BIOTECHNOLOGICAL PRODUCTION OF LNT, LNNT AND THEIR FUCOSYLATED DERIVATIVES

Description

[0001] The present invention relates to genetically modified microorganisms for the in vivo synthesis of lacto-N-tetraose (LNT) or lacto-N-neotetraose (LNnT) and their fucosylated derivatives in a microorganism, as well as uses of such microorganisms in processes for the production of lacto-N-tetraose or lacto-N-neotetraose and their fucosylated derivatives.

[0002] Human breast milk is considered to play an important role in healthy child development. The oligosaccharides found therein (human milk oligosaccharides (HMO)) are one of the main solid components of breast milk and have a core structure that features a lactose unit at the reducing end and is continued in a branched or chain-like manner by N-acetyllactosamine units. Structural variability is further enhanced by fucosyl or sialyl modifications at the terminal positions.

[0003] Bei Lacto-N-tetraose (LNT) handelt es sich um ein Tetrasaccharid der chemischen Formel N-[(2S,3R,4R,5S,6R)-2-{[(2R,3S,4S,5R,6S)-3,5-Dihydroxy-2-(hydroxymethyl)-6-{[(2R,3S,4R,5R)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy}oxan-4-yl]oxy}-5-hydroxy-6-(hydroxymethyl)-4-{[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxan-3-yl]acetamid mit folgender Struktur:

[0004] Lacto-N-neotetraose (LNnT) hat die chemische Formel N-[(2S,3R,4R,5S,6R)-2-{[(2R,3S,4S,5R,6S)-3,5-Dihydroxy-2-(hydroxymethyl)-6-{[(2R,3R,4R,5R)-1,2,4,5-tetrahydroxy-6-oxohexan-3-yl]oxy}oxan-4-yl]oxy}-4-hydroxy-6-(hydroxymethyl)-5-{[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxan-3-yl]acetamid bzw. folgende Struktur:

[0005] With regard to their health- and development-promoting effects, the biological function of HMOs is the subject of numerous studies, but requires the technically pure extraction of the compounds in sufficient quantities. The rather laborious extraction from breast milk is currently the most widely used method. Although biotechnological production processes have been described for HMOs (see Han et al., Biotechnol. Adv. 2012, 30, 1268-1278), lacto-N-tetraose, for example, one of the most common HMOs, is currently not available for research at a reasonable price. Both chemical and enzymatic syntheses for LNT are known from the literature (see Aly et al., Carbohydr. Res. 1999, 316, 121-132; Murata et al., Glycoconj. J. 1999, 16, 189-195).However, since chemical synthesis requires multiple steps of protection and deprotection of reactive groups and enzymatic synthesis is affected by unfavorable equilibrium product distributions and regioselectivities, these methods do not provide satisfactory results.

[0006] The primary object of the present invention was therefore to provide a system, preferably microorganisms, which is / are capable of producing LNT or LNnT and their fucosylated derivatives in high yield.

[0007] A further object of the present invention was to provide a corresponding process that enables the efficient and inexpensive biotechnological production of LNT and LNnT or their fucosylated derivatives.

[0008] The primary object is achieved according to the invention by a genetically modified microorganism for the in vivo synthesis of lacto-N-tetraose or lacto-N-neotetraose in a microorganism, wherein the microorganism (i) a first transgene encoding β 1,3-N-acetylglucosaminyltransferase, and (ii) a second transgene encoding β 1,3-galactosyltransferase (in the case of the synthesis of lacto-N-tetraose) or β 1,4-galactosyltransferase (in the case of the synthesis of lacto-N-neotetraose), and wherein the microorganism is further genetically modified so that the expression of LacZ and LacA is suppressed, wherein (i) the first Transgene is integrated into the LacZYA locus and the microorganism contains another transgene encoding LacY.

[0009] In this context, a genetically modified microorganism is understood to be a microorganism in which individual genes have been specifically deactivated using biotechnological methods and / or genes of a species or foreign to the species have been inserted (transgenes). A transgene within the meaning of the present invention can be an introduced gene from another organism or a gene naturally occurring in the microorganism in question that has been genetically integrated at a different location in the genome and is therefore expressed, for example, under a promoter other than the natural one.

[0010] In the context of the present invention, it was surprisingly found that standard genetically engineered microorganisms which produce β 1,3- N-Acetylglucosaminyltransferase and a β 1,3-galactosyltransferase or β 1,4-galactosyltransferase can be successfully used for the synthesis of LNT and LNnT, respectively. For example, Leloir glycosyltransferases (LgtA and LgtB) can be used, which initially convert lactose as a substrate for glycosylation to lacto-N-triose II (LNT II) as an intermediate and then elongate it to LNT in a nucleotide-activated sugar-dependent step (see Fig. 1 and Frey et al. FASEB J. 1996, 10, 461-70). Suitable transgenes within the scope of the invention include, for example, the gene coding for β 1,3- N -Acetylglucosaminyltransferase encoding IgtA gene from Neisseria meningitides and the wbgO gene encoding β 1,3-galactosyltransferase from E. coli The donor substrates of the recombinant glycosyltransferases LgtA (UDP-N-acetylglucosamine) and WbgO (UDP-galactose) are intermediates of the E. coliK12 metabolism and are continuously synthesized during growth (see Raetz et al., Annu. Rev. Biochem., 2002, 71, 635-700). UDP-N-acetylglucosamine is a precursor of peptidoglycan, lipopolysaccharide, and enterobacterial common antigen biosynthesis (see Neidhardt et al., Cellular and Molecular Biology, second edition 1996). It is produced from fructose-6-phosphate by the biosynthetic enzymes GlmS, GlmM, and GlmU (see Barreteau et al., FEMS Microbiol. Rev., 2008, 32, 168-207). UDP-galactose is a precursor substrate of lipopolysaccharide and colanic acid biosynthesis in E. coli and is formed from glucose-6-phosphate in three enzymatic steps catalyzed by Pgm, GalU, and GalE (see Frey, FASEB J., 1996, 10, 461-70). For cell growth and the intracellular delivery of the nucleotide-activated sugars, inexpensive substrates such as glycerol or glucose can be advantageously used.

