 39:1737-1747) and optimized for the purposes of this disclosure. More specifically, the strains disclosed in this embodiment were screened in 96-well plates using a 4-day protocol. During the first 24 hours, the pre-cultures were grown to high density (OD600 up to 5) and then transferred to a medium that allows for the induction of gene expression and product formation.

[0532] More specifically, during Day 1, fresh precultures were prepared using a basal minimal medium (BMM) (pH 7.0) supplemented with magnesium sulfate (0.12 g / L), thiamine (0.004 g / L), and glucose (5.5 g / L). The basal minimal medium had the following composition: NaOH (1 g / L), KOH (2.5 g / L), KH₂PO₄ (7 g / L), NH₄H₂PO₄ (7 g / L), citric acid (0.5 g / L), and a trace mineral stock solution (5 mL / L). The trace mineral stock solution contained: ZnSO₄·7H₂O 0.82 g / L, citric acid 20 g / L, MnSO₄·H₂O 0.98 g / L, FeSO₄·7H₂O 3.925 g / L, and CuSO₄·5H₂O 0.2 g / L. The pH of the basal minimal medium was adjusted to 7.0 with 5N NaOH and autoclaved. The preculture was incubated at 34°C and 1000 rpm for 24 hours with shaking, and then further transferred to 0.75 mL of fresh BMM (pH 7.5) to begin the master culture. The fresh BMM was supplemented with magnesium sulfate (0.12 g / L), thiamine (0.02 g / L), a first-dose glucose solution (0.1–0.15 g / L), and a first-dose lactose solution (5–20 g / L). Additionally, 20% sucrose stock solution (40–45 g / L) or maltodextrin (19–20 g / L) was used as the carbon source, with specific hydrolases, sucrose hydrolases, or glucoamylases added, respectively, to ensure glucose release at a rate suitable for carbon-limited growth, similar to a typical fed-batch fermentation process. The master culture was incubated at 28°C and 1000 rpm for 72 hours with shaking. To analyze the total fermentation broth, 96-well plates were boiled at 100°C, centrifuged, and the supernatant was analyzed by HPLC.

[0533] Fermentation

[0534] The *E. coli* strain was cultured in a 250 mL fermenter (Ambr250 HT Bioreactor system, Sartorius) starting with 100 mL of mineral medium consisting of 30 g / L glucose and NH4H2PO4, KH2PO4, MgSO4 x 7H2O, KOH, NaOH, citric acid, trace element solution, antifoaming agent, and thiamine. Dissolved oxygen levels were maintained at 20% by first stirring and then initiating a gas flow at 700 rpm (maximum 4500 rpm) and 1 VVM (maximum 3 VVM). The pH was maintained at 6.8 by titration with 8.5% NH4OH solution. The culture began with a 2% (v / v) inoculum from a preculture containing 10 g / L glucose, (NH4)2HPO4, KH2PO4, MgSO4 x 7H2O, KOH, NaOH, citric acid, trace element solution, antifoaming agent, and thiamine. After the glucose in the basal minimal medium was depleted, a feed solution containing glucose, MgSO4x7H2O, H3PO4, and trace minerals was continuously added to the fermenter at a rate maintaining carbon-limiting conditions. The temperature was initially set at 33°C but was reduced to 30°C in a linear ramp of 3 hours after 12 hours of feeding. Lactose was added in a push-feed manner as a 25% lactose monohydrate solution at 36 hours of feeding, and then every 19 hours, to prevent lactose from becoming a rate-limiting factor. Cell growth, metabolic activity, and metabolic state were tracked by online measurements of agitation, dissolved oxygen tension, reflectance, NH4OH base addition, O2 uptake rate, and CO2 release rate. Throughout fermentation, HPLC sampling was used to determine the concentrations of HMO products, lactose, and other minor byproducts.

[0535] Example 1 – In vivo LNFP-VI synthesis

[0536] We screened gene-modified cells expressing a single α-1,3-fucosyltransferase to assess their ability to produce fucosylated HMO LNFP-VI.

[0537] Four enzymes that produce fewer complex fucosylated oligosaccharide byproducts were identified and compared with enzymes that have broader specificity for the monosaccharide units present in LNnT. The ability of these enzymes to synthesize LNFP-VI was tested after being introduced into gene-modified cells that produce LNnT and GDP-fucose (Table 7).

