Novel sialyltransferase for in vivo synthesis of LST-c

Abstract

The present invention relates to the production of sialylated human milk oligosaccharides (HMOs), in particular to the production of sialyl-milk-N-neotetrasaccharides (LST-c) from precursor oligosaccharides, and the genetic engineering of suitable cells used for said production, as well as to methods of producing said sialylated human milk oligosaccharides (HMOs).

Description

Technical Field

[0001] The present invention relates to the production of sialylated human milk oligosaccharides (HMOs), in particular to the production of sialyl-lacto-N-tetraose c (LST-c), and genetically engineered cells suitable for said production. Background Art

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

[0003] To this end, rational strain engineering principles are often applied to individual bacterial cells. These principles generally refer to a) introducing the desired biosynthetic pathway into the host, b) increasing the cellular pool of the relevant activated sugars required as donors in the desired reaction, c) enhancing the import of lactose through the native lactose permease LacY, and d) introducing appropriate glycosyltransferases to promote the biosynthetic production of sialylated oligosaccharides (for review, see Bych et al. 2019, Current Opinion in Biotechnology 56:130–137).

[0004] For example, WO2007 / 101862 has disclosed the production of sialylated HMOs, describing the modifications required to produce, for example, 3'-SL from non-pathogenic microorganisms without providing sialic acid to the culture, thereby producing sialylated HMOs on a large scale in a cheaper manner.

[0005] WO2019 / 020707 in turn describes examples of sialyltransferases expressed in genetically modified cells that are capable of producing complex sialylated HMOs. However, the sialyltransferases disclosed therein only produce a small amount of complex sialylated HMOs and form a large amount of by-products.

[0006] The production of sialylated HMOs may be hampered by side activities of sialyltransferases in the producing strains, which may affect the ability of the cells to grow robustly even in the absence of substrate, which in turn is reflected in the low yield of sialylated HMO products.

[0007] In summary, the production of sialylated HMOs, especially the production of more complex sialylated human milk oligosaccharides (HMOs), is often affected by the low yield of the desired sialylated HMO compared to other HMO products (e.g., HMO precursor products) present after fermentation, as well as the co-formation of other sialylated HMO species (HMO byproducts), which in turn require laborious separation procedures. Therefore, there is a need for sialyltransferases that are more specific for one or more specific sialylated HMOs (particularly for one or more specific complex sialylated HMOs) to reduce byproduct formation and simplify product purification. Summary of the invention

[0008] The present invention relates to a genetically modified cell comprising a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity (α-2,6-sialyltransferase), which is capable of transferring sialic acid from an activated sugar to the terminal galactose of LNnT (acceptor) and / or the galactose of lactose (acceptor). The genetically modified cell is capable of producing one or more HMOs, wherein at least 10%, such as at least 11%, of the total molar HMO content produced by the cell is LST-c.

[0009] Specifically, the present invention relates to a genetically modified cell comprising a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, the enzyme being selected from Shal (SEQ ID NO: 1), HAC1268 (SEQ ID NO: 2), Valg2 (SEQ ID NO: 3) and a functional homologue of Shal (SEQ ID NO: 1), HAC1268 (SEQ ID NO: 2) or Valg2 (SEQ ID NO: 3), the amino acid sequence of which is at least 80% identical to the amino acid sequence selected from SEQ ID NO: 1, 2 and 3, respectively, and wherein the cell produces at least one sialylated human milk oligosaccharide (HMO), and wherein at least 10% of the total molar HMO content produced by the cell is LST-c. That is, at least 10% (e.g., at least 11%) of the total molar HMO content produced by the cell is LST-c. If lactose is used as the initial substrate, the genetically modified cell may also produce 6'SL. In embodiments, the level of 6'SL is less than 50% of the total molar HMOs produced by the cell.

[0010] The genetically modified cell according to the present invention may also comprise a promoter element, which controls the expression of a recombinant nucleic acid encoding an enzyme having α-2,6-sialyltransferase activity. The sialyltransferase may be controlled, for example, by a promoter selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR, PglpT_70UTR and variants thereof, the nucleic acid sequences of which are selected from SEQ ID NOs 28 to 51, respectively. Preferably, the recombinant nucleic acid encoding an enzyme having α-2,6-sialyltransferase is controlled by a strong promoter selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR, the nucleic acid sequences of which are shown in SEQ ID NOs 40, 49, 37, 38 or 39, respectively.

[0011] The genetically modified cells according to the invention may also comprise a nucleic acid sequence encoding an MFS transporter capable of exporting sialylated HMOs into the extracellular medium.

[0012] The genetically modified cells according to the present invention may also comprise 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 produce a precursor of a sialylated human milk oligosaccharide product, such as LNnT, or further modifying sialylated human milk oligosaccharides to produce more complex sialylated human milk oligosaccharides.

[0013] Furthermore, the genetically modified cells according to the invention typically comprise a recombinant nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyltransferase, such as LgtA from Neisseria meningitidis and / or a recombinant nucleic acid sequence encoding a β-1,4-galactosyltransferase, such as GalT from Helicobacter pylori.

[0014] The genetically modified cells according to the present invention may comprise a biosynthetic pathway for making sialic acid sugar nucleotides, such as CMP-Neu5Ac. The sialic acid sugar nucleotide pathway may be encoded by a nucleic acid sequence (SEQ ID NO:25) encoding neuBCA from Campylobacter jejuni. The nucleic acid sequence encoding neuBCA may be encoded by a high copy plasmid with a neuBCA operon. Alternatively, neuA, neuB and neuC genes may be integrated into the genome of the genetically modified cells, preferably independently controlled by a separate promoter.

[0015] Usually, the genetically modified cell according to the present invention is a microorganism, such as a bacterium or a fungus, wherein the fungus can be selected from a yeast cell, such as a Komagataella genus, a Kluyveromyces genus, a Yarrowia genus, a Pichia genus, a Saccaromyces genus, a Schizosaccharomyces genus or a Hansenula genus, or a filamentous fungus selected from the genus Aspargillus, the genus Fusarium or the genus Thricoderma, and the bacterium can be selected from the exemplified Escherichia sp., Bacillus sp., lactobacillus sp., Corynebacterium sp. and Campylobacter sp. Therefore, the genetically modified cell according to the present invention can be Escherichia coli.

[0016] The genetically modified cells of the invention can be used to produce sialylated HMOs.

[0017] Therefore, the present invention also relates to a method for producing sialylated human milk oligosaccharides (HMOs), said method comprising culturing a genetically modified cell according to the present invention.

[0018] In addition, the present invention also relates to a nucleic acid construct encoding an enzyme having α-2,6-sialyltransferase activity, such as an enzyme selected from Shal (SEQ ID NO: 1), HAC1268 (SEQ ID NO: 2) and Valg2 (SEQ ID NO: 3) or a functional homologue thereof, the amino acid sequence of which has at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 1, 2 or 3, respectively, wherein the enzyme coding sequence is preferably controlled by a promoter sequence, such as a promoter selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR and variants thereof (SEQ ID NOs 28 to 51). The nucleic acid construct is generally used in a host cell to produce sialylated HMOs, such as LST-c and / or 6'SL.

[0019] The present invention also relates to an HMO mixture consisting essentially of LST-c, LNnT, 6'SL and pLNnH. Thus, in an embodiment, the present invention relates to an HMO mixture wherein LST-c is in the range of 10-45 mol% of the mixture, such as in the range of 10-30 mol% of the mixture, LNnT is in the range of 25-70 mol% of the mixture, such as in the range of 40-70 mol% of the mixture, 6'SL is in the range of 0-65 mol% of the mixture, such as in the range of 0-30 mol% of the mixture, and pLNnH is in the range of 1-20 mol% of the mixture, such as in the range of 3-20 mol% of the mixture. In a further embodiment, the mixture is produced according to the method of the present invention and the HMO mixture is purified such that the purified mixture contains less than 15% (w / w) lactose.

[0020] Various exemplary embodiments and details will be described below with reference to the relevant drawings and sequences. It should be noted that the drawings are only used to facilitate the description of the embodiments. They are not intended to be an exhaustive description of the present disclosure or a limitation on the scope of the present disclosure. In addition, the illustrated embodiments do not necessarily have all the aspects or advantages shown. The aspects or advantages described in conjunction with a particular embodiment are not necessarily limited to that embodiment and may be practiced in any other embodiment, even if not shown or not so explicitly described. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Cells expressing an enzyme with α-2,6-sialyltransferase activity produce a molar content of LST-c (expressed as a percentage, % of total HMO) that is greater than the level of LST-c produced by cells expressing Pdam (SEQ ID NO: 8), which has been shown in the prior art to be able to sialylate LNnT.

[0022] Figure 2 : Overview of the synthesis of LST-c mixtures. DETAILED DESCRIPTION

[0023] The present invention addresses the biotechnological challenges of HMO production in vivo, particularly sialylated HMOs containing at least one sialic acid monosaccharide, such as the sialylated HMOs LST-c and 6'SL. The present invention provides a specific strain engineering solution to produce specific complex sialylated HMOs, particularly LST-c, by exploiting the substrate specificity for the terminal galactose moiety on LNnT and the activity of the α-2,6-sialyltransferase disclosed herein.

[0024] The genetically modified cells of the present invention express genes encoding key enzymes for the biosynthesis of sialylated HMOs, and in some embodiments, also express one or more genes encoding biosynthetic pathways for making sialic acid sugar nucleotides, such as the neuBCA operon from Campylobacter jejuni shown in SEQ ID NO: 25, thereby allowing the formation of CMP-N-acetylneuraminic acid, which enables the cell to produce sialylated oligosaccharides from one or more oligosaccharide substrates (such as lactose, LNT-II and / or LNnT). Depending on the substrate, one or more additional glycosyltransferases and pathways for making nucleotide activated sugars, such as glucose-UDP-GlcNac, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetyl-glucosamine, UDP-N-acetyl-galactosamine and CMP-N-acetylneuraminic acid, may also be present in the genetically modified cells.

[0025] In particular, the sialylated HMO produced is LST-c and / or 6'SL.

[0026] The advantage of using any of the α-2,6-sialyltransferases disclosed in the present context is that they are not only able to recognize and sialylate lactose to produce 6'SL, but are also able to recognize and sialylate larger oligosaccharides (such as LNnT) to produce LST-c. Specifically, the present disclosure describes enzymes having α-2,6-sialyltransferase activity (α-2,6-sialyltransferases) that have higher activity on the terminal galactose of LNnT than the α-2,6-sialyltransferases described in the prior art (e.g., Plst6_119 (see WO2019 / 020707) and Pdam (see WO2021 / 1123113)).

[0027] Therefore, the properties of the α-2,6-sialyltransferase described herein are well suited for high-level industrial production of LST-c as the major sialylated HMO, or with the simultaneous formation of other sialylated HMOs such as 6'SL and other byproduct HMOs to produce a desired sialylated oligosaccharide product mixture.

[0028] The genetically modified cells of the present invention express an α-2,6-sialyltransferase with high specificity for the terminal galactose of LNnT, and for the first time are able to produce high titers of LST-c. Thus, the present invention enables more efficient production of LST-c, which is very beneficial for the biotechnological production of more complex sialylated HMOs such as LST-c. In particular, some of the enzymes described herein are suitable for the production of LST-c with less 6'SL byproduct formation, while other enzymes are suitable for the production of a mixture of 6'SL and LST-c, which may also be beneficial for products requiring both HMOs.

[0029] In the following sections, various elements of the present invention, in particular various elements of genetically modified cells, are described, and it will be understood that these elements can be combined between the various sections.

[0030] Oligosaccharides

[0031] In this context, the term "oligosaccharide" refers to a sugar polymer containing at least three monosaccharide units, i.e., three, four, five, six or more oligosaccharides. Oligosaccharides can have a linear or branched structure comprising monosaccharide units interconnected by interglycosidic bonds. Specifically, oligosaccharides include a lactose residue at a reducing end and one or more naturally occurring monosaccharides of 5-9 carbon atoms, which are selected from aldoses (e.g., glucose, galactose, ribose, arabinose, xylose, etc.), ketoses (e.g., fructose, sorbose, tagatose, etc.), deoxy sugars (e.g., rhamnose, fucose, etc.), deoxy amino sugars (e.g., N-acetyl-glucosamine, N-acetyl-mannosamine, N-acetyl-galactosamine, etc.), uronic acid and ketouronic acid (e.g., N-acetylneuraminic acid). Preferably, the oligosaccharide is a HMO.