[0011] According to the present invention, the microorganism is genetically modified such that the expression of LacZ and LacA is suppressed. (i) The first transgene is integrated into the LacZYA locus, and the microorganism contains another transgene encoding LacY.

[0012] To prevent the metabolism and possible acetylation of lactose by LacZ and LacA, the expression of these genes is suppressed in a microorganism according to the invention. According to the invention, this is achieved by integrating the first transgene (i) into the LacZYA locus. However, to ensure continued lactose uptake by the microorganism, LacY is transgenically expressed elsewhere in the genome, for example, under a different promoter, preferably a P tac promoter. LacY is particularly preferably integrated into the fuclK locus, which encodes genes involved in fucose metabolism.

[0013] To ensure the highest possible yield of LNT or LNnT, it is advantageous to provide plenty of nucleotide-activated sugars, especially UDP-galactose, intracellularly so that the conversion of LNT II to LNT can proceed efficiently.

[0014] Accordingly, in a further preferred embodiment of the present invention, the microorganism contains a further transgene encoding a UDP-sugar pyrophosphorylase (USP).

[0015] Such a USP is, for example, represented by the open reading frame LmjF17.1160 in Leishmania major encoded (see Damerow et al., J. Biol. Chem. 2010, 285, 878-887). The USP catalyzes the production of UDP-galactose using galactose-1-phosphate. Advantageously, this reaction can also prevent the potentially cytotoxic accumulation of galactose-1-phosphate.

[0016] In a further preferred embodiment of the present invention, the microorganism is further genetically modified such that the expression of UDP-glucose-4-epimerase is suppressed.

[0017] Such suppression can be achieved, for example, by deleting the galE gene, which is preferably replaced by a T5 promoter, so that the following genes of the operon continue to be expressed. Advantageously, this also increases the intracellular UDP-galactose concentration. A combination of this embodiment with the microorganism containing a transgene encoding a UDP-sugar pyrophosphorylase (USP) (as described above) is particularly preferred.

[0018] According to the invention, furthermore, a microorganism according to one of the embodiments described above is particularly preferred, in which one or both or one, several or all transgenes are chromosomally integrated.

[0019] The use of a plasmid-free strain is particularly advantageous because no selective pressure (antibiotic resistance) is necessary to maintain productivity. Furthermore, the use of antibiotics in food-related or pharmaceutical production is undesirable.

[0020] According to a further preferred embodiment of the microorganism according to the invention, it contains a further transgene which codes for a bifunctional enzyme with L-fucose kinase activity and L-fucose-1-phosphate guanylyltransferase activity and at least one transgene which codes for an enzyme which is capable of α (alpha) 1,2-fucosylation, α (alpha) 1,3-fucosylation or α (alpha) 1,4-fucosylation.

[0021] Such a microorganism is capable of producing the fucosylated derivatives of these two compounds as a subsequent reaction of the synthesis of LNT or LNnT and thus expanding the application possibilities of the microorganism according to the invention with regard to the structural variability of the naturally occurring HMOs (see Fig. 2 For example, FKP can be used as a bifunctional enzyme with L-fucose kinase activity and L-fucose-1-phosphate guanylyltransferase activity. For fucosylation, for example, the expression of enzymes encoded by the genes futC (α 1,2-fucosylation), fucT14 (α 1,4-fucosylation), or futA (α 1,3-fucosylation) is suitable.

[0022] In a preferred embodiment of the microorganism according to the invention (as described above), the transgene encoding the bifunctional enzyme with L-fucose kinase activity and L-fucose-1-phosphate guanylyltransferase activity is chromosomally integrated and the at least one transgene encoding an enzyme capable of α 1,2-fucosylation, α 1,3-fucosylation or α 1,4-fucosylation is expressed on a plasmid vector.

[0023] In a particularly preferred embodiment of the microorganism according to the invention (as described above), both the transgene encoding the bifunctional enzyme with L-fucose kinase activity and L-fucose-1-phosphate guanylyltransferase activity and the at least one transgene encoding an enzyme capable of α 1,2-fucosylation, α 1,3-fucosylation or α 1,4-fucosylation are chromosomally integrated.

[0024] A further aspect of the present invention relates to the use of a genetically modified microorganism as described herein, preferably as described herein as preferred according to one of the embodiments described above, for the in vivo synthesis of lacto-N-tetraose or lacto-N-neotetraose or a fucosylated derivative of lacto-N-tetraose or lacto-N-neotetraose in a microorganism.

[0025] The use of such a genetically modified microorganism enables the efficient and inexpensive production of lacto-N-tetraose or lacto-N-neotetraose or a fucosylated derivative of lacto-N-tetraose or lacto-N-neotetraose on a scale that can be adapted to the intended use.

[0026] According to a further aspect, the present invention relates to a process for the preparation of lacto-N-tetraose or lacto-N-neotetraose or a fucosylated derivative of lacto-N-tetraose or lacto-N-neotetraose, comprising the following steps: (a) providing a genetically modified microorganism as described above, preferably as described above as preferred, (b) cultivating the genetically modified microorganism under conditions which allow the synthesis of lacto-N-tetraose or lacto-N-neotetraose, (c) optionally adding fucose, (d) optionally isolating the synthesized lacto-N-tetraose or lacto-N-neotetraose or the fucosylated derivative of lacto-N-tetraose or lacto-N-neotetraose.