[0538] Following the methods section, gene-modified strains expressing four α-1,3-fucosyltransferases (Table 5) were constructed. These cells were then screened in a deep-well assay apparatus following the methods section.

[0539] Table 6 lists the genotypes of strains capable of producing LNFP-VI. The molar amounts of various HMOs produced by each strain were calculated using HPLC analysis.

[0540] Table 7 shows the results of LNFP-VI cell production, expressed as the percentage (%) of HMO produced by each strain relative to the total HMO molar content.

[0541] Table 7: Percentage of each HMO produced by each strain in the total HMO molar content (mM) (results are the average of at least 3 replicate experiments).

[0542]

[0543] As shown in Table 7, four previously unreported enzymes capable of producing LNFP-VI—Bacbac1, Bacbac2, Para1, and CafF—can specifically transfer fucosylation units to the Glc region of LNnT. FutA, CafC, and FucT109, reported by Dumon et al. (2004), WO2016 / 040531, and WO2019 / 008133, respectively, and Prev1 reported in this paper, can transfer fucosylation units to the Glc and GlcNAc regions of LNnT via α-1,3 linkages, thereby producing LNFP-III and / or LNDFH-III. FutB, reported by Dumon et al. (2004), appears to be a low-efficiency α-1,3-fucosylation enzyme when using LNnT as the backbone.

[0544] As shown in Table 7, the three novel enzymes, Bacbac1, Bacbac2, and Para1, can specifically link fucosylation units to the Glc moiety of LNnT via α-1,3 linkages, forming LNFP-VI, which accounts for more than 25% of the total HMOs, without producing complex fucosylation byproducts HMOs, LNDFH-III, or LNFP-III. In contrast, the existing enzymes FutA and FucT109, and enzyme Prev1, produce LNFP-VI, accounting for 65%, 18%, and 30% of the total HMOs, respectively, while also producing a large amount of the unintended byproduct LNDFH-III, at 25%, 24%, and 41%, respectively. Furthermore, enzymes FucT109 and Prev1 also produce 27% and 15% of the unintended HMO byproduct LNFP-III, respectively.

[0545] None of the four enzymes, Bacbac1, Bacbac2, Para1, and CafF, produced any LNFP-III or LNDFH-III. This clearly demonstrates that these enzymes are highly specific for the Glc moiety of LNnT, but do not possess any fucosyltransferase activity for the GlcNAc or Gal moiety of LNnT. Therefore, enzymes Bacbac1, Bacbac2, Para1, and CafF can simplify the production of LNFP-VI without generating unwanted HMOs such as LNDFH-III and LNFP-III, thus simplifying the purification process, which is highly advantageous for large-scale production.

[0546] Interestingly, Dumon et al. (2004) proposed that FutB would produce a mixture of LNDFH-III, LNFP-III, LNFP-VI and 3FL, but in reality, FutB does not produce any LNDFH-III, only a small amount of LNFP-III and LNFP-VI.

[0547] Furthermore, as shown in Table 8 (which displays the total amount of HMOs in single-copy strains), strains expressing Bacbac1, Para1, CafF, CafC, and FucT109 produced a higher total amount of HMOs than strains expressing FutA.

[0548] Table 8: Comparison of total HMO production by each strain with that of FutA strain

[0549]

[0550] For purification of the produced LNFP-VI, the absence of other fucosylated species in the resulting mixture is highly advantageous. Furthermore, obtaining pure LNFP-VI through low-level LNnT expression of Bacbac1 and Para1 at high copy numbers is also beneficial. Additionally, the fact that these enzymes do not significantly affect the overall HMO production capacity of the cell is also highly advantageous. Even the lowest-yielding Bacbac2 strain still achieves approximately 87% of the HMO yield of the FutA strain, thus maintaining an advantage because the produced HMOs do not contain the unwanted byproducts LNFP-III and LNDFH-III, resulting in a higher overall LNFP-VI yield.