[0032] Human Milk Oligosaccharides (HMO)

[0033] Preferred oligosaccharides of the present disclosure are human milk oligosaccharides (HMOs).

[0034] The term "human milk oligosaccharide" or "HMO" in this context refers to a complex carbohydrate found in human milk. HMOs have a core structure whose reducing end comprises a lactose unit which may be extended by one or more β-N-acetyl-lactosamine units and / or one or more β-lacto-N-disaccharide units and which may be substituted by α-L-fucosyl and / or α-N-acetyl-neuraminic acid (sialic acid) moieties. The HMO structure is disclosed, for example, by Xi Chen in Advances in Carbohydrate Chemistry and Biochemistry 2015, Vol. 72, Chapter 4.

[0035] The present invention focuses on sialylated HMOs, which are generally acidic. Examples of acidic HMOs include 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), 3-fucosyl-3'-sialyllactose (FSL), 3'-O-sialyllacto-N-tetraose a (LST-a), fucosyl-LST-a (FLST-a), 6'-sialyllacto-N-tetraose b (LST-b), fucosyl-LSTb (FLSTb), 6'-sialyllacto-N-tetraose b (LST-b), fucosyl-LSTb (FLSTb), 6'-sialyllacto-N-tetraose b (LST-c), 6'-sialyllacto-N-tetraose b (LST-d ... -neotetraose (LST-c), fucosyl-LST-c (FLST-c), 3'-sialyllacto-N-neotetraose (LST-d), fucosyl-LSTd (FLST-d), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexose I (SLNH-I), sialyl-lacto-N-neohexose II (SLNH-II), and disialyllacto-N-tetraose (DSLNT).

[0036] In the context of the present invention, a complex HMO consists of at least 4 monosaccharide units, preferably at least 5 monosaccharide units. Preferably, in one embodiment, a complex HMO is an HMO that requires at least two different glycosyltransferase activities to be produced from lactose as an initial substrate, for example, the formation of LST-c requires α-2,6-sialyltransferase, β-1,3-N-acetyl-glucosaminyltransferase and β-1,4-galactosyltransferase.

[0037] In one aspect according to the invention, the human milk oligosaccharide (HMO) is an acidic HMO, such as a sialylated HMO. The sialylated HMO in one aspect comprises at least three monosaccharide units, such as three, four, five or six monosaccharide units.

[0038] In one aspect of the invention, the sialylated human milk oligosaccharide (HMO) produced by the cell is a sialylated HMO selected from 6'SL and LST-c. In another aspect of the invention, the sialylated human milk oligosaccharide (HMO) produced by the cell is an HMO of at least five monosaccharide units, such as LST-c.

[0039] In one embodiment, the one or more HMOs produced by the methods and / or genetically engineered cells disclosed herein are a mixture of one or more of the following HMOs: LNT-II, LNnT, pLNnH, 6'SL, and LST-c.

[0040] In one embodiment, LST-c is a desired product produced by the methods and / or genetically engineered cells disclosed herein, and Figure 2Other oligosaccharides (such as HMOs) produced by the process shown in are considered by-product oligosaccharides or by-product HMOs. In the case of high concentrations of LST-c, examples of by-product HMOs are 6'SL, LNT-II, LNnT, and pLNnH. The product HMO (in this case LST-c) can be purified to reduce or eliminate by-product HMOs in the final product.

[0041] In another embodiment, a mixture of sialylated HMOs (eg, a mixture of LST-c and 6'SL) is the desired product produced by the methods and / or genetically engineered cells disclosed herein, and Figure 2 Other oligosaccharides (such as HMOs) produced by the process shown in are considered by-product oligosaccharides or by-product HMOs. In the case where a mixture of sialylated HMOs is produced, examples of by-product HMOs are LNT-II, LNnT, and pLNnH. In this case, the product HMOs are LST-c and 6'SL, which can be purified to reduce or eliminate by-product HMOs in the final product.

[0042] The production of these HMOs may require the presence of two or more glycosyltransferase activities, particularly if starting from lactose as the acceptor oligosaccharide.

[0043] Receptor oligosaccharide

[0044] The genetically modified cell according to the present invention comprises a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, which is capable of transferring sialic acid from an activating sugar to the terminal galactose of an acceptor oligosaccharide.

[0045] In the context of the present invention, an acceptor oligosaccharide is an oligosaccharide that can serve as a substrate for a glycosyltransferase that is capable of transferring a glycosyl moiety from a glycosyl donor to the acceptor oligosaccharide. The glycosyl donor is preferably a nucleotide activated sugar as described in the "glycosyltransferase" section. Preferably, the acceptor oligosaccharide is a precursor for the preparation of a more complex HMO, which may also be referred to as a precursor molecule.

[0046] The acceptor oligosaccharide may be an intermediate product of the present fermentation process, the end product of a separate fermentation process employing separate genetically modified cells, or an enzymatically or chemically produced molecule.

[0047] In the present context, the acceptor oligosaccharide of the α-2,6-sialyltransferase is preferably lacto-N-neotetraose (LNnT), which is produced from the precursor molecules lactose (e.g., the acceptor of β-1,3-N-acetyl-glucosaminyltransferase) and / or lacto-N-triose II (LNT-II) (e.g., the acceptor of β-1,4-galactosyltransferase). The precursor molecules are preferably fed to a genetically modified cell capable of producing LNnT from the precursor.

[0048] Glycosyltransferase

[0049] The genetically modified cells according to the present invention comprise at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase (i.e., sialyltransferase) capable of transferring sialic acid residues from a sialic acid donor to an acceptor oligosaccharide, thereby synthesizing a sialylated human milk oligosaccharide product.

[0050] The genetically modified cell according to the present invention may comprise at least one additional recombinant nucleic acid sequence encoding at least one glycosyltransferase capable of transferring a glycosyl residue from a glycosyl donor to an acceptor oligosaccharide. Preferably, the additional glycosyltransferase enables the genetically modified cell to synthesize LNnT from a precursor molecule (e.g., lactose or LNT-II).

[0051] The additional glycosyltransferase is preferably selected from the group consisting of galactosyltransferase, glucosaminyltransferase, sialyltransferase, N-acetylglucosaminyltransferase and N-acetylglucosaminyltransferase.

[0052] In one aspect, the sialyltransferase in the genetically modified cell of the invention is an α-2,6-sialyltransferase. Preferably, the α-2,6-sialyltransferase is capable of transferring a sialic acid unit to the terminal galactose of the LNnT molecule.

[0053] In the present invention, at least one functional enzyme (α-2,6-sialyltransferase) capable of transferring a sialic acid moiety from a sialic acid donor to an acceptor oligosaccharide can be selected from the list consisting of Shal, HAC1268, Valg2, Pmult and Plst6_119 (Table 1). These enzymes can be used, for example, to produce 6'SL and / or LST-c, respectively.

[0054] In a preferred embodiment, the α-2,6-sialyltransferase is selected from the group consisting of Shal, HAC1268 and Valg2.

[0055] Without being bound by theory, an α-2,6-sialyltransferase with a higher affinity for the terminal galactose moiety of LNnT compared to the terminal galactose moiety of lactose may have an advantage because such an α-2,6-sialyltransferase would theoretically produce less 6'SL when the initial substrate is lactose and the availability of LNnT is not limited. In this case, a lower amount of 6'SL would result in a more favorable purification of LST-c because it would be simpler to purify LST-c from an HMO mixture consisting essentially of neutral HMO byproducts and LST-c because it is easier to separate neutral HMOs from acidic HMOs than to separate different acidic HMOs. Therefore, it is believed that a lower initial amount of 6'SL is favorable for the purification of LST-c.

[0056] In a preferred embodiment, when lactose is the initial substrate, the α-2,6-sialyltransferase of the invention results in greater LST-c formation than 6'SL formation. In a further preferred embodiment, the α-2,6-sialyltransferase is HAC1268.

[0057] In an embodiment, the expression of the α-2,6-sialyltransferase of the invention in the genetically modified cell is further combined with the expression of a β-1,4-galactosyltransferase (such as galT from Helicobacter pylori). In a further embodiment, a third enzyme is added, such as a β-1,3-N-acetyl-glucosaminyltransferase, for example LgtA from Neisseria meningitidis.

[0058] Exemplary glycosyltransferases are preferably selected from the glycosyltransferases described below.

[0059] α-2,6-Sialyltransferase

[0060] 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 in an α-2,6-linked manner. Preferably, the α-2,6-sialyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding the α-2,6-sialyltransferase is of heterologous origin and is selected from the α-2,6-sialyltransferases listed in Table 1. In the context of the present invention, the acceptor molecule of the α-2,6-sialyltransferase is lactose and / or an acceptor oligosaccharide of at least four monosaccharide units, such as LNnT. Heterologous α-2,6-sialyltransferases capable of transferring a sialic acid moiety to LNnT are known in the art, two of which are listed in Table 1.

[0061] The α-2,6-sialyltransferases studied in the present application are listed in Table 3. Among the α-2,6-sialyltransferases studied (Table 3), only the α-2,6-sialyltransferases listed in Table 1 are capable of producing LST-c. The sialyltransferase can be selected from an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with the amino acid sequence of any α-2,6-sialyltransferase listed in Table 1.

[0062] Table 1. List of α-2,6-sialyltransferases capable of producing LST-c

[0063]

[0064]

[0065] 1 The GenBank ID reflects the full-length enzyme, and truncated or mutated versions may be used in the present invention, which are represented by the sequences shown in SEQ ID NO.

[0066] 2 SEQ ID NO: 28 of WO 2019 / 020707 has 99.6% identity to SEQ ID NO: 5. There is no evidence that this enzyme can produce LST-c.

[0067] 3 SEQ ID NO:32 of WO2021 / 123113 is truncated at 93 amino acids at the N-terminus and 208 amino acids at the C-terminus, making it 59.1% identical to SEQ ID NO:11, but the overlapping region is 100% identical. In addition, SEQ ID NO:32 of WO2021 / 123113 is truncated at 90 amino acids at the N-terminus and 13 amino acids at the C-terminus, making it 75.1% identical to SEQ ID NO:8. The overlapping region has 94.9% identity with SEQ ID NO 8.

[0068] Example 1 of the present invention has identified heterologous α-2,6-sialyltransferases Shal, HAC1268 and Valg2 (SEQ ID NOs: 1, 2 and 3, respectively), which, when introduced into LNnT producing cells, are able to produce higher LST-c titers than the previously known α-2,6-sialyltransferases Plst6_116 and Pdam.

[0069] In addition, the experiments performed in Example 1 identified heterologous α-2,6-sialyltransferases Shal, HAC1268, Valg2, pmult, plst6_119 (SEQ ID NOs: 1, 2, 3, 4, and 5, respectively), which, when introduced into LNnT producing cells, were able to produce higher LST-c titers than the previously known α-2,6-sialyltransferase Pdam. In addition, although plst6_119 is known to produce 6'SL (WO 2019 / 020707), to our knowledge, it has never been used in methods for producing LST-c before.

[0070] In an example, Shal, HAC1268, Valg2, pmult and plst6_119 are used in combination with LgtA from Neisseria meningitidis and galT from Helicobacter pylori to produce a mixture of LST-c and 6'SL using lactose as a substrate. Shal, HAC1268, Valg2, pmult and plst6_119 can also be combined with galT from Helicobacter pylori to produce LST-c using LNT-II as a substrate, which can eliminate the formation of 6'SL. In addition, when starting from LNnT, Shal, HAC1268, Valg2, pmult and plst6_119 can be sufficient to produce LST-c.

[0071] If desired, the α-2,6-sialyltransferases listed in Table 1 can also be used in modified strains that lack β-1,3-N-acetyl-glucosaminyl-transferase and β-1,3-galactosyltransferase activities, thereby resulting in the production of 6'SL in the absence of LST-c when lactose is used as a substrate.

[0072] In embodiments, the genetic modification may comprise more than one copy, such as two copies or three copies, of a nucleic acid sequence encoding α-2,6-sialyltransferase. In a preferred embodiment, the nucleic acid sequence encoding α-2,6-sialyltransferase is integrated into the genome of the modified cell.

[0073] In one embodiment of the invention, the enzyme having α-2,6-sialyltransferase activity is Shal from Shewanella halifaxensis, comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO: 1. In an embodiment, when the Shal enzyme is introduced into a cell capable of producing LNnT from lactose, the enzyme is capable of producing a 6'SL:LST-c ratio of between 1:1 and 3:1. In an embodiment, the Shal enzyme is capable of producing at least 15%, such as at least 20% of LST-c of the total HMO and at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60% of 6'SL of the total HMO.