[0027] In the process according to the invention, a genetically modified microorganism is first provided as described above and cultivated, for example, in a shake flask under conditions that allow the synthesis of lacto-N-tetraose or lacto-N-neotetraose. Cell growth depends primarily on the microorganism used. The microorganism used is preferably a standard microorganism for biotechnological applications that is optimized for maximum productivity. Advantageously, in addition to the necessary lactose, inexpensive (other) carbon sources can be used, for example selected from the group consisting of glucose, glycerol, galactose and any mixtures thereof. In order to achieve the synthesis of LNT orTo enable LNnT, lactose must be present as a substrate and the expression of the transgenes (i) and (ii) (as described above) must be induced, if necessary depending on the promoter under which they are expressed. To ensure fucosylation of the products, fucose must also be added. Fucose is preferably added only after the genes for LNT or LNnT synthesis have been induce, ideally in such a way that sufficient corresponding substrate is available and not only lactose is fucosylated. Alternatively, fucose can already be present at the beginning of step (b) and expression can be placed under a different promoter than the one regulating the expression of the genes for LNT or LNnT synthesis. The induction of the fucosyltransferase genes then takes place at the desired time by adding the appropriate induction agent. In a preferred embodiment, the genes for LNT or LNnT are expressed in this case.LNnT synthesis under an IPTG-inducible promoter and the expression of the fucosyltransferase genes under a rhamnose-inducible promoter.

[0028] The resulting products are then optionally isolated. For this purpose, the cells are collected by centrifugation, for example, resuspended in water, and lysed. The resulting sugars can then be purified from the supernatant using standard methods.

[0029] In a preferred embodiment of the process according to the invention, in step (b) Galactose is used as a carbon source for the microorganism or glycerol and galactose are used as a carbon source for the microorganism.

[0030] Within the scope of the present invention, it was found that the yield of LNT in relation to the intermediate LNT II can be controlled via the carbon sources provided (see Fig. 3). Particularly high yields are therefore achieved, for example, when galactose is present as the primary carbon source. The (wt.) proportion of galactose, based on the total weight of the lactose required for the synthesis and any other carbon sources present, such as glycerol or glucose, is preferably at least 50%, preferably 70%, particularly preferably at least 90%. Particularly high yields are also achieved when glycerol is used as the primary carbon source and galactose is added at the start of the induction of the genes for LNT or LNnT synthesis. Again, the (wt.) proportion of glycerol, based on the total weight of the lactose required for the synthesis and any other carbon sources present, such as glucose, is at least 50%, preferably at least 70%, particularly preferably at least 90%.

[0031] Furthermore, a process according to the invention (as described above) is preferred in which in step (b) one or more carbon sources, preferably selected from the group consisting of lactose, glucose, glycerol, galactose and any mixtures thereof, preferably at least lactose, particularly preferably lactose and galactose or lactose, galactose and glycerol, are added continuously or stepwise.

[0032] Advantageously, by continuously or gradually adding the respective carbon source(s) when they have been completely or partially consumed, cytotoxic accumulations or undesirable inhibitions can be avoided.

[0033] Preferably, the genetically modified microorganism (as described above) or the microorganism to be used according to a use described herein or the microorganism to be used according to the invention in a method described herein is selected from the group consisting of bacteria, fungi and plants, preferably microorganisms of the genus Corynebacterium, in particular Corynebacterium glutamicum, Bevibacterium, in particular Bevibacterium flavum, Bacillus, Saccharomyces and Escherichia, in particular E. coli.

[0034] The use of standard genetically engineered microorganisms is particularly advantageous for carrying out the present invention, since they have been optimized for high productivity and genetic engineering methods for introducing transgenes and inducing them are known.

[0035] Within the scope of the present invention, it was demonstrated that the process according to the invention can be efficiently carried out in the form of a feed-batch process even on a liter scale (see Example 3). Therefore, a process as described above is preferred, wherein the process is carried out as a feed-batch process with a batch volume in the range of 2 to 30 L, preferably 3 to 20 L, particularly preferably 5 to 15 L.

[0036] In the following, the invention is explained in more detail using figures and examples. Short description of the characters:

[0037] Figure 1 : Scheme of the intracellular turnover of lactose to lacto-N-tetraose. Figure 2 . Scheme of the intracellular synthesis of fucosylated HMOs with LNT as the core structure. FucT stands for a fucosyltransferase and LNFX for the resulting products. Figure 3 :Proportion of the respective oligosaccharide in shake flask cultures in the culture supernatant relative to the total amount of oligosaccharide 24 hours after induction as a function of the carbon source. Induction with 0.5 mM IPTG and addition of 2 g of lactose and 2 g of the second-listed carbon source, and incubation at 30°C and 90 rpm. Figure 4 : LNT concentrations in shake flask cultures 24 hours after induction as a function of carbon sources. Induction with 0.5 mM IPTG and addition of 2 g of lactose and 2 g of the second carbon source listed, followed by incubation at 30°C and 90 rpm. Figure 5 : LNT II concentrations in shake flask cultures 24 hours after induction as a function of carbon sources. Induction with 0.5 mM IPTG and addition of 2 g of lactose and 2 g of the second carbon source listed, followed by incubation at 30°C and 90 rpm. Figure 6: Structure of LNF I (LNT with an α 1,2-linked fucosyl residue at the galactosyl residue at the non-reducing end). Figure 7 : Structure of LND II (LNT with an α 1,4-linked fucosyl residue at the N-acetylglucosaminyl residue and an α 1,3-linked fucosyl residue at the glycosyl residue at the reducing end). Figure 8 : Comparison of lactose consumption and product formation in shake flask experiments on different carbon sources 24 hours after induction. a) Concentration of lactose (white), LNT II (grey) and LNT (black). b) Product yields per biomass. c) Proportion of products in the culture supernatant in %. Figure 9 : Intracellular concentration of UDP-sugars during exponential growth on different carbon sources: UDP-glucose (gray), UDP-galactose (black), and UDP-acetylglucosamine (white). Values are given as means and SE for ≥ 2 independent experiments. Figure 10: LNT feed batch production. Vertical dashed lines (12.6 hours) indicate the addition of IPTG for the induction of protein expression and the first addition of lactose. Vertical dotted lines (20.5 hours) indicate the end of the batch phase and the start of galactose and nitrogen addition. a) Profile of the total carbon source added to the system: galactose (solid line), nitrogen source: ammonium phosphate (dotted line) and lactose (dashed line); b) Cell dry weight concentration (CDW); c) Concentration of LNT II (open triangles) and LNT (filled circles). Figure 11 : Structure of fucosylated lacto-N-triose II. Figure 12 : Structure of difucosylated lacto-N-pentaose. Example 1: Production of a genetically modified microorganism according to the invention