[0551] Example 2 – Production of LNFP-VI by fermentation using Bacbac1 and Bacbac2

[0552] To verify the HMO profile observed in the deep-well assay, particularly the proportion of LNFP-VI in the total HMO content produced, we fermented the strains expressing Bacbac1 and Bacbac2 from Example 1 (containing a single copy of the Bacbac1 genome or one or two copies of the Bacbac2 genome, respectively), as detailed in the "Methods" section above. The results are shown in Table 9.

[0553] Table 9: Percentage of each HMO content in the total HMO content produced by the strain

[0554]

[0555] As can be seen from the data in Table 9, the proportion of LNFP-VI produced by strain Bacbac1 was lower than that produced in deep well assays, while the amount of LNFP-VI produced by strain Bacbac2 was higher than that produced in deep well assays.

[0556] Both strains demonstrated that Bacbac1 and Bacbac2 are suitable for the production of LNFP-VI, producing extremely low amounts of similar complex fucosylation byproducts HMO (LNFP-III and LNDFH-III) during fermentation. In particular, strain Bacbac2 produces extremely low amounts of LNnT and 3FL, and does not produce LNFP-III or LNDFH-III, thus enabling the acquisition of high-purity LNFP-VI at a relatively low purification cost.

[0557] Example 3 – In vivo LNFP-V Synthesis

[0558] Genetically modified cells expressing one or two copies of the α-1,3-fucosyltransferase genome were screened to detect their ability to produce fucosylated HMO LNFP-V (genotypes are listed in Table 6).

[0559] Using the deep-well assay apparatus described in the “Methods” section, the ability of these cells and these enzymes to synthesize LNFP-V after being introduced into genetically modified cells capable of producing LNT and GDP-fucose was evaluated.

[0560] Table 10 shows the content of HMOs (including LNFP-V) produced by LNT scaffold cells under different α-1,3-fucosyltransferase expression conditions, expressed as a percentage (%) of the total molar content of HMOs produced by each strain.

[0561] Table 10: Percentage of various HMOs produced by LNT background strains relative to total HMO content

[0562]

[0563] As shown in Table 10, the Bacbac2 enzyme exhibits very similar activities towards LNT and LNnT. It specifically transfers fucosylation units to the Glc moiety of LNT, rather than the GlcNAc moiety, thus avoiding the formation of complex fucosylated HMO byproducts such as LNFP-II and LNDFH-II. This indicates that Bacbac2 does not possess α-1,4-fucosylation activity. The Bacbac1 and Para1 enzymes appear to have very low activity towards any part of LNT, acting in a manner essentially similar to that of FutB in LNnT strains. On the other hand, FutB exhibits some fucosylation activity and appears to be specific to the glucose moiety on LNT. CafF appears to have slightly higher activity towards lactose in LNT-background strains and produces only a small amount of complex fucosylated HMOs. The enzymes FutA, FucT109, and CafC reported by Dumon et al. in 2004, WO2019 / 008133, and WO2016 / 040531 respectively appear to have certain α-1,4-fucosyltransferase activity, and therefore can fucosylate the GlcNAc moiety in LNTs, thereby generating some LNFP-II or LNDFH-II in the LNT background.

[0564] As shown in Table 10, the novel enzyme Bacbac2 is the only enzyme capable of specifically transferring fucosylation units to the Glc moiety of LNT via α-1,3 linkages, thereby forming LNFP-V. Its content exceeds 50% of the total HMOs, and it does not produce LNDFH-II or LNFP-II. In contrast, the existing enzyme FutA produces 79% LNFP-V of the total HMOs, while also generating 13% of the unwanted product LNDFH-II.

[0565] The data also indicate that enzyme specificity varies depending on the HMO backbone produced by the expressing strain. Only Bacbac2 appears to maintain similar activity in both LNnT and LNT background strains.

[0566] When using the Bacbac2 enzyme in LNT-background strains, it is highly advantageous when purification of LNFP-V is required because the resulting mixture lacks other complex fucosylation byproducts. Therefore, it remains advantageous even if the percentage of LNFP-V in the total HMO yield is low, as no LNDFH-II or LNFP-II is produced. The product profile of another enzyme, FutB, shows a significant reduction in LNFP-V production compared to the Bacbac2 strain.

