Mixing process for the production of complex hmo

Abstract

The present disclosure relates to a method of producing oligosaccharides wherein the method combines a fermentation process with an enzymatic reaction in a fermentation medium, thereby enabling the formation of oligosaccharides in a continuously fermenting fermentation medium. The method is particularly suitable for producing complex oligosaccharides.

Description

Technical Field

[0001] The present disclosure relates to a method for producing complex oligosaccharides (e.g., complex fucosylated and / or sialylated HMOs or neutral HMOs of at least six monosaccharides). The hybrid method combines fermentation and enzymatic reactions in the fermentation medium, thereby allowing the formation of complex oligosaccharides in the medium of continuous fermentation. HMO mixtures derived from the hybrid process and uses of such mixtures are also described herein. Background Art

[0002] Efforts have been made to produce more complex human milk oligosaccharides (HMOs) through chemical synthesis routes, enzymatic routes and in vivo fermentation methods. Chemical routes are too complex and expensive for industrial use.

[0003] The enzymatic synthesis of HMOs relies on the use of donor oligosaccharides (HMOs) and acceptor oligosaccharides (HMOs), which are catalyzed by an enzyme with transglycosidase activity to produce a third oligosaccharide (complex HMO), but due to the nature of the enzymatic reaction, the HMOs produced by this route will always be a mixture of donor, acceptor and third oligosaccharides (HMOs), as well as byproducts (leaving groups, such as lactose) released from the donor substrate portion due to the equilibrium of the enzymatic reaction (see, for example, WO2012 / 156897, WO2012 / 156898 and WO2016 / 063262). In addition, the enzymatic process uses separately produced and purified donor and acceptor substrates, which increases the cost of the process. In addition, in the case where the byproduct released by the donor substrate is lactose, extensive purification is required to remove this large amount of unwanted lactose.

[0004] HMO bioproduction systems using in vivo fermentation are currently the preferred way to produce smaller fucosylated and sialylated and neutral core HMOs (for review, see Bych et al. 2019, Current Opinion in Biotechnology 56:130-137). However, for more complex HMOs, fermentation pathways may face challenges in exporting the HMOs from cells to the culture medium, which is necessary to achieve high yields on an industrial scale.

[0005] Since lactose is used as the initial substrate in many bioproduction processes, there is usually excess lactose at the end of the fermentation, which may interfere with the subsequent purification of the desired oligosaccharide product. WO2015 / 036138 describes the use of a suitable glycosidase (e.g., β-galactosidase) in the culture medium to degrade excess lactose.

[0006] Purpose of the Invention

[0007] The present disclosure has identified a hybrid process suitable for producing one or more oligosaccharides, such as HMOs. The process is particularly suitable for producing complex fucosylated and / or sialylated HMOs. The hybrid process is a combination of fermentation and enzymatic reaction of the fermentation product (first oligosaccharide) in the fermentation medium with the second oligosaccharide or disaccharide, so that the desired more complex third oligosaccharide is produced in the fermentation medium. Some benefits of this process are that it is not necessary or only requires at least one of the starting oligosaccharides (second oligosaccharides) to be in pure form, thereby reducing the cost of the process, and the by-products (e.g., lactose) produced during the enzymatic step are recycled in situ by the genetically modified microbial strain as raw materials for producing the first oligosaccharide and the second oligosaccharide that may be produced, thereby reducing the final by-product (e.g., lactose) level of the enzymatic process at the end of the process. In an embodiment in which the process includes only one genetically modified cell, the feed of lactose can be significantly reduced or completely omitted during the entire fermentation process of the first oligosaccharide. The recycling of lactose also shifts the balance of the enzymatic process toward the desired third oligosaccharide (bypassing the equilibrium barrier) to allow almost complete conversion of the first or second substrate (donor or acceptor) oligosaccharide. Therefore, a higher ratio of the desired third oligosaccharide can be produced relative to the first and / or second oligosaccharides than conventional enzymatic processes. In addition, since the production of the desired oligosaccharide occurs in the fermentation medium rather than inside the genetically modified microbial strain, the challenge of exporting complex / large oligosaccharides outside the cell is solved. Summary of the invention

[0008] The application relates to a hybrid method for producing oligosaccharides, wherein the method combines a fermentation process that produces a donor substrate (oligosaccharide) and / or an acceptor substrate (oligosaccharide or disaccharide) with an enzymatic transglycosidase reaction. The fermentation process produces a first substrate for the transglycosidase reaction and supplies a second substrate to the fermentation in progress, wherein a third (product) oligosaccharide is formed in the culture medium in the presence of the transglycosidase. The process further produces a byproduct that is input by the cell during the fermentation process via a byproduct import protein (e.g., lactose, milk-N-disaccharide or N-acetyllactosamine import protein) to produce more of the first substrate.

[0009] One aspect is a method for producing oligosaccharides from a donor oligosaccharide and an acceptor oligosaccharide or an acceptor disaccharide, the method comprising the steps of:

[0010] a) culturing a genetically modified cell capable of producing a first oligosaccharide of at least three monosaccharide units in a medium supplied with a carbon source, wherein the genetically modified cell comprises one or more nucleic acids encoding:

[0011] i) at least one by-product import protein, and

[0012] ii) at least one recombinant glycosyltransferase, and

[0013] iii) at least one pathway for producing a nucleotide activated sugar, and

[0014] b) supplying a second disaccharide or oligosaccharide to the culture medium, and

[0015] c) making an enzyme having transglycosidase activity available in the culture medium, and

[0016] d) incubating the first oligosaccharide, the second disaccharide or oligosaccharide and a transglycosidase in the medium in which the first oligosaccharide was produced to form a third oligosaccharide.

[0017] In an embodiment, the third oligosaccharide is a complex oligosaccharide of at least four monosaccharide units selected from the group consisting of DFL, FSL, Lewis B, Lewis Y, sialyl-Lewis A, sialyl-Lewis B, LNFP-I, LNFP-II, LNFP-III, LNFP-IV, LNFP-V, LNFP-VI, LST-a, LST-b, LST-c, DSLNT, LNDFH-I, LNDFH-II, LNDFH-III, FLST-a, FLST-b, FLST-c, pLNH, pLNnH, LNH, LNnH, FLNH-I, FLNH-II, FLNH-III, FpLNH-I, FpLNnH II, DF-LNF-I, DF-LNF-II, DF-LNF-III, DF-p-LNH, DF-p-LNnH, FLNnHa, FLNnHb, DFLNnH, TF-LNH, SLNH, FSLNH, SLNnH-I, FSLNnH-I, SLNnH-II and DS-FLNH-II.

[0018] In a further embodiment, the genetically modified cell produces a donor oligosaccharide selected from a fucosylated oligosaccharide of three to five monosaccharide units, a sialylated oligosaccharide of three to five monosaccharide units, and a neutral core oligosaccharide of three to four monosaccharide units. In a preferred embodiment, the donor oligosaccharide is selected from the HMO of 2'FL, 3FL, DFL, LNFP-I, FSL, 3'SL, 3'SLacNAc, 3'SLNB, 6'SL, LST-a, LNT-II, LNT, and LNnT.

[0019] In other embodiments, the genetically modified cells produce an acceptor oligosaccharide selected from 2'FL, 3FL, 2'FLacNAc, 2'FLNB, LewisA, Lewis X, LNT-II, LNT, LNnT, p-LNnH, LNFP-I, LNFP-II, LNFP-III, LNFP-IV, LNFP-V, LNFP-VI, 3'SL, 6'SL, LST-a and LST-c, which are preferably exported into the culture medium.

[0020] In a further embodiment, the second disaccharide or oligosaccharide is selected from LacNAc, LNB, 2'FL, 3FL, 2'FLacNAc, 2'FLNB, Lewis A, Lewis X, 3'SL, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LST-a, LST-c, LNH, F-LNH-II, F-LNH-III, DF-LNH-I, DF-LNH-II, DF-LNH-III, S-LNH, DS-LNH, FS-LNH, LNnH, p-LNH and p-LNnH. In embodiments where the second genetically engineered cell co-cultured in the same culture medium with the first genetically engineered cell supplies the second oligosaccharide to the culture medium, the oligosaccharide produced by the second genetically engineered cell is preferably exported to the culture medium.

[0021] In a further embodiment, the transglycosidase is selected from α-1,2-transfucosidase, α-1,3-transfucosidase, α-1,3 / 4-transfucosidase, α-2,3-transsialidase, α-2,6-transsialidase, β-N-acetylglucosaminidase, trans-lacto-N-biosidase and trans-β-galactosidase. The transglycosidase can be added to the culture medium during the fermentation process or can be expressed by genetically engineered cells.

[0022] Another aspect described herein is a composition produced by the mixing method disclosed in the examples. Specifically, the HMO composition consists essentially of:

[0023] a) at least 50 wt% FSL, 20 to 45 wt% 3FL, and 0.1 to 2 wt% 3'SL and 0 to 3 wt% lactose, or

[0024] b) at least 50 wt% LST-a, 15 to 40 wt% LNT, 0 to 15 wt% 3'SL and 0 to 2 wt% lactose, or

[0025] c) at least 50 wt% LST-c, 15 to 25 wt% LNnT, 15 to 25 wt% 6'SL and 0 to 7 wt% lactose, and

[0026] Each component in the total composition accounts for 100 wt%, and the composition is a mixture of at least two components.

[0027] Another aspect is the use of the HMO composition described herein in the manufacture of a nutritional composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1: A non-limiting description of a mixed fermentation-enzymatic process occurring in a fermentation bioreactor. A carbon source (e.g., glucose, glycerol, sucrose, etc.) is supplied to the strain producing the HMO in the reactor, and an initial amount of lactose (lac) is provided to produce the first HMO. A sterilized second HMO is fed into the fermentation broth containing the strain producing the first HMO. Once the first and second HMOs are present in sufficient amounts, a transglycosidase is added to the fermentation broth, in which the transglycosidase catalyzes the transfer of the glycosyl moiety from the donor HMO (e.g., sialyllactose or fucosyllactose, which may be the first or second HMO) to the acceptor HMO, thereby generating a third (glycosylated) HMO and lactose as a byproduct (leaving group of the enzymatic step). Lactose, in turn, is taken up (recycled) by the cells producing the first HMO, thereby pushing the balance toward the formation of the third complex HMO, and lactose does not need to be fed into the fermentation broth, which results in a lower lactose level at the end of the fermentation. If the rate of recycling of the leaving group (lactose) is faster than the rate of the enzymatic reaction, the reverse enzymatic reaction is very slow and express.

[0029] Figure 2 : The concentration profiles in the mixing process are shown as weight percentage (mass %) relative to the total moles of substrate and product, illustrating the process of forming 3FL in situ from lactose using the 3FL strain and synthesizing FSL by adding a sterilized 3'SL solution to the culture medium. The transsialylation of 3FL (acceptor) was initiated with 3'SL as the sialic acid donor by adding α-2,3-transsialidase at pH 6.7 and 25°C. The lactose concentration is shown as a dotted line with circles, the 3FL concentration as a short-dashed line with squares (hashed line), the 3'SL concentration as a long-dashed line with triangles, and the FSL concentration as a solid line with diamonds.

[0030] Figure 3 : The concentration profiles in the hybrid process are shown as weight percentage (mass %) relative to the total weight of substrate and product, illustrating the process of forming 3FL in situ from lactose using the 3FL strain and synthesizing LNDFH-I by adding an equimolar amount of LNFP-I to the culture medium. Transfucosylation of LNFP-I (acceptor) was initiated using 3FL as a fucosyl donor by adding sterile filtered α-1,3 / 4-transfucosidase (BiTF-641) at pH 6.8 and 25°C. Lactose concentration is shown as a dashed line with circles, 3FL concentration as a short dashed line with squares, LNFP-I concentration as a long dashed line with triangles, and LNDFH-I concentration as a solid line with diamonds.

[0031] Figure 4: Shows the progress curve of the in vitro enzymatic reaction expressed as weight percentage (mass fraction %) relative to the total weight of the substrate, starting with equimolar amounts of LNFP-I and 3FL and the addition of α-1,3 / 4-transfucosidase (BiTF-641) at pH 6.55 and 25°C, α-1,3 / 4-transfucosidase (BiTF-641) catalyzes the transfucosylation of LNFP-I (acceptor) using 3FL as a fucosyl donor to synthesize LNDFH-I. The lactose concentration is shown as a dotted line with circles, the 3FL concentration as a short dashed line with squares, the LNFP-I concentration as a long dashed line with triangles, and the LNDFH-I concentration as a solid line with diamonds.

[0032] Figure 5 : Concentration profiles in the hybrid process are shown as weight percentage (wt%) relative to the total weight of substrate and product, illustrating the process of forming 3FL in situ from lactose using the 3FL strain and synthesizing LNDFH-I by adding an excess molar ratio of LNFP-I and lactose (i.e., LNFP-1 / lactose 2:1, mol / mol) to the culture medium. Transfucosylation of LNFP-I (acceptor) was initiated using 3FL as a fucosyl donor by adding α-1,3 / 4-transfucosidase (BiTF-641) at pH 6.8 and 25°C. Lactose concentration is shown as a dashed line with circles, 3FL concentration as a short dashed line with squares, LNFP-I concentration as a long dashed line with triangles, and LNDFH-I concentration as a solid line with diamonds.

[0033] Figure 6 : Shows the progress curve of the in vitro enzymatic reaction expressed as weight percentage (mass fraction %) relative to the total weight of the substrate, starting with an excess molar ratio of LNFP-I and 3FL and the addition of α-1,3 / 4-transfucosidase (BiTF-641) at pH 6.5 and 37°C, α-1,3 / 4-transfucosidase (BiTF-641) catalyzes the transfucosylation of LNFP-I (acceptor) using 3FL as a fucosyl donor to synthesize LNDFH-I. The lactose concentration is shown as a dotted line with circles, the 3FL concentration as a short dashed line with squares, the LNFP-I concentration as a long dashed line with triangles, and the LNDFH-I concentration as a solid line with diamonds.

[0034] Figure 7: shows the concentration distribution curves expressed as weight percentage (mass fraction %) relative to the total weight of substrate and product in the dual strain mixing process, illustrating the process progress of the synthesis of 6'SL (strain MF5) and LNnT (strain MF6), and the decrease of lactose as the two HMOs are produced. In the transsialylation reaction, 6'SL acts as a donor substrate and LNnT is the acceptor. When α-2,6-transsialidase PITS-197 is added to the fermentation, the reaction is initiated at 113 hours after the start of fermentation, at which time LST-c begins to form, 6'SL and LNnT levels decrease, and lactose is formed to stabilize the production of 6'SL and LNnT. Lactose concentration is shown as a dotted line with circles, 6'SL concentration as a short dashed line with squares, LNnT concentration as a long dashed line with triangles, and LST-c concentration as a solid line with diamonds.

[0035] Figure 8 : Shows the progress curve of the in vitro enzymatic reaction expressed as weight percentage (mass fraction %) relative to the total weight of the substrate, α-2,6-trans-sialidase (PITS-197) catalyzes the transsialylation of LNnT (acceptor) using 6'SL as a fucosyl donor to synthesize LST-c. The lactose concentration is represented by a dotted line with circles, the 6'SL concentration is a short dashed line with squares, the LNnT concentration is a long dashed line with triangles, and the LST-c concentration is a solid line with diamonds.

[0036] Fig. 9: Non-limiting description of a dual strain mixed fermentation-enzymatic process occurring in a fermentation bioreactor. A first carbon source (e.g., glucose, glycerol, sucrose, fructose, galactose, arabinose, sorbitol, maltose, etc.) is provided to the strain producing the first oligosaccharide / HMO in the bioreactor, and an initial amount of lactose (lac) is provided to produce the first oligosaccharide / HMO. The bioreactor also contains a strain producing the second oligosaccharide / HMO, to which a second carbon source (e.g., glucose, glycerol, sucrose, fructose, galactose, arabinose, sorbitol, maltose, etc.) different from the first carbon source is provided, and the strain produces the second oligosaccharide / HMO from lactose. Preferably, the two strains are inoculated into the bioreactor at the beginning of the fermentation, and their growth and the production rate of their respective oligosaccharides can be controlled by feeding the two carbon sources at different rates. Once the first and second oligosaccharides / HMO produced by the cells are present in sufficient amounts in the culture medium, they can serve as donors and acceptors in the transglycosylation reaction. Transglycosylation is catalyzed by a transglycosidase, which can be provided to the culture medium, for example by addition or by production by one of the strains. Once the transglycosidase is present in the medium in which the fermentation is running, it catalyzes the transfer of the glycosyl moiety from the donor oligosaccharide / HMO (e.g., sialyllactose or fucosyllactose) to the acceptor oligosaccharide / HMO, thereby producing a third sialylated or fucosylated complex HMO and lactose as a byproduct (the leaving group of the enzymatic step). The lactose is in turn taken up (recycled) by the first and second strains, producing more of the first and second oligosaccharides / HMOs, thereby pushing the equilibrium towards the formation of the third complex HMO. At the end of the fermentation, the lactose feed to the fermentation broth can be stopped and the residual lactose consumed until the fermentation is terminated. If the rate of recycling of the leaving group (lactose) is faster than the rate of the enzymatic reaction, then the reverse enzymatic reaction is very slow and the lactose used is not sufficient. express.

[0037] Fig.10 : Concentration profiles expressed as weight percent (wt%) relative to the total weight of substrate and product in a single strain mixed process are shown, illustrating the process progression of adding 6'SL to the LNnT strain culture and the decline in lactose as LNnT is produced. In the transsialic acid reaction, 6'SL acts as a donor substrate and LNnT is the acceptor, which is initiated by adding α-2,6-transsialidase PITS-197 to the fermentation, at which time LST-c begins to form and 6'SL and LNnT levels decrease. Lactose concentration is shown as a dotted line with circles, 6'SL concentration as a short dashed line with squares, LNnT concentration as a long dashed line with triangles, and LST-c concentration as a solid line with diamonds.

[0038] Fig.11: Shows the progress of lactose, LNnT, 3FL and LNFP-III expressed as a weight percentage relative to the total weight of substrate and product in a single strain process. In the transfucosylation reaction, 3FL acts as a donor substrate (produced by the 3FL-S2 strain in Table 12), LNnT is the acceptor, and the reaction is initiated by adding α-1,3 / 4-transfucosidase BiTF-641 to the fermentation, at which time LNFP-III begins to form and 3FL and LNnT levels decrease. Lactose concentration is shown as a dotted line with a circle, 3FL concentration is a short dashed line with a square, LNnT concentration is a long dashed line with a triangle, and LNFP-III concentration is a solid line with a diamond.

[0039] Fig.12 : Shows the progress curve of the in vitro enzymatic reaction expressed as weight percentage (mass fraction %) relative to the total weight of the substrate. Starting from a molar ratio of LNnT to 3FL of 3:1, α-1,3 / 4-transfucosidase (BiTF-641) catalyzes the transfucosylation of LNnT (acceptor) using 3FL as a fucosyl donor to synthesize LNFP-III. The lactose concentration is shown as a dotted line with circles, the 3FL concentration is a short dashed line with squares, the LNnT concentration is a long dashed line with triangles, and the LNFP-III concentration is a solid line with diamonds.

[0040] Fig.13 : Shows the progress of lactose, LNT, 3FL and LNFP-II expressed as a weight percentage relative to the total weight of substrate and product in a single strain process. In the transfucosylation reaction, 3FL acts as a donor substrate (produced by the 3FL-S1 strain in Table 12), LNT is the acceptor, and the reaction is initiated by adding α1,3 / 4-transfucosidase BiTF-641 to the fermentation, at which time LNFP-II begins to form and 3FL and LNT levels decrease. Lactose concentration is shown as a dotted line with circles, 3FL concentration is a short dashed line with squares, LNnT concentration is a long dashed line with triangles, and LNFP-II concentration is a solid line with diamonds.

[0041] Fig.14 : Shows the progress curve of the in vitro enzymatic reaction expressed as weight percentage (mass fraction %) relative to the total weight of the substrate. Starting from a molar ratio of LNT to 3FL of 2:1, α-1,3 / 4-transfucosidase (BiTF-641) catalyzes the transfucosylation of LNT (acceptor) using 3FL as a fucosyl donor to synthesize LNFP-II. The lactose concentration is shown as a dotted line with circles, the 3FL concentration is a short dashed line with squares, the LNT concentration is a long dashed line with triangles, and the LNFP-III concentration is a solid line with diamonds.

[0042] Fig.15: Shows the progress of lactose, LNT, 3'SL and LST-a expressed as weight percentage relative to the total weight of substrate and product in a single strain process. In the transsialylation reaction, 3'SL acts as a donor substrate and LNT is the acceptor (produced by the LNT strain in Table 12), which is initiated by adding α-2,3-trans-sialidase TcTS to the fermentation, at which time LST-a begins to form and 3'SL and LNT levels decrease. Lactose concentration is shown as a dotted line with circles, 3'SL concentration as a short dashed line with squares, LNT concentration as a long dashed line with triangles, and LST-a concentration as a solid line with diamonds.

[0043] Fig.16 : Shows the progress curve of the in vitro enzymatic reaction expressed as weight percentage (mass fraction %) relative to the total weight of the substrate, starting with a molar ratio of LNT to 3'SL of 1:1, α-2,3-transsialidase TcTS catalyzes the transsialylation of LNT (acceptor) using 3'SL as a fucosyl donor to synthesize LST-a. The lactose concentration is shown as a dotted line with circles, the 3'SL concentration is a short dashed line with squares, the LNnT concentration is a long dashed line with triangles, and the LST-a concentration is a solid line with diamonds.

[0044] Fig.17 The progression of lactose, LNT, 3'SL, and LST-a in the dual-strain process is shown as weight percent relative to the total weight of substrate and product. 3'SL acts as the donor substrate and LNT is the acceptor in the transsialylation reaction, which is initiated by the addition of the α-2,3-transsialidase TcTS to the fermentation, at which point LST-a begins to form and 3'SL and LNT levels decrease. Lactose concentration is shown as a dashed line with circles, 3'SL concentration as a short dashed line with squares, LNT concentration as a long dashed line with triangles, and LST-a concentration as a solid line with diamonds.

[0045] Fig.18 : Experimental setup showing regeneration and viability assessment of freeze-dried probiotics under acidic conditions at pH 3.0.

[0046] Fig.19: Shows the regeneration and viability of freeze-dried Lactobacillus rhamnosus (DSM 32550), incubated at pH 3.0 for 3 hours and plated at two dilutions 1:100 (E-2), 1:1000 (E-3), A) is a control without HMO, B) is a combination of Lactobacillus rhamnosus (DSM 32550) and a HMO mixture containing 55% LST-a and 45% LNT (mix 5); C) is a combination of Lactobacillus rhamnosus (DSM 32550) and a HMO mixture containing 65% LST-a, 55% LNT and 10% 3'SL (mix 6); D) is a combination of Lactobacillus rhamnosus (DSM 32550) and a HMO mixture containing 45% 3FL and 55% FSL (mix 7); E) is a combination of Lactobacillus rhamnosus (DSM 32550) and a HMO mixture containing 25% Combination of LNnT and an HMO mixture of 50% LST-c and 25% 6'SL (mix 8).

[0047] Fig. 20 :show Fig.19 Results of the plates are presented as the mean (n=2) of the standard deviation (SD) of the colony forming units (CFU) per ml calculated from the Lactobacillus rhamnosus (DSM 32550) colonies on the agar plates. A) CFU of mix 5 and 6 (mixture of LST-a and LNT) counted on the agar plates of the dilution step E-2. B) CFU of mix 7 and 8 counted on the agar plates of the dilution step E-4.

[0048] Fig.21 : Shows the regeneration and viability of freeze-dried Bifidobacterium longum (DSM 32946), incubated at pH 3.0 for 30 minutes and plated undiluted, A) is a control without HMO, B) is a combination of Bifidobacterium longum (DSM32946) and an HMO mixture containing 55% LST-a and 45% LNT (mix 5); C) is a combination of Bifidobacterium longum (DSM32946) and an HMO mixture containing 65% LST-a and 55% LNT and 10% 3'SL (mix 6); D) is a combination of Bifidobacterium longum (DSM 32946) and an HMO mixture containing 45% 3FL and 55% FSL (mix 7); E) is a combination of Bifidobacterium longum (DSM 32946) and an HMO mixture containing 25% LNnT and 50% LST-c and 25% 6'SL (mix 8). DETAILED DESCRIPTION

[0049] The inventors of the present invention have ingeniously discovered a way to obtain the best properties from an in vivo biological production system and an in vitro enzymatic production system for oligosaccharides such as HMOs by combining them into a hybrid production system that combines a fermentation step and an enzymatic step in the same vessel. The fermentation step and the enzymatic step are combined into a recycle loop, where a second oligosaccharide substrate is added to the loop and converted into a third product oligosaccharide that leaves the loop.

[0050] The discovery that enzymatic transglycosylation reactions allow for the formation of complex HMOs can be efficiently performed in an ongoing fermentation process, as shown in Examples 1, 3, 5, 7, 8, 9, 11 and 12, is quite surprising. In a hybrid process, the conditions are determined by the fermentation conditions (e.g., temperature, pH, oxygen, carbon dioxide, agitation, etc.), the reaction environment is significantly more complex, the fermentation broth contains multiple substrates and metabolites, and may include proteases released from the cells, whereas the conventional in vitro enzymatic processes shown in Examples 2, 4, 6, 8, 9 and 11 have only two initial substrates (acceptor and donor) and enzymes. To our knowledge, this is the first time that an enzymatic reaction has been used to synthesize a larger molecule from two smaller molecules in an ongoing fermentation process, especially when the byproducts from the enzymatic process are recycled in situ to generate substrates, i.e., beyond the degradation of unwanted molecules.

[0051] Oligosaccharides

[0052] In this context, the term "oligosaccharide" refers to a sugar polymer containing at least three monosaccharide units, i.e., three, four, five, six or higher oligosaccharides. Oligosaccharides can have a linear or branched structure, which contains monosaccharide units interconnected by interglycosidic bonds. In a preferred embodiment, oligosaccharides include lactose, milk-N-disaccharide or N-acetyllactosamine residues / parts at the reducing end, and one or more naturally occurring monosaccharides of 5-9 carbon atoms, selected from aldose (e.g., glucose, galactose, ribose, arabinose, xylose, etc.), ketose (e.g., fructose, sorbose, tagatose, etc.), deoxy sugar (e.g., rhamnose, fucose, etc.), deoxy amino sugar (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acid and ketouronic acid (e.g., sialic acid).

[0053] In an embodiment of the present invention, the oligosaccharide produced by the method described herein may have a lactose (Galβ1-4Glc) moiety or a lacto-N-biose (LNB or Galβ1-3GlcNAc) moiety or an N-acetyllactosamine (LacNAc or Galβ1-4GlcNAc) moiety at the reducing end.

[0054] In an embodiment, the oligosaccharide produced by the method described herein comprises at least 3 monosaccharide units and a lacto-N-biose (LNB or Galβ1-3GlcNAc) moiety or an N-acetyllactosamine (LacNAc or Galβ1-4GlcNAc) moiety at the reducing end, such as Lewis A (LeA or Galβ1-3[fucα1-4]GlcNAc) or Lewis X (LeX or Galβ1-4[fucα1-3]GlcNAc), or Lewis Y (LeY or Fucα1-2Galβ1-4[Fucα1-3]GlcNAc) or Lewis-B (LeB or Fucα1-2Galβ1-3[Fucα1-4]GlcNAc) or 3' sialyl-LNB (Neu5Ac-α2-3Galβ1-3-GlcNAc) or 3' sialyl-LacNAc (Neu5Ac-α2-3Galβ1-4-GlcNAc) or 6' sialyl-LacNAc (Neu5Ac-α2-6Galβ1-4-GlcNAc) or 6' sialyl-LLNB (Neu5Ac-α2-6Galβ1-3-GlcNAc) or sialyl-Lewis A (SLeA or Neu5Ac-α2-3Galβ1-3[fucα1-4]GlcNAc) or sialyl-Lewis X (SLeX or Neu5Ac-α2-3Galβ1-4[fucα1-3]GlcNAc).

[0055] In the context of the present disclosure, complex oligosaccharides are divided into three categories: i) oligosaccharides consisting of at least four monosaccharide units, at least two of which are selected from fucosyl and / or sialic acid moieties, ii) oligosaccharides consisting of at least five monosaccharide units, preferably with at least one sialic acid or fucosyl monosaccharide, iii) oligosaccharides consisting of at least 6 monosaccharide units, preferably neutral non-fucosylated oligosaccharides. In the context of the present disclosure, a subclass of complex oligosaccharides are highly complex oligosaccharides, wherein at least one monosaccharide unit in the oligosaccharide comprises at least three glycosidic bonds to other monosaccharide units.

[0056] HMO

[0057] Preferred oligosaccharides of the present disclosure are human milk oligosaccharides (HMOs).

[0058] The term "human milk oligosaccharide" or "HMO" in this context refers to carbohydrates found in human milk. HMOs have a core structure comprising a lactose unit / moiety at the reducing end, which may be extended by one or more β-N-acetyl-lactosamine groups and / or one or more β-lacto-N-diosyl units, and which may be substituted by α-L-fucopyranosyl (fucosylation) and / or α-N-acetyl-neuraminic acid moieties (sialylation). The HMO structure is disclosed in, for example, Chapter 4 of Xi Chen's Advances in Carbohydrate Chemistry and Biochemistry 2015vol72.

[0059] In the context of this disclosure, lactose is not considered to be a HMO species, but rather a substrate for the process. It is preferred to reduce lactose as much as possible at the end of the process.

[0060] HMOs are neutral or acidic. In this regard, non-acidic (or neutral) HMOs lack sialic acid residues and acidic HMOs have at least one sialic acid residue in their structure. Non-acidic (or neutral) HMOs can be fucosylated or non-fucosylated. Non-fucosylated neutral HMOs are also called neutral core HMOs.

[0061] Examples of such neutral non-fucosylated HMOs (neutral core HMOs) include lacto-N-triose II (LNT-II), lacto-N-tetraose (LNT or Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-neotetraose (LNnT or Galβ1-4GlcNAcβ1-3Galβ1-4Glc), lacto-N-neohexaose (LNnH or Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and lacto-N-neohexaose (LNnH or Galβ1-4GlcNAcβ1-3Galβ1-4Glc). c), p-lacto-N-neohexaose (pLNnH or Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc), p-lacto-N-hexaose (pLNH or Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc) and lacto-N-hexaose (LNH or Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc).

[0062] Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2'FL or Fucα1-2Galβ1-4Glc), 3-fucosyllactose (3FL or Galβ1-4(Fucα1-3)Glc), difucosyllactose (DFL or LDFT or Fucα1-2Galβ1-4(Fucα1-3)Glc), lacto-N-fucopentaose I (LNFP-I or Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose II (LNFP-II or Galβ1-3[Fuc-α1-4]GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose II (LNFP-II or Galβ1-3[Fuc-α1-4]GlcNAcβ1-3Galβ1-4Glc), and lacto-N-fucopentaose II (LNFP-II or Galβ1-3[Fuc-α1-4]GlcNAcβ1-3Galβ1-4Glc). I (LNFP-III or Galβ1-4[Fuc-α1-3]GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose IV (LNFP-IV or Fuc-α1-2Galβ1-4GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose V (LNFP-V or Galβ1-3GlcNAcβ1-3Galβ1-4[Fuc-α1-3]Glc), lacto-N-fucopentaose VI (LNFP-VI or Galβ1-4GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc), lacto-N-difucohexaose I (LNFP-I or Fucα1-2Galβ1-4GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose V (LNFP-V or Galβ1-3GlcNAcβ1-3Galβ1-4[Fuc-α1-3]Glc), lacto-N-difucohexaose I (LNFP-II ... 1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4Glc), lacto-N-difucohexaose II (LNDFH-II or Galβ1-3[Fuc-(α1-4)]GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc), lacto-N-difucohexaose III (LNDFH-III or Galβ1-4[Fuc-(α1-3)]GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc), fucosyl-lacto-N-hexaose I (FLNH-I or Fuc-α1-2Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6Galβ1-4 Glc), fucosyl-lacto-N-hexaose II (FLNH-II or Galβ1-3(Fuc-α1-4)GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc), fucosyl-lacto-N-hexaose III (FLNH-III or Galβ1-3GlcNAcβ1-3((Gal(β1-4(Fucα1-3)GlcNAcβ1-6))Galβ1-4Glc), fucosyl-p-lacto-N-hexaose I (FpLNH-I or Galβ1-3GlcNAcβ1-3Galβ1-4[Fuc-(α1-3)]GlcNAcβ1-3Galβ1-4Glc),Fucosyl-p-lacto-N-neohexose II (FpLNnH-II or Galβ1-4GlcNAcβ1-3Gal(fucα1-3)β1-4GlcNAcβ1-3Galβ1-4Glc), difucosyl-lacto-N-hexaose I (DF-LNH-I or DF-LNHa or fuc-α1-2Galβ1-3GlcNAcβ1-3((Gal(β1-4(Fuc1-3)GlcNAcβ1-6))Galβ1-4Glc), difucosyl-lacto-N-hexaose II (DF-LNH-II or DF-LNHb or Galβ1-3 (fuc-α1-4)GlcNAcβ1-3((Gal(β1-4(Fuc1-3)GlcNAcβ1-6))Galβ1-4Glc), difucosyl-lacto-N-hexaose III (DF-LNH-III or DF-LNHc or fuc-α1-2Galβ1-3(fuc-α1-4)GlcNAcβ1-3(Gal(β1-4GlcNAcβ1-6)Galβ1-4Glc), difucosyl-p-lacto-N-hexaose (DF-p-LNH or Galβ1-3(fuc-α1-4)GlcNAcβ1-3Galβ1,

[0063] -4(fuc-α1-3)GlcNAcβ1-3Galβ1-4Glc), difucosyl-p-lacto-N-neohexose (DF-p-LNnH or Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4[Fuc-α1-3]-GlcNAcβ1

[0064] -3Galβ1-4Glc), fucosyl-lacto-N-neohexose a (FLNnHa), fucosyl-lacto-N-neohexose b (FLNnHb), difucosyl-lacto-N-neohexose (DFLNnH) and trifucosyl-lacto-N-hexaose (TF-LNH or fuc-α1-2Galβ1-3(fuc-α1-4)GlcNAcβ1-3((Gal(β1-4(Fuc1-3)GlcNAcβ1-6))Galβ1-4Glc).