[0038] The E. coliK-12 strain LJ110 was used as the starting strain for production. This plasmid-free strain was modified by knocking out gene loci for sugar degradation in the corresponding expression cassettes using homologous recombination. The β-galactosidase-encoding gene lacZ was removed, and the strain was grafted with the gene encoding β-1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis The gene encoding IgtA was added to allow the synthesis of LNT II. Finally, the strain was equipped with the wbgO gene, which encodes the β-1,3-galactosyltransferase WbgO. The genes were integrated chromosomally.

[0039] For this purpose, the IgtA gene was first cloned into an expression vector with an IPTG-inducible P tac promoter, which was then equipped with an FRT-flanked chloramphenicol resistance gene downstream of the IgtA gene. The expression cassette, including the P tac promoter, a ribosome binding site (Shine-Dalgarno sequence), IgtA, FRT-cat-FRT resistance marker, and a transcription terminator sequence from rrnB, was amplified by PCR. The cassette was then chromosomally integrated into the LacZYA locus.

[0040] The strain was further modified with an E. coli K12 lacY gene under the control of a P tac promoter to ensure lactose uptake. LacY was cloned into an expression vector, and then a resistance-tagged expression cassette was generated by downstream cloning of an FRT-kan-FRT resistance cassette. After amplification, this was chromosomally integrated into the fucIK locus.

[0041] For the intracellular conversion of LNT II to LNT, the wbgO gene from the E. coli strain O55:H7, which encodes a β 1,3-galactosyltransferase, was chromosomally integrated into the xylAB locus as described for IgtA. Example 2: Investigation of the formation of LNT or LNT II using different carbon sources

[0042] Despite catabolite repression of glucose by galactose as described in the literature (see McGinnis et al. J. Bacteriol. 1969, 100, 902-913), galactose was used in this experiment both in a mixture with glucose or glycerol, and as the sole usable carbon source in minimal medium to analyze product formation. The strain produced in Example 1 was used for the experiments. The culture size was 50 ml in each case. The main carbon sources, glucose, glycerol, and galactose, were each used at a final concentration of 10 g I -1<, while lactose was used at a final concentration of 2 g I -1< and the admixed galactose was also used at 2 g I -1< and was added at the time of induction at OD 600 = 0.4-0.6 (with 0.5 mM IPTG, final conc.).

[0043] LNT formation was determined 24 hours after induction in both the culture supernatants and the culture pellets using HPLC fluorescence photometry after derivatization with anthranilic acid (see Ruhaak et al., Proteomics 2010, 10, 2330-2336). For example, an improvement in LNT yield was observed when switching from glycerol to glucose. As expected, the addition of galactose to the culture with glucose showed no effect on growth or on product formation due to catabolite repression. When galactose was used exclusively as a carbon source alongside lactose, or when galactose was added to the culture with glycerol during induction, the LNT concentration in the cultures 24 hours after induction was significantly increased.By adding galactose to the culture with glycerol, the LNT concentration increased by a factor of 2.7 to 434.3 mg I -1<, thus demonstrating a product formation rate approximately twice as high as the value using glucose. When 10 g I -1< galactose was used in the culture medium without glucose or glycerol, an LNT concentration of 798.1 mg I -1< was achieved. Thus, the highest previous value achieved with glucose was increased by a factor of 3.6 (see ). Fig. 4 ).

[0044] When considering the production of the trisaccharide LNT II, it can be seen that glycerol as the carbon source leads to comparatively the highest synthesis of LNT II, while glucose and galactose lead to approximately 16.4% less LNT II synthesis. When glucose and galactose are used together, the LNT II concentration 24 h after induction is significantly lower at only 769 mg I -1< (see Fig. 5). This can possibly be explained by the lower cell density of the culture. However, it is also conceivable that there is an interaction between inducer exclusion through glucose uptake (see Nelson et al., EMBO J. 1983, 2, 715-720) and the inhibition of Lac permease by the present galactose (see Olsen et al., J. Biol. Chem. 1989, 264, 15982-15987), so that less lactose is taken up than in systems with only one type of Lac permease inhibition. This inhibition is also supported by the fact that, compared to cultures with other carbon sources, a significantly higher ratio of LNT to LNT II and lactose predominates in the pellet.

[0045] If one considers the LNT content in the culture supernatant (see Fig. 3), it becomes clear that the strain grown on galactose not only synthesizes significantly more LNT, but also that the proportion of LNT in the culture supernatant, at approximately 93.3%, is significantly higher than the proportion in the culture supernatant of the glucose cultures (approximately 54.6%). Since the isolation of LNT from the culture supernatant is advantageous compared to isolation from the culture pellet, and more product is formed with galactose as the carbon source, the synthesis of LNT with galactose is presumably particularly advantageous and therefore preferred despite the higher substrate price.

[0046] The formation of the tetrasaccharide LNT requires the glycosyl transfer of N-acetylglucosaminyl and galactosyl units to the acceptor substrate lactose. The respective donor substrates UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-galactose (UDP-Gal), which are required for the cytosolic glycosyltransferase reactions, are - as already mentioned - formed by the E. coli-metabolism. However, the incomplete conversion of lactose to LNT indicates a limitation in the supply of donor substrates, especially UDP-galactose.