[0074] In another embodiment of the invention, the enzyme having α-2,6-sialyltransferase activity is HAC1268 from Helicobacter acinonychis str. Sheeba, which comprises or consists of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO: 2. The genetically modified cell according to any of the preceding embodiments, wherein the α-2,6-sialyltransferase is HAC1268, and when introduced into a cell capable of producing LNnT from lactose, the LST-c:6'SL ratio is higher than 2:1, preferably higher than 3.5:1, preferably higher than 15:1. In an embodiment, the HAC1268 enzyme is capable of producing at least 15%, such as at least 20%, such as at least 30%, such as at least 40% of LST-c of the total HMO and less than 10%, such as less than 8%, such as less than 5% of 6'SL of the total HMO.

[0075] In another embodiment of the invention, the enzyme having α-2,6-sialyltransferase activity is Valg2 from Vibrio alginolyticus, comprising or consisting of the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO:3. In an embodiment, when the Valg2 enzyme is introduced into a cell capable of producing LNnT from lactose, it is capable of producing a 6'SL:LST-c ratio of between 1:1 and 3:1. In an embodiment, the Valg2 enzyme is capable of producing at least 10%, such as at least 15%, such as at least 20% of LST-c of the total HMOs and at least 10%, such as at least 15%, such as at least 20%, such as at least 25% of 6'SL of the total HMOs.

[0076] In another embodiment of the present invention, the enzyme having α-2,6-sialyltransferase activity is pmult from Pasteurela multocida, which comprises or consists of the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:4.

[0077] In another embodiment of the present invention, the enzyme having α-2,6-sialyltransferase activity is plst6_119 from Photobacterium leiognathid, which comprises or consists of the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO: 5. Plst6_119 is specifically introduced into a genetically modified cell that also comprises a β-1,4-galactosyltransferase and preferably also a β-1,3-N-acetyl-glucosaminyltransferase.

[0078] β-1,3-N-acetyl-glucosaminyltransferase

[0079] β-1,3-N-acetyl-glucosaminyltransferase is any protein that comprises the ability to transfer 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 a species derived from a genetically engineered cell, i.e., the gene encoding the β-1,3-galactosyltransferase is of heterologous origin. In the context of the present invention, the acceptor molecule is lactose or an oligosaccharide of at least four monosaccharide units, such as LNnT, or a more complex HMO structure.

[0080] Table 5 gives non-limiting examples of β-1,3-N-acetyl-glucosaminyl transferases. β-1,3-N-acetyl-glucosaminyl transferase variants may also be useful, preferably, these variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to the amino acid sequence of any β-1,3-N-acetyl-glucosaminyl transferase in Table 5.

[0081] Table 5. List of β-1,3-N-acetyl-glucosaminyltransferases

[0082]

[0083]

[0084] In one embodiment, the recombinant nucleic acid sequence encoding β-1,3-N-acetyl-glucosaminyltransferase includes the amino acid sequence of SEQ ID NO:26 (LgtA from Neisseria meningitidis (N. meningitidis)) or a functional homolog thereof or consists of the same, and the amino acid sequence of the functional homolog has at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:26.

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

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

[0087] β-1,4-galactosyltransferase

[0088] β-1,4-galactosyltransferase is any protein capable of transferring galactose of UDP-galactose to the N-acetyl-glucosaminyl part of the acceptor molecule with a β-1,4-bond. Preferably, the β-1,4-galactosyltransferase used herein is not a species derived from a genetically engineered cell, i.e. the gene encoding the β-1,4-galactosyltransferase is of heterologous origin. In the context of the present invention, the acceptor molecule is an acceptor sugar, such as LNT-II or a more complex HMO structure.

[0089] The following examples use a heterologous β-1,4-galactosyltransferase named GalT or a variant thereof, in combination with other glycosyltransferases to produce, for example, LST-c.

[0090] Table 6 provides non-limiting examples of β-1,4-galactosyltransferases. β-1,4-galactosyltransferase variants may also be useful, preferably, these variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to the amino acid sequence of any β-1,4-galactosyltransferase in Table 6.

[0091] Table 6. List of β-1,4-glycosyltransferases

[0092]

[0093] In one embodiment, the recombinant nucleic acid sequence encoding β-1,4-galactosyltransferase comprises the amino acid sequence of SEQ ID NO:27 (galT from Helicobacter pylori (H. pylori)) or a functional homolog thereof or consists of the same, and the amino acid sequence of the functional homolog has at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:27.

[0094] To produce LNnT from the LNT-II precursor, a β-1,4-galactosyltransferase is required. In one embodiment, the genetically modified cell comprises a β-1,4-galactosyltransferase gene or a functional homologue or fragment thereof.

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

[0096] In a non-limiting example, LgtA from Neisseria meningitidis is used in combination with galT from Helicobacter pylori and Shal from Shewanella halifaxensis to produce LST-c using lactose as the initial substrate.

[0097] In a non-limiting example, LgtA from Neisseria meningitidis is used in combination with galT from Helicobacter pylori and HAC1268 from Helicobacter acinonychisstr. Sheeba to produce LST-c using lactose as the initial substrate.

[0098] In a non-limiting example, LgtA from Neisseria meningitidis is used in combination with galT from Helicobacter pylori and Valg2 from Vibrio alginolyticus to produce LST-c using lactose as the initial substrate.

[0099] In a non-limiting example, LgtA from Neisseria meningitidis is used in combination with galT from Helicobacter pylori and pmult from Pasteurella multocida to produce LST-c using lactose as the initial substrate.

[0100] In a non-limiting example, LgtA from Neisseria meningitidis is used in combination with galT from Helicobacter pylori and plst6_119 from Photobacterium leiognathid to produce LST-c using lactose as the initial substrate.

[0101] In a non-limiting example, galT from Helicobacter pylori is used in combination with Shal from Shewanella halifaxensis to produce LST-c using LNT-II as the initial substrate.

[0102] In a non-limiting example, galT from Helicobacter pylori is used in combination with HAC1268 from Helicobacter acinonychis str. Sheeba to produce LST-c using LNT-II as the initial substrate.

[0103] In a non-limiting example, galT from Helicobacter pylori is used in combination with Valg2 from Vibrio alginolyticus to produce LST-c using LNT-II as the initial substrate.

[0104] In a non-limiting example, galT from Helicobacter pylori is used in combination with pmult from Pasteurela multocida to produce LST-c using LNT-II as the initial substrate.

[0105] In a non-limiting example, galT from Helicobacter pylori is used in combination with plst6_119 from Photobacterium leiognathid to produce LST-c using LNT-II as the initial substrate.

[0106] Glycosyl-donor-nucleotide-activating sugar pathway

[0107] When implementing the method of the present disclosure, preferably a glycosylation reaction mediated by a glycosyltransferase occurs, wherein the activated sugar nucleotide acts as a glycosyl donor. The activated sugar nucleotide usually has a phosphorylated glycosyl residue attached to the nucleoside. Specific glycosyltransferases only accept 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-acetylglucosamine (GlcNAc) and CMP-N-acetylneuraminic acid. Genetically modified cells according to the present disclosure can include one or more pathways to produce nucleotide activated sugars selected from glucose UDP-GlcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylglucosamine and CMP-N-acetylneuraminic acid.

[0108] In one embodiment of the method, the genetically modified cell can produce one or more of the above-mentioned activated sugar nucleotides by a de novo pathway. In this regard, the cell prepares activated sugar nucleotides with a stepwise reaction sequence starting from a simple carbon source such as glycerol, sucrose, fructose or glucose under the action of an enzyme participating in the de novo biosynthetic pathway of the corresponding sugar nucleotide (for a review of monosaccharide metabolism, see, for example, HH Freeze and AD Elbein: Chapter 4: Glycosylation precursors, in: Essentials of Glycobiology, Second Edition (A. Varki et al.), Cold Spring Harbour Laboratory Press (2009)).

[0109] The enzymes involved in the de novo biosynthetic pathway for activated sugar nucleotides may be naturally present in the cell or introduced into the cell by genetic or recombinant DNA techniques, all of which is part of the general knowledge of the person skilled in the art.

[0110] In another embodiment, the genetically modified cell can utilize the recovered monosaccharide as a sugar nucleotide. In the salvage pathway, the monosaccharide from the degraded oligosaccharide is phosphorylated by a kinase and converted into a nucleotide sugar by a pyrophosphorylase. The enzyme involved in this process can be a heterologous enzyme or a native enzyme of the host cell.

[0111] Sialyl sugar nucleotide synthesis pathway

[0112] Preferably, the genetically modified cell according to the present disclosure comprises sialic acid sugar nucleotide synthesis capability, ie, the genetically modified cell comprises a biosynthetic pathway for preparing sialic acid sugar nucleotides, such as CMP-N-acetylneuraminic acid as the glycosyl donor for the α-2,6-sialyltransferase of the present disclosure. For example, the genetically modified cells are provided with exogenous UDP-GlcNAc 2-dimerase (e.g., neuC of Campylobacter jejuni (GenBank AAK91727.1) or equivalent (e.g., (GenBank CAR04561.1)), Neu5Ac synthetase (e.g., neuB of C. jejuni (GenBank AAK91726.1) or equivalent (e.g., Flavobacterium limnosediminis sialic acid synthase, GenBank WP_023580510.1), and / or CMP-Neu5Ac synthetase (e.g., neuA of C. jejuni (GenBank AAK91728.1) or equivalent (e.g., Vibrio brasiliensis CMP sialic acid synthase, GenBank WP_023580510.1). WP_006881452.1) includes sialic acid synthesis capability.

[0113] In one or more examples, the UDP GlcNAc 2-dimerase, CMP-Neu5Ac synthetase, Neu5Ac synthetase from Campylobacter jejuni (also referred to as NeuBCA from Campylobacter jejuni or neuBCA operon for short) can be plasmid-borne or integrated into the genome of the genetically modified cell. In some embodiments, the sialic acid sugar nucleotide pathway is encoded by a nucleic acid sequence encoding NeuBCA from Campylobacter jejuni (SEQ ID NO: 25) or a functional variant thereof having a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, or such as at least 99% identity to SEQ ID NO: 25.

[0114] Furthermore, the nucleic acid sequence encoding neuBCA is preferably encoded by a high copy plasmid carrying the neuBCA operon. In an embodiment, the high copy plasmid is a BlueScribe M13 plasmid (pBS). With respect to the present invention, a high copy plasmid is a plasmid that replicates to more than 50 copies when introduced into a cell.

[0115] In other embodiments, the pathway for making sialic acid sugar nucleotides (CMP-Neu5Ac) is encoded by genomic integrated neuA, neuB and neuC nucleic acid sequences, wherein the three genes are preferably controlled by a single promoter. The neuA, neuB and neuC genes introduced into the genome of the genetically engineered cell can be, for example, codon-optimized neuC (GenBank AAK91727.1) of Campylobacter jejuni, codon-optimized neuB (GenBank AAK91726.1) of Campylobacter jejuni, and codon-optimized neuA (GenBank AAK91728.1) of Campylobacter jejuni, or equivalents thereof as shown above. In one embodiment, codon-optimized neuA is SEQ ID NO: 52, neuB is SEQ ID NO: 53, and neuC is SEQ ID NO: 54 or a functional variant thereof. Preferably, the integrated genes are independently controlled by a promoter selected from Table 2, more preferably a strong promoter selected from SEQ ID NOs 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 and 44. In one embodiment, the neuA, neuB and neuC genes are independently controlled by the PglpF promoter (SEQ ID NO: 40), and these genes are preferably heterologous.

[0116] In an embodiment, the genetically modified cell comprises both a genomic integration pathway for producing sialic acid sugar nucleotides (CMP-Neu5Ac) and a plasmid carrying the neuBCA operon for increasing the production of CMP-Neu5Ac.