[0065] Examples of acidic HMOs include 3'-sialyllactose (3'SL or Neu5Ac-α2-3Galβ1-4-Glc), 6'-sialyllactose (6'SL or Neu5Ac-α2-6Galβ1-4-Glc), 3-fucosyl-3'-sialyllactose (FSL or Neu5Ac-α2-3Galβ1-4(Fucα1-3)Glc), 3'-sialyllacto-N-tetraose a (LST a or Neu5Ac-α2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc), fucosyl-LST a (FLST a or Neu5Ac-α2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc), 6'-sialyllacto-N-tetraose b (LST b or Galβ1-3(Neu5Ac-α2-6)GlcNAcβ1-3Galβ1-4Glc), fucosyl-LST b (FLST b or Fucα1-2Galβ1-3(Neu5Ac-α2-6)GlcNAcβ1-3Galβ1-4Glc), 6'-sialyllacto-N-neotetraose (LST c or Neu5Ac-α2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc), fucosyl-LST c (FLST c or Neu5Ac-α2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc), 3'-sialyllacto-N-neotetraose (LST d), fucosyl-LST d (FLSTd), disialyl-lacto-N-tetraose (DSLNT or Neu5Ac-α2-3Galβ1-3(Neu5Ac-α2-6)GlcNAcβ1-3Galβ1-4Glc), sialic acid-p-lacto-N-neohexose (S-pLNnH or Neu5Ac-α2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc), sialic acid-lacto-N-hexaose (SLNH or N eu5Ac-α2-6Galβ1-4GlcNAcβ1-6(Galβ1-3GlcNAcβ1-3)Galβ1-4Glc), fucosyl-sialic acid-lacto-N-hexaose (FSLNH or Neu5Ac-α2-6Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-3GlcNAcβ1-3)Galβ1-4Glc), sialic acid-lacto-N-neohexaose I (SLNnH-I or Neu5Ac-α2-6Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-3GlcNAcβ1-3)Galβ1-4Glc), Ac-α2-3Galβ1-4GlcNAcβ1-6 (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), fucosyl-sialic acid-lacto-N-neohexose I (FSLNnH-I or Neu5Ac-α2-6Galβ1-4GlcNAcβ1-3 (Galβ1-4(Fucα1-3)GlcNAcβ1-6)Galβ1-4Glc), sialic acid-lacto-N-neohexose II (SLNnH-II or Neu5Ac-α2-6Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc) and disialo-fucosyl-lacto-N-hexaose II (DS-FLNH-II or Neu5Ac-α2-3Galβ1-3(Neu5Ac-α2-6)GlcNAcβ1-3(Galβ1-4(Fucα1-3)GlcNAcβ1-6)Galβ1-4Glc).

[0066] In the context of the present disclosure, complex HMOs are divided into three categories: i) HMOs consisting of at least four monosaccharide units, at least one of which is a fucosyl or sialic acid moiety, preferably, if a complex HMO consists of four monosaccharide units, it comprises at least two monosaccharide units selected from fucosyl and / or sialic acid moieties, such as DFL and FSL; ii) HMOs consisting of at least five monosaccharide units, preferably with at least one sialic acid or fucosyl monosaccharide, non-limiting examples are LNFP-I, LNFP-II, LNFP-V, LST-a, LST-c and many highly complex HMOs mentioned below; and iii) HMOs consisting of at least 6 monosaccharide units, preferably neutral non-fucosylated oligosaccharides, such as pLNH-I, pLNnH LNH and LNnH. In the context of the present disclosure, a subclass of complex HMOs are highly complex HMOs, wherein at least one monosaccharide unit in the oligosaccharide comprises at least three glycosidic bonds to other monosaccharide units. Non-limiting examples of highly complex HMOs are LNH, LNnH, LNFP-II, LNFP-III, LST-b, DSLNT, LNDFH-I, LNDFH-II, FLST-a, FLST-b, FpLNnH, FpLNnH-II, F-LNH-I, F-LNH-II, DF-LNH-I, DF-LNH-II, DF-LNH-III, TF-LNH, DFpLNH, DFpLNnH, S-LNFP-I, S-LNH, S-LNnH-I, FS-LNH, FS-LNnH-I, and DS-F-LNH-II.

[0067] Preferably, if produced by fermentation, the complex HMOs of the present invention are not easily exported from the cytoplasmic matrix to the supernatant. The complex HMOs produced by the hybrid method described herein require the action of at least two enzymes. The two enzymes can be, for example, at least one glycosyltransferase present in the cytoplasmic matrix of the genetically engineered cells used in the process and a transglycosidase present in the fermentation medium. For example, FSL produced using the hybrid system of the present invention requires the presence of α-1,3-fucosyltransferase in the genetically modified cells to form 3FL exported to the culture medium, and α-2,3-transsialidase in the culture medium to form FSL from the 3FL produced by the cells and the 3'SL added to the culture medium. Export from the genetically modified cells to the culture medium may require the presence of recombinant transport in the genetically modified cells. For non-limiting examples of suitable transporters, see, for example, WO2010 / 142305, WO2021 / 148615, WO2021 / 148614, WO2021 / 148611, WO2021 / 148610, WO2021 / 148620 and WO2021 / 148618.

[0068] In one method described herein, a complex oligosaccharide having at least four monosaccharide units, such as human milk oligosaccharide (HMO), is a fucosylated and / or sialylated HMO of four monosaccharide units, such as DFL or FSL. Preferably, the complex HMO of four monosaccharide units is FSL.

[0069] In one method described herein, a complex oligosaccharide having at least four monosaccharide units, such as human milk oligosaccharides (HMOs), is a fucosylated and / or sialylated oligosaccharide having five monosaccharide units, such as an oligosaccharide selected from LNFP-I, LNFP-II, LNFP-III, LNFP-IV, LNFP-V, LNFP-VI, LST-a, LST-b, LST-c and LST-d. Specifically, the fucosylated and / or sialylated HMO having five monosaccharide units can be an HMO selected from LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LST-a, LST-b and LST-c.

[0070] In one method described herein, a complex oligosaccharide having at least four monosaccharide units, such as human milk oligosaccharide (HMO), is a fucosylated and / or sialylated HMO having six monosaccharide units. Specifically, the fucosylated and / or sialylated HMO having six monosaccharide units can be selected from DSLNT, LNDFH-I, LNDFH-II, LNDFH-III, FLST-a, FLST-b and FLST-c.

[0071] In one method described herein, a complex oligosaccharide having at least four monosaccharide units, such as human milk oligosaccharides (HMOs), is a fucosylated and / or sialylated HMO of seven or eight monosaccharide units, such as an HMO selected from FLNH-I, FLNH-II, FLNH-III, FpLNH-I, FpLNnHII, DF-LNF-I, DF-LNF-II, DF-LNF-III, DF-p-LNH, DF-p-LNnH, FLNnHa, FLNnHb, DFLNnH, TF-LNH, SLNH, FSLNH, SLNnH-I, FSLNnH-I, SLNnH-II, and DS-FLNH-II. The production of these HMOs may require the presence of three or more glycosyltransferase and / or transglycosidase activities.

[0072] In one method described herein, the complex oligosaccharide having at least four monosaccharide units, such as human milk oligosaccharide (HMO), is a neutral core HMO of at least six monosaccharide units, such as an HMO selected from pLNH, pLNnH, LNH and LNnH.

[0073] Hybrid production method

[0074] The hybrid production system described in the present disclosure includes a fermentation step and an enzymatic step, which can be performed in the same container, wherein the container does not contain any means to separate the components in the container.

[0075] Figure 1 and Fig. 9 An exemplary non-limiting description of a hybrid process occurring in a fermentation bioreactor is provided. In a hybrid process, a genetically modified cell produces a first oligosaccharide, which is secreted / exported to a fermentation medium. The first oligosaccharide can serve as a donor oligosaccharide or an acceptor oligosaccharide. If the third (product) oligosaccharide is a fucosylated and / or sialylated oligosaccharide, and the first oligosaccharide is a donor oligosaccharide, it contains a fucosyl or sialic acid residue. The first oligosaccharide can also serve as an acceptor oligosaccharide in the transglycosylation reaction that occurs in the culture medium subsequently, in which case it serves as the backbone of the third (product) oligosaccharide. In an embodiment where the hybrid process is a dual strain process, the first and second oligosaccharides are independently produced by two genetically different strains in the same fermentation bioreactor (co-cultivation). In this case, one of the strains produces a donor oligosaccharide, and another strain produces an acceptor oligosaccharide.

[0076] In the context of the present invention, the term "donor oligosaccharide" is preferably understood as an oligosaccharide that provides a specific moiety to another compound (preferably an acceptor) in a chemical reaction (e.g., a nucleophilic or electrophilic substitution reaction). Similarly, the term "acceptor oligosaccharide" or "acceptor disaccharide" is preferably understood as an oligosaccharide or disaccharide that receives a specific moiety from a donor in a chemical reaction (e.g., a nucleophilic or electrophilic substitution reaction) to form a third compound.

[0077] Described herein is to produce oligosaccharides from donor oligosaccharides and acceptor oligosaccharides or disaccharides, by cultivating the genetically modified cells of the first oligosaccharide capable of producing at least three monosaccharide units in a culture medium and supplying the second disaccharide or oligosaccharide to the culture medium, and making the enzyme with transglycosidase activity available in the culture medium. By hatching the first oligosaccharide, the second disaccharide or oligosaccharide and transglycosidase in the culture medium producing the first oligosaccharide, the third oligosaccharide (product oligosaccharide) and by-product (for example, lactose, LNB or LAcNAc) are formed in the culture medium. When the second disaccharide or oligosaccharide is a disaccharide, it preferably serves as the acceptor in the transglycosidase process. In an embodiment, the acceptor is not lactose. In a further embodiment, the disaccharide acceptor can be milk-N-disaccharide (LNB) or N-acetyllactosamine (LacNAc), which are the skeletons based on Lewis A and Lewis X structures.

[0078] In the production of oligosaccharides (e.g., complex HMOs), according to the methods described herein, the first oligosaccharide is preferably an HMO. The genetically modified cell is engineered to efficiently produce the first oligosaccharide / HMO and preferably exported to the culture medium, where it is incubated with a second disaccharide / oligosaccharide / HMO in the culture medium (added to the culture medium or produced by a second genetically modified strain) and a transglycosidase to form a third complex oligosaccharide / HMO, such as a complex sialylated and / or fucosylated oligosaccharide / HMO of at least 4 monosaccharide units or a complex core oligosaccharide / HMO of at least 6 monosaccharide units.

[0079] One aspect described herein relates to a method for producing a complex oligosaccharide of at least four monosaccharide units from a donor oligosaccharide and an acceptor oligosaccharide, the method comprising the steps of:

[0080] a) culturing a genetically modified cell capable of producing a first oligosaccharide of at least three monosaccharide units in a medium supplied with a carbon source, and wherein the genetically modified cell comprises one or more nucleic acids encoding:

[0081] i) at least one by-product import protein, such as a lactose import protein, and

[0082] ii) at least one recombinant glycosyltransferase, and

[0083] iii) at least one pathway for producing a nucleotide activated sugar, and

[0084] b) supplying a second oligosaccharide to the culture medium, and

[0085] c) making an enzyme having transglycosidase activity available in the culture medium, and

[0086] d) incubating the first oligosaccharide, the second oligosaccharide and a transglycosidase in a medium in which the first oligosaccharide is produced to form a complex oligosaccharide of at least four monosaccharide units.

[0087] A further embodiment described herein relates to a method for producing sialylated and / or fucosylated oligosaccharides of at least four monosaccharide units from a donor oligosaccharide and an acceptor oligosaccharide, the method comprising the steps of:

[0088] a) culturing a genetically modified cell capable of producing a first oligosaccharide of at least three monosaccharide units in a medium supplied with a carbon source, and wherein the genetically modified cell comprises one or more nucleic acids encoding:

[0089] i) at least one lactose-infused protein, and

[0090] ii) at least one recombinant glycosyltransferase, and

[0091] iii) at least one pathway for producing a nucleotide activated sugar, and

[0092] b) supplying a second oligosaccharide to the culture medium,

[0093] wherein the first oligosaccharide or the second oligosaccharide is a fucosylated or sialylated donor oligosaccharide and the other oligosaccharide is an acceptor oligosaccharide, and

[0094] c) making an enzyme having transglycosidase activity available in the culture medium, wherein the enzyme having transglycosidase activity is

[0095] i) if the donor oligosaccharide is a fucosylated oligosaccharide, then a transfucosidase, or

[0096] ii) if the donor oligosaccharide is a sialylated oligosaccharide, then a trans-sialidase, and

[0097] d) incubating the first oligosaccharide, the second oligosaccharide and a transglycosidase in the medium in which the first oligosaccharide is produced to form a third sialylated and / or fucosylated oligosaccharide of at least four monosaccharide units.

[0098] In an embodiment, the donor oligosaccharide may be selected from a fucosylated oligosaccharide of three to five monosaccharide units, a sialylated oligosaccharide of three to five monosaccharide units, and a neutral core oligosaccharide of three to four monosaccharide units.

[0099] In the context of a hybrid production process, the term "first oligosaccharide" or "first HMO" refers to an oligosaccharide produced in situ by a first genetically modified cell and which constitutes the first substrate in the enzymatic (transglycosidase) step of the hybrid process. The term "second disaccharide", "second oligosaccharide" or "second HMO" refers to a disaccharide or oligosaccharide that constitutes the second substrate in the enzymatic (transglycosidase) step of the hybrid process. When the first oligosaccharide and the second disaccharide or oligosaccharide react with the aid of a transglycosidase, a third oligosaccharide (product oligosaccharide) and a byproduct (leaving group) are produced. The leaving group is recycled by the genetically modified cell to produce more first oligosaccharides and potential second oligosaccharides if this is supplied by a second genetically modified cell in the same culture as the first genetically modified cell. The third oligosaccharide is preferably an oligosaccharide desired by the process, such as a complex oligosaccharide, but it can also serve as an intermediate oligosaccharide for a second transglycosidase reaction that produces a fourth oligosaccharide, which is a complex oligosaccharide desired by the process. If the hybrid process comprises two enzymatic steps, it can be a two-step enzymatic process catalyzed by the same transglycosidase or by transglycosidases with different activities, e.g., one is a transfucosidase and the other is a transsialidase, depending on their selectivity. However, in case two different transglycosidases are used, it may be necessary to feed the process with another substrate for the second transglycosidase.

[0100] The enzymatic transglycosidase reaction occurs in the substratum, so the first oligosaccharide produced by the genetically modified cell needs to be available in the substratum. Preferably, the first oligosaccharide is exported to the extracellular without affecting the survival of the cell. In an alternative embodiment, the first oligosaccharide can be available in the substratum by the natural rupture of part of the cells during the fermentation, without stopping the culture growth.

[0101] In an embodiment, it is desirable that the genetically modified cell exports the first and potential second oligosaccharides produced by the cell to the culture medium so that they are readily available for transglycosidase reactions in the culture medium. The HMO that can be advantageously produced and exported to the culture medium by the genetically modified cell and can be used as a donor oligosaccharide in a transglycosylation reaction can be selected from 2'FL, 3FL, DFL, LNFP-I, 3'SL, 6'SL, 3'SLAcNAc, 3'SLNB, FSL, LST-a, LNT-II, LNT and LNnT.

[0102] In an embodiment, the HMO that can be advantageously produced by the genetically modified cells and exported into the culture medium and used as an acceptor oligosaccharide in a transglycosylation reaction can be selected from 2'FL, 3FL, 2'FLacNAc, 2'FLNB, Lewis A, Lewis X, 3'SL, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LST-a and LST-c.

[0103] The term "supplying a second oligosaccharide to the culture medium" is understood to mean making the second oligosaccharide available in the culture medium. This can be achieved, for example, by adding from an exogenous source or by supplying in situ from a second genetically modified cell grown in the same culture as the first genetically modified cell. In an alternative embodiment, the cell producing the first oligosaccharide can also produce the second oligosaccharide, thereby allowing one cell to supply the acceptor oligosaccharide and the donor oligosaccharide to the culture medium. In addition, such cells can also produce transglycosidase and supply it to the culture medium.

[0104] Basically, there is no restriction on which second oligosaccharide can be added to the culture medium during the fermentation process. Preferably, the second oligosaccharide is substantially free of other oligosaccharide compounds that can cause unwanted by-products. In the context of the present disclosure, substantially free of other oligosaccharide compounds means that at least 90% of the total oligosaccharides are second oligosaccharides, such as at least 95%, such as at least 98% of the total oligosaccharides are second oligosaccharides. Adding the second oligosaccharide to the fermentation is preferably carried out under aseptic conditions. The second oligosaccharide can act as a donor oligosaccharide, in which case the second oligosaccharide contains a fucosyl or sialic acid residue, or it can act as an acceptor oligosaccharide in a subsequent transglycosylation reaction. When producing complex HMOs, the second oligosaccharide is preferably an HMO.

[0105] In an embodiment, the oligosaccharide that can be advantageously supplied to the culture medium and can be used as a donor oligosaccharide in a transglycosylation reaction can be selected from 2'FL, 3FL, DFL, LNFP-I, 3'SL, 6'SL, 3'SLAcNAc, 3'SLNB, FSL, LST-a, LNT-II, LNT and LNnT.

[0106] In an embodiment, the oligosaccharide that can be advantageously supplied to the culture medium and used as an acceptor oligosaccharide in a transglycosylation reaction can be selected from 2'FL, 3FL, 2'FLacNAc, 2'FLNB, Lewis A, Lewis X, 3'SL, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-IV, LNFP-V, LNFP-VI, LST-a, LST-b, LST-c, LST-d, LNH, F-LNH-II, F-LNH-III, DF-LNH-I, DF-LNH-II, DF-LNH-III, S-LNH, DS-LNH, FS-LNH, LNnH, p-LNH and p-LNnH.

[0107] In an alternative embodiment, the second oligosaccharide is supplied to the culture medium by a second genetically modified cell capable of producing the second oligosaccharide. The second oligosaccharide may be in a more or less pure form, and may be in the form of a filtered (cell-free) fermentation broth obtained from the cultivation of the second genetically modified cell. In a preferred embodiment, the second genetically modified cell capable of producing the second oligosaccharide is co-cultured with the first genetically modified cell, i.e., the cells are grown in the same culture medium (see Fig. 9 ), thereby supplying the second oligosaccharide in the original position. The advantages of the original position generation of the first and second oligosaccharides. The advantage of the dual strain mixed system is that it can save production capacity, because both donor and acceptor oligosaccharides can be produced in a single fermentation, without having to produce the second oligosaccharide separately and then adding it to the fermentation. This process will also be more economical, because the oligosaccharides produced separately and purified are more expensive than the oligosaccharides produced in the original position.

[0108] In this specification, the term "culture" or "fermentation" or "fermentation process" can be used interchangeably and refers to the growth of genetically modified cells (strains) in a bioreactor to produce a first oligosaccharide and potentially a second oligosaccharide. The strain grows on a suitable carbon source, such as glucose, sucrose, galactose, arabinose, sorbitol, maltose, fructose, xylose and glycerol.

[0109] Genetically modified cells can produce the first or second oligosaccharide from substrate, and the substrate is preferably added to the culture medium and taken up by the cell to serve as the initial substrate for producing the first oligosaccharide (e.g., HMO). In the case where the reducing end of the third oligosaccharide has N-acetyllactosamine (LacNAc) milk-N-disaccharide (LNB), the initial substrate can be selected from N-acetyllactosamine (LacNAc) milk-N-disaccharide (LNB), then modified in the cell in a manner similar to lactose to produce, for example, Lewis A, Lewis X, 3'SLacNAc, 3'SLNB, 2'FLacNAc or 2'FLNB. Most commonly, lactose is used as the initial substrate, but LNT-II may also serve as the substrate for producing LNT or LNnT, or 2'FL or 3FL may serve as the substrate for producing DFL. In an embodiment, the substrate for producing the first HMO may be selected from lactose, 2'FL, 3FL or LNT-II. Preferably, the initial substrate is selected from lactose or 2'FL. In a preferred embodiment, the substrate for producing the first HMO is lactose. As an alternative to adding the initial substrate for producing the first oligosaccharide to the fermentation medium, the genetically modified cells can be further engineered to produce the initial substrate intracellularly (see, for example, WO2015 / 150328).

[0110] In order to achieve in situ recycling of by-products, it is advantageous if the genetically modified cell is able to internalize the initial substrate for the production of the first oligosaccharide.

[0111] In an embodiment, the genetically modified cell is capable of internalizing lactose, N-acetyllactosamine (LacNAc) lacto-N-biose (LNB), 2'FL, 3FL and / or LNT-II, depending on which compound is used as the initial substrate for making the first HMO and potentially the second HMO. This initial substrate internalized by the cell may correspond to a byproduct produced by the transglycosidase reaction, thereby recycling it. The internalization of the byproduct is preferably promoted by a byproduct import protein (e.g., lactose permease). Preferably, at least one of the genetically modified cells uses lactose as the initial substrate, and if the byproduct produced by the transglycosidase reaction is not lactose, lactose is fed to the culture during the fermentation. In addition, in embodiments where the fermentation is co-cultured and both cells use lactose as the initial substrate, lactose is preferably also fed to the culture during the fermentation to avoid exhaustion of the initial substrate.

[0112] In some embodiments, the genetically modified cells are preferably capable of internalizing lactose or 2'FL added to the culture medium, which are then utilized by the cells to produce the first oligosaccharide (e.g., HMO). Some microbial cells have an endogenous lactose uptake system, for example in the form of a lactose permease, which is also capable of importing 2'FL. If a higher lactose uptake rate is desired, the lactose permease can also be genetically engineered into the cell as a heterologous protein or an additional recombinant copy of the native gene.

[0113] There are four stages in natural microbial fermentation, namely the retardation phase, the growth phase, the stationary phase and the decay phase. In industrial fed-batch fermentation, cell growth is divided into two stages, the first stage cells grow rapidly in the culture medium, can obtain carbon sources without restriction, or restrict carbon sources after a rapidly increasing feed curve to obtain restrictively; the second stage cell growth is more controlled, usually producing industrial products. Carbon (sugar) restriction refers to the stage in which the growth rate is controlled by the kinetics of the carbon source (sugar) concentration in the culture medium during the fermentation process, and the carbon source concentration is in turn determined by the rate (sugar feed rate) of adding carbon to the fermentor. In the hybrid method described herein, the culture is preferably fed-batch or continuous fermentation. Preferably, the culture starts from the initial carbon source (batch phase), and when the carbon source is consumed, the carbon source is fed to the culture medium (feed phase) at a desired rate throughout the fermentation process. In some embodiments, the feed rate is adjusted to allow the culture to grow under carbon restriction, wherein the carbon source is continuously added to allow the product to form at this stage, while limiting the formation of additional biomass. Industrial fermentation will always stop before significant cell death occurs.

[0114] In an embodiment, the culture of the genetically modified cell begins when there is sufficient initial substrate to allow the cell to produce the desired oligosaccharide (e.g., the first oligosaccharide or the second oligosaccharide). In some embodiments, there is sufficient initial substrate at the beginning of the culture to produce the desired oligosaccharide of the desired amount so that no additional substrate is added to the culture medium after the culture begins. In an embodiment, the initial substrate for producing the desired oligosaccharide is selected from lactose, LacNAc, LNB, 2'FL, 3FL and LNT-II. In a preferred embodiment, the substrate for producing the first oligosaccharide and the potential second oligosaccharide is lactose or 2'FL, most preferably lactose.

[0115] In an alternative embodiment, when the initial carbon source is consumed, an initial substrate (e.g., lactose) for producing a desired oligosaccharide (e.g., a first oligosaccharide or a second oligosaccharide) is added to the culture medium, thereby allowing the cells to initially grow before starting to produce the first and / or second oligosaccharides. The initial substrate (e.g., lactose) can be added as a single portion, or it can be fed alone or together with a carbon source and / or a second oligosaccharide.

[0116] In the embodiment where the second oligosaccharide is added to the culture, it can be added at the beginning of the culture (batch phase) or at one or more time points during the culture period, or continuously fed during the culture period. Preferably, the addition of the second oligosaccharide does not continue to the end of the process, to allow exhaustion before the termination process. The amount of the second oligosaccharide is estimated based on the amount of the first oligosaccharide produced, so that the molar ratio of the first and second oligosaccharides is balanced to ensure the best formation of the third oligosaccharide. In an example, the molar ratio of the first and second oligosaccharides is 1:1 (equimolar). In some embodiments, the second oligosaccharide may be advantageous than the first oligosaccharide in excess, such as the ratio of the second oligosaccharide to the first oligosaccharide is 1.5:1-10:1, such as 1.5:1-5:1, such as 2:1, 3:1, 4:, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1. For example, in the double strain hybrid method for forming complex oligosaccharides, the affinity constants (Km's) of the enzyme to the donor and the acceptor may be different. Therefore, in order to maximize the enzymatic reaction efficiency, donor and acceptor can be advantageously supplied with a ratio reflecting Km. In the case of using a molecule of the first oligosaccharide to produce a molecule of the first oligosaccharide, the preferred ratio of the second oligosaccharide to the initial substrate (e.g., lactose) is identical to the ratio between the second oligosaccharide and the first oligosaccharide. In a preferred embodiment, the ratio of the second oligosaccharide to lactose is 1.5:1-10:1, such as 1.5:1-5:1, and the ratio of the second oligosaccharide to lactose is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1. In order to prevent contamination of the culture medium, the second oligosaccharide is preferably sterilized before being added to the culture medium.

[0117] In an embodiment, during the fermentation, the second oligosaccharide is continuously added to the substratum at a rate that is balanced with the output of the first oligosaccharide produced by the genetically modified cell. The advantage of continuously adding the second oligosaccharide to balance the formation of the first oligosaccharide is to ensure the desired ratio of the first and second oligosaccharides until transglycosidase is added, thereby ensuring the best formation of the third oligosaccharide. In other embodiments, the speed at which the second oligosaccharide is added to the substratum is faster than the formation rate of the first oligosaccharide.

[0118] In the transglycosylation process (enzymatic process) of the hybrid process, in order to be able to reduce the level of byproducts (e.g., lactose) produced as a leaving group, at the end of the process, it is desirable to stop feeding the substrate (e.g., lactose) used by the cells to produce the first oligosaccharide before the end of the process. When lactose is formed together with the third fucosylated or sialylated oligosaccharide as a result of the transglycosidase reaction, the genetically modified cells will be able to internalize the lactose, thereby removing any lactose produced in the enzymatic process and converting it to additional first oligosaccharides. In addition to removing undesirable lactose from the culture medium, this also helps to push the equilibrium towards the formation of additional third oligosaccharides / complex HMOs, so that the production ratio of the third oligosaccharide / complex HMO can be higher than the first and / or second oligosaccharides or both compared to conventional enzymatic processes.

[0119] In other embodiments, the byproduct from the transglycosidase reaction can be an oligosaccharide, such as an oligosaccharide with three monosaccharide units, such as an HMO. In this case, the genetically modified cell is preferably engineered so that it can take up the byproduct oligosaccharide and use it as a substrate for producing the first oligosaccharide that enters the transglycosylation process in the culture medium. For example, the genetically modified cell produces DFL, which acts as a fucosyl donor in the transglycosylation reaction and then produces 2'FL as a byproduct (leaving group). Then, 2'FL is taken up by the cell that produces DFL, and the cell can use 2'FL as a substrate for producing DFL. In this method, lactose will not be needed in this process, so lactose will be very little at the end of the culture, and similarly, the amount of 2'FL at the end of the culture is also very little because it is constantly reused by the cells.

[0120] In an embodiment, at the end of the process, the weight % of the third oligosaccharide / complex HMO exceeds the weight % of the donor oligosaccharide. Preferably, the ratio between the third oligosaccharide and the first oligosaccharide and / or the second oligosaccharide is greater than 1.5: 1, more preferably greater than 2: 1, and more preferably greater than 5: 1. Greater than a certain ratio means that the first number indicated in the ratio can be the indicated number or greater than the indicated number.

[0121] In the hybrid process described herein, a transglycosidase that mediates the transglycosylation of an acceptor oligosaccharide with a fucosyl or sialic acid moiety of a donor oligosaccharide is available in the fermentation medium.

[0122] In an embodiment, the transglycosidase is expressed by a recombinant nucleic acid in a genetically modified cell that produces a first oligosaccharide and is exported from the cell to the culture medium. For example, an appropriate signal peptide can be used to promote the export of the transglycosidase.

[0123] For expression in E. coli, the signal peptide can be selected, for example, from one of the following well-known signal peptides.

[0124]

[0125] In other embodiments, transglycosidase is exogenously added to the culture medium during the genetically modified cell culture. When the enzyme is added exogenously, it is preferably sterile filtered before adding to avoid culture contamination. Transglycosidase is added to the mixed process with sufficient activity to mediate the transglycosylation of acceptor oligosaccharides and donor oligosaccharides. If the activity of the enzyme decreases during the fermentation process, it may be advantageous to add enzyme when the genetically modified cells produce sufficient substrates, because it will not become the rate limitation of the process. It is also possible to add enzyme more than once in the culture process.

[0126] In some embodiments, the transglycosidase is added to the culture medium at a point in time when the genetically modified cell has converted at least 50% of the initial lactose (e.g., at least 75% of the initial lactose, e.g., at least 85% of the initial lactose, e.g., at least 90% of the initial lactose, e.g., between 95% and 100% of the initial lactose) into the first oligosaccharide. It is advantageous to allow a sufficient amount of the first oligosaccharide to form before starting the transglycosidation reaction to ensure that the substrate for the enzymatic process does not become rate limiting and that unconverted lactose does not inhibit the reaction.

[0127] In an embodiment, no additional lactose is added to the culture medium after the transglycosidase is added. At this point, the addition of lactose to the culture can be stopped because the transglycosidase reaction will produce lactose that the cells can take up to continue producing the first oligosaccharide.

[0128] At the end of fermentation, it is necessary to inactivate the transglycosidase to avoid a shift in the balance of the transglycosidase reaction, which will result in such a shift once the formation of the first oligosaccharide is stopped due to the cessation of the carbon source feed, which provides energy and carbon for the genetically modified cells, which is necessary for the continuous recycling of lactose. Preferably, the inactivation is performed immediately before or after the cells are harvested. Non-limiting examples of transglycosidase inactivation can be selected from: i) heating the fermentation broth to a temperature at which the enzyme is denatured, ii) adding a protease to the culture broth at the end of the fermentation to hydrolyze the enzyme, or iii) changing the pH of the culture to exceed the activity range of the enzyme or to denature the enzyme. If heating is used for inactivation, the fermentation broth is preferably heated to at least 60°C, such as at least 70°C, such as at least 80°C, such as at least 90°C, such as at least 95°C, for at least 5 minutes, such as at least 10 minutes, such as at least 15 minutes. If a protease is used to inactivate the enzyme, then preferably a protease with sufficient activity to hydrolyze all the enzyme is added at least 10 minutes, such as at least 20 minutes, such as at least 30 minutes before harvesting the cells. If a pH shift is used to inactivate the enzyme, then the pH is preferably lowered to below 5, preferably between 3 and 5, such as between 3.5 and 4.5.

[0129] The terms "manufacturing" or "manufacturing scale" or "large-scale production" or "large-scale fermentation" or "industrial production" are used interchangeably and are defined in the context of the present disclosure as fermenting a minimum volume of 100 L, such as 1000 L, such as 10,000 L, such as 100,000 L, such as 200,000 L of culture fluid. Typically, a "manufacturing scale" process is defined as being able to handle large volumes, producing the target HMO product quantity that meets (for example, in the case of a therapeutic compound or composition) the needs of toxicity testing, clinical trials, and market supply. In addition to the large volumes, the manufacturing scale process is characterized by the use of a technical system of a bioreactor (fermenter) equipped with stirring, aeration, nutrient feeding, process parameter (pH, temperature, dissolved oxygen tension, back pressure, etc.) monitoring and control devices compared to simple laboratory scale processes such as shake flask cultures. To a large extent, the behavior of the expression system in a laboratory scale process, such as the shake flask, benchtop bioreactor or deep well format described in the embodiments of the present disclosure, does allow for prediction of the behavior of the system in the complex environment of a bioreactor.