[0047] To improve the intracellular availability of UDP-galactose and thus achieve an increased LNT yield, minimal media with the various carbon sources were further investigated in detail with regard to the conversion of lactose and product formation as well as the release of the products into the medium. For this purpose, the strains prepared according to Example 1 were again used and cultivated in minimal media with either 1% glucose, 1% glycerol or 1% galactose, or 1% glucose or 1% glycerol supplemented with 0.2% galactose. The expression of the recombinant genes and the synthesis of LNT were initiated in each culture by adding IPTG (final concentration 2 g L -1 ) to the cells in the early exponential growth phase. 24 hours after induction, the concentrations of lactose, LNT II and LNT were determined in each culture.

[0048] Strains were cultured in LB medium containing 50 µg mL -1 < chloramphenicol (to avoid contamination) at 37 °C. A standard for lacto-N-tetraose was purchased from IsoSep (Tullinge, Sweden) with a purity of more than 95%. Standards of UDP-glucose disodium salt hydrate (≥ 98%) and UDP-N-acetylglucosamine sodium salt (≥ 98%) were purchased from Sigma Aldrich (Taufkirchen, Germany), and UDP-galactose disodium salt (≥ 95%) was purchased from Calbiochem (Merck, Darmstadt, Germany). Lactose monohydrate (Ph. Eur. grade), glucose monohydrate (≥ 99.5%), glycerol (≥ 98%), and galactose (≥ 98%) were purchased from Carl Roth (Karlsruhe, Germany). All other chemicals and reagents were purchased from either Carl Roth (Karlsruhe, Germany) or Sigma Aldrich (Taufkirchen, Germany) with the highest available purity.

[0049] The synthesis of LNT II and LNT was carried out at 30 °C and 90 rpm in two batches each in 500 mL shake flasks with 50 mL minimal medium containing 1% of the main carbon source (glycerol, glucose or galactose) and chloramphenicol (50 µg ml -1< to avoid contamination). The composition of the medium was: 2.68 g L -1< (NH 4 ) 2 SO 4 , 1 g L -1< (NH 4 ) 2 -H-citrate, 10 g L -1< main carbon source (glycerol, glucose or galactose), 14.6 g L -1< K 2 HPO 4 , 0.241 g L -1< MgSO 4 , 10 mg L -1< MnSO 4 • H 2 O, 2 g L -1< Na 2 SO 4 , 4 g L -1< NaH 2 PO 4 • H 2 O, 0.5 g L -1< NH 4 Cl, 10 mg L -1< thiamine hydrochloride, and trace element solution (3 mL L -1< : 0.5 g L -1< CaCl 2 • 2H 2 O, 16.7 g L -1< FeCl 3 • 6H 2 O, 20.1 g L -1< Na 2 -EDTA, 0.18 g L -1< ZnSO 4 • 7H 2 O, 0.1 g L -1< MnSO 4 • H 2 O, 0.16 g L -1< Cu-SO 4 • 5H 2 O, and 0.18 g L -1< CoCl 2 • 6H2O).The cultures were inoculated with a single colony grown on minimal medium agar plates containing 1% of the appropriate carbon source. After reaching an optical density at 600 nm (OD 600 ) of 0.4–0.6, the cultures were induced with 0.5 mM IPTG (final concentration), and 2 g L lactose was added at the time of induction. To determine the levels of galactose, lactose, LNT II, and LNT, 2 mL samples were centrifuged (15300 g, 2 min) 24 hours after induction. After centrifugation, the supernatants were stored at -20°C until derivatization; the pellets were washed with 1 mL ice-cold saline, centrifuged as before, and also stored at -20°C.

[0050] Cell dry weights (CDW) of cultures with a primary carbon source (glycerol, glucose, or galactose) were analyzed 24 hours after induction by centrifugation (5869 g, 4 °C, 20 min) of 10 mL of culture and drying of the cell pellet at 120 °C to constant weight (minimum of the two runs). CDW [g L -1< ] to OD 600 [-] correlations were determined in shake flasks (0.3 for glycerol as the primary carbon source, 0.37 for glucose as the primary carbon source, and 0.39 for galactose as the primary carbon source).

[0051] As in the Figures 8a and 8bAs shown, the conversion of lactose is significantly influenced by the provided carbon source, and the results suggest that the carbon sources used influence both the shift towards more UDP-activated sugars and lactose uptake. While cultures grown on glycerol, as described above, resulted in the lowest yield of LNT (0.152 ± 0.002 g L -1< ), the use of a glycerol plus galactose mixture increased the yield of LNT by a factor of almost 3. In contrast, the comparison of cultures grown on glucose or on a glucose / galactose mixture showed approximately equal yields of LNT, but in the case of the mixture, the conversion of lactose to LNT II was significantly lower. The highest conversion of lactose, as well as the highest yield of LNT (0.810 ± 0.013 g L -1< ) was observed in cultures grown on galactose alone.

[0052] In addition to the influence on the conversion of lactose to LNT II and LNT, the carbon sources used also showed an effect on the release of the product LNT (see Fig. 8c While cultures with glucose or a glucose / galactose mixture resulted in a release of about 50% of the produced LNT, in cultures with galactose or glycerol / galactose more than 90% of the formed LNT was found in the culture medium.

[0053] The shake flask experiments showed that the carbon source appears to influence LNT formation. To determine whether the carbon sources used can control the intercellular availability of the donor substrates and thus product formation, the concentrations of UDP-N-acetylglucosamine, UDP-glucose, and UDP-galactose were quantified. For this purpose, the strain prepared according to Example 1 was cultured in minimal medium containing either glycerol, glucose, or galactose, the cells were harvested in the late exponential growth phase, and the intercellular metabolites were analyzed by HPLC.