[0117] Defective sialic acid catabolic pathway

[0118] The genetically modified cells of the present invention preferably have a lacking sialic acid decomposition pathway. "Sialic acid decomposition pathway" refers to a series of reactions, usually controlled and catalyzed by enzymes, resulting in 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 isomerase), which are all encoded by the nanATEK-yhcH operon and inhibited by NanR (http: / / ecocyc.org / ECOLI). By introducing mutations in 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 isomerase, GI:947745), incorporated herein by reference), a lack of sialic acid decomposition pathways is provided in the E. coli host. Optionally, the nanT (N-acetylneuraminic acid transporter) gene is also inactivated or mutated. Other intermediates of sialic acid metabolism include: (ManNAc-6-P) N-acetylmannosamine-6-phosphate; (GlcNAc-6-P) N-acetylglucosamine-6-phosphate; (GlcN-6-P) glucosamine-6-phosphate and (Fruc-6-P) fructose-6-phosphate. In some preferred embodiments, nanA is mutated. In other preferred embodiments, nanA and nanK are mutated, while nanE remains functional. In another preferred embodiment, nanA and nanE are mutated, while nanK is not mutated, inactivated or deleted. Mutations are one or more changes in the nucleic acid sequence encoding the nanA, nanK, nanE and / or nanT gene products. For example, mutations can be 1, 2, at most 5, at most 10, at most 25, at most 50 or at most 100 changes in the nucleic acid sequence. For example, nanA, nanK, nanE and / or nanT genes are mutated by null mutations. Null mutations as described herein include amino acid substitutions, additions, deletions or insertions that result in loss of enzyme function (i.e., reduced activity or inactivity) or loss of enzyme (i.e., no gene product). "Deletion" refers to the complete or partial removal of a coding region, thereby not producing a (functional) gene product. Inactivation refers to changes in the coding sequence such that the resulting gene product is functionally inactive, or the activity of the encoded gene product is less than 100%, such as 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20% of the activity of a natural, naturally occurring endogenous gene product.Therefore, in the present invention, the nanA, nanK, nanE and / or nanT genes are preferably inactivated.

[0119] Major facilitator superfamily (MFS) transporters

[0120] Oligosaccharide products, such as HMOs produced by cells, can accumulate both within the cell and in the extracellular matrix. The product can be transported to the supernatant in a passive manner, i.e. it diffuses outward through the cell membrane. More complex HMO products may be retained in the cell, which may ultimately impair cell growth and thus affect the possible total yield of product from a single fermentation. HMO transport can be facilitated by major facilitator superfamily transporters, which facilitate the efflux of sugar derivatives from the cell to the supernatant. Major facilitator superfamily transporters can be present in exogenous or endogenous sources and overexpressed under fermentation conditions to facilitate the export of the produced oligosaccharide derivatives (HMOs). By known recombinant DNA techniques, the specificity for the sugar portion of the product to be secreted can be altered by mutation.

[0121] Thus, the genetically modified cell according to the present invention may further comprise a nucleic acid sequence encoding a major facilitator superfamily transporter capable of exporting sialylated human milk oligosaccharide product(s).

[0122] In recent years, several new and potent major facilitator superfamily transporters have been identified, each with specificity for a different recombinantly produced HMO, and the development of recombinant cells expressing said proteins has facilitated large-scale industrial HMO manufacturing. WO2021 / 123113 requires different E. coli and heterologous transporters for the export of 3'SL, 6'SL and LST-c.

[0123] Thus, in one or more exemplary embodiments, the genetically engineered cells according to the methods described herein further comprise a gene product that acts as a major facilitator superfamily transporter. The gene product that acts as a major facilitator superfamily transporter can be encoded by a recombinant nucleic acid sequence expressed in the genetically engineered cells. The recombinant nucleic acid sequence encoding the major facilitator superfamily transporter can be integrated into the genome of the genetically engineered cells or expressed using a plasmid.

[0124] In one embodiment, the genetically modified cell of the invention comprises a nucleic acid sequence encoding a major facilitator superfamily transporter capable of exporting sialylated human milk oligosaccharide products into the extracellular medium, in particular a transporter specific for LST-c and / or 6'SL.

[0125] HMO concentration

[0126] The genetically modified cells described herein that contain more than one glycosyltransferase typically produce a mixture of HMOs in a multi-step process toward a final HMO product. In the process of producing LST-c using lactose as the initial substrate, 6'SL (sialyllactose), LNT-II, LNnT, LST-c, and pLNnH are expected to be present at the end of the culture.

[0127] The HMO products produced by the methods disclosed herein can be described by their ratio in the HMO mixture. As used herein, "ratio" is understood to be the ratio between the amounts of two HMOs, such as, but not limited to, the amount of one HMO divided by the amount of another HMO, or the amount of one HMO divided by the total amount of HMOs.

[0128] In one embodiment described herein, after culturing the genetically modified cells described herein, the mole % of LST-c in the HMO mixture is between 10% and 45%, such as between 10% and 30%, and 6'SL is between 0 and 65%, such as between 4% and 50%, such as between 11% and 25% of LST-c and between 5% and 30% of 6'SL. In a preferred embodiment, the mole % of LST-c is higher than 11% of the total HMOs, such as higher than 15%, such as higher than 18%, such as higher than 25%, such as higher than 35%, such as higher than 40%. In a further preferred embodiment, the mole % of 6'SL is lower than 65% of the total HMOs, such as lower than 50%, such as lower than 40%, such as lower than 30%, such as lower than 20%, such as lower than 10%, such as lower than 5%.

[0129] In some embodiments described herein, upon culturing the genetically modified cells, a mixture of 6'SL and LST-c is produced, preferably, the cells produce a 6'SL:LST-c ratio between 1:1 and 4:1, such as 1.5:1 to 3:1.

[0130] In some embodiments described herein, upon culturing the genetically modified cells, LST-c is produced with a low amount of 6'SL byproduct, preferably the cells produce a LST-c:6'SL ratio greater than 2: 1, such as greater than 3.5: 1, preferably greater than 15: 1, such as between 5: 1 and 20: 1. In a more preferred embodiment, the cells do not produce any 6'SL at all.

[0131] In some embodiments, the genetically modified cells of the invention express Shal, which comprises or consists of an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 1, and the molar % content of LST-c produced by the genetically modified cells is higher than 11%, such as higher than 15%, such as higher than 25% of the total HMOs.

[0132] In some embodiments, the genetically modified cells of the invention express HAC1268 comprising or consisting of an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity thereto, and the genetically modified cells produce LST-c at a molar % content of greater than 11%, such as greater than 15%, such as greater than 20%, such as greater than 35%, such as greater than 40% of the total HMOs.

[0133] In some embodiments, the genetically modified cell of the invention expresses Valg2 comprising or consisting of an amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity thereto, and the genetically modified cell produces LST-c at a molar % content greater than 11% of total HMOs.

[0134] In some embodiments, the genetically modified cells of the invention express Pmult comprising or consisting of an amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity thereto, and the molar % content of LST-c produced by the genetically modified cells is greater than 10% of the total HMOs.

[0135] In some embodiments, the genetically modified cells of the present invention express Plst6_119, which comprises or consists of an amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO:5, and the molar % content of LST-c produced by the genetically modified cells is higher than 10% of the total HMOs.

[0136] The mole % ratios supported by the experimental data in the Examples show exemplary HMO composition ranges where the ratio of LST-c:6'SL ranges from 1:5 to 4:1.

[0137] In some embodiments, the ratio of LST-c:6'SL does not constitute more than 2 times of 6'SL of LST-c, and thus the ratio of LST-c:6'SL is not less than 1:2, preferably not less than 1:1.5. More preferably, the ratio of LST-c:6'SL is higher than 1:1, such as higher than 2:1, such as higher than 3:1.

[0138] In some embodiments, the genetically modified cell of the invention expresses HAC1268 comprising or consisting of an amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO:2, and the ratio of LST-c:6'SL is higher than 3:1, i.e., the genetically modified cell produces greater than 15% of LST-c of total HMOs and / or less than 5% of 6'SL of total HMOs, such as greater than 30% of LST-c of total HMOs and / or less than 10% of 6'SL of total HMOs.

[0139] In some embodiments, the genetically modified cells of the invention express HAC1268 comprising or consisting of an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity thereto, and the genetically modified cells produce 6'SL at a molar % content of less than 15%, such as less than 10%, such as less than 5%, of total HMOs.

[0140] Genetically modified cells

[0141] 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 with heterologous polynucleotide sequences, Crisper / Cas editing and / or random mutagenesis. In one embodiment, the genetically engineered cells have been transformed or transfected with a recombinant nucleic acid sequence.

[0142] The genetic modification may for example be selected from the inclusion of a glycosyltransferase, and / or metabolic pathway engineering and the inclusion of an MFS transporter, as described in the above sections, which a skilled person will know how to combine into a genetically modified cell capable of producing one or more sialylated HMOs.

[0143] In one aspect of the invention, the genetically modified cell comprises a recombinant nucleic acid sequence encoding an enzyme having alpha-2,6-sialyltransferase activity capable of producing LST-c that is at least 10% of the total molar HMO content produced by the cell.

[0144] In one embodiment, the genetically modified cell capable of producing sialylated HMOs comprises a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is selected from:

[0145] a. Shal, which comprises or consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80% identity with SEQ ID NO: 1,

[0146] b. HAC1268, which comprises or consists of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 80% identity with SEQ ID NO: 2, and

[0147] c. Valg2 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80% identity with SEQ ID NO: 3.

[0148] In this preferred embodiment, the genetically modified cell capable of producing sialylated HMOs comprises a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity as described herein, capable of producing LST-c at least 10% of the total molar HMO content produced by the cell.

[0149] In some embodiments, the genetically engineered cells are capable of producing a mixture of 6'SL and LST-c, preferably, the cells produce 6'SL:LST-c at a ratio between 1:1 and 4:1, such as 1.5:1 to 3:1.

[0150] In other embodiments, the genetically engineered cells are capable of producing LST-c with a low amount of 6'SL byproduct, preferably the cells produce a ratio of LST-c:6'SL, preferably the cells produce a ratio higher than 2: 1, such as higher than 3.5: 1, preferably higher than 15: 1, such as between 5: 1 and 20: 1. In even more preferred embodiments, the cells do not produce any 6'SL at all or less than 1% 6'SL.

[0151] The genetically engineered cells are preferably microbial cells, such as prokaryotic cells or eukaryotic cells. Suitable microbial cells that can be used as host cells include bacterial cells, archaeal cells, algae cells and fungal cells.

[0152] 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.

[0153] Host cells

[0154] With regard to bacterial host cells, there are in principle no restrictions; they may 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 cells have properties that allow cultivation to high cell densities. Non-limiting examples of bacterial host cells suitable for the recombinant industrial production of HMOs according to the invention may be Erwinia herbicola (Pantoea aggredens), Citrobacter freundii, Campylobacter sp, Pantoea citrea, Pectobacterium carotovorum or Xanthomonas campestris. Bacillus bacteria may also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans.Similarly, 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 suitable bacterial species for use in the disclosure described herein. Also included as part of the present invention are Enterococcus spp. (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium spp. (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas spp. (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa) engineered as described herein.

[0155] Non-limiting examples of fungal host cells suitable for the recombinant industrial production of heterologous products are, for example, yeast cells, such as Komagataella phaffii, Kluyveromyces lactis, Yarrowia lipolytica, Pichia pastoris and Saccaromyces cerevisiae, or filamentous fungi, such as Aspargillus sp., Fusarium sp., or Thricoderma sp., exemplary species being A. niger, A. nidulans, A. oryzae, F. solani, F. graminearum, and T. reesei.

[0156] In one or more exemplary embodiments, the genetically engineered cell is selected from Escherichia coli (E. coli), Corynebacterium glutamicum (C. glutamicum), Lactobacillus lactis (L. lactis), Bacillus subtilis (B. subtilis), Streptomyces lividans (S. lividans), Pichia pastoris (P. pastoris) and Saccharomyces cerevisiae (S. cerevisiae).

[0157] In one or more exemplary embodiments, the genetically engineered cell is B. subtilis.

[0158] In one or more exemplary embodiments, the genetically engineered cell is S. cerevisiae or P. pastoris.

[0159] In one or more exemplary embodiments, the genetically engineered cell is Escherichia coli.

[0160] In one or more exemplary embodiments, the present invention relates to genetically engineered cells, wherein the cells are derived from Escherichia coli (E. coli) K-12 strain or DE3.

[0161] Recombinant nucleic acid sequence

[0162] The present invention relates to a genetically modified cell comprising a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, such as an enzyme selected from the group consisting of Shal, HAC1268 and valg2, and wherein the cell produces human milk oligosaccharides (HMOs), in particular sialylated HMOs, and preferably the molar % content of LST-c is higher than or at least 10% of the total HMOs produced.