[0130] There is no restriction on the suitable culture medium used in the fermentation process. Culture medium can be semi-defined, i.e. containing complex culture medium compounds (e.g. yeast extract, soy peptone, tyrosine, etc.), or it can be chemically defined, without any complex compounds. Carbon source is optionally selected from glucose, sucrose, fructose, xylose and glycerol. In one or more exemplary embodiments, culture medium is supplemented with one or more carbon sources, and carbon source is selected from glycerol, sucrose, fructose, galactose, maltose, arabinose, sorbitol, xylose and glucose. Alternative carbon source can be selected from syrup, trans sugar, corn extract, yeast extract, tryptone, acetate, corn syrup, succinate, malate, pyruvate, lactate, ethanol, methanol, xylose (xylanose), citrate and cotton sugar.

[0131] Co-culture

[0132] The term "co-culture" or "co-cultivation" used in the present disclosure relates to two different genetically modified cells (strains) grown in the same culture container (e.g., shake flask, fermenter or bioreactor) to produce their products in the same culture medium. Preferably, two different strains grow simultaneously in the culture medium. For example, two different strains can be inoculated into the container at the beginning of the culture. This allows the strains to grow simultaneously in the same container and produce their products in the same culture medium. However, if it is desired to give the first strain an opportunity to increase its biomass before adding the second strain, the second strain can also be added to the container at a later time point. This will still result in the strains growing simultaneously during some culture times. If it is known that the initial growth rate of a strain is slower than that of another strain, if the oligosaccharide yield per mole of carbon source of the two strains is different, or if it is desired that the product ratios produced by the two strains are different, the two different strains can also be inoculated (delivered) at different ratios (cells / ml). The terms cell and strain can be used interchangeably in the present disclosure.

[0133] Embodiments described herein relate to a method for producing oligosaccharides (e.g., complex oligosaccharides of at least four monosaccharide units, e.g., at least five monosaccharide units) from donor oligosaccharides and acceptor oligosaccharides produced (supplied) by first and second genetically modified cells, the method comprising the steps of:

[0134] a) co-culturing a first and a second genetically modified cell in a culture medium, wherein

[0135] i) a first genetically modified cell capable of producing a first oligosaccharide of at least three monosaccharide units, and wherein the genetically modified cell

[0136] comprising one or more recombinant nucleic acid sequences encoding at least one glycosyltransferase; and

[0137] comprising at least one pathway for producing a nucleotide activated sugar from a first carbon source; and

[0138] preferably capable of exporting said first oligosaccharide into the culture medium; and

[0139] ii) a second genetically modified cell is capable of producing a second disaccharide or oligosaccharide, such as an oligosaccharide of at least three monosaccharide units, and wherein the genetically modified cell

[0140] comprising one or more recombinant nucleic acid sequences encoding at least one glycosyltransferase, and

[0141] comprising a biosynthetic pathway for producing the activated sugar nucleotide from a second carbon source;

[0142] preferably capable of exporting said second oligosaccharide into the culture medium, and

[0143] b) making available in the culture medium an enzyme having transglycosidase activity, and

[0144] c) incubating the first oligosaccharide, the second oligosaccharide and the transglycosidase in a culture medium, wherein the first and second oligosaccharides react to form a third complex oligosaccharide of at least four (eg, five) monosaccharide units.

[0145] Embodiments described herein relate to a method for producing sialylated and / or fucosylated oligosaccharides (e.g., sialylated and / or fucosylated oligosaccharides of at least four monosaccharide units) from a donor oligosaccharide and an acceptor disaccharide or acceptor oligosaccharide (produced by a first and a second genetically modified cell), the method comprising the steps of:

[0146] a) co-culturing a first and a second genetically modified cell in a culture medium, wherein

[0147] i) a first genetically modified microbial cell capable of producing a first oligosaccharide of at least three monosaccharide units, and wherein the genetically modified cell

[0148] comprising one or more recombinant nucleic acid sequences encoding at least one glycosyltransferase; and

[0149] comprising at least one pathway for producing a nucleotide activated sugar from a first carbon source; and

[0150] preferably capable of exporting said first oligosaccharide into the culture medium; and

[0151] ii) a second genetically modified microbial cell is capable of producing a second oligosaccharide of at least three monosaccharide units, and wherein the genetically modified cell

[0152] comprising one or more recombinant nucleic acid sequences encoding at least one glycosyltransferase, and

[0153] comprising a biosynthetic pathway for producing the activated sugar nucleotide from a second carbon source;

[0154] preferably capable of exporting said second oligosaccharide into the culture medium; and

[0155] wherein the first oligosaccharide or the second oligosaccharide is a fucosylated or sialylated donor oligosaccharide and the other disaccharide or oligosaccharide is an acceptor disaccharide or acceptor oligosaccharide, and

[0156] b) making an enzyme having transglycosidase activity available in the culture medium, wherein the enzyme having transglycosidase activity is

[0157] i) if the donor oligosaccharide is a fucosylated oligosaccharide, a transfucosidase, or

[0158] ii) if the donor oligosaccharide is a sialylated oligosaccharide, a trans-sialidase, and

[0159] c) incubating the first oligosaccharide, the second oligosaccharide and a transglycosidase in a medium in which the first and second oligosaccharides are produced to form a third sialylated and / or fucosylated oligosaccharide of at least four monosaccharide units.

[0160] To control the growth of two different genetically modified cells in the same culture medium, if they are Fig. 9 Ideally, the strain is i) unable to grow or ii) growth-restricted on the carbon source used by the other strain in the co-culture.

[0161] When using two different bacterial strains that feed on two different carbon sources, fermentation may basically comprise two batch stages that run simultaneously, one in every kind of bacterial strain / carbon source.Preferably, the amount of two carbon sources is adjusted so that the duration of two batch stages is similar.Based on the understanding of the individual growth rate and oligosaccharide product output of the first and second bacterial strains, the feed rate of two carbon sources can be predicted.If the oligosaccharides of a specific ratio are desired, then the ratio of two carbon sources in the feed can be achieved by balancing the oligosaccharide / carbon source output based on each bacterial strain.As mentioned above, if it is desired to start the growth of a bacterial strain before another kind of bacterial strain, the batch stage can also be staggered.Industrial fermentation will always stop before significant cell death occurs.

[0162] In order for the first and second genetically modified cells to grow on different carbon sources rather than on the same carbon source, it may be necessary to select cells having certain growth characteristics or to genetically modify the cells to have the desired growth characteristics.

[0163] Microorganisms are generally able to use a variety of carbon sources to promote their growth. In order to determine whether cells can grow on a certain carbon source, one can, for example, plate the microorganisms on agar plates with the selected carbon source and observe the formation of colonies.

[0164] Depending on the type of microbial strain used in the context of the present specification, different modifications may need to be made to ensure that the strain does not grow or has limited growth on the desired carbon source.

[0165] In the context of co-cultivation as described herein, the growth restriction or reduction of the genetically modified cells refers to the reduced affinity and uptake rate of the cells for a specific carbon source (low affinity strains), which means that it cannot effectively compete with strains with a higher affinity for the same specific carbon source. This is especially true when growth on a specific carbon source is under carbon-restricted conditions, such as in the feed phase of a carbon-restricted batch feed or continuous culture, in which case the strains with higher affinity will reduce the residual concentration of the specific carbon source in the culture medium to such a low level that the strains with lower affinity will hardly grow on the carbon source. In addition, even in the batch phase with excess carbon source, low affinity strains are at a significant disadvantage compared to high affinity strains because the maximum growth rate is also affected by the lack of the main carbon source uptake system for the specific carbon source (e.g., when the specific carbon source is glucose, ptsG is missing), because the maximum carbon source uptake rate is affected, and thus the maximum growth rate is also affected.

[0166] Gram-negative cells are known to have periplasmic space between the inner membrane of the cytoplasm and the outer membrane of the bacteria. Gram-positive bacteria may also have periplasmic space, although this is usually significantly smaller. In preventing or limiting the growth of microbial cells in a specific carbon source, one option is to prevent the carbon source from entering the cytoplasmic matrix of the microbial cells, so the carbon source can enter the periplasmic space, but if it is prevented from entering the cytoplasmic matrix, the cell may still not be able to grow thereon. Another option is to prevent the cell from further processing the carbon source once the carbon source enters the cytoplasmic matrix, so that the carbon source can not enter the energy generation pathway required for cell growth, such as glycolysis, pentose phosphate pathway or Krebs (Krebs) circulation. This can be achieved by preventing the cell from contacting the enzyme that for example makes the carbon source phosphorylation required. This needs to eliminate the uptake of the carbon source by the PTS-uptake system (if such a system exists for the carbon source), and remove the enzyme responsible for its phosphorylation inside the cytoplasmic matrix.

[0167] In an embodiment, the ability to grow on a first carbon source but not on a second carbon source can be achieved by ensuring that the cell expresses the correct transporter for the selected carbon source, while not having an efficient transporter for a second carbon source. If the desired transporter is naturally present or absent in the host cell, the cell can be genetically engineered to exhibit the desired carbon source utilization.

[0168] The following sections describe the transport and utilization of different carbon sources: glucose, glycerol, sucrose, galactose, arabinose, sorbitol, fructose and maltose. It should be understood that in the methods described herein, the genetically modified cells can have functional transport / utilization of sugars from one or more groups, or one or more transporters or utilization enzymes of sugars from one or more groups can be reduced or eliminated, for example, by mutation or deletion of the relevant genes described in the following sections.

[0169] In some embodiments, the first genetically modified microbial cell grows on sucrose and does not grow or has limited growth on glucose, and the second genetically modified microbial cell grows on glucose and does not grow or has limited growth on sucrose.

[0170] In some embodiments, the first genetically modified microbial cell grows on sucrose and does not grow or has limited growth on glycerol, and the second genetically modified microbial cell grows on glycerol and does not grow or has limited growth on sucrose.

[0171] In some embodiments, the first genetically modified microbial cell grows on sucrose and does not grow or is growth-limited on maltose, and preferably also grows on glucose, and the second genetically modified microbial cell grows on maltose and does not grow or is growth-limited on sucrose.

[0172] In some embodiments, the first genetically modified microbial cell grows on sucrose and does not grow or has limited growth on galactose, and the second genetically modified microbial cell grows on galactose and does not grow or has limited growth on sucrose.

[0173] In some embodiments, the first genetically modified microbial cell grows on sucrose and does not grow or has limited growth on fructose, and the second genetically modified microbial cell grows on fructose and does not grow or has limited growth on sucrose.

[0174] In some embodiments, the first genetically modified microbial cell grows on sucrose and does not grow or has limited growth on arabinose, and the second genetically modified microbial cell grows on arabinose and does not grow or has limited growth on sucrose.

[0175] In some embodiments, the first genetically modified microbial cell grows on sucrose and does not grow or has limited growth on sorbitol, and the second genetically modified microbial cell grows on sorbitol and does not grow or has limited growth on sucrose.

[0176] In some embodiments, the first genetically modified microbial cell grows on glucose and does not grow or has limited growth on sucrose, and the second genetically modified microbial cell grows on sucrose and does not grow or has limited growth on glucose.

[0177] In some embodiments, the first genetically modified microbial cell grows on glucose and does not grow or has limited growth on glycerol, and the second genetically modified microbial cell grows on glycerol and does not grow or has limited growth on glucose.

[0178] In some embodiments, the first genetically modified microbial cell grows on glucose and does not grow or has limited growth on galactose, and the second genetically modified microbial cell grows on galactose and does not grow or has limited growth on glucose.

[0179] In some embodiments, the first genetically modified microbial cell grows on glucose and does not grow or has limited growth on fructose, and the second genetically modified microbial cell grows on fructose and does not grow or has limited growth on glucose.

[0180] In some embodiments, the first genetically modified microbial cell grows on glucose and does not grow or has limited growth on arabinose, and the second genetically modified microbial cell grows on arabinose and does not grow or has limited growth on glucose.

[0181] In some embodiments, the first genetically modified microbial cell grows on glucose and does not grow or has limited growth on sorbitol, and the second genetically modified microbial cell grows on sorbitol and does not grow or has limited growth on glucose.

[0182] In some embodiments, the first genetically modified microbial cell grows on glycerol and does not grow or has limited growth on sucrose, and the second genetically modified microbial cell grows on sucrose and does not grow or has limited growth on glycerol.

[0183] In some embodiments, the first genetically modified microbial cell grows on glycerol and does not grow or has limited growth on glucose, and the second genetically modified microbial cell grows on glucose and does not grow or has limited growth on glycerol.

[0184] In some embodiments, the first genetically modified microbial cell grows on glycerol and does not grow or has limited growth on galactose, and the second genetically modified microbial cell grows on galactose and does not grow or has limited growth on glycerol.

[0185] In some embodiments, the first genetically modified microbial cell grows on glycerol and does not grow or has limited growth on fructose, and the second genetically modified microbial cell grows on fructose and does not grow or has limited growth on glycerol.

[0186] In some embodiments, the first genetically modified microbial cell grows on glycerol and does not grow or is growth-limited on maltose, and preferably also grows on glucose, and the second genetically modified microbial cell grows on maltose and does not grow or is growth-limited on glycerol.

[0187] In some embodiments, the first genetically modified microbial cell grows on glycerol and does not grow or has limited growth on arabinose, and the second genetically modified microbial cell grows on arabinose and does not grow or has limited growth on glycerol.

[0188] In some embodiments, the first genetically modified microbial cell grows on glycerol and does not grow or has limited growth on sorbitol, and the second genetically modified microbial cell grows on sorbitol and does not grow or has limited growth on glycerol.

[0189] In some embodiments, the first genetically modified microbial cell grows on galactose and does not grow or is growth-limited on maltose, and preferably also grows on glucose, and the second genetically modified microbial cell grows on maltose and does not grow or is growth-limited on galactose.

[0190] In some embodiments, the first genetically modified microbial cell grows on galactose and does not grow or has limited growth on fructose and preferably also grows on glucose, and the second genetically modified microbial cell grows on maltose and does not grow or has limited growth on galactose.

[0191] In some embodiments, the first genetically modified microbial cell grows on fructose and does not grow or is growth-limited on maltose, and preferably also grows on glucose, and the second genetically modified microbial cell grows on maltose and does not grow or is growth-limited on fructose.

[0192] In an embodiment, the one or more oligosaccharides produced by the co-culture are a mixture of at least two human milk oligosaccharides (HMOs).Preferably, at least two oligosaccharides are harvested from the co-culture. The HMO produced by the first and second genetically modified microbial cells can be independently selected from 2'FL, 3FL, 3'SL, 6'SL, DFL, LNT-II, LNT, LNnT, LNFP-I, LNFP-III, LNFP-V, LNFP-VI, FSL, LST-a, LST-b, LST-c, LST-d, LNDFH-II and LNDFH-III, DSLNT, pLNH, pLNnH, LNH, LNnH, (D)F-LNH-I, (D)F-LNH-II, (D)F-LNH-III, F-para-LNH-I, DF-para-LNH, DF-para-LNnH, TF-LNH, FLST b, FLST a, FLST-c, S-LNH, S-LNnH-I, FS-LNH, FS-LNnH-I, DS-F-LNH-II.

[0193] In a preferred embodiment, the HMO produced by the first and second genetically modified microbial cells can be independently selected from 2'FL, 3FL, 3'SL, 6'SL, DFL, LNT-II, LNT, LNnT, LNFP-I, LNFP-III, LNFP-V, LNFP-VI, LST-a, LST-c, LNDFH-II and LNDFH-III.

[0194] Glucose transport and utilization

[0195] Glucose is one of the most accepted carbon sources for microorganisms, and microbial cells can take up glucose and convert it into energy for growth through a variety of systems. Various glucose transport systems are well described, see for example Jaheris et al. 2008 FEMS Microbiol Rev 32: 891–907 (bacteria), Fuentes et al. 2013 Microbial Cell Factories 12: 42 (Escherichia coli), and Kim et al. 2013 Biochimica et Biophysica Acta 1830: 5204-5210 (yeast).

[0196] In embodiments described herein, cells grown on glucose have at least one glucose transport system. The glucose transport system can be selected from the systems described by Jaheris et al., Fuents et al., or Kim et al. Specifically, the glucose import system can be selected from a phosphoenolpyruvate:sugar phosphotransferase system (PTS), such as a PTS-dependent glucose (glc) utilization system, a PTS-dependent mannose (man) utilization system, a PTS-dependent maltose (mal) utilization system, a PTS-dependent β-glucosidase (bgl) utilization system, or a PTS-dependent N-acetylglucosamine (nag) utilization system. Alternative glucose transport systems are the galactose:H+ symporter GalP, the glucose uptake protein GlcU, the sodium / glucose transporter family (SGLT) or ABC transporters such as the galactose / glucose ABC transporter (mglABC) system, the trehalose / maltose / sucrose / palatinose (TMSP)-ABC transporter (malEFG) system and the glucose / mannose ABC transporter (glcEFG) system or MFS transporter systems such as the glucose proton symporter (glcP) and the glucose facilitator (glf) or the hexose transporter (HXT).

[0197] In other embodiments, reducing or preventing glucose from entering the cytoplasm of a microorganism can be achieved by mutating or deleting one or more sequences encoding proteins that affect glucose import capacity as described above.

[0198] In order to utilize glucose for energy production, once glucose enters the cytoplasm, the cell phosphorylates it into glucose-6-phosphate, which can enter energy-producing metabolic pathways such as glycolysis and the pentose phosphate pathway. Therefore, blocking the formation of glucose-6-phosphate can also be used to prevent cells from utilizing glucose as a carbon source for growth.

[0199] In an embodiment, the genetically modified microbial cell is a bacterium that grows reduced or no on glucose, wherein the functionality of one or more endogenous proteins involved in glucose import and utilization in the cell can be reduced or eliminated. Preferably, the protein is selected from the following:

[0200] i) Glucose PTS complex component IICB Glc (ptsG, e.g., Uniprot accession number P69786, or a functional variant thereof);

[0201] ii) β-Glucoside PTS complex component IIABC Bgl (bglF, e.g., Uniprot Accession No. P08722, or a functional variant thereof);

[0202] iii) Mannose PTS complex component - IICD Man (manX, e.g., Uniprot Accession No. P69797, or a functional variant thereof);

[0203] iv) N-acetylglucosamine PTS complex component IIABC Nag (nagE, e.g., Uniprot Accession No. P09323, or a functional variant thereof);

[0204] v) maltose / maltodextrin transport system (malX, e.g. Uniprot accession number P19642, or a functional variant thereof);

[0205] vi) a galactose / glucose high affinity ABC transporter component (mglC, e.g. Uniprot accession number P23200, or a functional variant thereof);

[0206] vii) trehalose / maltose / sucrose / palatinose (TMSP)-ABC transporter (malF, e.g. Uniprot accession number P02916, or a functional variant thereof);

[0207] viii) trehalose / maltose / sucrose / palatinose (TMSP)-ABC transporter (malG, e.g. Uniprot accession number P68183, or a functional variant thereof);

[0208] ix) galactose permease (galP, for example Uniprot accession number P0AEP1, or a functional variant thereof);

[0209] x) glucose proton symporter (glcP, e.g., Uniprot accession number 007563, or a functional variant thereof);

[0210] xi) glucose facilitator factor (glf, for example Uniprot accession number P37747 or P21906, or a functional variant thereof);

[0211] xii) a glucose uptake protein (glcU, e.g. Uniprot accession number P40420, or a functional variant thereof);

[0212] xiii) sodium / glucose transporter family (sglT, e.g. Uniprot accession number P96169, or a functional variant thereof); and

[0213] xiv) glucokinase (glk, e.g., Uniprot Accession No. P0A6V8, or a functional variant thereof); and

[0214] xv) Hexose transporter (HXT).

[0215] Wherein the proteins in i)-vi) and xv) are all part of various glucose import complexes, which are usually composed of multiple proteins. The proteins in items vii)-xiii) are single protein transporters identified in different bacterial species. The protein in xiv) is an example of a glucose utilization enzyme that phosphorylates glucose once glucose enters the cell. In the context of the present disclosure, it is preferred to reduce or eliminate the activity of membrane-bound transporters. Preferably, the genes to be mutated / deleted in the complex are italicized in brackets.

[0216] In an embodiment, the genetically modified microbial cell is an Escherichia coli, whose growth on glucose is reduced (restricted) or not grown, wherein the functionality of one or more endogenous proteins involved in glucose import and utilization in the cell can be reduced or eliminated. Preferably, the protein is selected from the following:

[0217] i) Glucose PTS complex component IICB Glc (ptsG)

[0218] ii) β-Glucoside PTS complex component IIABC Bgl (bglF)

[0219] iii) Mannose PTS complex component - IICD Man , (manX),

[0220] iv) N-acetylglucosamine PTS complex component IIABC Nag (nagE)

[0221] v) Maltose / maltodextrin transporter (malX)

[0222] vi) galactose / glucose high affinity ABC transporter component (mglC);

[0223] vii) galactose permease (galP); and / or

[0224] viii) Glucokinase (glk).

[0225] In embodiments described herein, at least one genetically modified cell has reduced or eliminated activity of at least one PTS-dependent sugar transport system selected from:

[0226] i) Glucose PTS complex component IICB Glc ;

[0227] ii) β-Glucoside PTS complex component - IIABC Bgl ;

[0228] iii) Mannose PTS complex component - IICD Man ;

[0229] iv) N-acetylglucosamine PTS complex component-IIABC Nag ;as well as

[0230] v) Maltose / Maltodextrin PTS Complex-IICB malX

[0231] vi) Sorbitol PTS Complex IICB slr .

[0232] Preferably, in bacteria that do not grow or have limited growth on glucose (e.g., E. coli), at least the glucose PTS complex component IICB is missing. Glc The ptsG gene.

[0233] Glycerol transport and utilization

[0234] In the embodiments described herein, the cells grown on glycerol have at least one glycerol transport system. The glycerol transport system can be selected from the group consisting of glycerol facilitator factor (glpF) or glycerol / H + -Symporter (stl1).

[0235] In other embodiments, reducing or preventing glycerol import into the cytoplasm of a microorganism can be achieved by mutating or deleting sequences encoding one or more proteins that affect glycerol import capacity, such as those encoding a glycerol facilitating factor (glpF) or a glycerol / H2O2 ... + - Deletion or mutation of the nucleic acid sequence of the symporter (stl1).

[0236] In order to utilize glycerol to produce energy, once glycerol enters the cytoplasmic matrix, the cell phosphorylates it into glycerol-3-phosphate, and phosphorylation is carried out by glycerol kinase (glpK). Therefore, blocking the formation of glycerol-3-phosphate can also help prevent cells from utilizing glycerol as a carbon source for growth. More information about glycerol utilization in various bacteria can be found in the review of LinAnn.Rev.Microbial.1976 30:535-78.

[0237] In an embodiment, the genetically modified cell grows less or not on glycerol, wherein the functionality of one or more endogenous proteins involved in glycerol import and utilization in the cell can be reduced or eliminated. Preferably, the reduction or elimination of protein activity is achieved by deleting or mutating one or more nucleic acid sequences encoding proteins selected from glpF (e.g., Uniprot Accession No., or a functional variant thereof), stl1 (e.g., Uniprot Accession No., or a functional variant thereof) and glpK (e.g., Uniprot Accession No., or a functional variant thereof).

[0238] If it is desired that bacteria (e.g., E. coli) grow less or no on glycerol, the functionality of the glycerol transporter (also known as glycerol facilitator (glpF)) is preferably reduced or eliminated, for example by mutating or deleting the glpF gene in the cell. In addition, the activity of glycerol kinase may be reduced or eliminated, for example, by mutating or deleting the glpK gene in the cell.

[0239] In an embodiment of the method described herein, the functionality of a protein involved in glucose and / or glycerol import and utilization in the genetically modified cell is reduced or eliminated by complete or partial inactivation of one or more genes selected from ptsG, bglF, manX, nagE, malX, mglC, glk and glpF.

[0240] Sucrose transport and utilization

[0241] In embodiments described herein, cells grown on sucrose have at least one sucrose transport system. The sucrose transport system can be a PTS-dependent sucrose (suc) utilization system. Alternatively, cells can grow on sucrose by having active sucrose invertase or sucrose hydrolase proteins in the outer membrane or periplasmic membrane (if present), which can decompose sucrose into glucose and fructose, which can then be taken up by cells through the relevant fructose and glucose transport systems (see, e.g., WO 2013 / 087884).

[0242] In an embodiment, the genetically modified microbial cell capable of growing on sucrose comprises one or more nucleic acid sequences encoding a PTS-dependent sucrose utilization system. The PTS-dependent sucrose utilization system can be, for example, encoded by scrY, scrA, scrB, and optionally scrR (see, for example, WO2015 / 197082), wherein the gene scrA encodes a sucrose transporter protease IIScr (e.g., SEQ ID NO: 48 or ncbi sequence ID: CAA40658.1 or a functional variant thereof), which provides intracellular sucrose-6-phosphate from extracellular sucrose by active transport across the cell membrane and concomitant phosphorylation. Sucrose-specific ScrY membrane pore protein (porin) (e.g., SEQ ID NO: 47 or ncbi sequence ID: CAA40657.1 or a functional variant thereof encoded by scrY) promotes the diffusion of sucrose through the outer membrane. ScrB invertase (e.g., SEQ ID NO: 49 or ncbi sequence ID: WP_000056853.1 or a functional variant thereof encoded by scrB) hydrolyzes the accumulated sucrose-6-phosphate into glucose-6-phosphate and fructose. scrR encodes a Lacl family DNA-binding transcriptional regulator (e.g., SEQ ID NO: 50 or ncbi sequence ID: WP_000851062.1 or a functional variant thereof).

[0243] WO2015 / 150328 describes an E. coli csc PTS-dependent sucrose system, which is expressed in the cscABKR gene cluster (SEQ ID NO: 52) encoding: a sucrose permease (e.g., cscB with UniProt accession number P30000.1 or a functional variant thereof), a fructokinase (e.g., csck with GenBank accession number EDV65567.1 or a functional variant thereof), a sucrose hydrolase (e.g., cscA with NCBI accession number WP_175214520.1 or a functional variant thereof), and a transcriptional repressor (e.g., cscR with GenBank accession number AJA27326.1 or a functional variant thereof).

[0244] Or, the genetically modified microbial cell that can grow on sucrose comprises the nucleic acid of coding invertase or sucrase, and described enzyme enables described cell to absorb sucrose.Invertase can be for example glycoside hydrolase and sucrose-6-phosphate hydrolase (for example, GeneBankID:SacC_Agal or its functional variant of WP_103853210.1) or beta-fructofuranosidase (for example, GeneBank ID:BW or its functional variant of BAD18121.1).Because sucrase or invertase can convert sucrose into glucose and fructose, for example in the periplasmic space of cell, cell should preferably can grow on glucose or fructose, this means that in double bacterial strain system, another kind of genetically modified cell should not grow or growth is restricted on fructose and / or glucose.

[0245] In embodiments where the carbon source is sucrose and it can be taken up by a sucrose invertase or sucrose hydrolase, the other cell of the dual strain system should preferably grow on glycerol or galactose.

[0246] In an embodiment, the genetically modified cell grows less or does not grow on sucrose, wherein the functionality of one or more endogenous proteins involved in sucrose input and utilization in the cell can be reduced or eliminated. Preferably, the reduction or elimination of protein activity is achieved by deleting or mutating one or more nucleic acid sequences encoding proteins selected from the PTS-dependent sucrose utilization system, such as by mutating or deleting the cscABKR gene cluster (SEQ ID NO: 52, or a functional variant thereof) (if the gene cluster is present in the cell and is functional). In particular, if the strain shows no growth or reduced growth on sucrose, then the deletion or mutation of the sucrose permease gene (e.g., cscB (e.g., Uniprot Accession No. P30000.1, or a functional variant thereof) or scrY (e.g., Uniprot Accession No. B1LQA1, or a functional variant thereof)) is relevant. Many non-pathogenic E. coli cells have lost the ability to grow on sucrose, so it is usually unnecessary to mutate the cell to prevent it from growing on sucrose because it no longer has the ability to do so.

[0247] Galactose transport and utilization

[0248] In embodiments described herein, cells grown on galactose have at least one galactose transport system. The galactose transport system can be selected from the galactose:H+ symporter GalP, the galactose / glucose ABC transporter (mglABC) system, the PTSLac (lacFE) system and / or the sodium / glucose transporter family (sglT).

[0249] Galactose imported into cells via GalP, mglABC or sglT is converted by galactokinase to galactose-1-phosphate (Gal1P), which in turn is metabolized to α-glucose-1-phosphate (G1P) via the Leloir pathway (galMKTE).

[0250] Galactose imported into cells via the PTSLac (lacFE) system is converted to galactose-6-phosphate (Gal6P) and further metabolized to trisaccharide phosphates via the Tag6P pathway (lacABCD).

[0251] Cells grown on galactose preferably also have a functional galactokinase (galK), a functional Leloir pathway and / or a Tag6P pathway.

[0252] In other embodiments, reducing or preventing galactose import into the cytoplasmic matrix of a microorganism can be achieved by mutating or deleting one or more sequences encoding proteins that affect galactose import capacity, such as deleting or mutating nucleic acid sequences of the galactose:H+ symporter GalP, the galactose / glucose ABC transporter (mglABC) system, the sodium / glucose transporter family (sglT) or the PTSLac (lacFE) system.

[0253] To utilize galactose for energy production, cells phosphorylate galactose to gal1P or gal6P. Therefore, mutating, deleting, or blocking the enzymes that convert galactose to gal1P or gal6P can also be used to prevent cells from utilizing galactose as a carbon source for growth.

[0254] In an embodiment, the genetically modified cell grows less or does not grow on galactose, wherein the functionality of one or more endogenous proteins involved in galactose import and utilization in the cell can be reduced or eliminated. Preferably, the reduction or elimination of protein activity is achieved by deletion or mutation of one or more nucleic acid sequences selected from encoding galP (e.g., Uniprot Accession No. P0AEP1, or a functional variant thereof), mglC (e.g., Uniprot Accession No. P23200, or a functional variant thereof), lacF (e.g., Uniprot Accession No. P24400, or a functional variant thereof), galK (e.g., Uniprot Accession No. P0A6T3, or a functional variant thereof) and / or sglT (e.g., Uniprot Accession No. P96169, or a functional variant thereof).

[0255] Fructose transport and utilization

[0256] In the embodiments described herein, the cells grown on fructose have at least one fructose transport system. The fructose transport system may be selected from the group consisting of fructose PTS complex components IIABC Fru, glucose PTS complex component IICB Glc , fructose transporter FruP.

[0257] Fructose introduced into the cell is converted to fructose-1-phosphate (fru1P) or fructose-6-phosphate (fru6P) by fructokinase.

[0258] In other embodiments, reducing or preventing fructose import into the cytoplasm of a microorganism can be achieved by mutating or deleting one or more sequences encoding proteins that affect fructose import capacity, such as deleting or mutating the sequences encoding fructose PTS complex component IIABC. Fru , glucose PTS complex component IICB Glc , the nucleic acid sequence of fructose transporter FruP.

[0259] To use fructose for energy production, cells phosphorylate fructose to fru1P or fru6P. Therefore, mutating, deleting, or blocking the enzymes that convert fructose to fru1P or fru6P can also be used to prevent cells from using fructose as a carbon source for growth.

[0260] In an embodiment, the genetically modified cell has reduced or no growth on fructose, wherein the functionality of one or more endogenous proteins involved in fructose import and utilization in the cell can be reduced or eliminated. Preferably, the reduction or elimination of protein activity is achieved by deleting or mutating one or more nucleic acid sequences selected from encoding fruA (e.g., Uniprot Accession No. P20966, or a functional variant thereof), ptsG (e.g., Uniprot Accession No. P69786, or a functional variant thereof), or FruP (e.g., Uniprot Accession No. F4TKS5, or a functional variant thereof).

[0261] Maltose transport and utilization

[0262] In the embodiments described herein, cells grown on maltose have at least one maltose transport system.

[0263] On top of this class of maltose transport systems is the MalFGK ABC superfamily transport system, which transports maltose to the cytoplasmic membrane of Escherichia coli. The MalFGK transport system is a heterotetrameric complex consisting of the integral membrane proteins MalF and MalG, which are associated with two units of the peripheral membrane protein MalK, which has ATP-binding properties and can therefore provide energy for the maltose permease encoded by malF and malG.

[0264] Alternatively, the maltose transport system may be selected from the maltose / maltodextrin PTS complex-IICB encoded by malX mal(For example, Uniprot Accession No. P19642, or a functional variant thereof.) The PTS enzyme-II protein encoded by malX is capable of recognizing glucose and maltose as substrates.