[0054] The strain was cultured at 30 °C and 90 rpm in 1 L shake flasks filled with 100 mL of minimal medium containing glycerol, glucose, or galactose, as described above. At an OD 600 of 0.4–0.6, expression was induced with 0.5 mM IPTG, and the cultures were further incubated at 30 °C and 90 rpm. 12 hours after induction, 25 ml samples were centrifuged (2876 rpm, 4 °C, 15 min). The pellets were then resuspended in quenching buffer (acetonitrile:methanol:H2O 4:4:2 with 0.1 M formic acid (Bennett et al. Nat. Chem. Biol., 2009, 5, 593-599)) and mixed vigorously on a vortex every 3 minutes during incubation on ice for 10 min at 4 °C. The suspension was then neutralized with 1 M NH4OH. The samples were subsequently centrifuged again (22410 g, 4 °C, 10 min). The supernatants were dried in a Speedvac CON-1000 (Fröbel, Lindau, Germany) and dissolved in H2O at 5% of the extraction volume prior to HPLC analysis.The UDP-sugars were analyzed using a Dionex HPLC instrument (Thermo Fisher Scientific, Dreieich, Germany) equipped with Chromeleon software, a Gina autosampler, P580 pumps, a UVD diode array detector, and a Luna C18(2) reverse phase column (250 mm x 4.5 mm, 5 µm, Phenomenex, Aschaffenburg, Germany). The following gradient, modified from Payne and Ames (Anal. Biochem., 1982, 123, 151-161) was applied at a flow rate of 1 mL min -1<: 0 to 30 min linear gradient from 100% solvent A / 0% solvent B to 80% solvent A / 20% solvent B, 30 to 30.5 min linear gradient from 80% solvent A / 20% solvent B to 100% solvent A / 0% solvent B, 30.5 to 35 min isocratic conditions with 100% solvent A to equilibrate the column for the next sample.Identification and quantification were analyzed at 262 nm by comparing retention times, spectra, and signal areas with commercial standards at seven different concentrations.

[0055] The result showed that the concentration of UDP-hexoses does indeed depend significantly on the carbon source used. Growth on galactose alone resulted in the highest intracellular amount of UDP-galactose (145.63 ± 20.52 nmol L -1< OD -1< ), approximately 3 times higher than that observed for growth on glucose (65.73 ± 5.63 nmol L -1< OD -1< ) or glycerol (45.87 ± 17.42 nmol L -1< OD -1< ). The highest amount of UDP-N-acetylglucosamine was observed during growth on glucose (334.03 ± 3.41 nmol L -1< OD -1< ) (see Fig. 9 ). Example 3: Demonstration of the scalability of LNT synthesis on Galacto se

[0056] The use of galactose as a carbon source for the whole-cell synthesis of LNT has, compared to the commonly used E. coliCarbon sources such as glucose or glycerol have an advantage due to the higher intracellular UDP-galactose concentration. To demonstrate the scalability of the synthesis of LNT on galactose, a feed-in cultivation (feed-batch process) was carried out in a high cell density bioreactor on a 10-liter scale. The process was started with an 8.45-liter batch, which reached a biomass concentration of approximately 13 g L -1 < CDW after consumption of the initially present galactose. During the following feed phase, the galactose supply was adjusted to maintain a constant growth rate (µ = 0.054), which ultimately led to a biomass of 55.7 g L -1 < CDW after a period of 47 hours (see Fig. 10a,b ).

[0057] The feedstock cultivation of the strain prepared in Example 1 was carried out using mineral salt medium and galactose as the main carbon source in a 30 L stirred-tank reactor (Bioengineering, Wald, Switzerland) at 30 °C with an initial volume of 8.45 L and a final volume of 13.63 L. The medium was modified compared to Wilms et al. (Biotechnol. Bioeng., 2001, 73, 95-103) and had the following composition: The 8 liters of batch medium consisted of 2.68 g L -1< (NH 4 ) 2 SO 4 , 1 g L -1< (NH 4 ) 2 -H-citrate, 25 g L -1< galactose, 3.9 g L -1< (NH 4 ) 2 HPO 4 , 14.6 g L -1< K 2 HPO 4 , 0.241 g L -1< MgSO 4 , 10 mg L -1< MnSO 4 • H 2 O, 2 g L -1< Na 2 SO 4 , 4 g L -1< NaH 2 PO 4 • H 2 O, 0.5 g L -1< NH 4 Cl, 10 mg L -1< Thiamine hydrochloride and trace element solution (3 mL L -1< , composition as described above). During the batch and feed phases, the pH was adjusted to 7.0 by titration with ammonia (25%).The relative dissolved oxygen (pO 2 ) was maintained above 40% by aeration and agitation at a reactor pressure of 500 hPa above atmospheric pressure. The batch medium was inoculated with 0.45 L of an overnight preculture to a cell dry weight concentration of 0.096 g L -1 < and cultured at 30 °C and 90 rpm in the mineral salt medium with 10 g L -1 < galactose, as described for the shake flasks above. 12.6 hours after inoculation, at a cell dry weight concentration of approximately 2.4 g L -1 < , the expression of the recombinant genes was induced by the addition of IPTG (0.5 mM final concentration). At the same time, lactose (16.9 g) was added to enable product formation. After the initially supplied galactose had been consumed (indicated by an increase in pO 2 ), the feed phase was started with 3 additions: Addition 1 consisted of 514.76 g L -1< galactose, 15.21 g L -1< MgSO 4 • 7H 2 O, 0.65 g L -1< thiamine hydrochloride and 100.89 ml L -1< trace element solution (composition as described above), while Addition 2 consisted of 335.59 g L -1< (NH 4 ) 2 HPO 4 and Addition 3 consisted of 150 g L -1< lactose for product formation. Additions 1 and 2 were added in a ratio of 81:19 with a galactose-limited growth rate according to formula (1). F t = μ set Y x s + m × c xo × V o c so × e μ set × t with F [L h -1< ] as the addition rate, t [h] the time of the feed phase, µ set [h -1< ] the desired growth rate (set to 0.1 in this formula), Y x / s [gg -1< ] the specific yield coefficient of biomass from the substrate (assumed to be 0.36 from previous shake flask experiments), m [gg -1< h -1< ] the specific constancy coefficient (assumed to be 0.04), c x0 [g L -1< ] the biomass concentration at the beginning of the feed phase (in this process 12.0), V 0 [L] the culture volume at the beginning of the feed phase (set to 8.25) and c 00 [g L -1< ] the galactose concentration of addition 1 (set to 514.76) (Wenzel et al., Appl. Environ. Microbiol., 2011, 77, 6419-6425). Lactose addition was manually adjusted based on consumption; a total of 200.4 g of lactose was added to the system. Cell growth was determined by measuring the OD 600 and calculating the CDW concentration using a correlation factor of 0.47 g I -1< (determined during fermentation) to a culture density of 40 OD units. CDW concentrations were then determined directly in duplicate by centrifuging 10 mL of culture and subsequently drying the cell pellets to constant weight in glass tubes.