[0163] In the present context, the terms "recombinant nucleic acid sequence", "recombinant gene / nucleic acid / nucleotide sequence / DNA code" or "coding nucleic acid sequence" are used interchangeably and are intended to refer to an artificial nucleic acid sequence (i.e., non-overlapping triplets (codons) that are transcribed into mRNA and translated into protein under the control of appropriate control sequences (i.e., promoter sequences).

[0164] The boundaries of the coding sequence are usually determined by the ribosome binding site, transcription start codon (AUG, GUG or UUG) and translation stop codon (UAA, UGA or UAG) located upstream of the open reading frame at the 5' end of the mRNA. The coding sequence may include, but is not limited to, genomic DNA, cDNA, synthetic and recombinant nucleic acid sequences.

[0165] The term "nucleic acid" includes RNA, DNA and cDNA molecules. It will be appreciated that as a result of the degeneration of the genetic code, a large number of nucleic acid sequences encoding a given protein can be generated.

[0166] The recombinant nucleic acid sequence can be a coding DNA sequence, such as a gene, or a non-coding DNA sequence such as regulatory DNA, for example a promoter sequence or other non-coding regulatory sequences.

[0167] The recombinant nucleic acid sequence can also be heterologous. As used herein, "heterologous" refers to a polypeptide, amino acid sequence, nucleic acid sequence or nucleotide sequence that is foreign to a cell or organism, i.e., a polypeptide, nucleic acid sequence, nucleic acid molecule or nucleotide sequence that is not naturally present in the cell or organism.

[0168] The present invention also relates to a nucleic acid construct comprising a coding nucleic acid sequence, i.e., a recombinant DNA sequence of a target gene (e.g., a sialyltransferase gene) and a non-coding regulatory DNA sequence, such as a promoter DNA sequence, such as a recombinant promoter sequence derived from a promoter sequence of a lac operon or a glp operon, or a promoter sequence derived from another genomic promoter DNA sequence or a synthetic promoter sequence, wherein the coding sequence and the promoter sequence are operably linked.

[0169] The term "operably linked" refers to the functional relationship between two or more nucleic acid (e.g., DNA) fragments. It refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, a promoter sequence is operably linked to a coding sequence if the promoter sequence stimulates or regulates transcription of the coding sequence in an appropriate host cell or other expression system.

[0170] Typically, promoter sequences that are operably linked to a transcribed sequence are physically adjacent to the transcribed sequence, ie, they are cis-acting.

[0171] In one exemplary embodiment, the nucleic acid construct of the present invention may be part of a vector DNA, in another embodiment, the construct is an expression cassette / cassette that is integrated into the host cell genome.

[0172] Therefore, the term "nucleic acid construct" refers to an artificially constructed nucleic acid fragment, particularly a DNA fragment, which is intended to be inserted into a target cell, such as a bacterial cell, to modify the expression of a genomic gene or the expression of a gene / coding DNA sequence that may be included in the construct. Therefore, in an embodiment, the present invention relates to a nucleic acid construct comprising a recombinant nucleic acid sequence encoding a sialyltransferase, wherein the recombinant nucleic acid sequence is selected from the nucleic acid sequences encoding Shal, HAC1268 and Valg2, such as SEQ ID NO: 13, 14 and 15, or a functional variant thereof.

[0173] One embodiment of the present invention is a nucleic acid construct comprising a recombinant nucleic acid sequence encoding a sialyltransferase, wherein the recombinant nucleic acid sequence is selected from: a) Shal, which comprises or consists of a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or for example at least 99% sequence identity with SEQ ID NO: 13; b) HAC1268, which comprises or consists of a nucleic acid sequence of SEQ ID NO: 14 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or for example at least 99% sequence identity with SEQ ID NO: 14; and / or c) Valg2, which comprises or consists of a nucleic acid sequence of SEQ ID NO: 15 or a nucleic acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or for example at least 99% sequence identity with SEQ ID NO: 15. Preferably, the sequence encoding the sialyltransferase is under the control of a promoter sequence selected from the promoter sequences having the nucleic acid sequence identified in Table 2.

[0174] Table 2 – Selected promoter sequences

[0175]

[0176]

[0177] *Promoter activity was assessed in the LacZ assay described below, with the PglpF promoter as a positive reference in the same assay. For comparison across multiple assays, activity was calculated relative to the PglpF promoter, and a range represents the results of multiple assays.

[0178] The promoter may be heterologous, native to the genetically modified cell, or it may be a recombinant promoter combining heterologous and / or native elements.

[0179] One approach to increasing product yields may be to modulate the production of a desired enzyme activity for the production of the product, such as a glycosyltransferase or an enzyme involved in a glycosyl donor biosynthetic pathway.

[0180] Increasing the strength of the promoter driving the expression of the desired enzyme may be a way to achieve this. The strength of the promoter can be assessed using the lacZ enzyme assay, wherein the β-galactosidase activity is as described previously (see, e.g., Miller J.H. Experiments in molecular genetics, Cold spring Harbor Laboratory Press, NY, 1972). In brief, cells are diluted in Z buffer and infiltrated with sodium dodecyl sulfate (0.1%) and chloroform. The LacZ assay is performed at 30°C. Preheat the sample, add 200 μl of o-nitrophenyl-β-galactosidase (4 mg / ml) to start the assay, and when the sample turns slightly yellow, add 500 μl of 1M Na2CO3 to stop the assay. The release of o-nitrophenol is then determined as the change in optical density at 420 nm. The specific activity is reported in Miller units (MU) [A420 / (min*ml*A600)]. Regulatory elements with an activity above 10,000 MU are considered strong, regulatory elements with an activity below 3,000 MU are considered weak, and regulatory elements in between have intermediate strength. An example of a strong regulatory element is the PglpF promoter, which has an activity of about 14,000 MU, and an example of a weak promoter is Plac, which has an activity of about 2,300 MU when induced with IPTG.

[0181] In an embodiment, the expression of the nucleic acid sequence of the present invention is controlled by a PglpF (SEQ ID NO: 40) or Plac (SEQ ID NO: 49) promoter or a PmglB_UTR70 (SEQ ID NO: 37) or a PglpA_70UTR (SEQ ID NO: 38) or a PglpT_70UTR (SEQ ID NO: 39) or a variant thereof (such as the promoters listed in Table 2, in particular a PglpF variant of SEQ ID NO: 35 or a Plac variant of SEQ ID NO: 31, or a PmglB_70UTR variant of SEQ ID NO: 28, 29, 32, 33, 34, 36 and 37). Further suitable variants of the PglpF, PglpA_70UTR, PglpT_70UTR and PmglB_70UTR promoter sequences are described in WO2019 / 123324 and WO2020 / 255054 (incorporated herein by reference).

[0182] Integration of the nucleic acid construct of interest contained in the construct (expression cassette) into the bacterial genome can be achieved by conventional methods, for example by using a linear cassette containing flanking sequences homologous to a specific site on the chromosome, as described for the attTn7 site (Waddell CS and Craig NL, Genes Dev. (1988) February; 2(2): 137-49.); methods for genomic integration of nucleic acid sequences, wherein recombination is mediated by the Red recombinase function of bacteriophage λ or the RecE / RecT recombinase function of the Rac prophage (Murphy, J Bacteriol. (1998); 180(8): 2063-7; Zhang et al., Nature Genetics (1998) 20: 123-128; Muyrers et al., EMBO Rep. (2000) 1(3): 239-243); methods based on Red / ET recombination (Wenzel et al., Chem. Biol. (2003); 1(4): 157-158; Muyrers et al., EMBO Rep. (2001); 1(4): 158-159; Muyrers et al., EMBO Rep. (2001); 1(4): 159-163; Muyrers et al., EMBO Rep. (2001); 1(4): 157-164; Muyrers et al., EMBO Rep. (2001); 1(4): 157-165; Muyrers et al., EMBO Rep. (2001); 1(4): 157-166; Muyrers et al., EMBO Rep. (2001); 1(4): 157-167; Muyrers et al., EMBO Biol. (2005), 12(3):349-56; Vetcher et al., Appl Environ Microbiol. (2005); 71(4):1829-35); or positive clones, ie clones carrying the expression cassette, can be selected, for example, by loss or gain of marker gene or gene function.

[0183] In one or more exemplary embodiments, the present disclosure relates to one or more recombinant nucleic acid sequences as shown in SEQ ID NOs: 13, 14 and 15 [nucleic acids encoding Sha1, HAC1268 or Valg2, respectively].

[0184] In particular, the present disclosure relates to one or more recombinant nucleic acid sequences and / or functional homologs thereof, which have a sequence that is at least 70% identical to SEQ ID NO: 13, 14 or 15 [nucleic acids encoding Shal, HAC1268 or Valg2, respectively], such as at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical or 100% identical.

[0185] Sequence identity

[0186] The term "sequence identity" used herein describes the correlation between two amino acid sequences or between two nucleotide sequences (i.e., between candidate sequence (e.g., sequence of the present invention) and reference sequence (e.g., prior art sequence)) based on their paired comparisons. 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) implemented in the Needle program of EMBOSS software 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 (visible https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). The parameters used are gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of 30BLOSUM62) substitution matrix. The output of Needle labeled "identity" (obtained using the -nobrief option) is used as the percent identity. In general, sequence identity can be calculated as follows: (identical residues x 100) / (aligned region).

[0187] For purposes of the present invention, the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) 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 preferably version 5.0.0 or higher. The parameters used are gap opening penalty 10, gap extension penalty 0.5 and DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of the Needle label "identity" (obtained using the -nobrief option) is used as percentage identity. Usually sequence identity can be calculated as follows: (identical deoxyribonucleotide × 100) / (aligned region).

[0188] Functional homologs

[0189] Functional homologues or functional variants of protein / nucleic acid sequences described herein are protein / nucleic acid sequences in which the genetic code has been altered but retains its original function. Functional homologues can be obtained by mutagenesis or can be naturally occurring variants from the same or other species. Compared with the functionality of the protein / nucleic acid sequence, the functional homologue should have at least 50% residual functionality, for example at least 60%, 70%, 80%, 90% or 100%.

[0190] Functional homologs of any of the disclosed amino acid or nucleic acid sequences may also have higher functionality. Functional homologs of any of the amino acid sequences shown in Table 1 or recombinant nucleic acids encoding any of the sequences in Table 4 should ideally be able to participate in the production of sialylated HMOs in terms of increased HMO yield, export of HMO products out of the cell or import of substrates for HMO production (e.g., acceptor oligosaccharides of at least three monosaccharide units), increased purity / byproduct formation, reduced biomass formation, viability of genetically engineered cells, robustness of genetically engineered cells according to the present disclosure, or reduction of consumables required for production.

[0191] Uses of genetically modified cells

[0192] The present disclosure also relates to any commercial use of the genetically modified cells or nucleic acid constructs disclosed herein, such as, but not limited to, methods for producing sialylated human milk oligosaccharides (HMOs).

[0193] In an exemplary embodiment, the genetically modified cells and / or nucleic acid constructs according to the invention are used to produce HMOs. Preferably, in the production of HMOs, the molar % content of LST-c produced by the genetically modified cells is higher than 10%, for example higher than 11%, of the total HMOs.

[0194] In an exemplary embodiment, the genetically modified cells and / or nucleic acid constructs according to the invention are used to produce one or more sialylated HMOs, wherein the sialylated HMO is 6'SL and / or LST-c.

[0195] In an exemplary embodiment, the genetically modified cells and / or nucleic acid constructs according to the present invention are used to produce a HMO mixture comprising at least two HMOs selected from 6'SL, LNT-II, LNnT, pLNnH and LST-c.

[0196] In an exemplary embodiment, the genetically modified cells and / or nucleic acid constructs according to the invention are used to produce a HMO mixture comprising 6'SL, LNnT, pLNnH and / or LST-c.

[0197] In an exemplary embodiment, the genetically modified cells and / or nucleic acid constructs according to the present invention are used to produce a HMO mixture consisting of 6'SL, LNnT, pLNnH and LST-c.

[0198] In an exemplary embodiment, the genetically modified cells and / or nucleic acid constructs according to the invention are used to produce a HMO mixture comprising 6'SL and LST-c.

[0199] In an exemplary embodiment, the genetically modified cells and / or nucleic acid constructs according to the invention are used to produce one or more sialylated HMOs, wherein the HMO is 6'SL and / or LST-c.

[0200] In one or more embodiments, genetically engineered cells and / or nucleic acid constructs are used to produce 6'SL.

[0201] In one or more exemplary embodiments, genetically engineered cells and / or nucleic acid constructs are used to produce LST-c.