[0265] In embodiments where the maltose / maltodextrin PTS complex is used for one strain to grow on maltose, it means that the second strain does not grow or has limited growth on glucose as well as on maltose.

[0266] Maltose imported into the cell is converted to glucose by an amylomaltase encoded by malQ (eg, UniProt Accession No. P15977.2 or a functional variant thereof) or an alternative maltase from another species. Glucose is in turn phosphorylated as described above in the "Glucose Transport" section.

[0267] In other embodiments, reducing or preventing maltose import into the cytoplasm of a microorganism can be achieved by mutating or deleting one or more sequences encoding proteins that affect maltose import capacity, such as deleting or mutating a component of the MalFGK ABC superfamily transport system or a component IICB of the maltose PTS complex. mal In addition, the deletion of glucokinase (glk, such as Uniprot accession number P0A6V8, or a functional variant thereof) will prevent the utilization of maltose as a carbon source, because glucose needs to be phosphorylated to be converted into energy by the cell.

[0268] In an embodiment, the genetically modified cell has reduced or no growth on maltose, wherein the functionality of one or more endogenous proteins involved in maltose import and utilization in the cell can be reduced or eliminated. Preferably, the reduction or elimination of protein activity is achieved by deleting or mutating one or more selected from the group consisting of encoding MalF (e.g., UniProt Accession No. P02916.1 or a functional variant thereof), MalG (e.g., GenBank: AAC77002.1 or a functional variant thereof), MalK (e.g., UniProt Accession No. P02916.1 or a functional variant thereof), PTS complex-IICB encoded by malX mal (e.g., Uniprot Accession No. P19642, or a functional variant thereof) and / or a nucleic acid sequence of a glucose kinase encoded by glk (e.g., Uniprot Accession No. P0A6V8, or a functional variant thereof).

[0269] Arabinose transport and utilization

[0270] In the embodiments described herein, cells grown on arabinose have at least one arabinose transport system.

[0271] On such an arabinose transport system is the AraFGH arabinose transporter, which is a member of the ATP cassette (ABC) transporter superfamily. AraF is a periplasmic binding protein (e.g., UniProt Accession No. P02924 or a functional variant thereof), AraH is a membrane component (e.g., UniProt Accession No. P0AE26 or a functional variant thereof), and AraG is the ATP-binding component of this ABC transporter (e.g., UniProt Accession No. P0AAF3 or a functional variant thereof).

[0272] Alternatively, the arabinose transport system may be selected from the arabinose-proton symporter AraE (eg, UniProt accession number P0AE24 or P96710 or a functional variant thereof).

[0273] In an embodiment, the genetically modified cell has reduced or no growth on arabinose, wherein the functionality of one or more endogenous proteins involved in arabinose import and utilization in the cell can be reduced or eliminated. For example, arabinose is prevented from being imported into the cytoplasmic matrix of the microorganism by mutating or deleting one or more sequences encoding proteins that affect arabinose import capacity, such as deleting or mutating nucleic acid sequences encoding components of the AraFGH ABC superfamily transport system or the maltose arabinose-proton symporter AraE.

[0274] Sorbitol transport and utilization

[0275] In embodiments described herein, cells grown on sorbitol have at least one sorbitol transport system.

[0276] The transport of sorbitol into prokaryotic cells is facilitated by the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Sorbitol-specific enzymes IIB and IIC (EIIBC srl ) components are responsible for binding to sorbitol and initiating its transport into the cell, and the enzymes are encoded by srlA (e.g., Uniprot Accession No. P56579 or O32333 or functional variants thereof) and srlE (e.g., Uniprot Accession No. P56580 or O32332 or functional variants thereof), respectively. As part of the PTS process, the input sorbitol molecules are simultaneously catalyzed by the sorbitol kinase (EIIA) encoded by the gene srlB (e.g., Uniprot Accession No. P05706 or A5I7D9 or functional variants thereof). srl ) phosphorylation.

[0277] In embodiments where the sorbitol PTS complex is used for one strain to grow on sorbitol, it is intended that the second strain does not grow or has limited growth on glucose as well as on maltose.

[0278] In embodiments, the genetically modified cells have reduced or no growth on sorbitol, wherein the functionality of one or more endogenous proteins involved in sorbitol import and utilization in the cells can be reduced or eliminated. For example, sorbitol is prevented from being imported into the cytoplasmic matrix of the microorganism by mutating or deleting one or more sequences encoding proteins that affect the sorbitol PTS system.

[0279] Whichever carbon source transport system is selected, it is expected that the first genetically modified cell exports the first oligosaccharide produced by the cell into the culture medium, and the second genetically modified cell exports the second oligosaccharide produced by the cell into the culture medium, so that they are easy to undergo transglycosidase reaction in the culture medium.

[0280] In an embodiment, the HMO that can be advantageously produced by one of the genetically modified cells and exported into the culture medium and can be used as a donor oligosaccharide in a transglycosylation reaction can be selected from 2'FL, 3FL, DFL, LNFP-I, 3'SL, 6'SL, FSL, LST-a, LNT.II, LNT and LNnT.

[0281] In an embodiment, the HMO produced by one of the genetically modified cells and used as an acceptor oligosaccharide in the transglycosylation reaction can be selected from 2'FL, 3FL, LNT-II, LNT, LNnT, p-LNnH, LNFP-I, LNFP-II, LNFP-III, LNFP-IV, LNFP-V, LNFP-VI, 3'SL, 6'SL, LST-a and LST-c.

[0282] In an embodiment, the HMO that can be advantageously produced by one of the genetically modified cells and exported into the culture medium and used as an acceptor oligosaccharide in the transglycosylation reaction can be selected from 2'FL, 3FL, LNT-II, LNT, LNnT and LNFP-I.

[0283] In order to achieve in situ recycling of by-products, it is advantageous if at least one genetically modified cell is able to internalize the initial substrate for the production of the first and / or second oligosaccharide.

[0284] In embodiments, at least one genetically modified cell is capable of internalizing lactose, 2'FL, 3FL and / or LNT-II, depending on which compound is used as the initial substrate for making the first or second HMO. The initial substrate internalized by the cell corresponds to the byproduct produced by the transglycosidase reaction, thereby recycling it.

[0285] In the embodiment where two different genetically modified cells are co-cultured and both use lactose as a substrate for producing the corresponding oligosaccharides, since the transglycosidase process only produces one lactose molecule, but two lactose molecules are required to produce more donor and acceptor oligosaccharides, lactose needs to be fed throughout the fermentation process. If the lactose feed is stopped at the end of the fermentation based on the design of achieving complete conversion of the acceptor oligosaccharide / HMO or a higher molar ratio of the third complex HMO to the acceptor oligosaccharide / HMO, it is preferred to inactivate the transglycosidase while reducing the lactose feed to prevent the accumulation of the enzymatic step leaving group (e.g., lactose) caused by the reverse reaction triggered by the side hydrolysis activity of the enzyme, and to maintain the designed product composition. This can be achieved by changing the pH, temperature, or adding a protease.

[0286] Transglycosidase

[0287] Glycoside hydrolases are carbohydrate-processing enzymes found in nature. In addition to their hydrolytic activity, some of them also exhibit high transglycosylation activity, also known as transglycosidases, which catalyze the transfer of sugar moieties between different glycosides and / or oligosaccharides.

[0288] In the context of the hybrid method described herein, it is advantageous if the transglycosidase has a hydrolytic activity as low as possible. Hydrolytic activity (e.g., trans-sialidase) causes the hydrolysis of donor oligosaccharides (in the case of 3'SL, the hydrolysis reaction produces lactose and sialic acid) and the hydrolysis of the third oligosaccharide to form an acceptor and sialic acid (e.g., 3FL and sialic acid are produced in the case of FSL). Hydrolytic activity (e.g., trans-fucosidase) causes the hydrolysis of donor oligosaccharides (in the case of 2'FL or 3FL, the hydrolysis reaction produces lactose and fucose) and the hydrolysis of the third oligosaccharide to form an acceptor and fucose (e.g., LNnT and fucose are produced in the case of LNFP-III). Typically, in the case where the acceptor substrate is sufficient relative to the donor substrate, the hydrolytic activity of the enzyme is suppressed. However, since the side hydrolyzate (e.g., lactose) is recycled to produce the first oligosaccharide in the genetically modified cell in the hybrid process, the impact on the formation of the third HMO product is very low, because the transglycosidase activity will regenerate the third oligosaccharide subsequently. The hydrolysis and transfucosylation activities of transfucosidases can be measured as described, for example, in Zeuner et al. 2018 Enzyme and Microbial Technology 115:37–44. Similar assays can be performed for transsialidases using 3'SL or 6'SL instead of 3FL. However, for functionality in the hybrid process described herein, it is preferred to compare the hydrolysis activities of potential transglycosidases in the actual process and then evaluate the amount of fucose or sialic acid generated by each enzyme. It is desirable to use a transfucosidase that produces as little fucose as possible in the hybrid method described herein. Similarly, in the hybrid method described herein, it is desirable to use a transsialidase that produces as little sialic acid as possible. Fucose and sialic acid levels can be measured by, for example, HPLC or alternative methods known to those skilled in the art.

[0289] In the mixing methods described herein, a transglycosidase is added to the mixing process in an amount sufficient to mediate transglycosylation of the acceptor oligosaccharide with a sugar moiety from the donor oligosaccharide. In the mixing methods where the transferred sugar moiety is a fucosyl or sialic acid moiety, the enzyme having transglycosidase activity is a transfucosidase or a transsialidase, respectively.

[0290] In the two-strain hybrid method, the transferred sugar moiety is a galactose or N-acetylglucosamine (GlcNAc) moiety, and the enzyme having transglycosidase activity is trans-β-galactosidase, trans-lacto-N-biosidase or β-N-acetylglucosaminidase.

[0291] In an embodiment, the transglycosidase is selected from α-1,2-transfucosidase, α-1,3-transfucosidase, α-1,3 / 4-transfucosidase, α-2,3-transsialidase, α-2,6-transsialidase, β-N-acetylglucosaminidase, trans-lacto-N-biosidase and trans-β-galactosidase.

[0292] It is advantageous if the transfucosidase is able to use fucosyllactose (e.g. 2'FL, 3FL or DFL) as a fucosyl donor and a second oligosaccharide as an acceptor. Likewise, it is advantageous if the transsialidase is able to use sialyl lactose (3'SL or 6'SL) as a sialic acid donor and a second oligosaccharide as an acceptor. It is advantageous if the β-1,3-N-acetylglucosaminidase is able to use LNT-II as a GlcNAc donor and a second oligosaccharide as an acceptor. It is advantageous if the β-1,3-galactosidase is able to use LNT as a galactose donor and a second oligosaccharide as an acceptor. It is advantageous if the trans-lacto-N-biosidase is able to use LNT as a lacto-N-biosaccharide and a second oligosaccharide as an acceptor.

[0293] In embodiments, wherein the complex oligosaccharide having at least four or five monosaccharide units produced by the hybrid method described herein is an HMO, the transglycosidase has substrate specificity for an oligosaccharide acceptor, preferably an HMO containing at least three monosaccharide units, such as four, five, six or seven monosaccharide units. In embodiments, the complex oligosaccharide has at least five monosaccharide units, such as at least six monosaccharide units, and is a neutral non-fucosylated complex oligosaccharide.

[0294] In embodiments wherein the sialylated and / or fucosylated oligosaccharide having at least four monosaccharide units produced by the hybrid methods described herein is an HMO, the transfucosidase or transsialidase has substrate specificity for an oligosaccharide acceptor, preferably an HMO containing at least three monosaccharide units, e.g., four, five, six or seven monosaccharide units.

[0295] In an embodiment, the transfucosidase and / or transsialidase has substrate specificity for at least one acceptor disaccharide or oligosaccharide selected from the group consisting of LNB, LAcNAc, 2'FL, 3FL, Lewis A, Lewis X, 2'FLacNAc, 2'FLNB, LNT, LNnT, LNH, LNnH, p-LNH, p-LNnH, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LSTa, and LSTc.

[0296] In a further embodiment, the transfucosidase and / or transsialidase has substrate specificity for at least one HMO acceptor oligosaccharide selected from 2'FL, 3FL, LNT, LNnT, LNH, LNnH, p-LNH, p-LNnH, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LSTa and LSTc.

[0297] One embodiment of the present application is a method for producing FSL, comprising the following steps:

[0298] a) culturing the genetically modified cells producing 3FL in a culture medium; and

[0299] b) supplying 3'SL to the culture medium of a); and

[0300] c) making an enzyme having trans-sialidase activity available in the culture medium; and

[0301] d) incubating 3FL, 3'SL, and trans-sialidase in a culture medium to form FSL and lactose; and

[0302] The lactose is then recycled by the cells to produce more 3FL.

[0303] Another embodiment of the present application is a method for producing LNDFH-I, comprising the following steps:

[0304] a) culturing the genetically modified cells producing 3FL in a culture medium, and

[0305] b) supplying LNFP-I to the culture medium of a), and

[0306] c) making an enzyme having transfucosidase activity available in the culture medium, and

[0307] d) incubating 3FL, LNFP-I and transfucosidase in a culture medium to form LNDFH-I and lactose, and

[0308] The lactose is then recycled by the cells to produce more 3FL.

[0309] Another embodiment of the present application is a method for producing LST-c, comprising the following steps:

[0310] a) culturing the genetically modified cell producing 6'SL in a culture medium, and

[0311] b) supplying LNnT to the culture medium of a), and

[0312] c) making an enzyme having trans-sialidase activity available in the culture medium, and

[0313] d) incubating 6'SL, LNnT and trans-sialidase in a culture medium to form LST-c and lactose, and

[0314] The lactose is recycled by the cell to produce more 6'SL.

[0315] Another embodiment of the present application is a method for producing LST-c, the method comprising the following steps:

[0316] a) culturing a first genetically modified cell producing LNnT in a culture medium; and

[0317] b) supplying 6'SL to the culture medium of a); and

[0318] c) making an enzyme having trans-sialidase activity available in the culture medium; and

[0319] d) incubating 6'SL, LNnT and trans-sialidase in a culture medium to form LST-c and lactose; and

[0320] The lactose is then recycled by the cells to produce more LNnT.

[0321] Another embodiment of the present application is a method for producing LNFP-III, comprising the following steps:

[0322] a) culturing the genetically modified cells producing 3FL in a culture medium, and

[0323] b) supplying LNnT to the culture medium of a), and

[0324] c) making an enzyme having transfucosidase activity available in the culture medium, and

[0325] d) incubating 3FL, LNnT and transfucosidase in a culture medium to form LNFP-III and lactose, and

[0326] The lactose is then recycled by the cells to produce more 3FL.

[0327] Another embodiment of the present application is a method for producing LST-c, comprising the following steps:

[0328] a) co-culturing a first genetically modified cell that grows on the first carbon source and produces 6'SL and a second genetically modified cell that grows on the second carbon source and produces LNnT in a medium supplied with first and second carbon sources; and

[0329] b) making an enzyme having trans-sialidase activity available in the culture medium, and

[0330] c) incubating 6'SL, LNnT and trans-sialidase in a culture medium to form LST-c and lactose, and

[0331] The lactose is recycled by the first and second genetically modified cells to produce more 6'SL and LNnT.

[0332] Another embodiment of the present application is a method for producing LST-a, the method comprising the following steps:

[0333] a) culturing a first genetically modified cell that produces LNT in a culture medium; and

[0334] b) supplying 3'SL to the culture medium of a); and

[0335] c) making an enzyme having trans-sialidase activity available in the culture medium; and

[0336] d) incubating 3'SL, LNT and trans-sialidase in a culture medium to form LST-c and lactose; and

[0337] The lactose is then recycled by the cells to produce more LNT.

[0338] Another embodiment of the present application is a method for producing LST-a, the method comprising the following steps:

[0339] a) culturing a first genetically modified cell that produces a 3'SL in a culture medium; and

[0340] b) supplying LNT to the culture medium of a); and

[0341] c) making an enzyme having trans-sialidase activity available in the culture medium; and

[0342] d) incubating 3'SL, LNT and trans-sialidase in a culture medium to form LST-c and lactose; and

[0343] The lactose is recycled by the cell to produce more 3'SL.

[0344] Another embodiment of the present application is a method for producing LST-a, comprising the following steps:

[0345] a) co-culturing a first genetically modified cell that grows on the first carbon source and produces 3'SL and a second genetically modified cell that grows on the second carbon source and produces LNT in a medium supplied with first and second carbon sources; and

[0346] b) making an enzyme having trans-sialidase activity available in the culture medium, and

[0347] c) incubating 3'SL, LNT and trans-sialidase in a culture medium to form LST-a and lactose, and

[0348] The lactose is recycled by the first and second genetically modified cells to produce more 3'SL and LNT.

[0349] To expand upon the above specific embodiments and to specifically illustrate methods for producing specific complex oligosaccharides or oligosaccharides having a LacNAc or LNB moiety at the reducing end, Table 1 below is a non-limiting list of sialylated and / or fucosylated oligosaccharides of at least four monosaccharide units that may be obtained using different transglycosidase activities, wherein fucosyllactose or sialyllactose serves as the donor oligosaccharide and a second oligosaccharide serves as the acceptor oligosaccharide.

[0350] Table 1: Non-limiting examples of complex oligosaccharides obtainable using the mixing process

[0351]

[0352]

[0353]

[0354] Examples of trans-lacto-N-biosidase from Bifidobacterium longum JCM1217 (LnbX, Sakamura et al., J. Biol. Chem. 288, 25194 (2013), GenBank No. DAA64542) and truncated functional analogs thereof can be used to make linear lacto-N-biose containing oligosaccharides. In one embodiment, a variant having 70% identity to the sequence at amino acid positions 45 to 625 in GenBank No. DAA64542 is mutated at least at one or more amino acid positions selected from 410, 416, 439 and 442, which are used to transfer the lacto-N-biose moiety from the donor oligosaccharide to the acceptor oligosaccharide according to the amino acid numbering of GenBank No. DAA64542. The advantages of the variants are described in PA202201151, where they are shown to function in an in vitro process for generating pLNH from LNT and LNnT.

[0355] Additionally, Tables 4 and 5 below show non-limiting examples of relevant trans-sialidases and trans-fucosidases.

[0356] Trans-sialidase

[0357] The enzyme having trans-sialidase activity and suitable for the purpose of the method for preparing sialylated oligosaccharides using the hybrid process described herein may be selected from sialidases and trans-sialidases.

[0358] Sialidases or neuraminidase (EC 3.2.1.18) and trans-sialidases (EC 2.4.1.-) are both classified in the GH33 family defined by the CAZY nomenclature (http: / / www.cazy.org) and have the ability to hydrolyze the α-linkage of the terminal sialic acid of various sialic acid glycoconjugates (exo-α-sialidases), said sialic acid being bound to galactose or glucose in α-2,3 or α-2,6 linkages. These enzymes are found, inter alia, in various viral families and bacteria, but also in protozoa, some invertebrates and mammals. Although sialidases have hydrolytic activity, they are able to act as catalysts for trans-sialylation reactions due to their α-2,3 and / or α-2,6 selective trans-sialidase activity.

[0359] In order to improve the trans-sialidase activity of sialidases, they can be modified by various engineering techniques. Preferably, under the conditions of the hybrid method described herein, the formation of sialic acid is low. Preferably, the amount of sialic acid is less than 5% of the total mole % of the donor oligosaccharide and the third oligosaccharide, more preferably less than 3% of the total mole % of the donor oligosaccharide and the third oligosaccharide. WO2012 / 007588 describes a series of suitable trans-sialidases.

[0360] Table 2: Suitable trans-sialidases

[0361]

[0362] In an embodiment, the trans-sialidase is selected from a suitable trans-sialidase in Table 2 or a functional homologue thereof, the amino acid sequence of which is at least 70% identical, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or even 97%, 98% or 99% identical to the sequence of a single trans-sialidase in Table 2.

[0363] In one embodiment, the transfucosidase comprises or consists of the amino acid sequence of SEQ ID NO: 13, 14, 40, 41 or 60.

[0364] In embodiments where the trans-sialidase is added to the fermentation broth, it is sterile filtered prior to its introduction into the mixing process.The trans-sialidase is added to the mixing process with sufficient activity to mediate transsialylation of the acceptor oligosaccharide with the donor oligosaccharide.

[0365] In an alternative embodiment, the genetically modified cell capable of producing the first oligosaccharide is further modified by introducing a heterologous nucleic acid encoding a trans-sialidase. Preferably, the trans-sialidase is secreted / exported into the culture medium by the further genetically modified cell. The heterologous nucleic acid encoding the trans-sialidase can be expressed from an inducible promoter so that the expression of the trans-sialidase is delayed compared to the formation of the first oligosaccharide produced by the same cell. The advantage of delayed expression of the trans-sialidase is that the first oligosaccharide will not become rate limiting in the enzymatic step of the hybrid process.

[0366] Transfucosidase

[0367] The enzyme having transfucosidase activity and suitable for the purpose of the method for preparing fucosylated oligosaccharides using the hybrid process described herein may be selected from the group consisting of fucosidase and transfucosidase.

[0368] α-L-fucosidases are classified according to EC 3.2.1.38 and EC 3.2.1.51 and belong to glycoside hydrolase families 29 and 95 (GH29 and GH95) as defined by the CAZY nomenclature (http: / / www.cazy.org). The substrate specificity of the GH29 family is broad, while the GH95 family has a strict specificity for α-1,2-linked fucosyl residues. The GH29 family appears to be divided into two subfamilies. One subfamily generally has a strict specificity for α-1,3- and α-1,4-fucosidic bonds. Members of the other subfamily have a broader specificity, covering two or three α-fucosyl bonds. α-L-fucosidases generally hydrolyze terminal fucosyl residues from glycans. These enzymes are also able to act as catalysts for fucosylation reactions due to their transfucosylation activity and can therefore be used in the context of the hybrid methods described herein.

[0369] In order to improve the transfucosidase activity, the fucosidase can be modified by various engineering techniques. WO2016 / 063261 and Zeuner et al. (2018Enzyme and Microbial Technology 115:37-44) describe mutants of α-1-3 / 4 transfucosidase from Bifidobacterium longum infantis subspecies ATCC 15697 (NCBI accession number WP_012578573) or Bifidobacterium bifidum JCM1254 (GenBank BAH80310.1), whose transfucosidase activity is increased and the hydrolase activity is reduced. Preferably, under the conditions of the hybrid method described herein, the formation of fucose is less. Preferably, the amount of fucose is less than 5% of the total mole % of the donor oligosaccharide and the third oligosaccharide, more preferably less than 3% of the total mole % of the donor oligosaccharide and the third oligosaccharide.

[0370] Table 3: Suitable transfucosidases

[0371]

[0372] In an embodiment, the transfucosidase is selected from a suitable transfucosidase in Table 3 or a functional homolog thereof, the amino acid sequence of which has at least 70% identity, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or even 97%, 98% or 99% identity compared to a single transfucosidase sequence in Table 3.

[0373] In an embodiment, the transfucosidase is derived from Bifidobacterium bifidum or Bifidobacterium longum.

[0374] In one embodiment, the transfucosidase comprises or consists of the amino acid sequence of SEQ ID NO: 19, 30, 39 or 59.

[0375] In an embodiment, the transfucosidase is added to the mixing process and is sterile filtered prior to introduction into the mixing process. The transfucosidase is added to the mixing process with sufficient activity to mediate transfucosylation of the acceptor oligosaccharide with the donor oligosaccharide.

[0376] In an alternative embodiment, the genetically modified cell capable of producing the first oligosaccharide is further modified by introducing a heterologous nucleic acid encoding a transfucosidase. Preferably, the transfucosidase is secreted / exported into the culture medium by the further genetically modified cell. The heterologous nucleic acid encoding the transfucosidase can be expressed from an inducible promoter so that expression of the transfucosidase is delayed compared to the formation of the first oligosaccharide produced by the same cell. The advantage of delayed expression of the transfucosidase is that the first oligosaccharide will not become rate limiting in the enzymatic step of the mixing process.

[0377] Genetically modified cells

[0378] In this context, the terms "genetically modified cells" and "genetically engineered cells" are used interchangeably. As used herein, "genetically modified cells" refer to host cells whose genetic material has been altered by human intervention using genetic engineering techniques, such as, but not limited to, transformation or transfection, such as using heterologous polynucleotide sequences, Crisper / Cas editing and / or random mutations. In one embodiment, the genetically engineered cells have been transformed or transfected using a recombinant nucleic acid sequence.

[0379] The genetic modification may for example be selected from the addition of glycosyltransferases, transglycosidases and / or metabolic pathway engineering and the addition of transporters, including importins and exportins as described in this application, the skilled person will know how to combine them all into a genetically modified cell capable of producing the desired HMO.

[0380] The genetically modified cells are preferably microbial cells, such as prokaryotic cells or eukaryotic cells. Suitable microbial cells that can function as host cells include bacterial cells, archaeal cells, algae cells and fungal cells.

[0381] The genetically engineered cell can be, for example, a bacterial or yeast cell. In a preferred embodiment, the genetically engineered cell is a bacterial cell.

[0382] Host cells

[0383] Regarding bacterial host cells, there are in principle no restrictions; they can be eubacteria (Gram-positive or Gram-negative) or archaea, as long as they allow genetic manipulation to insert the gene of interest and can be cultured on a manufacturing scale. Preferably, the host cell has properties that allow cultivation to high cell densities. Non-limiting examples of bacterial host cells suitable for the recombinant industrial production of HMOs according to the invention can be Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Campylobacter sp, Corynebacterium sp, Pantoea citrea, Pectobacterium carotovorum or Xanthomonas campestris. Bacillus bacteria, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans, can also be used.Similarly, bacteria of the genera Lactobacillus and Lactococcus can be engineered using the methods of the invention, including, but not limited to, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also bacterial species suitable for use in the invention described herein. Also included as part of the invention are strains engineered as described herein, such as from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomosporas spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa). aeruginosa)).

[0384] Non-limiting examples of fungal host cells suitable for the recombinant industrial production of heterologous products are yeast cells of the genera Komagataella, Kluyveromyces, Yarrowia, Pichia, Saccaromyces, Schizosaccharomyces or Hansenula, or filamentous fungi from the genera Aspargillus, Fusarium or Thricoderma. More specifically, yeast cell species such as Komagataella phaffii, Kluyveromyces lactis, Yarrowia lipolytica, Pichia pastoris, and Saccaromyces cerevisiae, or filamentous fungal species such as A. niger, A. nidulans, A. oryzae, F. solani, F. graminearum, and T. reesei.

[0385] In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of Escherichia, Bacillus, Lactobacillus, Corynebacterium, and Campylobacter.

[0386] In one or more exemplary embodiments, the genetically engineered cell is selected from Escherichia coli, Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

[0387] In one or more exemplary embodiments, the genetically engineered cell is Bacillus subtilis.

[0388] In one or more exemplary embodiments, the genetically engineered cell is Saccharomyces cerevisiae or Pichia pastoris.

[0389] In one or more exemplary embodiments, the genetically engineered cell is Escherichia coli.

[0390] In one or more exemplary embodiments, the present invention relates to a genetically engineered cell, wherein the cell is derived from E. coli K-12 strain or DE3.

[0391] Glycosyltransferase

[0392] The genetically modified cell according to the present invention comprises at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase capable of transferring a glycosyl residue from a glycosyl donor to an acceptor oligosaccharide to synthesize an oligosaccharide product, such as a human milk oligosaccharide product. The nucleic acid sequence encoding one or more expressed glycosyltransferases can be integrated into the genome of the genetically engineered cell (via chromosomal integration), or it can be contained in a plasmid and expressed in a plasmid-borne form as described in the present disclosure.

[0393] The genetically modified cell according to the invention may comprise at least two recombinant nucleic acid sequences encoding two different glycosyltransferases capable of transferring a glycosyl residue from a glycosyl donor to an acceptor oligosaccharide.

[0394] The one or more glycosyltransferases are preferably selected from enzymes having α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3 / 4-fucosyltransferase, α-1,4-fucosyltransferase, α-2,3-sialyltransferase, α-2,6-sialyltransferase, β-1,3-N-acetylglucosaminyltransferase, β-1,6-N-acetylglucosaminyltransferase, β-1,3-galactosyltransferase and β-1,4-galactosyltransferase activity, described in more detail below.

[0395] β-1,3-N-acetyl-glucosaminyltransferase

[0396] β-1,3-N-acetyl-glucosaminyltransferase is any protein capable of transferring N-acetyl-glucosamine of UDP-N-acetyl-glucosamine to lactose or another acceptor molecule with a β-1,3-bond. Preferably, the β-1,3-N-acetyl-glucosaminyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding the β-1,3-galactosyltransferase is of heterologous origin. Non-limiting examples of β-1,3-N-acetyl-glucosaminyltransferase are given in Table 4. β-1,3-N-acetyl-glucosaminyltransferase variants may also be useful, preferably, such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity with one of the β-1,3-N-acetyl-glucosaminyltransferases in Table 4.

[0397] Table 4. List of β-1,3-N-acetyl-glucosaminyltransferases

[0398]

[0399]

[0400] In an embodiment, the glycosyltransferase encoded by the genetically engineered cell is a β-1,3-N-acetyl-glucosaminyltransferase from Table 6. Preferably, the glycosyltransferase in the genetically engineered cell is a β-1,3-N-acetyl-glucosaminyltransferase from Neisseria meningitidis, such as the β-1,3-N-acetyl-glucosaminyltransferase of SEQ ID NO: 45 or a functional variant thereof.

[0401] β-1,3-galactosyltransferase

[0402] β-1,3-galactosyltransferase is any protein having the ability to transfer the galactose of UDP-galactose to the N-acetylglucosamine moiety with a β-1,3-bond and thereby to an acceptor molecule. Preferably, the β-1,3-galactosyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding the β-1,3-galactosyltransferase is of heterologous origin. Non-limiting examples of β-1,3-galactosyltransferases are given in Table 5. β-1,3-galactosyltransferase variants may also be useful, preferably, such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity with one of the β-1,3-galactosyltransferases in Table 5.

[0403] Table 5. List of β-1,3-galactosyltransferases

[0404]

[0405] In an embodiment, at least one glycosyltransferase encoded by the genetically engineered cell is a β-1,3-N-acetylglucosaminyltransferase and a β-1,3-galactosyltransferase. Preferably, the glycosyltransferase in the genetically engineered cell is a β-1,3-N-acetylglucosaminyltransferase selected from Table 4 and a β-1,3-galactosyltransferase selected from Table 5. More preferably, the 1,3-N-acetylglucosaminyltransferase is from Neisseria and the β-1,3-galactosyltransferase is from Helicobacter pylori, such as the β-1,3-N-acetylglucosaminyltransferase with GenBank reference number WP_002248149.1 and the β-1,3-galactosyltransferase with GenBank reference number WP_111735921.1.

[0406] β-1,4-galactosyltransferase

[0407] β-1,4-galactosyltransferase is any protein with the ability to transfer galactose of UDP-galactose to the N-acetylglucosamine moiety. Preferably, the β-1,4-galactosyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding the β-1,4-galactosyltransferase is of heterologous origin. Non-limiting examples of β-1,4-galactosyltransferases are given in Table 6. β-1,4-galactosyltransferase variants may also be useful, preferably, such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity with one of the β-1,4-galactosyltransferases in Table 6.

[0408] Table 6. List of β-1,4-galactosyltransferases

[0409] Protein name GenBank ID source GalT WP_001262061.1 Helicobacter pylori LqCy AAF42257.1 Neisseria meningitidis MC58

[0410] In an embodiment, at least one glycosyltransferase encoded by the genetically engineered cell is a β-1,3-N-acetylglucosaminyltransferase and a β-1,4-galactosyltransferase. Preferably, the glycosyltransferase in the genetically engineered cell is a β-1,3-N-acetylglucosaminyltransferase selected from Table 4 and a β-1,4-galactosyltransferase selected from Table 6. More preferably, the 1,3-N-acetylglucosaminyltransferase is from Neisseria and the β-1,4-galactosyltransferase is from Helicobacter pylori, such as the β-1,3-N-acetylglucosaminyltransferase with GenBank reference number WP_002248149.1 and the β-1,4-galactosyltransferase with GenBank reference number WP_001262061.1 or SEQ ID NO: 46, or a functional variant thereof.

[0411] α-1,2-fucosyltransferase

[0412] α-1,2-fucosyltransferase is a protein having the ability to catalyze the transfer of fucose from a donor substrate (e.g., GDP-fucose) to an acceptor molecule with an α-1,2-bond. Preferably, the α-1,2-fucosyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding the α-1,2-fucosyltransferase is of heterologous origin. Non-limiting examples of α-1,2-fucosyltransferases are given in Table 7. α-1,2-fucosyltransferase variants may also be useful, preferably, such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity with one of the α-1,2-fucosyltransferases in Table 7.

[0413] Table 7. List of α-1,2-fucosyltransferases

[0414]

[0415] In an embodiment, the glycosyltransferase encoded by the genetically engineered cell is an α-1,2-fucosyltransferase from Table 7. Preferably, the glycosyltransferase in the α-1,2-fucosyltransferase is from Helicobacter pylori, such as the α-1,2-fucosyltransferase having GenBank Accession No. WP_080473865.1.