[0058] The added lactose was completely consumed and converted to LNT II and LNT during cultivation. Both LNT II and LNT concentrations increased during the process, reaching final yields of 12.72 ± 0.21 g L -1 (LNT) and 13.70 ± 0.10 g L -1 (LNT II), respectively. The highest LNT II concentration was reached after 44 hours (15.78 ± 0.29 g L -1 ) and subsequently decreased due to lactose consumption and dilution by the galactose addition (see Fig. Fig. 10a,cTo ensure complete consumption of lactose at the end of the fermentation process, the lactose feed was stopped 44 hours after inoculation. The yield per time of LNT formation was 0.37 g L -1 h -1 , and the final amount of LNT produced in the feed-batch process was 173.37 ± 2.86 g, with the vast majority of the products (88.91 ± 0.06% of LNT II and 64.86 ± 0.12% of LNT) found in the culture supernatant. Example 4: Synthesis of fucosylated oligosaccharides with LNT or LNnT core structure

[0059] For the synthesis of fucosylated oligosaccharides containing LNT or LNnT as the core structure, the strain prepared in Example 1 was equipped with the corresponding fucosyltransferases for GDP-L-fucose synthesis using a recombinant route. While in the synthesis of the trisaccharide 2'-fucosyllactose, the de novoWhile the GDP-L-fucose synthesis pathway is preferred due to the costly addition of fucose in the salvage synthesis pathway (see Baumgärtner et al., Microb. Cell Fact. 2013, 12, 40), the salvage synthesis pathway is preferred for the synthesis of larger oligosaccharides. This is because the additional costs no longer have a significant effect on the potentially desired penta- and hexasaccharides, and because the salvage synthesis pathway cannot produce GDP-L-fucose without the addition of fucose, which allows for better control of the initiation of fucosylation reactions during cultivation. This is intended to prevent fucosylation from starting shortly after induction when using the same promoters for all recombinantly introduced genes, resulting in predominantly fucosylated lactose. For this purpose, the bifunctional enzyme FKP with L-fucose kinase activity and L-fucose-1-phosphate guanylyltransferase activity was isolated from Bacteroides fragilisused (see Coyne et al., Science (80-. ). 2005, 307, 1778- 1781; WO2010070104 A1). Since the gene fkp was already confirmed as functional on an expression plasmid, it was inserted into the arabinose degradation locus araBAD of the existing tribe.

[0060] For the synthesis of fucosylated LNTs, the strain was treated with plasmids containing the genes futC (for α 1,2-fucosylation, see Albermann et al., Carbohydr. Res. 2001, 334, 97- 103) or fucT14 (for α 1,4-fucosylation, see Rabbani et al., Glycobiology, 2005, 15, 1076-83; Rabbani et al., Biometals, 2009, 22, 1011-7, not yet described for in vivo applications in E. coli ) or transformed with the corresponding empty plasmid. For the synthesis of fucosylated LNnTs, the strain used was also transformed with plasmids containing the genes futC or futA(For α 1,3-fucosylation, see Ge et al. J. Biol. Chem. 1997, 272, 21357-63). To test product formation, the strains were each cultured in shake flasks in minimal medium containing glucose (10 g I -1< ) and casamino acids (1 g I -1< final conc., Difco, for more reliable growth). Protein expression was induced at OD 600 = 0.4-0.6 with IPTG (0.5 mM final conc.), and lactose (2 g I -1< final conc.) was added simultaneously. Fucose (2 g I -1 < final conc.) was added 26 hours after induction with IPTG and after prior sampling and addition of 0.5 culture volumes of minimal medium with glucose (10 g I -1 < ) to ensure continued carbon supply and the prior synthesis of sufficient amounts of LNT or LNnT. The cultures were then further incubated at 30°C and 90 rpm, and a second sampling was performed 65 hours after the first induction.Compared to the control with empty plasmid, the strains each showed products after 65 hours that were due to the successful fucosylation of the core structures. Example 5: Synthesis of fucosylated LNT or LNnT core structures using strains in which the fucosyltransferase genes were placed under the control of a rhamnose-inducible promoter

[0061] The same experiments were carried out again with strains that did not carry the fucosyltransferase genes on vectors with an IPTG-inducible tac promoter, but in plasmids that placed the fucosyltransferase genes (futC) under the control of a rhamnose promoter (see Wiese, A. Molecular genetic and functional characterization of the hydantoin operon from Arthrobacter aurescensDSM 3747. (2000)). The expression of the fucosyltransferase genes was first induced with L-rhamnose (2 g I -1< ) when L-fucose was added to provide more protein production resources for the glycosyltransferases LgtA and LgtB / WbgO. The shake flask cultures, which were otherwise carried out analogously to the previous experiments, showed stronger signals with fucosyltransferases in the samples 65 h after induction. For the antibiotic-free synthesis of these structures, the fucosyltransferase genes were also chromosomally inserted into the rhamnose operon. rhaBAD The integrated fucosyltransferase genes are each located under a tac promoter. The ability to synthesize fucosylated LNT or LNnT was demonstrated for all strains in shake flask experiments using HPLC and mass spectrometry. Example 6: Synthesis and isolation of larger fucosylated oligosaccharides