[0567] Furthermore, as shown in Table 11, the total HMO produced by the 2x Bacbac2 strain (relative to the total yield of 127%) was higher than that produced by the strain carrying 2x FutA, which resulted in the Bacbac2 strain producing 95% of the LNFP-V of the FutA strain. Interestingly, the total HMO produced by the FutB strain appeared to be even higher than that of the Bacbac2 strain, but its LNFP-V yield was still lower than that of the Bacbac2 strain.

[0568] Table 11. Percentage of total HMO and LNFP-V produced by each strain relative to the 2x FutA strain.

[0569]

[0570] Therefore, the amount of LNFP-V produced by strain Bacbac2 is almost the same as that of strain FutA, while it does not produce the byproduct LNDFH-II, which is very advantageous.

[0571] Example 4 – Regeneration and Viability of Lyophilized Lactobacillus rhamnosus

[0572] Probiotics can be consumed in live or dried (e.g., freeze-dried) form. Regardless of the drying method, rehydration is a crucial step in the recovery of dehydrated bacteria; inadequate rehydration / regeneration leads to poor cell viability and ultimately low survival rates. Therefore, rehydration is a critical step in the recovery process of freeze-dried cultures. For both live and rehydrated bacteria, survival under acidic conditions is essential, as they require the acidic environment of the stomach and may also need to be stored in acidic foods (shelf life).

[0573] In this example, we tested whether an HMO mixture similar to the HMO mixtures produced by the strains described in Examples 1 and 2 could promote the rehydration (regeneration) and survival of the probiotic strain *Lactobacillus rhamnosus* Probio-Tec® LGG® - DSM 33156. This test was conducted under acidic conditions to simulate the survival environment of bacteria when passing through the stomach or ingesting acidic beverages.

[0574] Add the lyophilized probiotics (0.4 mg / ml) alone (control) or mixed with the HMO mixture (5% w / v) shown in Table 13 to a test tube. Dissolve the mixture in sterile phosphate-buffered saline (PBS, pH=3), heat to 37°C, and stir vigorously for about 30 seconds until no visible lumps remain. Incubate the test tubes at 37°C for 3 hours. Further dilute the sample, spread 100 µl in duplicate onto MRS agar plates, and incubate in an anaerobic incubator at 37°C.

[0575] Table 13: HMO components tested in this embodiment

[0576]

[0577] The CFU / ml calculation is based on the colony count (average of two plates) after 48 hours of incubation. Figure 4 The plate photographs of Lactobacillus rhamnosus DSM 33156 colonies after 48 hours of incubation are shown. Results for all three strains are summarized in Table 14.

[0578] Table 14: Average CFU / ml of the specified strain after 3 hours of acid treatment and 48 hours of further culture at 37°C.

[0579]

[0580] The freeze-dried Lactobacillus strains dissolved in a human milk oligosaccharide (HMO) mixture described in this article exhibited significantly improved regeneration capacity and survival rate compared to the control group without the addition of an HMO mixture. These data clearly demonstrate that under low pH conditions (e.g., in the stomach or acidic beverages), any HMO mixture can enhance the regeneration capacity and survival rate of Lactobacillus rhamnosus strains.

[0581] To the best of our knowledge, no previous studies have shown that the tested mixture can improve the regeneration capacity and survival rate of Lactobacillus strains in acidic environments.

Claims

1. A method for producing human milk oligosaccharide (HMO) lacto-N-neofofucopentose VI (LNFP-VI), wherein, The method comprises the following steps: The content of fucosylated byproduct oligosaccharides having 5 or 6 monosaccharide units in the total molar content of HMO is less than 5%. a) Provide genetically engineered cells having a recombinant nucleic acid sequence encoding an α-1,3-fucosyltransferase derived from Bacteroidales bacteria; b) Culture the gene-modified cells under conditions that allow for the formation of LNFP-VI; and c) Optionally, the LNFP-VI is purified to remove byproducts such as 3FL and / or LNnT.

2. The method according to claim 1, wherein, The α-1,3-fucosyltransferase exhibits high specificity for the glucose (Glc) moiety in lact-N-neotetrasaccharide (LNnT), but low or no specificity for the N-acetylglucosamine (GlcNAc) or galactose (Gal) moiety in LNnT.