[0416] α-1,3-fucosyltransferase

[0417] α-1,3-fucosyltransferase refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate (e.g., GDP-fucose) to an acceptor molecule with an α-1,3-bond. Preferably, the α-1,3-fucosyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding the α-1,3-fucosyltransferase is of heterologous origin. Non-limiting examples of α-1,3-fucosyltransferases are given in Table 8. α-1,3-fucosyltransferase variants may also be useful, preferably, such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity with one of the α-1,3-fucosyltransferases in Table 8.

[0418] Table 8. List of α-1,3-fucosyltransferases

[0419]

[0420] In an embodiment, the glycosyltransferase encoded by the genetically engineered cell is an α-1,3-fucosyltransferase from Table 8. Preferably, the glycosyltransferase in the genetically engineered cell is an α-1,3-fucosyltransferase from Helicobacter pylori, such as the α-1,3-fucosyltransferase of SEQ ID NO: 1 or a functional homolog thereof.

[0421] α-1,3 / 4-fucosyltransferase

[0422] α-1,3 / 4-fucosyltransferase refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate (e.g., GDP-fucose) to an acceptor molecule with an α-1,3- or α-1,4-bond. Preferably, the α-1,3 / 4-fucosyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding the α-1,3 / 4-fucosyltransferase is of heterologous origin. Non-limiting examples of α-1,3 / 4-fucosyltransferase are given in Table 9. α-1,3 / 4-fucosyltransferase variants may also be useful, preferably, such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity with one of the α-1,3 / 4-fucosyltransferases in Table 9.

[0423] Table 9. List of α-1,3 / 4-fucosyltransferases

[0424] Protein name GenBank ID source FucTIII AY450598.1 Helicobacter pylori strain DSM 6709 FutA WP_000487428.1 Helicobacter pylori ATCC 26695

[0425] In an embodiment, the glycosyltransferase encoded by the genetically engineered cell is an α-1,3 / 4-fucosyltransferase from Table 11. Preferably, the glycosyltransferase in the genetically engineered cell is an α-1,3 / 4-fucosyltransferase FutA from Helicobacter pylori, such as the α-1,3 / 4-fucosyltransferase of SEQ ID NO: 1 or a functional variant thereof.

[0426] α-2,3-Sialyltransferase

[0427] α-2,3-sialyltransferase refers to a glycosyltransferase that catalyzes the transfer of sialic acid from a donor substrate (e.g., CMP-N-acetylneuraminic acid) to an acceptor molecule with an α-2,3-bond. Preferably, the α-2,3-sialyltransferase used herein is not a species derived from a genetically engineered cell, i.e., the gene encoding the 2,3-sialyltransferase is of heterologous origin. Non-limiting examples of α-2,3-sialyltransferases are given in Table 10. α-2,3-sialyltransferase variants may also be useful, preferably, such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity with one of the α-2,3-sialyltransferases in Table 10.

[0428] Table 10. List of α-2,3-sialyltransferases

[0429]

[0430]

[0431] In an embodiment, the glycosyltransferase encoded by the genetically engineered cell is an α-2,3-sialyltransferase from Table 10. Preferably, the glycosyltransferase in the genetically engineered cell is an α-2,3-sialyltransferase from Campylobacter gullus, Neisseria meningitidis, or Pasteurella oralis, such as an α-2,3-sialyltransferase with GenBank accession number EGK8106227.1, AAC44541.1, or WP_101774487.1.

[0432] α-2,6-Sialyltransferase

[0433] α-2,6-sialyltransferase refers to a glycosyltransferase that catalyzes the transfer of sialic acid from a donor substrate (e.g., CMP-N-acetylneuraminic acid) to an acceptor molecule with an α-2,6-bond. Preferably, the α-2,6-sialyltransferase used herein is not a species derived from a genetically engineered cell, i.e., the gene encoding the 2,6-sialyltransferase is of heterologous origin. Non-limiting examples of α-2,6-sialyltransferases are given in Table 11. α-2,6-sialyltransferase variants may also be useful, preferably, such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity with one of the α-2,6-sialyltransferases in Table 11.

[0434] Table 11. List of α-2,6-sialyltransferases

[0435]

[0436] In an embodiment, the glycosyltransferase encoded by the genetically engineered cell is an α-2,6-sialyltransferase from Table 11. The glycosyltransferase in the genetically engineered cell is an α-2,6-sialyltransferase from Photorhabdus spp., such as an α-2,6-sialyltransferase with GenBank Accession No. AB500947.1 or BAF92026.1 or an α-2,6-sialyltransferase of SEQ ID NO: 43, or a functional variant thereof.

[0437] Nucleotide activated sugar pathway

[0438] In the genetically engineered cells used in the hybrid method described herein, the glycosylation reaction mediated by glycosyltransferase occurs in the cell, wherein the activated sugar nucleotide is used as a glycosyl donor. The activated sugar nucleotide usually has a phosphorylated glycosyl residue attached to a nucleoside. Specific glycosyltransferase only accepts specific sugar nucleotides. Therefore, preferably the following activated sugar nucleotides participate in glycosyl transfer: glucose-UDP-GlcNAc, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine (GlcNAc) and CMP-N-acetylneuraminic acid. Genetically modified cells according to the present invention can include one or more pathways that produce nucleotide activated sugars, and the nucleotide activated sugars are selected from glucose-UDP-GlcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and CMP-N-acetylneuraminic acid (CMP-Neu5Ac).

[0439] In an embodiment, the genetically modified cell can produce one or more of the above-mentioned activated sugar nucleotides by de novo pathways. In this regard, activated sugar nucleotides are made by cells under the action of enzymes participating in respective de novo biosynthetic pathways of sugar nucleotides, starting from simple carbon sources (such as glycerol, sucrose, fructose or glucose), according to a stepwise reaction sequence (for a review of monosaccharide metabolism, see, for example, HH Freeze and AD Elbein: in: Essentials of Glycobiology, Chapter 4 in the 2nd edition (Eds. A. Varki et al.): Glycosylation precursors, Cold Spring Harbour Laboratory Press (2009)).

[0440] The enzymes involved in the activated de novo sugar nucleotide biosynthesis pathway may be naturally present in the cell or may be introduced into the cell by means of genetic technology or recombinant DNA technology, all of which are part of the general knowledge of the person skilled in the art.

[0441] In embodiments, the pathway that produces the nucleotide activating sugar is the de novo GDP-fucose pathway (gmd, wcaG, manB, manC, and manA) and / or the sialic acid sugar nucleotide pathway (neuB, neuC, and neuA) as described below.

[0442] In another embodiment, the genetically modified cells can synthesize sugar nucleotides using salvage monosaccharides. In the salvage pathway, monosaccharides from degraded oligosaccharides are phosphorylated by kinases and converted into nucleotide sugars by pyrophosphorylases. The enzymes involved in this process can be heterologous or native to the host cell.

[0443] Colanic acid gene cluster

[0444] For the production of fucosylated oligosaccharides / HMOs, the colanic acid gene cluster is very important to ensure the presence of sufficient GDP-fucose. In E. coli, GDP-fucose is an intermediate in the production of the extracellular polysaccharide colanic acid, which is the major oligosaccharide of the bacterial cell wall. In the context of the present invention, the colanic acid gene cluster encodes most of the enzymes involved in the de novo synthesis of GDP-fucose (gmd, wcaG, wcaH, wcaI, manB, manC), while one or more genes downstream of GDP-L-fucose (e.g. wcaJ) can be deleted to prevent the conversion of GDP-fucose to colanic acid.

[0445] The de novo GDP-fucose pathway genes responsible for GDP-fucose formation include or consist of the following genes: i) manA, which encodes a protein mannose-6-phosphate isomerase (EC 5.3.1.8, UniProt accession number P00946), which promotes the interconversion of fructose-6-phosphate (F6P) and mannose-6-phosphate; ii) manB, which encodes a protein phosphomannose mutase (EC 5.4.2.8, UniProt accession number P24175), which is involved in the biosynthesis of GDP-mannose by catalyzing the conversion of mannose-6-phosphate to mannose-1-phosphate; iii) manC, which encodes the protein mannose-1-phosphate guanosyltransferase (EC:2.7.7.13, UniProt accession number P24174), which is involved in the biosynthesis of GDP-mannose by synthesizing GDP-mannose from GTP and α-D-mannose-1-phosphate; iv) gmd, which encodes the protein GDP-mannose-4,6-dehydratase (UniProt accession number P0AC88), which catalyzes the conversion of GDP-mannose to GDP-4-dehydro-6-deoxy-D-mannose; v) wcaG (fcl), which encodes the protein GDP-L-fucose synthase (EC 1.1.1.271, UniProt accession number P32055), which catalyzes the two-step NADP-dependent conversion of GDP-4-dehydro-6-deoxy-D-mannose to GDP-fucose.

[0446] Therefore, it is preferred that when the genetically engineered cell produces one or more fucosylated heterologous products, the entire colanic acid gene cluster and / or one or more genes of the de novo GDP-fucose pathway are overexpressed, said genes being selected from manA, manB, manC, gmd and wcaG.

[0447] In one or more exemplary embodiments, the colanic acid gene cluster responsible for GDP-fucose formation can be expressed from its native genomic site. Expression can be actively regulated to increase the formation of GDP-fucose. Expression can be regulated by exchanging the native promoter with the target promoter, and / or by expressing a gene cluster from another non-native genomic site, or by additionally expressing the colanic acid gene cluster or its specific gene to increase the copy number of the colanic acid gene encoding the protein.

[0448] With respect to the present disclosure, the term "natural genomic site" in relation to the colanic acid gene cluster relates to the original and natural location of the gene cluster in the genome of a genetically engineered cell.

[0449] Sialyl sugar nucleotide synthesis pathway

[0450] If the genetically modified cell will produce sialylated oligosaccharides / HMOs, then the genetically modified cell includes sialic acid sugar nucleotide synthesis capability, ie, the genetically modified cell includes a biosynthetic pathway for making sialic acid sugar nucleotides, such as CMP-N-acetylneuraminic acid as the glycosyl donor for the sialyltransferase. For example, the genetically modified cell comprises providing an exogenous UDP-GlcNAc 2-epimerase (e.g., neuC of Campylobacter jejuni (GenBank AAK91727.1) or an equivalent (e.g., (GenBank CAR04561.1)), a Neu5Ac synthase (e.g., neuB of Campylobacter jejuni (GenBank AAK91726.1) or an equivalent (e.g., Flavobacterium limnosediminis sialic acid synthase, GenBank WP_023580510.1) and / or a CMP-Neu5Ac synthetase (e.g., neuA of Campylobacter jejuni (GenBank AAK91728.1) or an equivalent (e.g., Vibrio brasiliensis CMP-sialic acid synthase, GenBank WP_006881452.1). SEQ ID NOs disclosed herein NO:44 is an example of a neuBCA gene cluster from Campylobacter jejuni where alternative functional variants are also suitable for making sialic acid sugar nucleotides in genetically modified cells.

[0451] In addition, the genetically modified cells preferably have a sialic acid decomposition pathway of deletion. "Sialic acid decomposition pathway" refers to a series of reactions usually controlled and catalyzed by an enzyme, which causes sialic acid degradation. The exemplary sialic acid decomposition pathway described below is an Escherichia coli pathway. In this pathway, sialic acid (Neu5Ac; N-acetylneuraminic acid) is degraded by enzymes NanA (N-acetylneuraminic acid lyase) and NanK (N-acetylmannosamine kinase) and NanE (N-acetylmannosamine-6-phosphate epimerase), which are all encoded by the nanATEK-yhcH operon and suppressed by NanR (http: / / ecocyc.org / ECOLI). Inactivation of the sialic acid catabolic pathway in an E. coli host can be achieved by introducing one or more mutations in the endogenous nanA (N-acetylneuraminic acid lyase) (e.g., GenBank Accession No. D00067.1 (GL216588)) and / or nanK (N-acetylmannosamine kinase) genes (e.g., GenBank Accession No. (amino acid) BAE77265.1 (GL85676015)) and / or nanE (N-acetylmannosamine-6-phosphate epimerase, GI:947745) (incorporated herein for reference). Optionally, the nanT (N-acetylneuraminic acid transporter) gene is also inactivated or mutated. Inactivation refers to altering the coding sequence so that the resulting gene product is functionally inactivated or the encoded gene product activity is less than 100%, such as 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20% of the native, naturally occurring, endogenous gene product. Therefore, in the present invention, the nanA, nanK, nanE and / or nanT genes are preferably inactivated.

[0452] Lactose importin

[0453] The genetically modified cells described herein are capable of importing into the cell a byproduct generated as a leaving group during the transglycosylation process (enzymatic process) of the hybrid process.

[0454] In an embodiment, the genetically modified cell comprises a byproduct importer. Preferably, the byproduct importer may import one or more of the following byproducts: lactose, 2'FL and / or 3FL.

[0455] Most lactose importers are capable of importing both lactose and 2'FL. In embodiments where the byproduct is lactose or 2'FL, the genetically modified cell has a functional lactose importer or 2'FL importer. Lactose importers are common in a variety of species including bacteria and yeast.

[0456] The lactose import protein may be, for example, a lactose permease. The lactose permease may be an endogenous lactose permease naturally expressed by the cell used to produce the first oligosaccharide.

[0457] In one or more embodiments, the genetically modified cell comprises one or more overexpressed lactose permease genes.

[0458] The genetically modified cell can include at least one (eg, at least two, three, four) nucleic acid sequence encoding a lactose permease.

[0459] In one or more further exemplary embodiments, one or more lactose permeases are encoded by heterologous and / or recombinant nucleic acid sequences. The native lactose permease can be genetically engineered, for example, by placing it under the control of a stronger promoter than the native promoter, thereby generating a recombinant lactose permease gene that overexpresses the native lactose permease protein.

[0460] In one or more preferred exemplary embodiments, the nucleic acid sequence encoding one or more lactose permeases is a native gene of the genetically modified cell.

[0461] In E. coli, the lactose permease is encoded by the lacY gene in the lactose lac operon. In an exemplary embodiment, the lactose permease in the genetically modified cell is LacY from E. coli. Preferably, the lactose permease comprises or consists of the amino acid sequence of SEQ ID NO: 3, or a functional homolog thereof, such as a lactose permease having an amino acid sequence of at least 80%, such as at least 90%, such as at least 95%, such as at least 99% or 100% identity to SEQ ID NO: 3.

[0462] Transporter

[0463] The genetically modified cells of the present disclosure include at least one nucleic acid sequence encoding one or more transporters capable of exporting the first oligosaccharide from the cell to the culture medium. The genetically modified cells of the present disclosure preferably express a heterologous transporter of the major facilitator superfamily (MFS).

[0464] Transport proteins of the major facilitator superfamily (MFS) facilitate the transport of molecules such as, but not limited to, oligosaccharides across cell membranes.

[0465] In this context, the term "MFS transporter" refers to a protein that facilitates the transport of oligosaccharides, preferably HMOs, from the cytoplasmic matrix through or across the cell membrane to the periplasm and / or culture medium. Preferably, the MFS transporter transports HMOs / oligosaccharides synthesized by the genetically modified cells described herein. In addition, or alternatively, the MFS transporter can also facilitate the efflux of molecules that are not considered HMOs or oligosaccharides, such as lactose, glucose, cellular metabolites and / or toxins. In a preferred embodiment, the MFS transporter is capable of exporting 2'FL, 3FL, 3'SL, 6'SL, LNT-II, LNT and / or LNnT from the cytoplasmic matrix to the cell culture medium.

[0466] In the context of the present invention, lactose permease is not considered as a heterologous MFS transporter.

[0467] In one or more exemplary embodiments, the MFS transporter is selected from the group consisting of Bad, Nec, YberC, Fred, Vag, and Marc.

[0468] Thus, in one or more exemplary embodiments, the genetically modified cells of the present disclosure express a heterologous MFS transporter selected from the group consisting of Vag, Nec, Fred, Marc, YberC, Bad, and a functional homolog of any one of Vag, Nec, Fred, Marc, YberC, or Bad, the amino acid sequence of which has 80% identity to the heterologous MFS transporter.

[0469] Bad

[0470] The MFS transporter referred to herein as "Bad protein" or "Bad transporter" or "Bad" has an amino acid sequence corresponding to GenBank Accession ID WP_017489914.1.

[0471] In one or more embodiments of the invention, the genetically engineered cells express a heterologous MFS transporter protein bad or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 90%, such as at least 95%, such as at least 99% or 100% identical to GenBank Accession ID WP_017489914.1.

[0472] Nec

[0473] The MFS transporter, which may be referred to interchangeably herein as a "Nec protein" or a "Nec transporter" or "Nec," has an amino acid sequence corresponding to GenBank Accession ID WP_092672081.1.

[0474] In one or more embodiments of the invention, the genetically engineered cells express a heterologous MFS transporter Nec or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to GenBank Accession ID WP_092672081.1.

[0475] Nec is particularly suitable for transporting 2'FL, DFL, 3'SL, 6'SL and LNFP-I.

[0476] YberC

[0477] The MFS transporter, which may be referred to interchangeably herein as "YberC protein" or "YberC transporter" or "YberC," has an amino acid sequence corresponding to GenBank Accession ID EEQ08298.1.

[0478] In one or more embodiments of the invention, the genetically engineered cells express a heterologous MFS transporter YberC or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to GenBank Accession ID EEQ08298.1.

[0479] YberC is particularly useful in transporting LNT.

[0480] Fred

[0481] The MFS transporter, which may be referred to interchangeably herein as "Fred protein" or "Fred transporter" or "Fred," has an amino acid sequence corresponding to GenBank Accession ID WP_087817556.1.

[0482] In one or more exemplary embodiments, the MFS transporter expressed according to the present disclosure is Fred. Thus, in one or more exemplary embodiments, the genetically engineered cells of the present disclosure express a heterologous MFS transporter Fred.

[0483] In one or more embodiments of the invention, the genetically engineered cells express a heterologous MFS transporter fred or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to GenBank Accession ID WP_087817556.1.

[0484] Fred is particularly useful for transporting 3'SL and 6'SL.

[0485] Vag

[0486] The MFS transporter, which may be referred to interchangeably herein as "Vag protein" or "Vag transporter" or "Vag," has an amino acid sequence corresponding to SEQ ID NO:51 or GenBank Accession ID WP_048785139.1.

[0487] In one or more embodiments of the invention, the genetically engineered cells express a heterologous MFS transporter protein vag or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to SEQ ID NO:51 or GenBank Accession ID WP_048785139.1.

[0488] Vag is particularly useful in transporting LNnT.

[0489] Marc

[0490] The MFS transporter, which may be referred to interchangeably herein as "Marc protein" or "Marc transporter" or "Marc," has an amino acid sequence corresponding to SEQ ID NO: 2 or GenBank accession WP_060448169.1.

[0491] In one or more embodiments of the invention, the genetically engineered cells express a heterologous MFS transporter marc or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to SEQ ID NO: 2 or GenBank accession WP_060448169.1.

[0492] Marc is particularly useful in transshipping 3FL.

[0493] Thus, in one or more exemplary embodiments, the genetically engineered cells of the present disclosure express a heterologous MFS transporter that is Vag, Nec, Fred, Marc, YberC, or Bad.

[0494] In one or more exemplary embodiments, the genetically engineered cells of the present disclosure express a functional homolog of Vag, Nec, Fred, Marc, YberC and / or Bad, the amino acid sequence of which is at least 70%, 80%, 85%, 90%, 95% or at least 99% identical to the GenBank accession number of the above-mentioned Vag, Nec, Fred, Marc, YberC and / or Bad.

[0495] In a currently preferred embodiment, the MFS transporter expressed is Nec.

[0496] In a particularly preferred embodiment, the expressed MFS transporter is YberC.

[0497] In a particularly preferred embodiment, the expressed MFS transporter is Marc.

[0498] In a particularly preferred embodiment, the expressed MFS transporter is Vag.

[0499] In a particularly preferred embodiment, the MFS transporter expressed is Fred.

[0500] Sequence identity

[0501] The term "sequence identity" as used herein describes the association between two amino acid sequences or two nucleotide sequences based on their paired comparison, i.e., a candidate sequence (e.g., a sequence of the present invention) and a reference sequence (e.g., a prior art sequence). For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo / . Biol. 48: 443-453), which is implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or higher (available at https: / / www.ebi.ac.uk / Tools / psa / emboss needle / ). The parameters used are gap open penalty (gap open penalty) 10, gap extension penalty (gap extension penalty) 0.5 and EBLOSUM62 (EMBOSS version of 30BLOSUM62) substitution matrix. The Needle output (obtained using the -nobrief option) labeled as "identity" is used as percentage identity. Usually, sequence identity can be calculated as follows: (same residue × 100) / (length of comparison region).

[0502] For the purpose of the present invention, the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), which is implemented in the Needle program of the EMBOSS package (EMBOSS:The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277), 10 preferred versions 5.0.0 or higher. The parameters used are gap open penalty 10, gap extension penalty 0.5 and DNAFULL (EMBOSS version is NCBI NUC4.4) substitution matrix. The Needle output (obtained using the -nobrief option) labeled as "identity" is used as percentage identity. Typically, sequence identity can be calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment region).

[0503] Functional homologs

[0504] Functional homologues or functional variants of protein / nucleic acid sequences described herein are protein / nucleic acid sequences that have changed genetic code or amino acid sequence but retain their original functionality. Functional homologues can be obtained by mutation, or can be natural variants from the same or other species. Compared with the functionality of protein / nucleic acid sequences, functional homologues should have at least 50% remaining functionality, such as at least 60%, 70%, 80%, 90% or 100%. In an embodiment of the present invention, functional homologues have at least 80% identity, such as at least 85% identity, such as at least 90% identity, such as at least 95% identity with the protein / nucleic acid sequences shown in a given protein, nucleic acid or gene.

[0505] Functional variants of proteins or peptides can contain conservative amino acid substitutions compared to their native sequences (i.e., physiological sequences that have not mutated). Those amino acid sequences and their encoding nucleotide sequences particularly belong to the term functional variants defined herein. The substitutions derived from the same class of amino acids that are mutually exchanged are called conservative substitutions. Specifically, these are amino acids with aliphatic side chains, amino acids with positively or negatively charged side chains, amino acids with aromatic groups in the side chains, or amino acids whose side chains can enter hydrogen bridges, such as side chains with hydroxyl functions. This means that, for example, an amino acid with a polar side chain is replaced by another amino acid with the same polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is replaced by another amino acid with the same hydrophobic side chain (e.g., serine (threonine) is replaced by threonine (serine) or leucine (isoleucine) is replaced by isoleucine (leucine)). Truncations, insertions, and substitutions are possible, particularly at sequence positions that do not cause changes in the three-dimensional structure or do not affect the binding region. Changes in the three-dimensional structure caused by insertions or deletions can be easily determined, for example, using CD spectroscopy (circular dichroism) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry, Neuberger et al. (ed.), Elsevier, Amsterdam).

[0506] In addition, the functional variants of the proteins or peptides defined in this article may also include those sequences in which the nucleotides of the nucleic acid are replaced according to the degeneration of the genetic code without causing changes in the corresponding amino acid sequence of the protein or peptide, that is, in the above sense, in one or more mutations, the amino acid sequence or at least part of it may not differ from the original sequence.

[0507] Recycling / Harvesting

[0508] The sialylated and / or fucosylated oligosaccharides of at least four monosaccharides produced by the hybrid method described herein can be recovered from the culture medium of the process. In this context, the term "recovery" is used interchangeably with the term "harvesting". Both "recovery" and "harvesting" in this context refer to the collection of the produced HMOs from the culture / fermentation broth after the process is completed. Preferably, the biomass is discarded because it contains only the first oligosaccharide.

[0509] Preferably, the transglycosidase is inactivated before the cells are harvested. Non-limiting methods suitable for inactivating the transglycosidase may be selected from: i) heating the fermentation broth to a temperature that denatures the enzyme, ii) adding a protease to the culture broth at the end of the fermentation to hydrolyze the enzyme, or iii) changing the pH of the culture to exceed the activity range of the enzyme.

[0510] The separation of cells from the culture medium can be carried out by any method well known to those skilled in the art, such as any suitable type of centrifugation or filtration. The separation of cells from the culture medium can be carried out immediately after the fermentation broth is harvested, or can be carried out after the fermentation broth is stored under appropriate conditions.

[0511] After recovering the culture medium of the mixing process, the HMO mixture / composition can be used for further processing and purification. It may be desirable to separate individual HMOs from the HMO mixture to obtain, for example, purified or enriched sialylated and / or fucosylated oligosaccharides of at least four monosaccharides. Alternatively, the HMO mixture produced by the mixing process can be purified to remove lactose and other non-HMO byproducts of metabolism (e.g., by ultrafiltration and / or nanofiltration), and the HMO mixture or HMO composition can be used as is.

[0512] Purification of specific components of an HMO composition or HMO mixture can be performed according to procedures known to those skilled in the art. For example, HMOs can be purified according to procedures known in the art, for example, as described in WO2015 / 188834, WO2017 / 182965 or WO2017 / 152918, wherein the latter describes the purification of HMOs.

[0513] Mixtures of oligosaccharides such as HMOs and compositions thereof

[0514] As described in the section on the uses of HMO mixtures described in this application, complex fucosylated and / or sialylated HMOs, such as FSL, LNFP-III, LNDFH-I, LST-a and LST-c, and mixtures comprising FSL, LNFP-III, LNDFH-I, LST-a or LST-c, are of high relevance as nutritional supplements or therapeutic agents.

[0515] One aspect of the present disclosure is a mixture or composition of HMOs consisting essentially of:

[0516] a) at least 50 wt% FSL, less than 45 wt% 3FL, less than 2 wt% 3'SL and less than 3 wt% lactose, or

[0517] b) at least 60 wt% LNDFH-I, less than 5 wt% LNFP-I, less than 35 wt% 3FL and less than 5 wt% lactose, or

[0518] c) at least 70 wt% LNDFH-I, less than 10 wt% LNFP-I, less than 25 wt% 3FL and less than 3 wt% lactose, or

[0519] d) at least 40 wt% LST-c, less than 25 wt% LNnT, less than 25 wt% 6'SL and less than 10 wt% lactose, or

[0520] e) at least 55 wt% LST-a, less than 40 wt% LNT, less than 15 wt% 3'SL and less than 2 wt% lactose, and

[0521] Each component in the total composition accounts for 100 wt%, and the composition is a mixture of at least two components.

[0522] In an embodiment, the composition or mixture of HMOs consists essentially of:

[0523] a) at least 50 wt% FSL, 20 to 45 wt% 3FL, and 0.1 to 2 wt% 3'SL and 0 to 3 wt% lactose, or

[0524] b) at least 60 wt% LNDFH-I, 0.1 to 8 wt% LNFP-I, 15 to 35 wt% 3FL and 0 to 5 wt% lactose, or

[0525] c) at least 70 wt% LNDFH-I, 0.1 to 10 wt% LNFP-I, 5 to 20 wt% 3FL and 0 to 3 wt% lactose, or

[0526] d) at least 50 wt% LST-c, 15 to 25 wt% LNnT, 15 to 25 wt% 6'SL and 0 to 7 wt% lactose,

[0527] e) at least 50 wt% LST-a, 15 to 40 wt% LNT, 0 to 15 wt% 3'SL and 0 to 2 wt% lactose, and

[0528] Each component in the total composition accounts for 100 wt%.

[0529] In other embodiments, the HMO mixture described herein consists essentially of 40-50 wt% 3FL and 50-60 wt% FSL, wherein each component accounts for 100 wt% of the total composition.

[0530] In other embodiments, the HMO mixture described herein consists essentially of 45 wt% 3FL and 55 wt% FSL.

[0531] In other embodiments, the HMO mixture described herein consists essentially of 45-55 wt% LST-c, 20-30 wt% LNnT, and 20-30 wt% 6'SL, wherein each component accounts for 100 wt% of the total composition.

[0532] In other embodiments, the HMO mixture described herein consists essentially of 50 wt% LST-c, 25 wt% LNnT, and 25 wt% 6'SL.

[0533] In other embodiments, the HMO mixture described herein consists essentially of 60 to 80 wt% LST-a, 20 to 30 wt% LNT, and 5 to 15 wt% 3'SL, wherein each component accounts for 100 wt% of the total composition.

[0534] In other embodiments, the HMO mixture described herein consists essentially of 65 wt% LST-a, 25 wt% LNT, and 10 wt% 3'SL.

[0535] In other embodiments, the HMO mixtures described herein consist essentially of 50 to 60 wt% LST-a and 40 to 50 wt% LNT, wherein each component comprises 100 wt% of the total composition.

[0536] In other embodiments, the HMO mixtures described herein consist essentially of 55 wt% LST-a and 45 wt% LNT.

[0537] As shown in the examples, the hybrid method of the present disclosure allows for an improved ratio of desired complex fucosylated or sialylated HMOs to donor and / or acceptor HMOs compared to conventional in vitro processes, in which the ratio between the individual HMOs is limited by the kinetic barriers of the enzymatic reaction, thereby preventing a shift in the equilibrium between the components of the in vitro enzymatic reaction.

[0538] In an embodiment, the molar ratio of FSL:3'SL in the HMO composition and / or mixture is higher than 100:1 and the molar ratio of FSL:3FL is higher than 1:1.

[0539] In other embodiments, the molar ratio of LNDFH-I:LNFP-I in the HMO composition and / or mixture is greater than 50:1, and the molar ratio of LNDFH-I:3FL is in the range of 1.5:1 to 2.6:1.

[0540] In other embodiments, the molar ratio of LST-c:LNnT in the HMO composition and / or mixture is greater than 2.5:1 and the molar ratio of LST-c:6'SL is greater than 2.5:1.

[0541] In other embodiments, the molar ratio of LST-a:LNT in the HMO composition and / or mixture is greater than 1.5:1 and the molar ratio of LST-a:3'SL is greater than 8:1.

[0542] In an embodiment, the composition comprising the HMO mixture is a nutritional composition. The nutritional composition is, for example, an infant formula or a medical nutritional composition.

[0543] In an embodiment, the composition comprising the HMO mixture is a dietary supplement.

[0544] In an embodiment, the composition comprising the HMO mixture is a pharmaceutical composition.

[0545] Uses of HMO mixtures and compositions

[0546] Clinical data from infants suggests that human milk oligosaccharide supplementation may help develop a desired microbiota by serving as a food source for beneficial bacteria in the gut. HMOs are naturally present in human milk and have evolved over thousands of years, and existing research (clinical and preclinical) on HMOs suggests that supplementation with appropriate levels of specific HMOs may provide us with unique health benefits. In particular, human milk oligosaccharide supplementation may help support immunity and gut health, including supporting a balanced microbiota, with a potential role in cognitive development, which may open the door to future innovation opportunities.

[0547] Thus, in an embodiment, the present invention relates to the use of a mixture or composition disclosed herein in infant nutrition.

[0548] The present invention also relates to the use of the mixture or composition disclosed herein as a dietary supplement or a medical nutritional product or a pharmaceutical composition.

[0549] The HMO mixture or composition produced according to the methods described herein can be used to enhance the beneficial bacteria in the intestinal microbiota. Beneficial bacteria are, for example, bacteria of the genus Bifidobacterium, Lactobacillus or Barnesiella sp. The enhancement of beneficial bacteria in turn may lead to increased production of short-chain fatty acids (SCFAs), such as acetate, propionate and butyrate, which have been shown to have many benefits for infants and young children, such as inhibiting pathogens, preventing infection and diarrhea, reducing the risk of allergies and metabolic disorders (see, for example, WO2006 / 130205, WO2017 / 129644, WO2017 / 129649).

[0550] The HMO mixtures or compositions produced according to the methods described herein can be used to reduce the number of harmful viruses and bacteria in the intestinal microbiota. Examples of pathogenic bacteria and viruses that can be reduced by the HMO mixtures described herein include Candida albicans, Clostridium difficile, Enterococcus faecium, Escherichia coli, Helicobacter pylori, Streptococcus agalactiae, Shigella dysenteriae, Staphylococcus aureus, nora virus, and rotavirus. Each of the compositions described herein can also be used to treat and / or reduce the risk of infection of humans with a variety of bacteria.