[0202] The production of these HMOs may require the presence of two or more glycosyltransferase activities.

[0203] A method for producing sialylated human milk oligosaccharides (HMO)

[0204] The present invention also relates to a method for producing sialylated human milk oligosaccharides (HMOs), said method comprising culturing a genetically modified cell according to the present invention.

[0205] The present invention relates to a method for producing human milk oligosaccharides (HMOs), wherein the LST-c content produced by genetically modified cells is higher than 10% by mole of the total HMOs.

[0206] Accordingly, the present invention relates to a method for producing sialylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically modified cell comprising:

[0207] A recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is selected from:

[0208] a. Shal, which comprises or consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO: 1,

[0209] b. HAC1268, which comprises or consists of the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity to SEQ ID NO: 2, and / or

[0210] c. Valg2, which comprises or consists of the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 3; and

[0211] wherein the cells produce sialylated HMOs.

[0212] Another embodiment of the present invention is a method for producing one or more sialylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically modified cell comprising

[0213] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetyl-glucosaminyl transferase activity; and

[0214] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,4-galactosyltransferase activity; and

[0215] c. A recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is selected from:

[0216] i. Shal, which comprises or consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80% identity with SEQ ID NO: 1,

[0217] ii. HAC1268, which comprises or consists of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 80% identity with SEQ ID NO: 2, and

[0218] iii. Valg2 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80% identity with SEQ ID NO: 3; or

[0219] iv. Pmult comprising or consisting of the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 80% identity with SEQ ID NO:4; or

[0220] v. Plst6_119, which comprises or consists of the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 80% identity with SEQ ID NO:5; and

[0221] Wherein at least one sialylated HMO is LST-c.

[0222] In one or more exemplary embodiments, the α-2,6-sialyltransferase of the present invention is controlled by PglpF, Plac or PmglB_70UTR, PglpA_70UTR or PglpT_70UTR promoter. Therefore, in an exemplary embodiment, the α-2,6-sialyltransferase of the present invention is controlled by the PglpF promoter or its variant (Table 2). In another exemplary embodiment, the α-2,6-sialyltransferase of the present invention is controlled by the PmglB promoter or its variant (Table 2). Preferably, the recombinant nucleic acid encoding the enzyme with α-2,6-sialyltransferase is controlled by a strong promoter selected from SEQ ID NO: 28 to 44.

[0223] Further genetic modifications may be selected, for example, from the inclusion of additional glycosyltransferases and / or metabolic pathway engineering, and the inclusion of an MFS transporter, as described in the above section, which a skilled person will know how to combine into a genetically modified cell capable of producing one or more sialylated HMOs.

[0224] The method specifically comprises culturing a genetically modified cell that produces sialylated HMOs, wherein the cell produces LST-c at a level that is at least 10% of the total HMO content produced by the cell. In addition, the method comprises culturing a genetically modified cell that produces sialylated HMOs.

[0225] The method comprises culturing a genetically modified cell that produces a sialylated HMO, and further comprises culturing the genetically modified cell in the presence of an energy source (carbon source) selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.

[0226] In one aspect, sialylated human milk oligosaccharides (HMOs), such as 6'SL and / or LST-c, are produced according to the methods of the invention.

[0227] In one aspect, one or more HMOs are produced according to the methods of the invention, wherein the HMO is 6'SL, LNnT, pLNnH and / or LST-c.

[0228] In one aspect, the method according to the invention produces a HMO mixture comprising at least two HMOs, such as at least three HMOs selected from 6'SL, LNnT and LST-c.

[0229] In one aspect, the method according to the invention produces a HMO mixture comprising at least two HMOs selected from 6'SL, LNnT and LST-c.

[0230] In one aspect, the method according to the invention produces a HMO mixture comprising or consisting of 6'SL, LNnT, pLNnH and LST-c.

[0231] In one aspect, the methods according to the invention produce a HMO mixture comprising 6'SL and LST-c.

[0232] In one aspect, one or more sialylated HMOs are produced according to the methods of the invention, wherein the HMO is 6'SL and / or LST-c.

[0233] In one aspect, one or more sialylated HMOs are produced according to the methods of the invention, wherein the HMO is 6'SL and / or LST-c.

[0234] In one aspect, a 6'SL is produced according to the methods of the invention.

[0235] In one aspect, LST-c is produced according to the methods of the invention.

[0236] To enable production of sialylated HMOs in the methods of the invention, the genetically modified cells may contain a biosynthetic pathway for making sialic acid sugar nucleotides, or sialic acid may be added during cell culture.

[0237] In a preferred embodiment of the inventive method, the genetically modified cell comprises a biosynthetic pathway for the manufacture of sialic acid sugar nucleotides. Preferably, in the method of the present invention, sialic acid sugar nucleotides are CMP-Neu5Ac. Therefore, in the method of the present invention, sugar nucleotide pathways are expressed by genetically modified cells, wherein the CMP-Neu5Ac pathway is encoded by the neuBCA operon from Campylobacter jejuni of SEQ ID NO:25. In the method of the present invention, sialic acid sugar nucleotide pathways are encoded by high-copy plasmids with neuBCA operons. In alternative embodiments, pathways for the manufacture of sialic acid sugar nucleotides are encoded by cell genomes, preferably by inserting heterologous DNA encoding neuA, neuB and neuC, respectively, under the control of each promoter sequence.

[0238] The methods of the invention include providing a glycosyl donor that is synthesized separately by one or more genetically engineered cells and / or added exogenously to the culture medium from an alternative source.

[0239] In one aspect, the method of the present invention further comprises providing an acceptor sugar as a substrate for HMO formation, the acceptor sugar comprising at least two monosaccharide units, the acceptor sugar being exogenously added to the culture medium and / or produced by fermentation, such as separate microbial fermentation.

[0240] In one aspect, the method of the present invention comprises providing an acceptor sugar comprising at least two monosaccharide units, which is exogenously added to the culture medium and / or produced by fermentation, such as fermentation of a separate microorganism, and is selected from lactose, LNT-II and LNnT. In a preferred embodiment, the substrate for HMO formation is lactose, which is supplied to the culture during fermentation of the genetically engineered cells.

[0241] Sialyl human milk oligosaccharides (HMOs) are recovered from the culture, whether from the culture medium or from the genetically modified cells. Preferably, the product oligosaccharide, such as LST-c, is purified to produce at least 75% pure LST-c, such as at least 85% pure LST-c, such as at least 95% pure LST-c.

[0242] Specifically, the present invention relates to a method for producing LST-c, the method comprising:

[0243] a) obtaining a genetically modified cell comprising

[0244] i. A recombinant nucleic acid sequence, preferably under the control of a PglpF promoter, encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is selected from: Shal comprising or consisting of an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or for example at least 99% sequence identity to SEQ ID NO: 1; HAC1268 comprising or consisting of an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or for example at least 99% sequence identity to SEQ ID NO: 2; Valg2 comprising or consisting of an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or for example at least 99% sequence identity to SEQ ID NO: 3; Pmult comprising or consisting of an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or for example at least 99% sequence identity to SEQ ID NO: 5. NO:4 having at least 80% sequence identity or consisting of an amino acid sequence, and Plst6_119, which comprises or consists of an amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:5; and

[0245] ii. at least one nucleic acid sequence encoding a heterologous β-1,4-galactosyltransferase, such as GalT from Helicobacter pylori, and

[0246] iii. Optionally, preferably a nucleic acid sequence encoding β-1,3-N-acetyl-glucosaminyltransferase, such as LgtA from Neisseria meningitidis; and

[0247] b) culturing the genetically modified cell in a medium containing a carbon source and in the presence of lactose or LNT-II, and

[0248] c) producing said sialylated human milk oligosaccharides (HMO), in particular LST-c, by said genetically modified cells, and

[0249] d) recovering sialylated human milk oligosaccharides (HMO), in particular LST-c, from the culture medium and / or genetically modified cells.

[0250] Specifically, the present invention relates to a method for producing LST-c, the method comprising:

[0251] a) obtaining a genetically modified cell, said cell comprising

[0252] i. a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is Shal, which comprises or consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO: 1, and

[0253] ii. at least one nucleic acid sequence encoding a heterologous β-1,4-galactosyltransferase, the enzyme being GalT from Helicobacter pylori,

[0254] iii. at least one nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyltransferase, the enzyme being LgtA from Neisseria meningitidis, and

[0255] b) culturing the genetically modified cell in a medium containing a carbon source and in the presence of lactose or LNT-II, and

[0256] c) producing said sialylated human milk oligosaccharides (HMO), in particular LST-c, by said genetically modified cells, and

[0257] d) recovering sialylated human milk oligosaccharides (HMO), in particular LST-c, from the culture medium and / or genetically modified cells.

[0258] Specifically, the present invention relates to a method for producing LST-c, the method comprising:

[0259] a) obtaining a genetically modified cell, said cell comprising

[0260] i. a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is HAC1268, which comprises or consists of the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO: 2; and

[0261] ii. at least one nucleic acid sequence encoding a heterologous β-1,4-galactosyltransferase, the enzyme being GalT from Helicobacter,

[0262] iii. at least one nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyltransferase, the enzyme being LgtA from Neisseria meningitidis, and

[0263] b) culturing the genetically modified cell in a medium containing a carbon source and in the presence of lactose or LNT-II, and

[0264] c) producing said sialylated human milk oligosaccharides (HMO), in particular LST-c, by said genetically modified cells, and

[0265] d) recovering sialylated human milk oligosaccharides (HMO), in particular LST-c, from the culture medium and / or genetically modified cells.

[0266] Specifically, the present invention relates to a method for producing LST-c, the method comprising:

[0267] a) obtaining a genetically modified cell, said cell comprising

[0268] i. a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is Valg2, which comprises or consists of the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO: 3, and

[0269] ii. at least one nucleic acid sequence encoding a heterologous β-1,4-galactosyltransferase, the enzyme being GalT from Helicobacter pylori,

[0270] iii. at least one nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyltransferase, the enzyme being LgtA from Neisseria meningitidis, and

[0271] b) culturing the genetically modified cell in a medium containing a carbon source and in the presence of lactose or LNT-II, and

[0272] c) producing said sialylated human milk oligosaccharides (HMO), in particular LST-c, by said genetically modified cells, and

[0273] d) recovering sialylated human milk oligosaccharides (HMO), in particular LST-c, from the culture medium and / or genetically modified cells.

[0274] Specifically, the present invention relates to a method for producing LST-c, the method comprising:

[0275] a) obtaining a genetically modified cell, said cell comprising

[0276] i. a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme comprises or consists of the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO: 4, and

[0277] ii. at least one nucleic acid sequence encoding a heterologous β-1,4-galactosyltransferase, the enzyme being GalT from Helicobacter pylori,

[0278] iii. at least one nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyltransferase, the enzyme being LgtA from Neisseria meningitidis, and

[0279] b) culturing the genetically modified cell in a medium containing a carbon source and in the presence of lactose or LNT-II, and

[0280] c) producing said sialylated human milk oligosaccharides (HMO), in particular LST-c, by said genetically modified cells, and

[0281] d) recovering sialylated human milk oligosaccharides (HMO), in particular LST-c, from the culture medium and / or genetically modified cells.

[0282] Specifically, the present invention relates to a method for producing LST-c, the method comprising:

[0283] a) obtaining a genetically modified cell, said cell comprising

[0284] i. a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is plst6_119, which comprises or consists of the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:5, and

[0285] ii. at least one nucleic acid sequence encoding a heterologous β-1,4-galactosyltransferase, the enzyme being GalT from Helicobacter pylori,

[0286] iii. at least one nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyltransferase, the enzyme being LgtA from Neisseria meningitidis, and

[0287] b) culturing the genetically modified cell in a medium containing a carbon source and in the presence of lactose or LNT-II, and

[0288] c) producing said sialylated human milk oligosaccharides (HMO), in particular LST-c, by said genetically modified cells, and

[0289] d) recovering sialylated human milk oligosaccharides (HMO), in particular LST-c, from the culture medium and / or genetically modified cells.