3. The method according to claim 1 or 2, wherein, The α-1,3-fucosyltransferase is selected from: a) Bacbac2 or a functional homolog thereof comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 2, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2; or b) Bacbac1 or a functional homolog thereof comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO:

1.

4. The method according to any one of claims 1 to 3, wherein, In the total molar content of HMOs produced in culture step (b), the content of LNDFH-III and / or LNFP-III is less than 5%, for example less than 2.5%, for example less than 1%, for example less than 0.2%.

5. The method according to any one of the preceding claims, wherein, The cells also produce one or more HMOs selected from 3FL, LNT-II, LNnT and pLNnH.

6. The method according to any one of the preceding claims, wherein, Of the total molar content of HMOs produced in culture step (b), the content of LNFP-VI was higher than 25%.

7. The method according to any one of the preceding claims, wherein, The genetically engineered cells were cultured in the presence of receptor substrates selected from lactose, LNT-II, and LNnT.

8. The method according to any one of the preceding claims, wherein, The genetically engineered cell also contains a recombinant nucleic acid sequence encoding β-1,4-galactosyltransferase, and optionally also contains a recombinant nucleic acid sequence encoding β-1,3-N-acetylglucosamine transferase.

9. A genetically engineered cell capable of producing human lactose oligosaccharides (HMOs) selected from lacto-N-neofucopentose VI (LNFP-VI) and lacto-N-fucopentose V (LNFP-V), comprising a recombinant nucleic acid sequence encoding α-1,3-fucosyltransferase Bacbac2 or a functional homolog thereof, wherein Bacbac2 comprises or is composed of an amino acid sequence as shown in SEQ ID NO: 2, and the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO:

2.

10. A genetically engineered cell capable of producing human milk oligosaccharide (HMO) lact-N-neofucopentose VI (LNFP-VI), comprising a recombinant nucleic acid encoding an α-1,3-fucosyltransferase selected from: a) Bacbac2 or a functional homolog thereof comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 2, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 2; b) Bacbac1 or a functional homolog thereof comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO:

1. in, The genetically engineered cells also contain recombinant nucleic acid sequences encoding β-1,3-N-acetylglucosamine transferase and β-1,4-galactosyltransferase.

11. The genetically engineered cell according to claim 9 or 10, wherein, The content of fucosylated byproducts with 5 or 6 monosaccharide units, such as LNDFH-III and / or LNFP-III, produced by the genetically engineered cells is less than 5% of the total molar content of HMO, for example less than 2.5% or less than 1%.

12. The genetically engineered cell according to any one of claims 9 to 11, wherein, The cells also produce one or more HMOs selected from 3FL, LNT-II, and LNnT.

13. The genetically engineered cell according to any one of claims 9 to 12, wherein, Of the total molar content of HMOs produced by the cells, the content of LNFP-VI produced by the cells is higher than 25%.

14. The genetically engineered cell according to any one of claims 9 to 13, wherein the genetically engineered cell is selected from Escherichia coli, Bacillus subtilis, lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

15. Use of α-1,3-fucosyltransferase in the production of LNFP-VI, wherein the production of LNFP-VI is substantially free of fucosylated byproducts having 5 or 6 monosaccharide units, wherein the α-1,3-fucosyltransferase is selected from Bacbac1 or Bacbac2 comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 2, or a functional homolog thereof, wherein the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 1 or 2.

16. An HMO mixture, which is basically composed of the following: a) LNFP-VI and 3-FL, or b) LNFP-VI and LNnT, or c) LNFP-VI, 3-FL and LNnT, or d) LNFP-V, 3-FL and LNT.

17. The HMO mixture according to claim 16, which is substantially composed of the following: a) 25-70 mol% LNFP-VI, 35-70 mol% 3FL, 0-5% LNnT; or b) 55-90 mol% LNFP-VI, 0-15 mol% 3FL, 0-35% LNnT, and 0-10 mol% pLNnH; or c) 80 mol% LNFP-VI, 10 mol% 3FL, 10 mol% LNnT, or d) 60 mol% LNFP-VI and 40 mol% 3FL; The total HMO content is 100%.

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