[0551] The HMO mixture or composition produced according to the method described herein can be used to improve the regeneration and vitality of freeze-dried probiotics (including probiotics of the genera Bifidobacterium and Lactobacillus), especially in acidic environments (such as the stomach or acidic foods). Improved regeneration and / or vitality and / or shelf life are advantages of using the HMO mixture described herein. Examples of Bifidobacteria that may have increased regenerative capacity and vitality are Bifidobacterium animals lactis BB12 DSM 32269, Bifidobacterium animals lactis BIF6, Bifidobacterium longum DSM 32946, Bifidobacterium longum BB536, Bifidobacterium bifidum DSMZ 32403, Bifidobacterium infantis, Bifidobacterium breve DSM 33789, Bifidobacterium infantis SP37 DSM 32687, Bifidobacterium adolescentis DSM 34065 and / or Bifidobacterium animalis subsp. animalis DSM 16284. Examples of Lactobacillus species that may have increased regenerative capacity and vitality are Lactobacillus rhamnosus GG DSM 32550, Lactobacillus rhamnosus 19070-2 DSM 26357, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus LBrGG, Lactobacillus reuteri DSM 12246, Lactobacillus plantarum TIFN101, Lactobacillus gasseri Lg-36200BFloraFit Danisco, Lactobacillus casei DSM 32382, Lactobacillus paracasei, Lactobacillus plantarum PS128, Lactobacillus plantarum (Sacco) DSM 32383, Lactococcus lactis PAREVE, Lactobacillus paracasei ssp. Paracasei and / or Lactobacillus Limosilactobacillus reuteri S12 DSM 33752.

[0552] In the context of this application, "regeneration" refers to the process of regaining / restoring the vitality of dried bacteria (i.e. "reviving" bacterial cells by rehydration, where "rehydration" means restoring liquid). This process is sometimes also called "reconstitution".

[0553] In the context of this application, "viability" is the ability of bacterial cells to survive and function as living cells. One method of determining bacterial cell viability is to plate them on agar plates with a suitable growth medium and count the number of colonies formed after incubation for a predetermined time (plate count). Alternatively, FACS analysis can be used.

[0554] In the context of the present application, "improved regeneration" of Bifidobacterium and / or Lactobacillus bacteria means an increase in the number (number) of successfully regenerated / resuscitated Bifidobacterium and / or Lactobacillus bacteria compared to a corresponding control (i.e. the number / number of Bifidobacterium and / or Lactobacillus bacteria without the addition of HMO).

[0555] In the context of the present application, "improved viability" of Bifidobacterium and / or Lactobacillus bacteria means an increase in the number (number) of Bifidobacterium and / or Lactobacillus bacteria compared to a corresponding control (i.e. the number / number of Bifidobacterium and / or Lactobacillus bacteria without addition of HMO).

[0556] In the context of this application, "acidic" means a pH value below 7.0 (e.g., a pH value ≤ 6.0, or ≤ 5.0, or ≤ 4.0, or ≤ 3.0, or in the range of 1.0-6.0, such as 2.0 to 5.0). The pH value measured in the stomach is in the range of about 1.5-3.5. The pH value measured in a healthy vagina is in the range of about 3.8-5.0. The pH value of fruit juice is in the range of about 2.0-4.5.

[0557] The HMO mixture or composition produced according to the methods described herein can be used to extend the shelf life of probiotics (eg, Bifidobacterium and / or Lactobacillus).

[0558] One embodiment of the invention is a composition comprising a HMO mixture as described herein (particularly in the "HMO mixture" section) and one or more probiotics. Preferably, the probiotics are of the genus Bifidobacterium and / or Lactobacillus, such as any of the specific species mentioned above.

[0559] The HMO mixtures or compositions produced according to the methods described herein can be used to improve the flow properties of powders or to reduce the viscosity of liquids.

[0560] The HMO mixture or composition produced according to the methods described herein is used in nutritional compositions. The nutritional composition is, for example, infant formula, rehydration, or dietary maintenance, medical nutrition or supplements for the elderly or immunocompromised population. Such anti-infective compositions may also include macronutrients such as edible fats, carbohydrates and proteins. Edible fats include, for example, coconut oil, soybean oil, and mono- and diglycerides. Carbohydrates include, for example, glucose, edible lactose and hydrolyzed corn starch. Proteins include, for example, soy protein, whey and skim milk. Such anti-infective compositions may also include vitamins and minerals (e.g., calcium, phosphorus, potassium, sodium, chlorine, magnesium, manganese, iron, copper, zinc, selenium, iodine and vitamins A, E, D, C and B complex).

[0561] Implementation

[0562] The following embodiments of the invention may be used in combination with any other embodiments described herein.

[0563] 1. A method for producing oligosaccharides from donor oligosaccharides and acceptor oligosaccharides or disaccharides, the method comprising the following steps:

[0564] a) culturing a first genetically modified cell capable of producing a first oligosaccharide in a medium supplied with a carbon source, wherein the genetically modified cell comprises one or more nucleic acids encoding:

[0565] i) at least one recombinant glycosyltransferase, and

[0566] ii) at least one pathway for producing a nucleotide activated sugar, and

[0567] b) supplying a second disaccharide or oligosaccharide to the culture medium, and

[0568] c) making available in the culture medium an enzyme having transglycosidase activity,

[0569] d) incubating the first oligosaccharide, the second oligosaccharide and a transglycosidase in a culture medium to form a third complex oligosaccharide of at least four monosaccharide units.

[0570] 2. A method for producing sialylated and / or fucosylated oligosaccharides from donor oligosaccharides and acceptor oligosaccharides, the method comprising the following steps:

[0571] a) culturing a first genetically modified cell capable of producing a first oligosaccharide in a medium supplied with a carbon source, wherein the genetically modified cell comprises one or more nucleic acids encoding:

[0572] i) at least one recombinant glycosyltransferase, and

[0573] ii) at least one pathway for producing a nucleotide activated sugar, and

[0574] b) supplying a second disaccharide or oligosaccharide to the culture medium,

[0575] wherein the first oligosaccharide or the second oligosaccharide is a fucosyl donor or a sialic acid donor or a lacto-N-biose donor oligosaccharide; and

[0576] c) making available in the culture medium an enzyme having transglycosidase activity,

[0577] d) incubating the first oligosaccharide, the second oligosaccharide and a transglycosidase in a culture medium to form a third sialylated and / or fucosylated oligosaccharide of at least four monosaccharide units.

[0578] 3. The method according to item 1 or 2, wherein the first oligosaccharide or the second oligosaccharide is a fucosylated or sialylated donor oligosaccharide, and the other disaccharide or oligosaccharide is an acceptor oligosaccharide.

[0579] 4. A method according to item 1 or 3, wherein if the second disaccharide or oligosaccharide is a disaccharide, it is not lactose.

[0580] 5. The method according to item 1 or 4, wherein if the second disaccharide or oligosaccharide is a disaccharide, it is lacto-N-biose (LNB) or N-acetyllactosamine (LacNAc).

[0581] 6. The method according to any of the preceding items, wherein the oligosaccharide is a complex oligosaccharide of at least four monosaccharide units or an oligosaccharide of at least three monosaccharide units, wherein the reducing end has a lacto-N-biose (LNB) moiety or an N-acetyllactosamine (LacNAc) moiety.

[0582] 7. The method according to item 6, wherein the oligosaccharide of at least three monosaccharide units is Lewis X or Lewis A.

[0583] 8. The method according to item 1 or 7, wherein the second disaccharide or oligosaccharide is supplied to the culture medium by adding it.

[0584] 9. The method according to item 1 or 7, wherein the first genetically modified cell is capable of producing the first and second oligosaccharides.

[0585] 10. The method according to items 1 to 7, wherein the second oligosaccharide is supplied to the culture medium by a second genetically modified cell capable of producing the second oligosaccharide.

[0586] 11. The method according to item 10, wherein the second genetically modified cell is co-cultured with the first genetically modified cell in item 1a).

[0587] 12. The method according to item 10 or 11, wherein the second genetically modified cell

[0588] a) comprises at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase, and

[0589] b) comprises at least one biosynthetic pathway producing a nucleotide activated sugar,

[0590] c) capable of growing on a second carbon source, while growth is limited or does not grow on the carbon source (the first carbon source) on which the genetically modified cell in step a) of item 1 is capable of growing.

[0591] 13. The method according to item 11 or 12, wherein the first genetically modified cell of item 1a) has limited growth or does not grow on the second carbon source.

[0592] 14. A method according to items 11 to 13, wherein one of the genetically modified microbial cells is capable of growth on sucrose and comprises one or more nucleic acid sequences encoding a PTS-dependent sucrose utilization system or a nucleic acid encoding a sucrose invertase or a sucrose hydrolase, such that the cell is able to absorb sucrose.

[0593] 15. The method according to item 14, wherein the PTS-dependent sucrose utilization system is encoded by scrY (SEQ ID NO: 47), scrA (SEQ ID NO: 48), scrB (SEQ ID NO: 49) and optionally scrR (SEQ ID NO: 50) or by the cscABKR gene cluster (SEQ ID NO: 52).

[0594] 16. The method according to item 14 or 15, wherein the cell has a reduced ability to grow or is unable to grow on one or more carbon sources selected from the group consisting of glucose, glycerol, galactose, maltose, sorbitol, arabinose and fructose.

[0595] 17. The method according to items 11 to 13, wherein one of the genetically modified microbial cells is capable of growth on glucose and comprises one or more nucleic acids encoding one or more glucose transport systems.

[0596] 18. The method according to item 17, wherein the glucose transport system is a PTS-dependent glucose transport system selected from:

[0597] i) Glucose PTS complex component IICB Glc ;

[0598] ii) β-Glucoside PTS complex component - IIABC Bgl ;

[0599] iii) Mannose PTS complex component - IICD Man ;

[0600] iv) N-acetylglucosamine PTS complex component-IIABC Nag ;as well as

[0601] v) Maltose / Maltodextrin PTS Complex-IICB malX

[0602] 19. The method according to item 17, wherein the glucose transport system is selected from:

[0603] i) Galactose:H+ symporter GalP;

[0604] ii) glucose uptake protein GlcU;

[0605] iii) sodium / glucose transporter family (SGLT);

[0606] iv) galactose / glucose ABC transporter (mglABC) system;

[0607] v) trehalose / maltose / sucrose / palatinose (TMSP)-ABC transporter (malEFG) system;

[0608] vi) glucose / mannose ABC transporter (glcEFG) system;

[0609] vii) glucose proton symporter (glcP);

[0610] viii) glucose facilitator factor (glf); and

[0611] ix) Hexose transporter (HXT).

[0612] 20. The method according to items 17 to 19, wherein the cell has a reduced ability to grow or is unable to grow on one or more carbon sources selected from sucrose, glycerol, galactose, maltose, sorbitol, arabinose and fructose.

[0613] 21. The method according to items 11 to 13, wherein the genetically modified microbial cell capable of growing on glycerol comprises one or more nucleic acids encoding one or more glycerol transport systems.

[0614] 22. The method according to item 21, wherein the glycerol transport system is selected from a glycerol facilitator or a glycerol / H+ symporter.

[0615] 23. The method according to items 21 to 22, wherein the cell has a reduced ability to grow or is unable to grow on one or more carbon sources selected from sucrose, glucose, galactose, maltose, sorbitol, arabinose and fructose.

[0616] 24. The method according to items 11 to 23, wherein the first genetically modified microbial cell is grown on glucose or glycerol and the second genetically modified microbial cell is grown on sucrose.

[0617] 25. The method according to items 11 to 13, wherein the genetically modified microbial cell capable of growth on galactose comprises one or more nucleic acids encoding one or more galactose transport systems.

[0618] 26. The method according to item 25, wherein the galactose transport system is selected from the group consisting of galactose:H+ symporter, galactose / glucose ABC transporter (mglABC) system, PTSLac (lacFE) system and / or sodium / glucose transporter family (sglT).

[0619] 27. The method according to item 25 or 26, wherein the cell has a reduced ability to grow or is unable to grow on one or more carbon sources selected from sucrose, glucose, glycerol, maltose, sorbitol, arabinose and fructose.

[0620] 28. The method according to items 11 to 13, wherein the genetically modified microbial cell capable of growth on fructose comprises one or more nucleic acids encoding one or more fructose transport systems.

[0621] 29. The method according to item 28, wherein the galactose transport system is selected from PTS complex component IIABC Fru , glucose PTS complex component IICB Glc and the fructose transporter FruP.

[0622] 30. The method according to item 28 or 29, wherein the cell has a reduced ability to grow or is unable to grow on one or more carbon sources selected from sucrose, glucose, glycerol, maltose, sorbitol, arabinose and galactose.

[0623] 31. The method according to items 11 to 13, wherein the genetically modified microbial cell capable of growing on maltose comprises one or more nucleic acids encoding one or more maltose transport systems.

[0624] 32. The method according to item 31, wherein the galactose transport system is selected from the group consisting of the MalFGK ABC superfamily transport system and / or the maltose / maltodextrin PTS complex.

[0625] 33. The method according to item 31 or 32, wherein the cell has reduced growth ability or is unable to grow on one or more carbon sources selected from sucrose, glucose, glycerol, fructose, sorbitol, arabinose and galactose.

[0626] 34. The method according to items 11 to 13, wherein the genetically modified microbial cell capable of growing on arabinose comprises one or more nucleic acids encoding one or more arabinose transport systems.

[0627] 35. The method according to item 34, wherein the arabinose transport system is selected from the group consisting of the AraFGH ABC superfamily transport system and / or the arabinose-proton symporter AraE.

[0628] 36. The method according to item 34 or 35, wherein the cell has a reduced ability to grow or is unable to grow on one or more carbon sources selected from sucrose, glucose, glycerol, fructose, sorbitol and galactose.

[0629] 37. The method according to items 11 to 13, wherein the genetically modified microbial cell capable of growing on sorbitol comprises one or more nucleic acids encoding one or more sorbitol transport systems.

[0630] 38. The method according to item 37, wherein the sorbitol transport system is selected from the group consisting of the sorbitol-PTS system (EIIBC srl ).

[0631] 39. The method according to item 37 or 38, wherein the cell has a reduced ability to grow or is unable to grow on one or more carbon sources selected from sucrose, glucose, glycerol, fructose, arabinose and galactose.

[0632] 40. The method according to items 1 to 39, wherein said genetically modified cell exports said oligosaccharides produced by said cell into the culture medium.

[0633] 41. The method according to items 1 to 40, wherein the first genetically modified cell is capable of producing a first oligosaccharide of at least three monosaccharide units into the culture medium.

[0634] 42. The method according to items 8 to 41, wherein the second genetically modified cell is capable of producing the second oligosaccharide or disaccharide in situ in the medium in which the first genetically modified cell is cultured.

[0635] 43. The method according to item 42, wherein the second genetically modified cell produces a second oligosaccharide of at least three monosaccharide units.

[0636] 44. The method according to item 42, wherein the second genetically modified cell produces a disaccharide selected from lacto-N-biose (LNB) or N-acetyllactosamine (LacNAc).

[0637] 45. The method according to any one of items 1 to 44, wherein at least one genetically modified cell further comprises a nucleic acid encoding a byproduct importin.

[0638] 46. ​​The method according to item 45, wherein the by-product import protein is a lactose import protein.

[0639] 47. The method according to any one of items 1 to 43 or 45 or 46, wherein the first and / or second oligosaccharide is produced from lactose as initial substrate.

[0640] 48. The method according to any one of items 1 to 47, wherein the donor oligosaccharide is selected from fucosylated oligosaccharides of three to five monosaccharide units, sialylated oligosaccharides of three to five monosaccharide units and neutral core oligosaccharides of three to four monosaccharide units.

[0641] 49. The method according to any one of items 1 to 48, wherein the donor oligosaccharide is a fucosyl or sialic acid donor oligosaccharide selected from fucosyllactose or sialyllactose or sialyl-N-acetyllactosamine or sialyl-lacto-N-biose, and the other oligosaccharide is an acceptor oligosaccharide.

[0642] 50. The method according to items 1 to 45, wherein the first genetically modified cell is capable of importing 2'FL or 3FL.

[0643] 51. The method according to item 50, wherein the first genetically modified cell is capable of producing a first oligosaccharide of four monosaccharide units.

[0644] 52. The method according to item 50 or 51, wherein the first oligosaccharide is selected from DFL or FSL.

[0645] 53. The method according to items 1 to 45, wherein the first genetically modified cell is capable of importing N-acetyllactosamine or lacto-N-biose.

[0646] 54. The method according to any of the preceding items, wherein the transglycosidase is selected from the group consisting of α-1,2-transfucosidase, α-1,3-transfucosidase, α-1,3 / 4-transfucosidase, α-2,3-transsialidase, α-2,6-transsialidase, β-N-acetylglucosaminidase, trans-lacto-N-biosidase and trans-β-galactosidase.

[0647] 55. The method according to item 54, wherein the transglycosidase is selected from the trans-sialidases in Table 2 or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with a single trans-sialidase sequence in Table 2.

[0648] 56. The method according to item 54 or 55, wherein the trans-sialidase is an enzyme selected from SEQ ID NO: 13, 14, 40, 41 or 60.

[0649] 57. The method according to any of the preceding items, wherein the transglycosidase is a transfucosidase selected from Table 3 or a functional homologue thereof, the amino acid sequence of which has at least 80% identity with a single transsialidase sequence in Table 3.

[0650] 58. The method according to items 54 to 57, wherein the transfucosidase is an enzyme selected from SEQ ID NO: 19, 30, 39 or 59.

[0651] 59. The method according to any of the preceding items, wherein the enzyme having transglycosidase activity is i) a transfucosidase if the donor oligosaccharide is a fucosyl oligosaccharide, or

[0652] ii) if the donor oligosaccharide is a sialylo-oligosaccharide, then a trans-sialidase, or

[0653] iii) trans-lacto-N-biosidase.

[0654] 60. The method according to any of the preceding items, wherein a transglycosidase is added to the culture medium during cultivation or is expressed by a recombinant nucleic acid in said genetically modified cell.

[0655] 61. The method according to item 60, wherein the transglycosidase is expressed by the recombinant nucleic acid in the genetically modified cell and exported from the cell into the culture medium.

[0656] 62. A method according to any of the preceding items, wherein the incubation in step d) of item 1 or 2 allows transglycosylation of the acceptor oligosaccharide with the fucosyl moiety or sialic acid moiety or lacto-N-biose moiety of the donor oligosaccharide, mediated by the transglycosidase activity provided in step c) of item 1, to form a third sialylated and / or fucosylated oligosaccharide of at least four monosaccharide units.

[0657] 63. The method according to any one of items 54 to 62, wherein the hydrolytic activity of the transfucosidase results in fucose in the culture being less than 5% of the total molar % of the donor oligosaccharide and the third oligosaccharide at the end of the process.

[0658] 64. The method according to any one of items 54 to 62, wherein the hydrolytic activity of the trans-sialidase results in sialic acid in the culture being less than 5% of the total molar % of the donor oligosaccharide and the third oligosaccharide at the end of the process.

[0659] 65. The method according to any of the preceding items, wherein the genetically modified cells are cultured in the presence of one or more carbon sources selected from the group consisting of glucose, sucrose, fructose, arabinose, sorbitol, xylose, galactose, maltose and glycerol.

[0660] 66. The method according to any of the preceding items, wherein the culturing in step a) is initiated in the presence of sufficient initial substrate for the cells to produce the first oligosaccharide.

[0661] 67. The method according to item 66, wherein no additional initial substrate is added to the culture medium after initiation of the culture.

[0662] 68. The method according to items 1 to 66, wherein the initial substrate for the cells to produce the first oligosaccharide is added to the culture medium when the initial carbon source is consumed.

[0663] 69. The method according to any one of items 60 to 68, wherein the transglycosidase is added to the culture medium and no additional initial substrate is added to the culture medium after the addition of the transglycosidase.

[0664] 70. The method according to any one of items 67 to 69, wherein the transglycosidase is added to the culture at a point in time at which the genetically modified cell converts at least 50% of the initial substrate into the first oligosaccharide.

[0665] 71. The method according to items 67 to 70, wherein the transglycosidase is added to the culture when less than 10% of the initial substrate initially added to the culture remains.

[0666] 72. The method according to items 15 to 71, wherein the initial substrate for producing the first oligosaccharide is selected from lactose, N-acetyllactosamine, lacto-N-biose, 2'FL, 3FL or LNT-11.

[0667] 73. The method according to items 15 to 72, wherein the initial substrate for producing the first and / or second oligosaccharide is lactose.

[0668] 74. The method according to items 15 to 72, wherein the initial substrate for producing the first oligosaccharide is 2'FL.

[0669] 75. The method according to items 15 to 72, wherein the initial substrate for producing the first oligosaccharide is N-acetyllactosamine (LacNAc).

[0670] 76. The method according to items 15 to 72, wherein the initial substrate for producing the first oligosaccharide is lacto-N-biose (LNB).

[0671] 77. The method according to items 15 to 72, wherein the initial substrate for producing the first oligosaccharide is LNT-II.

[0672] 78. The method according to any one of items 1 to 8 or 40 to 42 or 45 to 74, wherein the second oligosaccharide is added to the culture medium at a rate corresponding to the rate of formation of the first oligosaccharide.

[0673] 79. The method according to any one of items 1 to 8 or 40 to 42 or 45 to 77, wherein the second oligosaccharide is added to the culture medium at a rate faster than the rate of formation of the first oligosaccharide.

[0674] 80. A method according to any one of items 1 to 8 or 40 to 42 or 45 to 77, wherein the second oligosaccharide or disaccharide is added to the culture medium in a molar amount of at least 2:1 (eg at least 3:1) to the initial substrate used to produce the first oligosaccharide.

[0675] 81. The method according to any of the preceding items, wherein the cultivation is a fed-batch or continuous fed-batch fermentation, wherein the carbon source is fed into the culture medium during the entire fermentation after the initial carbon source is consumed.

[0676] 82. The method according to item 81, wherein the carbon source is fed at a rate that enables sustained growth of the genetically modified cells.

[0677] 83. A method according to item 81, wherein the carbon source is fed at a rate that allows the culture to grow under carbon limitation.

[0678] 84. The method according to any one of items 47 to 83, wherein the by-product import protein is lactose permease.

[0679] 85. The method according to item 84, wherein the genetically modified cells express one or more recombinant lactose permeases.

[0680] 86. The method according to item 84 or 85, wherein the lactose permease is LacY comprising or consisting of SEQ ID NO: 3, or a functional variant thereof.

[0681] 87. The method according to any one of items 1 to 86, wherein the third oligosaccharide is a complex oligosaccharide of at least four monosaccharide units selected from DFL, FSL, Lewis B, Lewis Y, sialyl-Lewis A, sialyl-Lewis X, LNFP-I, LNFP-II, LNFP-III, LNFP-IV, LNFP-V, LNFP-VI, LST-a, LST-b, LST-c, DSLNT, LNDFH-I, LNDFH-II, LNDFH-III, FLST-a, FLST-b, FLST-c, pLNH, pLNnH, LNH, LNnH, FLNH-I, FLNH-II, FLNH-III, FpLNH-I, FpLNnH II, DF-LNF-I, DF-LNF-II, DF-LNF-III, DF-p-LNH, DF-p-LNnH, FLNnHa, FLNnHb, DFLNnH, TF-LNH, SLNH, FSLNH, SLNnH-I, FSLNnH-I, SLNnH-II and DS-FLNH-II.

[0682] 88. The method according to any one of items 1 to 87, wherein at least one of the first and second oligosaccharides is an HMO.

[0683] 89. The method according to any one of items 1 to 88, wherein the first, second and third oligosaccharides are HMOs.

[0684] 90. The method according to any of the preceding items, wherein the third oligosaccharide is selected from DFL, FSL, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LST-a, LST-b, LST-c, DSLNT, LNDFH-I, LNDFH-II, LNDFH-III, FLST-a, FLST-b, FLST-c, pLNH, pLNnH, LNH, LNnH, FLNH-I, FLNH-II, FLNH-III, FpLNH-I, FpLNnH HMOs of II, DF-LNF-I, DF-LNF-II, DF-LNF-III, DF-para-LNH, DF-para-LNnH, FLNnHa, FLNnHb, DFLNnH, TF-LNH, SLNH, FSLNH, SLNnH-I, FSLNnH-I, SLNnH-II, and DS-FLNH-II.

[0685] 91. The method according to any of the preceding items, wherein the first genetically modified cell produces a donor oligosaccharide selected from 2'FL, 3FL, DFL, LNFP-I, FSL, 3'SL, 6'SL, 3'SLacNAc, 3'SLNB, LST-a, LNT-II, LNT and LNnT.

[0686] 92. The method according to any one of items 1 to 51 or 54 to 73 or 78 to 91, wherein the complex oligosaccharide is FSL and the method comprises the following steps:

[0687] a) culturing a first genetically modified cell producing 3FL in a culture medium; and

[0688] b) supplying 3'SL to the culture medium of a); and

[0689] c) making an enzyme having trans-sialidase activity available in the culture medium; and

[0690] d) incubating 3FL, 3'SL, and trans-sialidase in a culture medium to form FSL and lactose; and

[0691] The lactose is then recycled by the cells to produce more 3FL.

[0692] 93. The method according to any one of items 1 to 51 or 54 to 73 or 78 to 91, wherein the complex oligosaccharide is LNDFH-I, and the method comprises the following steps:

[0693] a) culturing a first genetically modified cell producing 3FL in a culture medium; and

[0694] b) supplying LNFP-I to the culture medium of a); and

[0695] c) making an enzyme having transfucosidase activity available in the culture medium; and

[0696] d) incubating 3FL, LNFP-I and transfucosidase in a culture medium to form LNDFH-I and lactose; and

[0697] The lactose is then recycled by the cells to produce more 3FL.

[0698] 94. The method according to any one of items 1 to 51 or 54 to 73 or 78 to 91, wherein the complex oligosaccharide is LNFP-III, comprising the steps of:

[0699] a) culturing a first genetically modified cell producing 3FL in a culture medium, and

[0700] b) supplying LNnT to the culture medium of a), and

[0701] c) making an enzyme having transfucosidase activity available in the culture medium, and

[0702] d) incubating 3FL, LNnT and transfucosidase in a culture medium to form LNFP-III and lactose, and

[0703] The lactose is then recycled by the cells to produce more 3FL.

[0704] 95. The method according to any one of items 1 to 5151 or 54 to 73 or 78 to 91, wherein the complex oligosaccharide is LST-c, and the method comprises the following steps:

[0705] a) culturing a first genetically modified cell producing LNnT in a culture medium; and

[0706] b) supplying 6'SL to the culture medium of a); and

[0707] c) making an enzyme having trans-sialidase activity available in the culture medium; and

[0708] d) incubating 6'SL, LNnT and trans-sialidase in a culture medium to form LST-c and lactose; and

[0709] The lactose is then recycled by the cells to produce more LNnT.

[0710] 96. The method according to any one of items 1 to 51 or 54 to 73 or 78 to 91, wherein the complex oligosaccharide is LST-a, and the method comprises the following steps:

[0711] a) culturing a first genetically modified cell that produces LNT in a culture medium; and

[0712] b) supplying 3'SL to the culture medium of a); and

[0713] c) making an enzyme having trans-sialidase activity available in the culture medium; and

[0714] d) incubating 3'SL, LNT and trans-sialidase in a culture medium to form LST-c and lactose; and

[0715] The lactose is then recycled by the cells to produce more LNT.

[0716] 97. A method according to any one of items 1 to 90, wherein the first genetically modified cell produces an acceptor oligosaccharide selected from 2'FL, 3FL, 2'FLacNAc, 2'FLNB, Lewis A, Lewis X, LNT-II, LNT, LNnT, p-LNnH, LNFP-I, LNFP-II, LNFP-III, LNFP-IV, LNFP-V, LNFP-VI, 3'SL, 6'SL, LST-a and LST-c.

[0717] 98. The method according to any of the preceding items, wherein at least one recombinant glycosyltransferase in the genetically engineered cell is selected from the group consisting of α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3 / 4-fucosyltransferase, α-2,3-sialyltransferase, α-2,6-sialyltransferase, β-1,3-N-acetylglucosaminyltransferase, β-1,6-N-acetylglucosaminyltransferase, β-1,3-galactosyltransferase, β-1,4-galactosyltransferase, β-N-acetylglucosaminyltransferase and trans-β-galactosidase.

[0718] 99. The method according to item 98, wherein the glycosyltransferase in the genetically engineered cell is an α-1,3-fucosyltransferase from Table 8.

[0719] 100. The method according to item 98 or 99, wherein the glycosyltransferase in the genetically engineered cell is an α-1,3-fucosyltransferase from Helicobacter pylori, such as the α-1,3-fucosyltransferase of SEQ ID NO: 1 or a functional homologue thereof.

[0720] 101. The method according to item 98, wherein the glycosyltransferase in the genetically engineered cell is an α-1,2-fucosyltransferase from Table 7.

[0721] 102. The method according to item 98 or 101101, wherein the glycosyltransferase in the α-1,2-fucosyltransferase is derived from Helicobacter pylori.

[0722] 103. The method according to item 98, wherein the glycosyltransferase in the genetically engineered cell is an α-2,3-sialyltransferase from Table 10.

[0723] 104. The method according to item 98, wherein the glycosyltransferase in the genetically engineered cell is an α-2,6-sialyltransferase from Table 11.

[0724] 105. The method according to item 98, wherein at least one glycosyltransferase in the genetically engineered cell is β-1,3-N-acetylglucosaminyltransferase and β-1,3-galactosyltransferase.

[0725] 106. The method according to item 105, wherein the β-1,3-N-acetylglucosaminyltransferase is selected from Table 4 and the β-1,3-galactosyltransferase is selected from Table 5.

[0726] 107. The method according to item 98, wherein at least one glycosyltransferase in the genetically engineered cell is β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase.

[0727] 108. The method according to item 107, wherein the β-1,3-N-acetylglucosaminyltransferase is selected from Table 4 and the β-1,4-galactosyltransferase is selected from Table 6.

[0728] 109. A method according to any of the preceding items, wherein the pathway that produces a nucleotide activating sugar is a pathway that is capable of producing at least one sugar nucleotide selected from GDP-fucose, UDP-GlcNAc, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine (GlcNAc) and CMP-N-acetylneuraminic acid.

[0729] 110. The method according to any of the preceding items, wherein the pathway for producing nucleotide activating sugars is the de novo GDP-fucose pathway (gmd, wcaG, manB, manC and manA) and / or the sialic acid sugar nucleotide pathway (neuB, neuC and neuA).

[0730] 111. The method according to item 110, wherein the genetically modified cell overexpresses

[0731] a) the entire colanic acid gene cluster; and / or

[0732] b) one or more genes of the de novo GDP-fucose pathway selected from the group consisting of manA, manB, manC, gmd and wcaG.

[0733] 112. The method according to item 110, wherein the genetically modified cell expresses

[0734] a) Recombinant UDP-GlcNAc 2-epimerase

[0735] b) recombinant Neu5Ac synthase; and

[0736] c) Recombinant CMP-Neu5Ac synthetase.

[0737] 113. The method according to any of the preceding items, wherein the first genetically modified cell comprises a nucleic acid encoding a transporter protein capable of exporting the first oligosaccharide from the cell.

[0738] 114. The method according to any one of items 10 to 113, wherein the second genetically modified cell comprises a nucleic acid encoding a transporter protein capable of exporting a second oligosaccharide from the cell.

[0739] 115. The method according to item 113 or 114, wherein the transporter is a sugar efflux transporter or a major facilitator superfamily (MFS) transporter, preferably selected from the group consisting of setA, yberC, nec, vag, marc, bad and fred.

[0740] 116. The method according to any of the preceding items, wherein the second disaccharide or oligosaccharide is selected from LacNAc, LNB, 2'FL, 3FL, 2'FLacNAc, 2'FLNB, Lewis A, Lewis X, 3'SL, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LSTa, LSTc, LNH, F-LNH-II, F-LNH-III, DF-LNH-I, DF-LNH-II, DF-LNH-III, S-LNH, DS-LNH, FS-LNH, LNnH, p-LNH and p-LNnH.

[0741] 117. The method according to any of the preceding items, wherein the second oligosaccharide is selected from 2'FL, 3FL, 3'SL, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LST-a and LST-c.

[0742] 118. The method according to item 116 or 117, wherein the second oligosaccharide is produced by a second genetically modified cell.

[0743] 119. An HMO composition consisting essentially of:

[0744] a) at least 50 wt% FSL, less than 45 wt% 3FL, less than 2 wt% 3'SL and less than 3 wt% lactose, or

[0745] b) at least 60 wt% LNDFH-I, less than 10 wt% LNFP-I, less than 35 wt% 3FL and less than 5 wt% lactose, or

[0746] c) at least 40 wt% LST-c, less than 25 wt% LNnT, less than 25 wt% 6'SL and less than 10 wt% lactose, or

[0747] d) at least 55 wt% LST-a, less than 40 wt% LNT, less than 10 wt% 3'SL and less than 2 wt% lactose, and

[0748] Each component in the total composition accounts for 100 wt%, and the composition is a mixture of at least two components.

[0749] 120. The composition according to item 119, wherein the composition consists essentially of:

[0750] a) at least 50 wt% FSL, 20 to 45 wt% 3FL, 0.1 to 2 wt% 3'SL and 0 to 3 wt% lactose, or

[0751] b) at least 70 wt% LNDFH-I, 0.1 to 8 wt% LNFP-I, 15 to 25 wt% 3FL and 0 to 3 wt% lactose

[0752] c) at least 50 wt% LST-c, 15 to 25 wt% LNnT, 15 to 25 wt% 6'SL and 0 to 7 wt% lactose

[0753] d) e) at least 50 wt% LST-a, 15 to 40 wt% LNT, 0 to 15 wt% 3'SL and 0 to 2 wt% lactose, and

[0754] Each component in the total composition accounts for 100 wt%, and the composition is a mixture of at least two components.