[0290] Specifically, the present invention relates to a method for producing 6'SL and LST-c, the method comprising:

[0291] a) obtaining a genetically modified cell, said cell comprising

[0292] i. A recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is selected from: Shal, comprising or consisting of an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 1; HAC1268, comprising or consisting of an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 2; Valg2, comprising or consisting of an amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 3; Pmult, comprising or consisting of an amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 5. NO:4 having at least 80% identity or consisting of an amino acid sequence, and Plst6_119 comprising or consisting of an amino acid sequence of SEQ ID NO:5 having at least 80% identity with SEQ ID NO:5, and

[0293] ii. at least one nucleic acid sequence encoding a heterologous β-1,4-galactosyltransferase, such as GalT from Helicobacter pylori,

[0294] iii. Optionally, a nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyltransferase, such as LgtA from Neisseria meningitidis, and

[0295] b) culturing the genetically modified cell in a medium containing a carbon source and in the presence of lactose, and

[0296] c) producing said sialylated human milk oligosaccharides (HMO) 6'SL and LST-c by said genetically modified cells, and

[0297] d) recovering sialylated human milk oligosaccharides (HMO) 6'SL and LST-c from the culture medium and / or genetically modified cells.

[0298] Specifically, the present invention relates to a method for producing 6'SL and LST-c, the method comprising:

[0299] a) obtaining a genetically modified cell, said cell comprising

[0300] i. a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is Shal, which comprises or consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO: 1, and

[0301] ii. at least one nucleic acid sequence encoding a heterologous β-1,4-galactosyltransferase, the enzyme being GalT from Helicobacter pylori,

[0302] iii. at least one nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyltransferase, the enzyme being LgtA from Neisseria meningitidis, and

[0303] b) culturing the genetically modified cell in a medium containing a carbon source and in the presence of lactose, and

[0304] c) producing said sialylated human milk oligosaccharides (HMO) 6'SL and LST-c by said genetically modified cells, and

[0305] d) recovering sialylated human milk oligosaccharides (HMO) 6'SL and LST-c from the culture medium and / or genetically modified cells.

[0306] Specifically, the present invention relates to a method for producing 6'SL and LST-c, the method comprising:

[0307] a) obtaining a genetically modified cell, said cell comprising

[0308] i. a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is HAC1268, which comprises or consists of the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO: 2; and

[0309] ii. at least one nucleic acid sequence encoding a heterologous β-1,4-galactosyltransferase, the enzyme being GalT from Helicobacter pylori,

[0310] iii. at least one nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyltransferase, the enzyme being LgtA from Neisseria meningitidis, and

[0311] b) culturing the genetically modified cell in a medium containing a carbon source and in the presence of lactose, and

[0312] c) producing said sialylated human milk oligosaccharides (HMO) 6'SL and LST-c by said genetically modified cells, and

[0313] d) recovering sialylated human milk oligosaccharides (HMO) 6'SL and LST-c from the culture medium and / or genetically modified cells.

[0314] Specifically, the present invention relates to a method for producing 6'SL and LST-c, the method comprising:

[0315] a) obtaining a genetically modified cell, said cell comprising

[0316] i. a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is Valg2, which comprises or consists of the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO: 3, and

[0317] ii. at least one nucleic acid sequence encoding a heterologous β-1,4-galactosyltransferase, the enzyme being GalT from Helicobacter pylori,

[0318] iii. at least one nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyltransferase, the enzyme being LgtA from Neisseria meningitidis, and

[0319] b) culturing the genetically modified cell in a medium containing a carbon source and in the presence of lactose, and

[0320] c) producing said sialylated human milk oligosaccharides (HMO) 6'SL and LST-c by said genetically modified cells, and

[0321] d) recovering sialylated human milk oligosaccharides (HMO) 6'SL and LST-c from the culture medium and / or genetically modified cells.

[0322] Specifically, the present invention relates to a method for producing 6'SL and LST-c, the method comprising:

[0323] a) obtaining a genetically modified cell, said cell comprising

[0324] i. a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is pmult, which comprises or consists of the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO: 4, and

[0325] ii. at least one nucleic acid sequence encoding a heterologous β-1,4-galactosyltransferase, the enzyme being GalT from Helicobacter pylori,

[0326] iii. at least one nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyltransferase, the enzyme being LgtA from Neisseria meningitidis, and

[0327] b) culturing the genetically modified cell in a medium containing a carbon source and in the presence of lactose, and

[0328] c) producing said sialylated human milk oligosaccharides (HMO) 6'SL and LST-c by said genetically modified cells, and

[0329] d) recovering sialylated human milk oligosaccharides (HMO) 6'SL and LST-c from the culture medium and / or genetically modified cells.

[0330] Specifically, the present invention relates to a method for producing 6'SL and LST-c, the method comprising:

[0331] a) obtaining a genetically modified cell, said cell comprising

[0332] i. a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is plst6_119, which comprises or consists of the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO:5, and

[0333] ii. at least one nucleic acid sequence encoding a heterologous β-1,4-galactosyltransferase, the enzyme being GalT from Helicobacter pylori,

[0334] iii. at least one nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyltransferase, the enzyme being LgtA from Neisseria meningitidis, and

[0335] b) culturing the genetically modified cell in a medium containing a carbon source and in the presence of lactose, and

[0336] c) producing said sialylated human milk oligosaccharides (HMO) 6'SL and LST-c by said genetically modified cells, and

[0337] d) recovering sialylated human milk oligosaccharides (HMO) 6'SL and LST-c from the culture medium and / or genetically modified cells.

[0338] Cultivation or fermentation (used interchangeably herein) in a controlled bioreactor generally includes (a) a first phase of exponential cell growth in a carbon source-assured medium, and (b) a second phase of cell growth in a medium operated under carbon limitation, wherein a carbon source is continuously added along with an acceptor oligosaccharide (e.g., lactose) to allow the formation of HMO products during this phase. Carbon (sugar) limitation refers to the phase in the fermentation where the growth rate is dynamically controlled by the carbon source (sugar) concentration in the culture broth, which in turn is determined by the rate of carbon addition to the fermentor (sugar feed-rate).

[0339] The terms "manufacturing" or "manufacturing scale" or "large-scale production" or "large-scale fermentation" are used interchangeably and in the meaning of the invention define fermentations with a minimum volume of 100 L, such as 1000 L, such as 10000 L, such as 100000 L, such as 200000 L of culture broth. In general, a "manufacturing scale" process is defined as one capable of processing large production quantities of the HMO product of interest, which, for example, in the case of therapeutic compounds or compositions, meets the following requirements: toxicity testing, clinical trials and market supply. In addition to the large volume, the manufacturing scale process is characterized by the use of a technical system of a bioreactor (fermenter) equipped with means for stirring, aeration, nutrient feeding, monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.) compared to simple laboratory scale processes such as shake flask cultivation. 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 to predict the behavior of the system in the complex environment of a bioreactor.

[0340] At the end of the culture, the oligosaccharides as products can be accumulated in the intracellular and extracellular matrix, or exported into the culture medium.

[0341] The method according to the invention comprises culturing genetically engineered microbial cells in a culture medium designed to support the growth of microorganisms and comprising one or more carbohydrate sources or only carbon sources, for example selected from glucose, sucrose, fructose, xylose and glycerol. In one or more exemplary embodiments, the culture medium is supplemented with one or more energy and carbon sources selected from glycerol, sucrose and glucose.

[0342] With regard to suitable cell culture media used in the fermentation process, there are no restrictions. The culture medium may be semi-defined, ie, containing complex media compounds (eg, yeast extract, soy peptone, casamino acids, etc.), or may be chemically defined, without any complexing compounds.

[0343] In one or more exemplary embodiments, the culture medium contains sucrose as the sole carbon and energy source. In one or more exemplary embodiments, the genetically engineered cells contain one or more heterologous nucleic acid sequences encoding one or more heterologous polypeptides that enable the use of sucrose as the sole carbon and energy source for the genetically engineered cells.

[0344] In one or more exemplary embodiments, the genetically engineered cell comprises a PTS-dependent sucrose utilization system further comprising scrYA and scrBR operons as described in WO2015 / 197082 (incorporated herein by reference).

[0345] After carrying out the method of the present invention, the produced sialylated HMOs can be collected from the cell culture or fermentation broth in a conventional manner.

[0346] Recycling / Harvesting

[0347] Sialyl human milk oligosaccharides (HMOs) are recovered from culture medium and / or genetically modified cells. In this article, the term "recovery" is used interchangeably with the term "harvest". "Recovery" and "harvest" in this context both refer to the collection of produced HMOs from the culture / fermentation broth after fermentation is terminated. In one or more exemplary embodiments, it may include collecting HMOs contained in both the biomass (i.e., host cells) and the culture medium, i.e., before / without separation of the fermentation broth from the biomass. In other embodiments, the produced HMOs can be collected separately from the biomass and the fermentation broth, i.e., after / after the separation of the biomass from the culture medium (i.e., the fermentation broth).

[0348] The separation of cells from the culture medium can be performed by any method known to those skilled in the art, such as any suitable type of centrifugation or filtration. The separation of cells from the culture medium can be performed immediately after harvesting the fermentation broth, or at a later stage after storing the fermentation broth under appropriate conditions. Recovering the produced HMO from the remaining biomass (or total fermentation broth) includes extraction from the biomass (i.e., the production cells).

[0349] After recovery from fermentation, HMOs can be used for further processing and purification.

[0350] The HMOs may be purified according to procedures known in the art, such as those described in WO2017 / 182965 or WO2017 / 152918, the latter of which describes the purification of sialylated HMOs. In an embodiment, LST-c is purified to produce at least 75% pure LST-c, such as at least 80% pure LST-c, such as at least 85% pure LST-c, such as at least 90% pure LST-c, such as at least 95% pure LST-c. The purified HMOs may be used as nutritional products, such as early life nutritional products, dietary supplements or other nutritional products, pharmaceuticals, or for any other purpose, such as for research.

[0351] manufactures

[0352] The term "manufactured product" refers to one or more HMOs intended for use as one or more product HMOs, depending on the use of the genetically engineered cell or nucleic acid construct. The various products are described above.

[0353] Thus, the finished product can be a HMO mixture comprising or consisting essentially of LST-c, LNnT, 6'SL, and pLNnH. Thus, in an embodiment, LST-c is in the range of 10 to 45 mol %, such as 10-30 mol %, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 1, 42, 43, 44 or 45 mol %, LNnT is in the range of 25-70 mol %, for example 40-70 mol %, such as 25, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 mol %, 6'SL is in the range of 0 to 65 mol %, such as 0-30 mol %, such as in the range of 0.1-25 mol %, such as 0, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 mol % and pLNnH is in the range of 1-20 mol %, such as 3-20 mol %, for example 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mol %.

[0354] In one embodiment, the HMO mixture comprises or consists essentially of LST-c at 15-45 mol % of the mixture, LNnT at 25-75 mol % of the mixture, 6'SL at 0 to 8 mol % of the mixture, and pLNnH at 1-20 mol % of the mixture.

[0355] In another embodiment, the HMO mixture comprises or consists essentially of LST-c at 15-30 mol % of the mixture, LNnT at 25-65 mol % of the mixture, 6'SL at 15 to 65 mol % of the mixture, and pLNnH at 1-10 mol % of the mixture.

[0356] In a further embodiment, the mixture is produced according to the method of the invention, wherein the HMO mixture is purified such that it contains less than 15% (w / w) lactose, such as less than 10% (w / w) lactose, such as less than 5% (w / w) lactose, such as less than 2% (w / w) lactose.

[0357] Advantageously, the methods disclosed herein both reduce the ratio of byproducts to products and increase the overall yield of products (and / or total HMOs). The lower byproduct formation relative to product formation helps increase product yields and improves the efficiency of the production and product recovery processes, thereby providing a superior HMO manufacturing process.

[0358] The finished product may be a powder, composition, suspension or gel comprising one or more HMOs.

[0359] sequence

[0360] This application contains a Sequence Listing in both text and electronic formats, which is incorporated herein by reference.

[0361] An overview of the SEQ ID NOs used in this application can be found in Table 1 (α-2,6-sialyltransferase protein sequences), Table 2 (promoter sequences) and Table 4 (α-2,6-sialyltransferase DNA sequences), additional sequences described in this application are the DNA sequence encoding the neuBCA operon from Campylobacter jejuni (SEQ ID NO:25) and the β-1,3-N-acetyl-glucosaminyltransferase LgtA from N. meningitidis (SEQ ID NO:26), the β-1,4-galactosyltransferase galT from Helicobacter pylori (H. pylori) (SEQ ID NO:27) and the DNA sequences encoding the codon-optimized individual members of the neuBCA operon from Campylobacter jejuni, namely neuA (SEQ ID NO:52), neuB (SEQ ID NO:53) and neuC (SEQ ID NO:54).