[0062] Since the aforementioned experiments also demonstrated that fucosylation is effective for LNnT, and since α 1,3- and α 1,2-fucosylated compounds based on LNnT have already been prepared (see Drouillard et al., Angew Chem Int Ed Engl 2006, 45, 1778-1780; Dumon et al., Glycoconj. J. 2001, 18, 465-474), the produced fucosylated compounds were to be isolated and further characterized. For this purpose, the above cultivations with the L-rhamnose-inducible plasmids were repeated on a 750 ml scale in 3-liter shake flasks with baffles under otherwise identical conditions as before. The products were then recovered from the cell pellets by resuspension in H2O, incubation at 100°C for 20 minutes and centrifugation and then isolated by preparative activated carbon / Celite545 chromatography and gel filtration chromatography.In addition to Bio-Rad's Bio-Gel P2, Bio-Gel P4 (extra fine) with a narrower particle size distribution and larger cutoff size was also used, as this had already been successfully used for the separation of larger neutral oligosaccharides (see Priem et al., Glycobiology 2002, 12, 235-240). Through these isolation steps, a total of 59.4 mg of LNFI (see structure) was isolated from the cultures. Fig. 6 ) were isolated. Portions of this were analyzed by mass spectrometry and NMR to fully elucidate the structure. The mass spectrum of the product shows, in addition to a few impurities, primarily the signals of the proton adduct, sodium adduct, and disodium adduct of LNFI.

[0063] The oligosaccharide isolation from a strain equipped with FucT14 yielded a total of 133.7 mg LNDII (structure see Fig. 7). In addition, 71.5 mg of fucosylated lacto-N-triose II were isolated. The mass spectra also showed the masses of the expected adducts and hardly any impurities. These substances are fucosylated or difucosylated compounds with LNT as the core structure, which have not yet been tested in vivo in E. coli synthesized compounds have been described (other synthesis routes with equivalent product quantities are also unknown). Another compound isolated is a lacto-N-pentaose with two fucosyl residues, which probably arose from the elongation of LNT with another N-acetylglucosaminyl group.

Claims

1. A genetically modified microorganism for in vivo synthesis of lacto-N-tetrose or lacto-N-neotetrose in a microorganism, said microorganism comprising (i) a first transgene coding for β1,3-N-acetylglucosaminyltransferase, and (ii) a second transgene coding for β1,3-galactosyltransferase or for β1,4-galactosyltransferase, and said microorganism in addition being genetically modified so as to suppress expression of LacZ and LacA, wherein the (i) first transgene has been integrated into the LacZYA locus and said microorganism comprises a further transgene coding for LacY.

2. The genetically modified microorganism according to claim 1, wherein the transgene coding for LacY has been integrated into the FucIK locus.

3. The genetically modified microorganism according to either of claims 1 and 2, said microorganism comprising a further transgene coding for UDP-sugar pyrophosphorylase.

4. The genetically modified microorganism according to any of claims 1 to 3, said microorganism in addition being genetically modified so as to suppress expression of UDP-glucose 4-epimerase.

5. The genetically modified microorganism according to any of claims 1 to 4, wherein one or both, or one, multiple or all, transgenes is / are chromosomally integrated.

6. The genetically modified microorganism according to any of claims 1 to 5, said microorganism comprising a further transgene coding for a bifunctional enzyme having L-fucokinase activity and L-fucose-1-phosphate guanylyltransferase activity, and at least one transgene coding for an enzyme capable of alpha1,2-fucosylation, alpha1,3-fucosylation or alpha1,4-fucosylation.

7. The genetically modified microorganism according to claim 6, wherein the transgene coding for said bifunctional enzyme having L-fucokinase activity and L-fucose-1-phosphate guanylyltransferase activity is chromosomally integrated, and the at least one transgene coding for an enzyme capable of alpha1,2-fucosylation, alpha1,3-fucosylation or alpha1,4-fucosylation is expressed on a plasmid vector.

8. The genetically modified microorganism according to claim 6, wherein both the transgene coding for said bifunctional enzyme having L-fucokinase activity and L-fucose-1-phosphate guanylyltransferase activity and the at least one transgene coding for an enzyme capable of alpha1,2-fucosylation, alpha1,3-fucosylation or alpha1,4-fucosylation are chromosomally integrated.

9. The use of a genetically modified microorganism according to any of the preceding claims for in vivo synthesis of lacto-N-tetrose or lacto-N-neotetrose or a fucosylated derivative of lacto-N-tetrose or lacto-N-neotetrose in a microorganism.

10. A method of preparing lacto-N-tetrose or lacto-N-neotetrose or a fucosylated derivative of lacto-N-tetrose or lacto-N-neotetrose, comprising the following steps: (a) providing a genetically modified microorganism according to any of claims 1 to 8, (b) culturing said genetically modified microorganism under conditions that permit synthesis of lacto-N-tetrose and lacto-N-neotetrose, (c) optionally adding fucose, (d) optionally isolating the synthesized lacto-N-tetrose or lacto-N-neotetrose or fucosylated derivative of lacto-N-tetrose or lacto-N-neotetrose.

11. The method according to claim 10, wherein step (b) comprises - using galactose as carbon source for said microorganism or - using glycerol and galactose as carbon source for said microorganism.

12. The method according to either of claims 10 and 11, wherein step (b) comprises adding one or more carbon source(s), preferably selected from the group consisting of lactose, glucose, glycerol, galactose, and any mixtures thereof, continuously or in batches.

13. The method according to any of claims 10 to 12, said method being carried out by way of a fed batch process with a batch volume in the range from 2 to 30 L, preferably from 3 to 20 L, particularly preferably from 5 to 15 L.

14. The genetically modified microorganism according to any of claims 1 to 8 or the use according to claim 9 or the method according to any of claims 10 to 13, wherein the genetically modified microorganism is selected from the group consisting of bacteria, fungi, and plants, preferably microorganisms of the genera Corynebacterium, Bevibacterium, Bacillus, Saccharomyces and Escherichia.