[0755] 121. The composition according to item 119 or 120, wherein the molar ratio of FSL:3'SL is higher than 100:1 and the molar ratio of FSL:3FL is higher than 1:1.

[0756] 122. The composition according to item 119 or 120, wherein the molar ratio of LNDFH-I:LNFP-I is higher than 50:1, and the molar ratio of LNDFH-I:3FL is in the range of 1.5:1 to 5:1.

[0757] 123. The composition according to item 119 or 120, wherein the molar ratio of LST-a:LNT is higher than 1.5:1 and the molar ratio of LST-a:3'SL is higher than 9:1.

[0758] 124. Use of the composition according to items 119 to 123 for the regeneration and viability of freeze-dried probiotics.

[0759] 125. A composition comprising the HMO composition according to items 119 to 123, wherein the composition further comprises one or more probiotics.

[0760] 126. The composition according to item 125, wherein the probiotic is of the genus Bifidobacterium and / or Lactobacillus.

[0761] 127. The composition according to item 125 or 126, wherein the probiotic is selected from Bifidobacterium animalis BB12 DSM32269, Bifidobacterium animalis BIF6, Bifidobacterium longum DSM 32946, Bifidobacterium longum BB536, Bifidobacterium bifidum DSMZ32403, Bifidobacterium infantis, Bifidobacterium breve DSM 33789, Bifidobacterium infantis SP37 DSM 32687, Bifidobacterium adolescentis DSM 34065, Bifidobacterium animalis subsp. animalis DSM 16284, Lactobacillus rhamnosus GG DSM 32550, Lactobacillus rhamnosus 19070-2 DSM 26357, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus LBrGG, Lactobacillus reuteri DSM 12246, Lactobacillus plantarum TIFN101, Lactobacillus gasseri Lg-36 200B FloraFit Danisco, Lactobacillus casei FloraActive PAREVE DSMZ 32382, Lactobacillus paracasei, Lactobacillus plantarum PS128, Lactobacillus plantarum (Sacco) DSM 32383, Lactococcus lactis PAREVE, Lactobacillus paracasei subsp. paracasei and Lactobacillus Lactobacillus reuteri S12 DSM 33752.

[0762] 128. Use of a composition according to items 119 to 127 for the production of a nutritional composition.

[0763] 129. The use according to item 128, wherein the nutritional composition is a dietary supplement and / or a medical nutritional product.

[0764] 130. The use according to item 128, wherein the nutritional composition is an infant nutrition product.

[0765] Sequence Listing

[0766] This application contains a Sequence Listing in both text and electronic formats, which is incorporated herein by reference.

[0767] A summary of the SEQ ID NOs used in this application is shown in the table below.

[0768] Summary of sequences listed in this application

[0769]

[0770]

[0771]

[0772] Example

[0773] method

[0774] strain

[0775] The strain (genetically engineered cell) constructed in this application is based on Escherichia coli K-12DH1, and its genotype is: Fˉ, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. The E. coli K-12DH1 strain was additionally modified to generate the MDO strain with the following modifications: lacZ: 1.5 kbp deletion, lacA: 0.5 kbp deletion, nanKETA: 3.3 kbp deletion, melA: 0.9 kbp deletion, wcaJ: 0.5 kbp deletion, mdoH: 0.5 kbp deletion, and the Plac promoter was inserted upstream of the gmd gene.

[0776] Methods for inserting a target gene into the E. coli genome are well known to those skilled in the art. Genetic cassettes can be inserted into the E. coli chromosome using gene gorging (see, e.g., Herring and Blattner 2004 J. Bacteriol. 186: 2673-81 and Warming et al. 2005 Nucleic Acids Res. 33 (4): e36) and specific selection marker genes and screening methods.

[0777] The MDO strain was further modified to generate a strain producing the desired HMO. For example, a 3FL production strain was generated by exchanging the native promoter in front of the colanic acid cluster with the PglpF promoter (wcaF::PglpF). In addition, the strain has three copies of the futA gene encoding the α-1,3-fucosyltransferase of SEQ ID NO: 1 under the control of the PglpF promoter, and one copy of the marc gene encoding the MFS transporter Marc of SEQ ID NO: 2 under the control of the PglpF promoter. The genotypes of all strains used in this example are shown in Table 12.

[0778] Table 12: Genotypes of E. coli strains used in the examples

[0779]

[0780] 1 ST6, Pd2 - one genomic copy of the gene encoding the alpha-2,6-sialyltransferase of SEQ ID NO: 43 under the control of the PglpF promoter (SEQ ID NO: 42).

[0781] 2ΔnadC—quinoline phosphoribosyltransferase lacking WP_101348535.1, for more details see WO2017 / 101958.

[0782] 3 pBS-nadC-Plac-neuBCA—a plasmid expressing the neuBCA gene cluster (SEQ ID NO: 44). For more details about nadC, see WO 2017 / 101958.

[0783] 4 lgtA-PglpF—two copies of a gene encoding β-1,3-N-acetylglucosaminyltransferase (SEQ ID NO: 45) under the control of the PglpF promoter (SEQ ID NO: 42) inserted into the genome.

[0784] 5 galT-PglpF—a gene encoding β-1,4-galactosyltransferase (SEQ ID NO:46) under the control of the PglpF promoter (SEQ ID NO:42) inserted into the genome.

[0785] 6 scrYA, scrBR—two operons encoding the sequences of SEQ ID NOs: 47 and 48 and SEQ ID NOs: 49 and 50, respectively, and under the control of PglpF_SD1 promoter (SEQ ID NO: 53) and Pscr promoter (SEQ ID NO: 54), respectively.

[0786] 7 vag - a copy of the gene encoding the MFS transporter of SEQ ID NO: 50 under the control of the PglpF promoter (SEQ ID NO: 42) inserted into the genome.

[0787] 8 wcaF::PglpF - Insertion of the PglpF promoter (SEQ ID NO: 42) in front of the endogenous colanic acid operon. This can also be written as CA::CA(PglpF).

[0788] 9 FutA - three copies of a gene encoding alpha-1,3-fucosyltransferase (SEQ ID NO: 1) under the control of the PglpF promoter (SEQ ID NO: 19) inserted separately into the genome.

[0789] 10 marc - a copy of the gene encoding the MFS transporter (SEQ ID NO: 2) under the control of the PglpF promoter (SEQ ID NO: 42) inserted into the genome.

[0790] 11 ΔptsG—missing the native glucose PTS complex component IICB Glc - Reduced ability of strains to grow on glucose.

[0791] 12 PglpF-galTK—a copy of the gene encoding the beta-1,3-galactosyltransferase of SEQ ID NO:56 under the control of the PglpF promoter (SEQ ID NO:42) inserted into the genome.

[0792] 13 Plac-YberC—a gene copy inserted into the genome encoding the MFS transporter of SEQ ID NO:57 under the control of the PglpF promoter (SEQ ID NO:42).

[0793] 14 PglpF-LacY—an additional copy of the gene encoding the E. coli LacY transporter of SEQ ID NO: 3 under the control of the PglpF promoter (SEQ ID NO: 42) inserted into the genome.

[0794] 15 Δglf - missing the native glucose facilitator factor - reduces growth of the strain on glucose.

[0795] 16 Plac-D29nst—two copies of the gene encoding the α-2,3-sialyltransferase of SEQ ID NO: 58 under the control of the Plac promoter (SEQ ID NO: 55) inserted separately into the genome.

[0796] 17 Deletion of the Δlacl-lac repressor eliminates the use of IPTG.

[0797] 18 ΔrpoS—missing the σ factor S—increases product yield (for more details, see PCT / EP2023 / 065491).

[0798] Fermentation

[0799] The fermentation-enzymatic hybrid process was carried out in a 2L Sartorious B-stat bioreactor, starting with 700-1000 g of sterile mineral medium consisting of glucose and / or sucrose as carbon sources (sterilized separately), lactose monohydrate (sterilized separately, the amount of lactose monohydrate is sufficient to produce the desired amount of oligosaccharides produced by the cells, but not more than the amount that the cells can convert into the desired oligosaccharides produced by the cells, for example in the range of 10-80 g / kg), (NH4)2HPO4, KH2PO4, MgSO4 x 7H2O (sterilized separately), KOH, NaOH, citric acid, trace element solution, defoamer and thiamine (added by sterile filtration). The dissolved oxygen level was maintained above 20%, for example 23%, by a cascade operation of first stirring (1000-2000 rpm) and then air flow (1-3 VVM). The pH was maintained at 6.8 by titration with NH4OH solution. Cultures were started with a 2% (v / v) inoculum grown in similar medium containing glucose and / or sucrose to an OD of 600 For a pre-culture of 2.5-5.

[0800] After the carbon source contained in the batch medium is exhausted, a sterile mineral feed solution containing a predetermined amount of carbon source (glucose and / or sucrose), MgSO4 x 7H2O, H3PO4, trace metals and defoamers is continuously fed using a predetermined feeding method to maintain culture carbon limitation and perform sufficient respiratory metabolism. In addition, in some embodiments, a sterile second HMO (defined in the following embodiments) is added by feeding or from the beginning of fermentation. The temperature is initially set to 33 or 34°C and then dropped to a preset temperature between 30°C and 25°C (defined in the following embodiments). The growth and state of the cells are tracked by biological wet weight (the weight of the cell pellet after centrifugation for 3 minutes at 14,000g / medium weight), optical density at 600nm, and online detection of CO2 release rate, stirring, NH4OH base addition, dissolved oxygen and temperature. Lactose and oligosaccharide concentrations are determined once or more per day by HPLC. When collecting samples for HPLC analysis, heat treatment is performed at 90°C for 20 minutes to stop the enzymatic process.

[0801] Example 1 Synthesis of FSL by using the 3FL strain and adding α-2,3-trans-sialidase and pure 3'SL to the culture broth through a fermentation-enzymatic hybrid process

[0802] This example demonstrates that it is possible to form FSL directly in the fermentation broth using cells that produce 3FL from lactose and by adding α-2,3-transsialidase and a second substrate, 3'SL, to the broth.

[0803] In this example, the 3FL strain was fermented as described in the method section above, starting in 700 g of medium after the addition of inoculum. After an initial batch phase of 14 hours, the batch feeding phase was started by continuously feeding a glucose solution containing minerals, with an initial glucose feed rate of 1.35 g glucose / hour, which rose linearly to 2.7 g / hour within 5 hours and remained constant thereafter. 3'SL was fed with a feed curve corresponding to a fraction of the glucose feed rate, which reflected the yield of the 3FL strain on glucose to roughly match the accumulation rate of 3'SL and 3FL. 24 hours after the start of feeding, the fermentation temperature dropped linearly to 25°C within 5 hours. Transsialylation of 3FL was started by adding 115 mg / L of sterile filtered α-2,3-trans-sialidase (TcTS, SEQ ID NO: 60) of the fermentation broth at 89 hours and adding an additional 0.175 mg / ml TcTS at 118 hours. Batch feeding continued until 139 hours after the start of fermentation. In addition, the 3'SL (second HMO) feed was stopped 119 hours after the start of the fermentation.

[0804] The formation of 3FL from lactose, the formation of FSL and the consumption of 3'SL were monitored by measuring the composition by HPLC. The samples for HPLC analysis were heat treated at the time of collection to stop the enzymatic process. Figure 2 The progress of the process is shown as weight percent relative to the total weight of substrate and product. Figure 2 As can be seen in Figure 5, at hour 89 (i.e., addition of α-2,3-transsialidase), almost all lactose was consumed by the cells to form 3FL, while 3'SL was fed into the system in proportion to the mass of 3FL formed. By adding α-2,3-transsialidase at hour 89 of the process, transsialylation of 3FL to form FSL was initiated using 3'SL as a sialic acid donor. Figure 2The formation of FSL and the decrease of 3'SL can be seen in the figure. The transsialylation process consumes 3FL and 3'SL, and forms equimolar amounts of byproduct lactose. Lactose is in turn taken up by the E. coli strain and recycled to 3FL; therefore, after the start of the enzymatic reaction, the levels of lactose and 3FL are close to a steady state, because a 1:1 molar stoichiometric amount of 3FL is consumed in the FSL reaction, while a 1:1 molar amount of byproduct lactose is formed, which is recycled to 3FL in a 1:1 molar ratio. On the other hand, FSL continues to increase until 3'SL is almost completely converted. Therefore, at the end of the mixing process, an 82% yield of FSL is achieved relative to the total amount of 3'SL added, while the conversion of 3'SL is 99.5%. It can be observed that 3'SL is not 100% converted to FSL, which may be due to factors such as the stability of 3'SL during heat sterilization and storage, and / or due to the side hydrolysis activity of the enzyme, which does not have 100% transsialylation selectivity. Furthermore, with the complete conversion of 3′SL and recycling of lactose for 3FL formation, the hybrid process produced a final solution composed of 3FL and FSL as the major HMOs and a very small amount of residual lactose.

[0805] Example 2 is a comparative example of an in vitro process (strictly enzymatic) using α-2,3-transsialidase on purified 3FL and 3'SL. Table 13 below shows the relative yields of each component at the end of the in vitro or mixed process, as well as the conversion rate of the sialic acid donor (3'SL) and the yield of FSL relative to 3'SL.

[0806] Table 13: Comparison of the final product composition, 3'SL conversion rate and FSL yield relative to the amount of 3'SL added of FSL synthesized by the hybrid process (Example 1) and the in vitro process (Example 2).

[0807]

[0808] The comparison showed that the hybrid process had a significantly higher yield of FSL relative to 3'SL compared to the in vitro process. In addition, the amount of 3'SL and lactose remaining in the final product composition was very low for the hybrid process. This suggests that the hybrid process provides a further purification advantage because it is easier to separate FSL from 3FL than from 3'SL due to the difference in electrostatic charge (FSL and 3'SL are negatively charged in the conjugate base form, while 3FL is neutral).

[0809] Example 2 - Comparative Example - Synthesis of FSL using an in vitro process

[0810] The use of in vitro transsialylation reactions to form sialylo-oligosaccharides is known, for example, from WO 2012 / 007588. In this example, such reactions were performed using pure 3FL and 3'SL as acceptor and donor, respectively, to illustrate the difference between the in vitro system and the mixed system in Example 1.

[0811] In vitro synthesis of FSL was performed using α-2,3-trans-sialidase (TcTS, SEQ ID NO: 60) in Example 1 to catalyze the trans-sialylation of 3FL using 3'SL as a sialic acid donor. A substrate solution consisting of 107.33 mM 3FL and 107.33 mM 3'SL was prepared at pH 6.85. The reaction was started by adding 1 mg / ml TcTS at 25°C. The reaction progress was monitored by measuring the concentration of substrate and product by HPLC. Samples for HPLC measurements were collected by treating at 95°C for 5 minutes to denature the TcTS enzyme, thereby terminating the reaction. Table 14 shows the relative amounts of different products relative to the total amount of product over time.

[0812] Table 14: Relative amounts of various components in the in vitro synthesis of FSL from 3FL and 3'SL catalyzed by α-2,3-transsialidase over time

[0813]

[0814] From the results in Table 14, it can be seen that as the concentration of 3'SL decreases, the concentration of FSL increases, indicating that 3'SL is converted into FSL. In addition, as sialic acid is removed from 3'SL, the concentration of lactose also increases. The last column of Table 2 shows the yield of FSL relative to the starting amount of 3'SL. At reaction equilibrium, the yield of FSL relative to the starting amount of 3'SL is about 54%.

[0815] Example 3: Synthesis of LNDFH-I using a hybrid process

[0816] The synthesis of LNDFH-I using a hybrid process was carried out by fermentation from lactose using the 3FL production strain 3FL-S1 in Table 12, which started with 1000 g of culture medium after adding the inoculum. In this example, the fermentation of the 3FL strain was as described in the method section above. After an initial batch phase of 14 hours, the batch feeding phase was started by continuously feeding a glucose solution containing minerals, with an initial feed rate of 2.19 g glucose / hour, and the feed rate rose linearly to 4.37 g / hour within 5 hours, and then remained constant. Three hours after the start of the feed, the fermentation temperature dropped linearly to 30°C within 1 hour. In this example, equimolar amounts of lactose and LNFP-I were added at the start of the fermentation. Transfucosylation of LNFP-I was started by adding 68 mg / L of sterile filtered mutant recombinant α1,3 / 4-transfucosidase (from Bifidobacterium longum infantis subspecies (BiTF-641, SEQ ID NO: 59)) of the fermentation broth at 90 hours after the start of the fermentation. In this hybrid process, the acceptor molecule (LNFP-I) was monitored by measuring the individual components by HPLC. Transfucosylation of the culture medium using 3FL formed in vivo (from lactose) as a fucosyl donor to form LNDFH-I was performed. Samples for HPLC analysis were heat treated at the time of collection to terminate the enzymatic process. Figure 3 The progress of lactose, LNFP-I, 3FL and LNDFH-I is shown as weight percentage relative to the total weight of substrate and product. Figure 3 It can be seen that the formation of LNDFH-I did not occur until 90 hours into the process (i.e., the enzyme was added), while lactose was completely converted to 3FL through the recycling activity of the genetically modified cells. By adding BiTF-641 to the process at the 90th hour, transfucosylation of LNFP-I began using 3FL as a fucosyl donor. After the addition of the enzyme, an increase in LNDFH-I was observed, and the molar equivalent of LNFP-I decreased. After the transfucosylation reaction began, the lactose concentration increased as it was released from the fucosyl donor 3FL, but it was subsequently taken up by the cells and converted back to 3FL, indicating that lactose can be recycled. Figure 3 As can be seen in the figure, the recycling of lactose forms 3FL in situ, bypassing the transfucosylation kinetic equilibrium barrier and resulting in a conversion rate of 98.7% from LNFP-I to LNDFH-I.

[0817] Example 4 is a comparative example of applying α1,3 / 4-transfucosidase to an in vitro process (strictly enzymatic) of purified 3FL and LNFP-I. Table 15 below shows the relative composition of the components at the end of the in vitro and mixed processes and the conversion rate of LNFP-I.

[0818] Table 15: Comparison of final product composition and LNFP-I conversion of the hybrid and in vitro processes for the synthesis of LNDFH-I starting from equimolar acceptor (LNFP-I) to donor (lactose in the hybrid process or 3FL in the in vitro process) ratios.

[0819]

[0820] Due to the recycling of lactose, 3FL is continuously formed, so only the conversion rate of LNFP-I is used as a process comparison indicator. The hybrid process can not only improve the conversion rate of LNFP-I, but also the lactose content of the final product mixture is very low. Therefore, the advantage of the hybrid process is that the final product obtained is composed of 3FL and LNDFH-I as the main components and very low lactose and LNFP-I, as shown in Table 15. If pure LNDFH-I is desired, this is an advantage because it is easier to separate 3FL and LNDFH-I than to separate LNFP-I and LNDFH-I due to the greater difference in molecular size. In addition, the advantage of the hybrid process is that the expensive raw material 3FL can be replaced by cheap lactose, and a higher conversion rate of the more expensive raw material LNFP-I can be achieved compared with the in vitro process, so overall, it is a more economical process.

[0821] Example 4: Comparative Example - Synthesis of LNDFH-I using an in vitro process

[0822] In vitro synthesis of LNDFH-I was performed by preparing a substrate solution consisting of equimolar donor and acceptor substrates, using 3FL as a fucosyl donor, and using BiTF-641 (SEQ ID NO: 59) to catalyze the transfucosylation of LNFP-I. A substrate solution consisting of 150 mM LNFP-I and 150 mM 3FL was prepared at pH 6.55. The transfucosylation reaction was started by adding 0.51 mg / ml BiTF-641 at 25°C. The reaction progress was monitored by measuring the concentration of substrate and product by HPLC. The samples for HPLC were treated at 95°C for 5 minutes to denature the BiTF-641 enzyme, thereby terminating the reaction and collecting the samples. Figure 4 The reaction progress curve of LNDFH-I synthesized in vitro is shown as a weight percentage relative to the total weight of substrate and product, and indicates that the reaction has reached equilibrium. Starting from an equimolar ratio of acceptor (i.e., LNFP-I) to donor (i.e., lactose in the hybrid process or 3FL in the in vitro process), the final product composition and LNFP-I conversion rate of LNDFH-I synthesized in vitro and hybrid processes are shown in Table 15 in Example 3.

[0823] exist Figure 4As can be seen from the results, compared to the hybrid process with very low and stable lactose concentration and 98.7% LNFP-I conversion (i.e. Figure 3 As shown in Figure 2 ), the in vitro process produced at least stoichiometrically equimolar or more amounts of lactose to LNDFH-I and a 42.5% conversion rate to LNFP-I. Due to the side hydrolytic activity of the BiTF-641 enzyme, it can cleave 3FL, thereby increasing the level of lactose without forming LNDFH-I, so lactose is generally formed in excess of the stoichiometric amount relative to LNDFH-I in the in vitro process.

[0824] Example 5: Synthesis of LNDFH-I using a hybrid process starting from a high ratio of acceptor to donor

[0825] LNDFH-I synthesis in a hybrid process was performed using the 3FL-S1 strain in Table 12 by lactose fermentation starting from 900 g of culture medium after addition of inoculum.

[0826] In this example, the 3FL strain was fermented as described in the method section above. After an initial batch phase of 14 hours, the batch feeding phase was started with an initial feed rate of 1.96 g glucose / hour, which rose linearly to 3.92 g / hour within 5 hours and remained constant thereafter. Three hours after the start of feeding, the fermentation temperature dropped linearly to 30°C within 1 hour. In this example, the total amount of lactose used in the process was added at the start of fermentation, while LNFP-I was fed at a fixed rate 15 hours after the start of the batch feeding phase and continued for 48 hours. 88 hours after the start of fermentation, transfucosylation of LNFP-I was started by adding 112 mg / L of a sterile filtered mutant recombinant α1,3 / 4-transfucosidase fermentation broth from Bifidobacterium longum infantis subspecies (BiTF-641, SEQ ID NO: 59). In this hybrid process, 3FL formed in vivo (from lactose) was used as a fucosyl donor, and the formation of LNDFH-I caused by transfucosylation of the acceptor molecule LNFP-I in the culture medium was monitored over time by measuring the individual components by HPLC. Samples for HPLC analysis were heat treated at the time of collection to terminate the enzymatic process.

[0827] Figure 5The progress of the process is shown in weight percentage relative to the total weight of substrate and product. The formation of LNDFH-I did not occur until the 88th hour of the process (i.e., the time of enzyme addition), but lactose was observed to be completely consumed, which was confirmed by the formation of 3-FL in vivo. The amount of LNFP-I continued to increase until the feeding was stopped and dropped rapidly after the addition of the enzyme. As expected, the level of LNDFH-I increased after the addition of the enzyme, while the corresponding LNFP-I (receptor) decreased. However, due to the recycling of lactose released from the transfucosylation reaction for the formation of 3FL, the levels of lactose and 3FL remained basically constant. The recycling of lactose to form 3FL in situ bypasses the transfucosylation kinetic equilibrium barrier, which makes the conversion rate of LNFP-I to LNDFH-I 97.8%.

[0828] Example 6 is a comparative example of an in vitro process (strictly enzymatic) using α1,3 / 4-transfucosidase for purified 3FL and LNFP-I. Table 16 below shows the relative composition of the components at the end of the in vitro and mixed processes and the conversion rate of LNFP-I.

[0829] Table 16: Comparison of the final product composition of LNDFH-I synthesized by the hybrid process and the in vitro process, starting from a molar ratio of acceptor (LNFP-I) to donor (3-FL) of 2.

[0830]

[0831] Since 3FL is continuously formed due to the recycling of lactose, only the conversion rate of LNFP-I was used as a process comparison indicator.

[0832] As can be seen from Table 16, the hybrid process can not only improve the conversion rate of LNFP-I, but also the content of lactose and LNFP-I in the final product mixture is very low. Therefore, the advantage of the hybrid process is that the final mixture obtained is composed of 3FL and LNDFH-I as the main components and very low lactose and LNFP-I, as shown in Table 16. If it is desired to obtain pure LNDFH-I, this is an advantage because it is easier to separate 3FL and LNDFH-I than to separate LNFP-I and LNDFH-I. In addition, the advantage of the hybrid process is that the expensive raw material 3FL can be replaced by cheap lactose, and the conversion rate of the more expensive raw material LNFP-I can be greatly improved compared with the in vitro process, so overall, it is a more economical process.

[0833] Examples 3 and 5 were used to study the effect of excess of the second oligosaccharide (in this case the acceptor) relative to the first oligosaccharide in a mixed process, and Examples 4 and 6 were used to study the same situation in an in vitro process. For the mixed process, the ratio of acceptor to donor was essentially between LNFP-I and lactose, since lactose was completely converted to 3FL. Table 17 compiles the results of Tables 15 and 16.

[0834] Table 17: Comparison of the final product composition of the hybrid process and the in vitro process for the synthesis of LNDFH-I starting from different molar ratios of acceptor (LNFP-I) to donor (3-FL)

[0835]

[0836] The results clearly showed that in the hybrid process, increasing the amount of the second oligosaccharide (fed to the culture (acceptor)) relative to the first oligosaccharide (produced by the strain (donor)) had a positive effect, where LNDFH-I levels increased by 35% and 3FL levels decreased by 50% when the acceptor was in excess. For the in vitro process, the increased acceptor:donor ratio did not have any positive effect on LNDFH-I formation, and LNFP-I levels remained significantly higher than 3FL and lactose, indicating that it was limited by the kinetic barrier of the enzymatic reaction. However, in the hybrid process, avoiding the oligosaccharide added in the limiting process (the second oligosaccharide) can increase the formation of the third oligosaccharide, because as long as the second oligosaccharide (3FL) is present, the first oligosaccharide (LNFP-I) will continue to be produced, and lactose will be generated from the transfucosylation reaction, thereby continuously feeding the strain with the initial substrate to renew the formation of LNFP-I and eliminating the kinetic barrier observed in the in vitro process.

[0837] Example 6: Comparative Example - Synthesis of LNDFH-I Using an In Vitro Process Starting with a High Ratio of Acceptor to Donor

[0838] In vitro synthesis of LNDFH-I was performed using 3FL as a fucosyl donor, starting from a ratio of 2:1 mol / mol of LNFP-I:3FL, using BiTF-641 (SEQ ID NO:59) to catalyze the transfucosylation of LNFP-I. A substrate solution consisting of 200 mM LNFP-I and 100 mM 3FL was prepared at pH 6.5. The transfucosylation reaction was started by adding 0.25 mg / ml BiTF-641 at 37°C. The reaction progress was monitored by measuring the concentration of substrate and product by HPLC. The samples for HPLC were treated at 95°C for 5 minutes to denature the BiTF-641 enzyme, thereby terminating the reaction and collecting the samples.

[0839] Figure 6The reaction progress curve showing the in vitro synthesis of LNDFH-I when the molar ratio of acceptor to donor was 2:1 clearly indicated that the reaction had reached thermodynamic equilibrium.

[0840] from Figure 6 As can be seen from the results, compared to the hybrid process (i.e. Figure 5 The in vitro process resulted in at least stoichiometric equimolar lactose compared to LNDFH-I and a limited conversion of LNFP-I of 37%. The results are summarized in Table 17.

[0841] Example 7: Synthesis of LST-c using a dual-strain hybrid process

[0842] In this embodiment, two strains, LNnT-S1, which produces LNnT from lactose (substrate) and sucrose (carbon source), and 6'SL-S1, which produces 6'SL from lactose (substrate) and glucose (carbon source) (see Table 12), were co-cultured. Due to the absence of the ptsG transporter, the growth of LNnT-S1 on glucose was reduced. The 6'SL-S1 strain cannot grow on sucrose. 6'SL is produced as a sialic acid donor substrate and LNnT is produced as an acceptor substrate for subsequent transsialic acid reactions. By adding α2,6-trans-sialidase to the fermentation medium, LST-c is formed in the fermentation medium. This process is also called a dual strain mixing process.

[0843] Fermentation

[0844] The dual strain hybrid process was performed in a 2L Sartorious B-stat bioreactor starting with 1000 g of mineral medium consisting of 15 g / kg glucose (sterilized separately) and 15 g / kg sucrose (sterilized separately), lactose monohydrate (sterilized separately), (NH4)2HPO4, KH2PO4, MgSO4 x 7H2O (sterilized separately), KOH, NaOH, citric acid, trace element solution, defoamer and thiamine (filter sterilized). The dissolved oxygen level was maintained at 20% by cascading operation of first stirring and then 1 VVM (maximum 3 VVM) of air flow. The pH was maintained at 6.8 by titration with NH4OH solution. The culture was started with a 1% (v / v) inoculum grown to OD 50 in a similar containing medium. 600The pre-culture of 2.5-5, glucose as the carbon source of 6'SL strain, sucrose as the carbon source of LNnT strain. After about 14 hours, after the glucose and sucrose contained in the batch medium are exhausted, a mineral feed solution containing glucose and sucrose in a 1:2 ratio (w / w) (sterilized separately with minerals), MgSO4 x 7H2O, H3PO4, trace metals and defoamers is continuously fed using a constant feeding method, which provides 1g / hour of glucose and 2g / hour of sucrose, so that the culture is kept limited carbon. The temperature was initially set to 34°C, but dropped to 28°C after 3 hours of feeding with a linear slope of 1 hour. The growth and state of the cells are tracked by biological wet weight (cell sediment weight / fermentation broth weight after 14,000g centrifugation for 3 minutes), optical density at 600nm, and online measurement of CO2 release rate, stirring, alkali addition, dissolved oxygen and temperature.

[0845] 113 hours after the start of fermentation, transsialylation was started by adding 3390 mg / L of α-2,6-trans-sialidase (from Photorhabdus luminescens JT-SHIZ-119 (PITS-197, SEQ ID NO: 41)) fermentation broth, at which time almost all lactose had been converted to 6'SL and LNnT. The process was monitored by measuring the concentration of substrates and products by HPLC.

[0846] result

[0847] Figure 7 Concentration data are shown as weight percent relative to the total weight of substrate and product to illustrate process progress.

[0848] It can be seen that by the 113th hour, all lactose was consumed and the trans-sialidase was added to the culture, with the LNnT and 6'SL strains producing 6'SL and LNnT in nearly equimolar ratios. After the addition of the trans-sialidase, LST-c began to form by consuming 6'SL and LNnT (as can be seen from their reduced amounts). The formation of LST-c also generates an equimolar amount of lactose as a leaving group for the trans-sialylation reaction. However, since lactose is taken up by the LNnT and 6'SL strains and reused to form 6'SL and LNnT, its levels remain low.

[0849] This example shows (see Figure 7), the second oligosaccharide can be obtained from a second genetically modified organism co-cultured with a genetically modified organism (genetically modified strain) that produces the first oligosaccharide. The two strains are co-cultured on two different carbon sources, which allows the first and second oligosaccharides to be stably formed, and the first and second oligosaccharides are used for the transsialic acid reaction to form complex sialylated oligosaccharides. It also shows that the huge difference in product yield (grams of LNnT or 6'SL per gram of carbon source) of the two strains can be compensated by feeding with a modified carbon source ratio to achieve a desired product ratio, in this case, a glucose: sucrose ratio of 1:2 (g / g) to achieve a 6'SL: LNnT ratio of 1:1.

[0850] As with the above-mentioned single-strain mixed process example, the dual-strain mixed process was also compared with a conventional in vitro enzymatic process.

[0851] The conventional in vitro enzymatic process was performed by the same α-2,6-transsialidase PITS-197, which catalyzes the transsialylation of LNnT using 6'SL as a sialic acid donor. A substrate solution containing 116.8 mM LNnT and 116.8 mM 6'SL was prepared at pH 6.87. The transsialylation reaction was started by adding 1.57 mg / ml PITS-197 at 25°C. The progress of the reaction was monitored by measuring the concentration of substrate and product by HPLC. The sample for HPLC was treated at 95°C for 5 minutes to denature the PITS-197 enzyme, thereby stopping the reaction and collecting the sample. Figure 8 The reaction progress curve showing the in vitro synthesis of LST-c. Figure 8 It can be seen that compared with the dual-strain mixing process, it produces very low lactose concentration (such as Figure 7 Table 18 shows the final product composition comparison of LST-c synthesized using the dual strain mixed process and the in vitro process.

[0852] Table 18: Comparison of the final product composition of LST-c synthesized by the dual-strain mixed process and the in vitro process starting from an equimolar ratio of acceptor (LNnT) to donor (6'SL).

[0853]

[0854] Table 18 shows that the formation of LST-c in the dual-strain mixed process is significantly increased due to the higher conversion rate of LNnT. In addition, the final product mixture obtained from the dual-strain mixed process has a lower level of lactose. Therefore, the dual-strain mixed process provides the advantages of obtaining a higher level of LST-c and a reduced lactose level. In addition, compared to the in vitro process, the advantage of the mixed process is that the expensive raw materials 6'SL and LNnT are replaced by cheap lactose, and the conversion rate of LNnT into the desired product LST-c is higher, so overall, this is a more economical process.

[0855] Example 8: Synthesis of LST-c using a single strain hybrid process

[0856] In addition to the dual strain mixing process described in Example 7, this example describes the synthesis of LST-c using a single strain mixing process. In this single strain mixing process, the acceptor substrate LNnT is produced in situ from lactose by the LNnT strain (LNnT-S2 in Table 12), and the purified sialic acid donor substrate 6'SL is added during the culture process. The transsialylation reaction is catalyzed by the addition of α-2,6-trans-sialidase from Photorhabditis elegans JT-SHIZ-119 (PITS-197, SEQ ID NO: 41).

[0857] As described in the method section above, cultivation started with 700 g of mineral medium containing lactose and 25 g / kg sucrose. After about 15 hours, the sucrose contained in the batch medium was exhausted, and then a feed solution containing sucrose and minerals was continuously fed in a manner to keep the culture carbon-limited, initially starting with a sucrose feed rate of 1.43 g / hour, gradually increasing to 2.93 g / hour within 5 hours, and then kept constant. The temperature was initially set to 33 ° C, then dropped to 30 ° C after 3 hours of feeding at a linear rate of 1 hour. Sterile 6'SL was fed alone at a constant rate for 24 hours after 15 hours of the batch feeding phase (approximately 30 hours after inoculation).

[0858] At 69.5 hours after inoculation (start of fermentation), transsialylation was initiated by adding 115 mg / L of α-2,6-trans-sialidase fermentation broth from Photorhabdus luminescens JT-SHIZ-119 (PITS-197, SEQ ID NO: 41), at which time almost all lactose had been converted to LNnT and 6'SL had been added. Additional enzyme solution pulses were added at 99 hours (366 mg / L), 121 hours (281 mg / L), and 146 hours (542 mg / L).

[0859] Fig.10The process progress curve is shown as the mass fraction of substrate and product relative to the total mass of substrate and product. As shown, until the addition of enzyme, lactose is almost completely converted to LNnT by the E. coli strain. The increased 6'SL level is due to the continued addition of 6'SL solution until the addition of enzyme. Starting from the enzyme addition point at 69.5 hours, the results of enzymatic transsialylation, namely the formation of LST-c and the consumption of 6'SL, can be seen. In addition, the lactose concentration is maintained at a stable low level because the lactose byproduct released by the transsialylation reaction is rapidly recycled through the formation of LNnT in vivo. As described in Example 7, the performance of the single strain hybrid production of LST-c was compared with the performance of LST-c synthesized in vitro from 6'SL and LNnT, and Figure 8 Table 19 shows the comparison of the final product composition of LST-c synthesized using the single strain mixed process and the in vitro process.

[0860] Table 19: Comparison of the final product composition of LST-c synthesized by single strain mixed process and in vitro process

[0861]

[0862] As can be seen from Table 19, the single strain mixed LST-c process produces a lower fraction of lactose and a higher fraction of LST-c. Therefore, like the double strain mixed process, the advantage of the single strain mixed process is that it provides efficient process economy by obtaining a higher fraction of the third oligosaccharide product LST-c and easier purification due to the reduced lactose level.

[0863] Example 9: Synthesis of LNFP-III using a single strain hybrid process

[0864] The synthesis of LNFP-III in a single strain mixed process was carried out using the 3FL production strain 3FL-S2 in Table 12 and externally added LNnT and α1,3 / 4-transfucosidase.

[0865] In this example, as described in the method section above, the 3FL strain was cultured in 700 g of culture medium, lactose and 25 g / kg glucose. After approximately 15 hours, the glucose contained in the batch culture medium was exhausted, and a feed solution containing glucose and minerals was continuously fed by keeping the culture carbon-limited, initially starting with a glucose feed rate of 0.88 g / hour, which was gradually increased to 1.76 g / hour within 5 hours. This rate was maintained for 92 hours, after which it was increased to 3.61 g / hour and maintained until the end of the culture. The temperature was initially set to 33 ° C and then dropped to 30 ° C after 3 hours of feeding at a linear rate of 1 hour. In this example, an excess of LNnT relative to lactose was added to the fermentor at 3:1 (mol / mol). LNnT was added 15 hours after the start of the batch feeding phase (approximately 30 hours after inoculation) and continued for 24 hours. At 42 hours after inoculation (fermentation start), 67 mg / L sterile filtered mutant recombinant α1,3 / 4-transfucosidase (from Bifidobacterium longum subsp. infantis (BiTF-641, SEQ ID NO: 59)) fermentation broth was added to start transfucosylation of LNnT. Additional enzyme solution pulses were added at 97 hours (48 mg / L) and 121 hours (42 mg / L).

[0866] Fig.11 The progress of lactose, LNnT, 3FL and LNFP-III is shown in weight percentage relative to the total weight of substrate and product. 3FL3FL The transfucosylation of LNnT (acceptor) using 3FL as a fucosyl donor begins by adding BiTF-641 at the 42nd hour, which corresponds to the beginning of the formation of LNFP-III. From this time on, LNFP-III begins to accumulate, while LNnT levels decline. In addition, since the byproduct lactose is subsequently taken up by cells and recycled as 3FL, the process maintains a stable level of 3FL while reducing the lactose initially added. The single strain mixed synthesis of LNPF-III makes the LNnT conversion rate 93.1%. Table 20 shows the composition 3FL of lactose and HMO at the end of the single strain mixed process.

[0867] As in the previous examples, the single strain mixing process for forming LNFP-III was also compared to the conventional in vitro enzymatic process for forming LNFP-III described herein.

[0868] Pure in vitro enzymatic synthesis of LNFP-III was performed using 3FL as a fucosyl donor, starting from a 3:1 mol / mol ratio of LNnT:3FL, using BiTF-641 (SEQ ID NO: 59) to catalyze the transfucosylation of LNnT. A substrate solution consisting of 200 mM LNnT and 66.7 mM 3FL was prepared at pH 6.5. The transfucosylation reaction was started by adding 0.4 mg / ml BiTF-641 at 30°C. The reaction progress was monitored by measuring the concentrations of substrate and product by HPLC. The samples for HPLC were treated at 95°C for 5 minutes to denature the BiTF-641 enzyme, thereby terminating the reaction and collecting the samples. Fig.12 The in vitro reaction progress of the synthesis of LNFP-III starting from a 3:1 mol / mol LNnT:3FL ratio is shown as the mass fraction of substrate and product relative to the total mass of substrate and product. The in vitro reaction progress was stopped at equilibrium after 34.6%. Fig.12 It can be seen that for the in vitro process carried out at a high ratio of acceptor to donor substrate, the extent of product formation is limited by the conversion of the limiting substrate, resulting in a very high fraction of the unconverted acceptor substrate LNnT in the final product mixture as shown in Table 20, and a very low fraction of the third product oligosaccharide LNFP-III. On the other hand, in the hybrid process, the byproduct lactose is recycled back to the donor substrate in the circulation loop of the combined fermentation and enzymatic steps, which makes the conversion rate of the excess acceptor substrate (LNnT) very high, thereby producing a high fraction of the third oligosaccharide product (LNFP-III). Table 20 shows a comparison of the final product composition of LNFP-III synthesized using a single strain hybrid process and an in vitro process, starting with a 3-fold molar amount of LNnT relative to lactose in the hybrid process and a 3-fold molar amount of LNnT relative to 3FL in the in vitro process.

[0869] Table 20: Comparison of the final product composition of LNFP-III synthesized by the single strain mixed process and the in vitro process. At the beginning, the molar amount of LNnT in the mixed process was 3 times that of lactose, and the molar amount of LNnT in the in vitro process was 3 times that of 3FL

[0870]

[0871] Table 20 shows that when the molar amount of LNnT in the mixed process was 3 times relative to lactose at the beginning, the molar amount of LNnT in the in vitro process was 3 times relative to 3FL, and the single strain mixed process produced 3.5 times more LNFP-III than the conventional in vitro process. In addition, the byproduct lactose in the single strain mixed process was 15 times lower than that in the in vitro process.

[0872] Specifically, single-strain mixed LNPF-III synthesis resulted in a 93.1% conversion of LNnT, with LNnT added in a 3-fold (mol / mol) excess relative to lactose. In contrast, in vitro enzymatic LNFP-III synthesis starting from a 3:1 mol / mol ratio of LNnT:3FL, as described in the comparative example, only provided a 34.6% conversion of LNnT.

[0873] The large difference in conversion between the two processes is due to the fact that lactose, a byproduct of the transfucosylation reaction, is recycled back to the cells producing the donor substrate (3FL) in the recycle loop of the combined fermentation and enzymatic steps, which results in a high conversion of the excess acceptor substrate (LNnT), resulting in a high fraction of the third oligosaccharide product (LNFP-III). Therefore, the advantage of the single-strain hybrid process is that it provides efficient process economy by obtaining a higher fraction of the third oligosaccharide product LNFP-III and easier purification due to reduced lactose and LNnT levels.

[0874] Example 10: Synthesis of LNFP-II using a single strain hybrid process

[0875] The synthesis of LNFP-II in a single strain mixed process was carried out using the 3FL production strain 3FL-S1 in Table 12 and externally added LNT and α1,3 / 4-transfucosidase.

[0876] In this example, as described in the method section above, the 3FL strain started fermentation in 700 g of culture medium, lactose and 25 g / kg glucose. After about 13 hours, the glucose contained in the batch culture was exhausted, and a feed solution containing glucose and minerals was continuously fed in a manner that kept the culture carbon-limited, initially starting with a glucose feed rate of 1.5 g / hour, gradually increasing to 3 g / hour over 5 hours, and maintaining this rate until the end of the culture. The temperature was initially set to 33°C and then dropped to 30°C after 3 hours of feeding at a linear rate of 1 hour. In this example, a two-fold molar excess of LNT relative to lactose was added to the fermentor during the fermentation process. While lactose was fully supplied from the beginning, LNT was added starting at the 15th hour after the start of the batch feeding phase and continued for 24 hours. At 43 hours after the start of fermentation, 66 mg / L sterile-filtered mutant recombinant α1,3 / 4-transfucosidase (from Bifidobacterium longum subsp. infantis (BiTF-641, SEQ ID NO: 59)) fermentation broth was added to start transfucosylation of LNT.

[0877] Fig.13The progress of lactose, LNT, 3FL and LNFP-II is illustrated as weight percentage relative to the total weight of substrate and product. Transfucosylation of LNT by BiTF-641 using 3FL as a fucosyl donor began after the addition of the enzyme at 42 hours, and an accumulation of LNFP-II levels and a decrease in LNT levels can be observed. After the start of the transfucosylation reaction, the concentration of 3FL immediately decreased due to the very fast initial enzymatic rate when the product concentration was still low, and the lactose concentration immediately increased as it was released from the fucosyl donor 3FL. The single-strain mixed LNPF-II synthesis resulted in a conversion rate of 95.5% for LNT, starting at a molar ratio of LNT to lactose of 2:1. Table 21 shows the composition of lactose and HMO at the end of the single-strain mixed process.

[0878] As in the previous examples, the single strain mixing process for forming LNFP-II was also compared to a conventional in vitro enzymatic process for forming LNFP-II, as described herein.

[0879] The 3FLA pure in vitro enzymatic synthesis of LNFP-II was performed using 3FL as a fucosyl donor, starting from a 1:1 or 2:1 mol / mol ratio of LNT:3FL, using BiTF-641 (SEQ ID NO:59) to catalyze the transfucosylation of LNT. A substrate solution consisting of 200 mM LNT and 100 mM 3FL or 150 mM LNT and 150 mM 3FL was prepared at pH 6.7. The transfucosylation reaction was started by adding 0.51 mg / ml BiTF-641 at 25°C. The reaction progress was monitored by measuring the concentration of substrate and product by HPLC. The samples for HPLC were treated at 95°C for 5 minutes to denature the BiTF-641 enzyme, thereby stopping the reaction and collecting the samples. Fig.14 The progress of the in vitro synthesis of LNFP-II starting from a 2:1 mol / mol LNnT:3FL ratio is shown as the mass fraction of substrate and product relative to the total mass of substrate and product. The in vitro process with the same acceptor:donor ratio as the hybrid process (LNT:3FL is 2:1) only achieved a 28.5% conversion of the supplied LNT. Table 21 shows a comparison of the final product composition of the synthesis of LNFP-II using a single strain hybrid process and an in vitro process, starting with a molar amount of LNT in the hybrid process that was 2 times relative to lactose and an LNT / 3FL ratio of 1:1 or 2:1 in the in vitro process.

[0880] Table 21: Comparison of the final product composition of LNFP-II synthesized by single strain mixed process and in vitro process

[0881]

[0882] Table 21 shows that the single strain mixed process produced at least 3 times more LNFP-II than the conventional in vitro process when the acceptor: donor ratio was 1: 1 and 2: 1. In addition, the byproduct lactose in the single strain mixed process was at least 16 times lower than that in the conventional in vitro process.

[0883] Specifically, single-strain mixed LNPF-II synthesis resulted in a conversion of 95.5% of LNT, with LNT added in a 2-fold (mol / mol) excess relative to lactose. In contrast, in vitro enzymatic LNFP-II synthesis starting from a 2:1 mol / mol LNT:3FL ratio, as described in the comparative example, resulted in only a 28.5% conversion of the supplied LNT. When run at a 1:1 ratio in vitro, the conversion of LNT improved significantly, clearly demonstrating that the in vitro process is kinetically limited because it reaches equilibrium, and this cannot be significantly altered by increasing the amount of acceptor in the process.

[0884] Therefore, the advantage of the single-strain mixed process is that it provides efficient process economy by obtaining a higher fraction of the third oligosaccharide product LNFP-II and easier purification due to reduced lactose and LNT levels.

[0885] Example 11: Synthesis of LST-a using a single strain hybrid process

[0886] This example describes the synthesis of LST-a using a single strain mixed process. The process was carried out using the LNT production strain LNT-S1 in Table 12 and by adding purified sialic acid donor substrate 3'SL and α-2,3-trans-sialidase during the culture process.

[0887] The LNT-S1 strain in Table 12 was used for LST-a synthesis in a single strain mixed process. In this example, as described in the method section above, the LNT strain was cultured in 700 g of culture medium, lactose and 25 g / kg sucrose. After about 12 hours, the sucrose contained in the batch culture medium was exhausted, and a feed solution containing sucrose and minerals was continuously fed in a manner that kept the culture carbon-limited, initially starting with a sucrose feed rate of 1.46 g / hour, gradually increasing to 2.9 g / hour within 5 hours, and maintaining this rate until the end of the reaction. The temperature was initially set to 33°C and then dropped to 30°C after 3 hours of feeding at a linear rate of 1 hour. In this example, 3'SL was added to the fermentor in a total molar amount relative to lactose of 2:1. 3'SL was added at the 15th hour after the start of the batch feeding phase (approximately 27 hours after the start of inoculation / fermentation) and continued for 24 hours. At 49 hours after the start of fermentation, transsialylation of LNT was initiated in 264 mg / L sterile-filtered α-2,3-transsialidase (TcTS, SEQ ID NO: 60) fermentation broth.

[0888] Fig.15 The progress of lactose, LNT, 3'SL and LST-a is shown as weight percentage relative to the total weight of substrate and product. Sialylation of LNT by TcTS using 3'SL as sialic acid donor began immediately after the addition of enzyme at 49 hours, and accumulation of LST-a levels and decrease in LNT and 3'SL levels can be observed. After the addition of enzyme, LST-a began to form, which can also be seen from the decrease in 3'SL levels and its final complete depletion. After the start of the transsialylation reaction, the lactose concentration increased immediately as it was released from the sialic acid donor 3'SL due to the very fast enzymatic reaction rate when the product concentration is low. Single strain mixed LST-a synthesis resulted in a conversion rate of 100% for 3'SL, starting at a molar ratio of 3'SL to lactose of 2:1. Table 22 shows the composition of lactose and HMO at the end of the single strain mixed process.

[0889] As in the previous examples, the single strain mixing process for forming LST-a was also compared to a conventional in vitro enzymatic process for forming LST-a, as described herein.

[0890] In vitro comparative experiments were performed using purified LNT and 3'SL as substrates. A substrate solution consisting of 150 mM LNT and 150 mM 3'SL was prepared at pH 6.5 and 25°C. The transsialylation reaction was started by adding 0.51 mg / mL α-2,3-trans-sialidase (TcTS, SEQ ID NO: 60). The reaction progress was monitored by measuring the concentration of substrate and product by HPLC. Samples for HPLC analysis were collected and immediately heated at 90°C for 5 minutes to terminate the reaction. Fig.16 The progress of in vitro transsialic acidation of LNT to synthesize LST-a using 3'SL as a sialic acid donor is shown. Compared to the hybrid process, which fully converted 3'SL even when the 3'SL level was two times higher (mol / mol) than lactose, the in vitro process only achieved 57% 3'SL conversion at a 3'SL / LNT ratio of 1:1 mol / mol. Table 22 shows the final product composition comparison of the synthesis of LST-a using a single strain hybrid process and an in vitro process, starting with a 2:1 molar ratio of 3'SL to lactose in the hybrid process and a 1:1 molar ratio of 3'SL to LNT in the in vitro process.

[0891] Table 22: Comparison of the final product composition of LST-a synthesized by the single strain mixed process and the in vitro process. At the beginning, the molar amount of 3'SL in the mixed process was twice that of lactose, and the molar ratio of 3'SL to LNT in the in vitro process was 1:1.

[0892]

[0893] Table 22 shows that the single strain mixed process produced 1.5 times more LST-a than the conventional in vitro process. In addition, the byproduct lactose was reduced to 0 in the single strain mixed process, while it remained at 18% in the in vitro process.

[0894] Specifically, single-strain mixed LST-a synthesis resulted in 100% conversion of 3'SL with a 2-fold (mol / mol) excess of 3'SL relative to lactose. In contrast, in vitro enzymatic LST-a synthesis starting from a 1:1 mol / mol ratio of 3'SL:LNT, as described in the comparative example, resulted in only 57% conversion of the supplied LNT.

[0895] Since the hybrid process can completely convert the acidic (ie, ionizable) substrate 3'SL, it has a great advantage in terms of purification, because the acidic (ie, ionizable) product LST-a can be easily separated from the neutral LNT.

[0896] Example 12: Synthesis of LST-a using a dual-strain hybrid process

[0897] In addition to the single strain mixing process for synthesizing LST-a described in Example 11, this example describes the synthesis of LST-a using a dual strain mixing process. A first strain that produces LNT from lactose (substrate) and sucrose (carbon source) and a second strain that produces 3-'SL from lactose (substrate) and glucose (carbon source) are co-cultured. 3'SL is produced as a sialic acid donor substrate and LNT is produced as an acceptor substrate for subsequent transsialylation reactions catalyzed by α-2,3-transsialidase (TcTS, SEQ ID NO: 60) added to the culture medium to produce LST-a.

[0898] LST-a was synthesized in a dual strain mixed process using the LNT producing strain LNT-S1 and the 3'SL producing strain 3'SL-S1 in Table 12. In this example, the strains were co-cultured in 700 g of medium, lactose, 15 g / kg glucose, and 15 g / kg sucrose as described in the methods section above. The co-culture was started with a 1% (v / v) inoculum from each strain grown in a pre-culture with similar medium containing sucrose for the LNT strain and glucose for the 3'SL strain, and both were grown to an OD of 1. 6002.5-5. After about 18 hours, the glucose and sucrose contained in the batch medium were exhausted, and separate glucose and sucrose mineral feed solutions were continuously fed at a rate of 1.17 g glucose / hour and 1.17 g sucrose / hour by keeping the co-culture carbon limited. The temperature was initially set to 33°C and dropped to 28°C at a linear rate of 1 hour at the beginning of the batch feeding phase. At 68 hours after the start of the fermentation, transsialylation of LNT was started by adding 339 mg / L sterile filtered α-2,3-transsialidase (TcTS, SEQ ID NO: 60) fermentation broth, and another pulse of enzyme solution was added again at 90 hours.

[0899] Fig.17 The progress of lactose, LNT, 3'SL and LST-a is shown as weight percentage relative to the total weight of substrate and product. Data until the 68th hour of the process (i.e., enzyme addition) showed that the LNT strain and the 3'SL strain converted lactose into LNT and 3'SL, respectively, without forming LST-a. After the addition of enzymes at the 68th hour, LNT and 3'SL decreased as the amount of LST-a formed increased. In addition, although lactose is released as a by-product of the enzymatic reaction, its concentration continues to decrease because it is rapidly recycled into LNT and 3'SL by the corresponding strains. As shown in Table 23, compared with the pure in vitro LST-a process, the dual-strain mixed process for synthesizing LST-a achieves full utilization of lactose and a higher LST-a score. Table 23 shows a comparison of the final product composition of LST-a synthesized using the dual-strain mixed process and the in vitro process.

[0900] Table 23: Comparison of the final product composition of LST-a synthesized by the dual-strain mixed process and the in vitro process

[0901]

[0902] As can be seen from Table 23, compared with the pure in vitro LST-a process, the dual-strain mixed process for synthesizing LST-a achieved full utilization of lactose and a higher LST-a fraction. In addition, the amount of 3'SL in the final mixture produced by the dual-strain mixed process was 2.5 times lower, which has advantages in terms of purification, as separating 3'SL from LST-a is more challenging than separating it from neutral LNT.

[0903] Example 13 - Regeneration and viability of freeze-dried Lactobacillus species

[0904] Probiotics can be consumed as live bacteria or dried (e.g., freeze-dried) products. Regardless of the drying method, rehydration is an important step in the recovery of dehydrated bacteria; an insufficient rehydration / regeneration step may result in poor cell viability and low final survival. Therefore, rehydration is a highly critical step in the recovery process of freeze-dried cultures. For live and regenerated bacteria, bacterial survival under acidic conditions is critical because they need to pass through the acidic environment of the stomach and may also face storage (shelf life) issues in acidic foods.

[0905] In this example, it was tested whether a mixture similar to the HMO mixture produced by the mixing process described in Examples 1, 7, 8, 11 and 12 could provide benefits for the rehydration (regeneration) and vitality of probiotics. The test was conducted under acidic conditions to simulate the conditions that the bacteria must survive when passing through the stomach or stored in an acidic beverage.

[0906] As shown in Table 24, freeze-dried probiotic Lactobacillus rhamnosus DSM 32550 (0.4 mg / ml) used alone (control) or in combination with a HMO mixture (5% w / v) was dissolved in sterile phosphate buffered saline (PBS, pH=3), heated to 37°C and mixed vigorously for about 30 seconds until no visible clumps remained. The tubes were incubated at 37°C for 3 hours. The samples were further diluted and 100 μl samples were plated in duplicate on MRS agar plates and incubated at 37°C in an anaerobic chamber. Experimental setup see Fig.18 .

[0907] Table 24: HMO compositions tested in this example

[0908] HMO C Mix5 Mix6 Mix7 Mix8 LST-a 0 55 65 0 0 LNT 0 45 25 0 0 3'SL 0 0 10 0 0 3FL 0 0 0 45 0 FSL 0 0 0 55 0 LqCy 0 0 0 0 25 LST-c 0 0 0 0 50 6'SL 0 0 0 0 25

[0909] Fig.19 The image of the plate containing colonies of Lactobacillus rhamnosus DSM 32550 after 6 days of incubation is shown. CFU / ml was calculated based on the colony count after 72 hours of incubation (average of two plates). The E-2 dilution plate was used to count mix 5 and mix 6, and the results are shown in Fig. 20 As shown in A (mixture containing LST-a), the E-4 dilution plate was used to count mix 7 and mix 8, and the results were as follows Fig. 20 As shown in B.

[0910] Compared to the control without HMO mixture (whose survival rate was 0), freeze-dried Lactobacillus rhamnosus DSM 32550 dissolved with the HMO mixture described herein showed enhanced regeneration and survival ability. These data clearly show that in the presence of any HMO mixture, the regeneration and vitality of Lactobacillus rhamnosus DSM 32550 can be improved after exposure to low pH conditions (such as in the stomach or in acidic beverages). It can also be seen that the combination of more amounts of LST-a with some 3'SL is more effective than the mixture of LST-a and LNT alone. Compared to mixtures 5 and 6, mixtures 7 (3FL and FSL) and mixtures 8 (LST-c, LNnT and 6'SL) are more effective in the regeneration and vitality of Lactobacillus rhamnosus DSM 32550.

[0911] To our knowledge, the tested mixture has not been previously shown to have the benefit of improving the ability of probiotics to reproduce and survive in an acidic environment.

[0912] Example 14 - Regeneration and viability of freeze-dried Bifidobacterium species

[0913] As in Example 13 above, the ability of the HMO mixture in Table 24 to provide benefits for improving the regeneration and survival ability of Bifidobacterium longum DSM 32946 in an acidic environment was also tested.

[0914] The freeze-dried probiotic Bifidobacterium longum DSM 32946 (0.4 mg / ml), used alone or in combination with a HMO mixture (5% w / v) as indicated in Table 24, was dissolved in sterile water at pH 3.0, heated to 37°C and mixed vigorously for about 30 seconds until no visible clumps remained. The tubes were incubated at 37°C for 30 minutes. 100 μl samples were then plated in duplicate on MRS cysteine ​​agar plates and incubated at 37°C in an anaerobic chamber for 48 hours. The regeneration and viability of the probiotics were determined by counting the colonies on the plates after 48 hours of incubation.

[0915] Fig.21 A picture of a plate containing colonies of Bifidobacterium longum DSM 32946 after 2 days of incubation is shown. CFU / ml was calculated as the colony count on the undiluted plate after 48 hours of incubation (average of two plates). The results are shown in Table 25.

[0916] Table 25: Mean CFU / ml of Bifidobacterium longum DSM 32946 after 30 min acid treatment and subsequent incubation at 37°C for 48 h

[0917] mixture Comparison mix5 mix6 mix7 mix8 Average CFU / ml 0 85 95 165 3245

[0918] As can be seen from Table 25, the mixture was able to enable some Bifidobacterium longum DSM32946 strains to survive the acid treatment, compared to a survival rate of 0 in the control.

Claims

1. A method for producing oligosaccharides from donor oligosaccharides and acceptor oligosaccharides or acceptor disaccharides, the method comprising the following steps: a) culturing a genetically modified cell capable of producing a first oligosaccharide of at least three monosaccharide units in a medium supplied with a carbon source, wherein the genetically modified cell comprises one or more nucleic acids encoding: i) at least one by-product import protein, and ii) at least one recombinant glycosyltransferase, and iii) at least one pathway for producing a nucleotide activated sugar, and b) supplying a second disaccharide or oligosaccharide to the culture medium, and c) making an enzyme having transglycosidase activity available in said culture medium, and d) incubating the first oligosaccharide, the second disaccharide or oligosaccharide and the transglycosidase in the culture medium producing the first oligosaccharide to form a third oligosaccharide.

2. The method according to claim 1, wherein the second oligosaccharide or disaccharide supplied to the culture medium is produced in a separate process or is produced by the genetically modified cell in step a).

3. The method of claim 1, wherein the second oligosaccharide or disaccharide is supplied to the culture medium by a second genetically modified cell present in the same culture medium as the genetically modified cell in step a), and the second genetically modified cell is capable of producing the second oligosaccharide or disaccharide.

4. The method of claims 1 to 3, wherein the genetically modified cell exports the first and / or second oligosaccharide or disaccharide produced by the cell into the culture medium.

5. The method according to any one of claims 1 to 4, wherein the transglycosidase is added to the culture medium during the culturing or is expressed by a recombinant nucleic acid in the genetically modified cell.

6. The method according to any one of the preceding claims, wherein the transglycosidase is selected from the group consisting of α-1,2-transfucosidase, α-1,3-transfucosidase, α-1,3 / 4-transfucosidase, α-2,3-transsialidase, α-2,6-transsialidase, β-N-acetylglucosaminidase, trans-lacto-N-biosidase and trans-β-galactosidase.

7. The method according to any one of the preceding claims, wherein the donor oligosaccharide is selected from fucosylated oligosaccharides of three to five monosaccharide units, sialylated oligosaccharides of three to five monosaccharide units and neutral core oligosaccharides of three to four monosaccharide units.

8. The method according to any one of the preceding claims, wherein the culturing in step a) is initiated in the presence of sufficient initial substrate for the production of the first oligosaccharide.

9. The method of any one of claims 5 to 8, wherein the transglycosidase is added to the culture at a time point when the genetically modified cell has converted at least 50% of the initial substrate used to produce the first oligosaccharide into the first oligosaccharide.

10. The method according to any one of claims 8 or 9, wherein the initial substrate for producing the first oligosaccharide is lactose, N-acetyllactosamine, lacto-N-biose or 2'FL.

11. The method according to any one of claims 8 to 10, wherein the second oligosaccharide or disaccharide is added to the culture medium, and the molar amount of the second oligosaccharide and the initial substrate added for producing the first oligosaccharide is at least 2:1, such as at least 3:

1.

12. The method according to any one of the preceding claims, wherein the byproduct import protein is a lactose import protein, such as LacY comprising or consisting of SEQ ID NO: 3, or a functional variant thereof.

13. The method according to any one of the preceding claims, wherein the third oligosaccharide is a complex oligosaccharide of at least four monosaccharide units selected from DFL, FSL, Lewis B, Lewis Y, sialyl-Lewis A, sialyl-Lewis X, LNFP-I, LNFP-II, LNFP-III, LNFP-IV, LNFP-V, LNFP-VI, LST-a, LST-b, LST-c, DSLNT, LNDFH-I, LNDFH-II, LNDFH-III, FLST-a, FLST-b, FLST-c, pLNH, pLNnH, LNH, LNnH, FLNH-I, FLNH-II, FLNH-III, FpLNH-I, FpLNnH II, DF-LNF-I, DF-LNF-II, DF-LNF-III, DF-p-LNH, DF-p-LNnH, FLNnHa, FLNnHb, DFLNnH, TF-LNH, SLNH, FSLNH, SLNnH-I, FSLNnH-I, SLNnH-II and DS-FLNH-II.

14. The method of any one of the preceding claims, wherein the genetically modified cell produces a donor oligosaccharide selected from 2'FL, 3FL, DFL, LNFP-I, 3'SL, 6'SL, 3'SLacNAc, 3'SLNB, FSL, LST-a, LNT-II, LNT and LNnT.

15. The method of any one of claims 1 to 13, wherein the genetically modified cell produces an acceptor oligosaccharide selected from the group consisting of 2'FL, 3FL, 2'FLacNAc, 2'FLNB, Lewis A, Lewis X, LNT-II, LNT, LNnT, p-LNnH, LNFP-I, LNFP-II, LNFP-III, LNFP-IV, LNFP-V, LNFP-VI, 3'SL, 6'SL, LST-a, and LST-c.

16. The method of any one of the preceding claims, wherein the first, second and third oligosaccharides are HMOs.

17. The method according to any one of the preceding claims, wherein the at least one recombinant glycosyltransferase is selected from the group consisting of α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3 / 4-fucosyltransferase, α-2,3-sialyltransferase, α-2,6-sialyltransferase, β-1,3-N-acetylglucosaminyltransferase, β-1,6-N-acetylglucosaminyltransferase, β-1,3-galactosyltransferase and β-1,4-galactosyltransferase.

18. The method of any one of the preceding claims, wherein the pathway that produces nucleotide activating sugars is the de novo GDP-fucose pathway (gmd, wcaG, manB, manC, and manA) and / or the sialic acid sugar nucleotide pathway (neuB, neuC, and neuA).

19. The method of any one of the preceding claims, wherein the second disaccharide or oligosaccharide is selected from LacNAc, LNB, 2'FL, 3FL, 2'FLacNAc, 2'FLNB, Lewis A, Lewis X, 3'SL, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LSTa, LSTc, LNH, F-LNH-II, F-LNH-III, DF-LNH-I, DF-LNH-II, DF-LNH-III, S-LNH, DS-LNH, FS-LNH, LNnH, p-LNH and p-LNnH.

20. An HMO composition consisting essentially of: a) at least 50 wt% FSL, less than 45 wt% 3FL, less than 2 wt% 3'SL and less than 3 wt% lactose, or b) at least 55 wt% LST-a, less than 40 wt% LNT, less than 10 wt% 3'SL and less than 2 wt% lactose, or c) at least 40 wt% LST-c, less than 25 wt% LNnT, less than 25 wt% 6'SL and less than 10 wt% lactose, Each component accounts for 100 wt% in the total composition, and the composition is a mixture of at least two components.

21. The composition of claim 20, wherein the composition consists essentially of: a) at least 50 wt% FSL, 20 to 45 wt% 3FL, 0.1 to 2 wt% 3'SL and 0 to 3 wt% lactose, or b) at least 50 wt% LST-a, 15 to 40 wt% LNT, 0 to 15 wt% 3'SL and 0 to 2 wt% lactose, or c) at least 50 wt% LST-c, 15 to 25 wt% LNnT, 15 to 25 wt% 6'SL and 0 to 7 wt% lactose, and Each component accounts for 100 wt% in the total composition, and the composition is a mixture of at least two components.

22. Use of a composition according to claims 20 to 21 for the production of a nutritional composition.

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