[0362] project

[0363] The following items describe various embodiments of the present disclosure.

[0364] 1. A genetically modified cell comprising a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the cell is capable of producing one or more HMOs, and wherein at least 10% of the total molar HMO content produced by the cell is LST-c.

[0365] 2. The genetically modified cell according to embodiment 1, wherein the α-2,6-sialyltransferase is selected from the group consisting of:

[0366] a. Shal, which comprises or consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80% identity with SEQ ID NO: 1,

[0367] b. HAC1268, which comprises or consists of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 80% identity with SEQ ID NO: 2, and

[0368] c. Valg2 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80% identity with SEQ ID NO: 3.

[0369] 3. The genetically modified cell according to any one of embodiments 1 or 2, wherein the cell produces LST-c and 6'SL when lactose is used as the initial substrate for HMO formation.

[0370] 4. The genetically modified cell according to any one of the preceding embodiments, wherein the recombinant nucleic acid sequence encoding α-2,6-sialyltransferase is present in more than one copy, such as two copies, three copies, four copies.

[0371] 5. The genetically modified cell according to any one of the preceding embodiments, wherein the recombinant nucleic acid sequence encoding α-2,6-sialyltransferase is integrated into the genome of the genetically modified cell.

[0372] 6. The genetically modified cell according to any one of the preceding embodiments, wherein the α-2,6-sialyltransferase is Valg2 or Sha1, and the 6'SL:LST-c ratio is between 1:1 and 3:1.

[0373] 7. The genetically modified cell according to any of the preceding embodiments, wherein the α-2,6-sialyltransferase is HAC1268 and the LST-c:6'SL ratio is higher than 2:1, preferably higher than 3.5:1, preferably higher than 15:1.

[0374] 8. A genetically modified cell according to any of the preceding embodiments, wherein the sialyltransferase is controlled by a promoter selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 40, 49, 37, 38 and 39, respectively) and variants thereof, and wherein the promoter is preferably a strong promoter selected from SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 and 44.

[0375] 9. The genetically modified cell according to any one of the preceding embodiments, wherein the cell further comprises a recombinant nucleic acid sequence encoding β-1,4-galactosyltransferase.

[0376] 10. The genetically modified cell according to embodiment 9, wherein the genetically modified cell further comprises a recombinant nucleic acid sequence encoding β-1,3-N-acetyl-glucosaminyltransferase.

[0377] 11. A genetically modified cell according to any one of embodiments 9 or 10, wherein the β-1,3-N-acetyl-glucosaminyltransferase is LgtA from Neisseria meningitidis (SEQ ID NO: 26) and the β-1,4-galactosyltransferase is GalT from Helicobacter pylori (SEQ ID NO: 27).

[0378] 12. The genetically modified cell according to any one of the preceding embodiments, wherein the cell comprises a biosynthetic pathway for producing sialic acid sugar nucleotides.

[0379] 13. The genetically modified cell according to embodiment 12, wherein the sialic acid sugar nucleotide is CMP-Neu5Ac, and the pathway for producing sialic acid sugar nucleotide is encoded by a nucleic acid sequence encoding neuBCA of Campylobacter jejuni (SEQ ID NO: 25).

[0380] 14. The genetically modified cell according to embodiment 12 or 13, wherein the sialic acid sugar nucleotide pathway is encoded by a high copy plasmid carrying the neuBCA operon.

[0381] 15. The genetically modified cell according to embodiments 12 to 14, wherein the pathway for producing sialic acid sugar nucleotides is encoded by genomically integrated neuA, neuB and neuC, wherein these three genes are preferably under the control of separate promoters.

[0382] 16. The genetically modified cell according to any one of the preceding embodiments, wherein the modified cell is a microorganism.

[0383] 17. A genetically modified cell according to any one of the preceding embodiments, wherein the modified cell is a bacterium or a fungus.

[0384] 18. A genetically modified cell according to embodiment 17, wherein the fungus is selected from a yeast cell of the genera Komagataella, Kluyveromyces, Yarrowia, Pichia, Saccaromyces, Schizosaccharomyces or Hansenula, or a filamentous fungus selected from the genera Aspargillus, Fusarium or Thricoderma.

[0385] 19. The genetically modified cell according to embodiment 17, wherein the bacterium is selected from the group consisting of Escherichia sp., Bacillus sp., Lactobacillus sp., Corynebacterium sp. and Campylobacter sp.

[0386] 20. The genetically modified cell according to embodiment 19, wherein the bacterium is Escherichia coli (E. coli).

[0387] 21. A method for producing one or more sialylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically modified cell comprising,

[0388] a. a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetyl-glucosaminyl transferase activity; and

[0389] b. a recombinant nucleic acid sequence encoding an enzyme having β-1,4-galactosyltransferase activity; and

[0390] c. A recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is selected from:

[0391] i. Shal, which comprises or consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80% identity with SEQ ID NO: 1,

[0392] ii. HAC1268, which comprises or consists of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 80% identity with SEQ ID NO: 2,

[0393] iii. Valg2 comprising or consisting of the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 80% identity with SEQ ID NO: 3;

[0394] iv. Pmult comprising or consisting of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 80% identity with SEQ ID NO:4; and

[0395] v. Plst6_119, which comprises or consists of the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 80% identity with SEQ ID NO:5, and

[0396] Wherein at least one sialylated HMO is LST-c.

[0397] 22. The method according to embodiment 21, wherein the genetically modified cell comprises at least one additional modification according to any one of embodiments 8 to 14.

[0398] 23. The method of embodiment 21 or 0, wherein at least 10% of the total molar HMO content produced by the method is LST-c.

[0399] 24. The method according to any one of embodiments 21 to 22, wherein the genetically modified cell is a microorganism according to any one of embodiments 17 to 20.

[0400] 25. The method according to any one of embodiments 21 to 24, wherein the sialylated human milk oligosaccharide (HMO) produced is LST-c and optionally 6'SL.

[0401] 26. The method according to any one of embodiments 21 to 25, wherein the α-2,6-sialyltransferase is Valg2 or Sha1, and the 6'SL:LST-c ratio is between 1:1 and 3:1.

[0402] 27. The method according to any one of embodiments 21 to 25, wherein the α-2,6-sialyltransferase is HAC1268 and the LST-c:6'SL ratio is higher than 2:1, preferably higher than 3.5:1, preferably higher than 15:1.

[0403] 28. The method according to embodiments 21 to 27, wherein the content of 6'SL produced by the cells is less than 50% of the total HMO content produced by the cells.

[0404] 29. The method according to any one of embodiments 21 to 25, wherein the method comprises culturing the genetically engineered cell in the presence of an energy source selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.

[0405] 30. The method according to any one of embodiments 21 to 28, wherein lactose is added as substrate for HMO formation during the cultivation of the genetically engineered cells.

[0406] 31. The method according to any one of embodiments 21 to 28, wherein LNT-II is added as a substrate for HMO formation during the cultivation of the genetically engineered cells.

[0407] 32. The method according to any one of embodiments 21 to 31, wherein sialylated human milk oligosaccharides (HMOs) are recovered from the culture medium and / or the genetically modified cells.

[0408] 33. A method according to claim 32, wherein LST-c is purified to produce at least 75% pure LST-c, such as at least 85%, such as at least 95% pure LST-c.

[0409] 34. A nucleic acid construct comprising a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the recombinant nucleic acid sequence is selected from:

[0410] a. Shal, which comprises or consists of the nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence having at least 80% identity with SEQ ID NO: 13,

[0411] b. HAC1268, which comprises or consists of the nucleic acid sequence of SEQ ID NO: 14 or a nucleic acid sequence having at least 80% identity with SEQ ID NO: 14, and / or

[0412] c. Valg2, which comprises or consists of the nucleic acid sequence of SEQ ID NO: 15 or a nucleic acid sequence having at least 80% identity with SEQ ID NO: 15; and

[0413] The enzyme coding sequence is controlled by a promoter sequence selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 40, 49, 37, 38 and 39, respectively) and variants thereof.

[0414] 35. A nucleic acid construct comprising a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity according to embodiment 34, for producing sialylated HMO in a host cell, wherein at least 10% of the total molar HMO content produced by the method is LST-c.

[0415] 36. The genetically modified cell according to any one of embodiments 1 to 20, for producing at least one sialylated HMO.

[0416] 37. An HMO mixture consisting essentially of LST-c, LNnT, 6'SL and pLNnH.

[0417] 38. The HMO mixture according to embodiment 36, wherein

[0418] a. LST-c is in the range of 10-45 mol % of the mixture,

[0419] b. LNnT in the range of 25-70 mol % of the mixture,

[0420] c. 6'SL is in the range of 0-65 mol % of the mixture, and

[0421] d. pLNnH is in the range of 1-20 mol % of the mixture.

[0422] 39. The HMO mixture according to embodiment 36 or 37, wherein

[0423] a. LST-c is in the range of 15-45 mol % of the mixture,

[0424] b. LNnT in the range of 25-75 mol % of the mixture,

[0425] c. 6'SL is in the range of 0-8 mol% of the mixture, and

[0426] d. pLNnH is in the range of 1-20 mol % of the mixture.

[0427] 40. The HMO mixture according to embodiment 36 or 37, wherein

[0428] a. LST-c is in the range of 10-30 mol % of the mixture,

[0429] b. LNnT in the range of 25-65 mol % of the mixture,

[0430] c. 6'SL is in the range of 15-65 mol % of the mixture, and

[0431] d. pLNnH is in the range of 1-10 mol % of the mixture.

[0432] 41. The HMO mixture according to embodiments 36 to 40, wherein the mixture is produced according to the method of embodiments 21 to 33, and wherein the HMO mixture is purified such that it contains less than 15% (w / w) lactose.

[0433] Example

[0434] method

[0435] Unless otherwise indicated, standard techniques, vectors, control sequence elements and other expression system elements known in the field of molecular biology are used for nucleic acid manipulation, transformation and expression. Such standard techniques, vectors and elements can be found below, for example: Ausubel et al. (eds.), Current Protocols in Molecular Biology (1995) (John Wiley & Sons); Sambrook, Fritsch, & Maniatis (eds.), Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, NY); Berger & Kimmel, Methods in Enzymology 152: Guide to Molecular Cloning Techniques (1987) (Academic Press); Bukhari et al. (eds.), DNA Insertion Elements, Plasmids and Episomes (1977) (Cold Spring Harbor Laboratory Press, NY); Miller, JH Experiments in molecular genetics (1972.) (Cold spring Harbor Laboratory Press, NY).

[0436] The embodiments described below are chosen to illustrate the present invention and are not intended to limit the present invention in any way.

[0437] Enzymes:

[0438] Using known α-2,6-sialyltransferases as queries and utilizing information sources such as scientific articles or databases (e.g., KEGG and CAZY databases), 19 enzymes were collected by a computer selection method based on protein BLAST search.

[0439] Table 3. List of enzymes tested within the framework of the present invention

[0440]

[0441] *Compared to the GenBank sequence, the sequences used in this application may be truncated at the N-terminus or the C-terminus.

[0442] strain

[0443] 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.

[0444] Methods for inserting a gene of interest into the E. coli genome are well known to those skilled in the art. Gene cassettes can be inserted into the E. coli chromosome by gene gorging using specific selection marker genes and screening methods (see, e.g., Herring and Blattner 2004 J. Bacteriol. 186: 2673-81 and Warming et al. 2005 Nucleic Acids Res. 33 (4): e36).

[0445] The MDO strain was further engineered to produce a LNnT producing strain by chromosomal integration of β-1,3-GlcNAc transferase (LgtA from Neisseria meningitidis, homologous to NCBI Accession No. WP_033911473.1, shown as SEQ ID NO: 26) and β-1,4-galactosyltransferase (GalT from Helicobacter pylori, homologous to GenBank ID WP_001262061.1, shown as SEQ ID NO: 27), both of which were controlled by the PglpF promoter (SEQ ID NO: 40), and the strain was named LNnT strain.

[0446] A codon-optimized DNA sequence encoding a single α-2,6-sialyltransferase was genomically integrated into the LNnT strain. In addition, each strain was transformed with a high-copy plasmid under the control of the Plac promoter carrying the neuBCA operon from Campylobacter jejuni (SEQ ID NO: 25). The neuBCA operon encodes all enzymes required to form activated sialic acid sugar nucleotides (CMP-Neu5Ac). CMP-Neu5Ac serves as a donor for the expected glycosyltransferase reaction, which is promoted by the studied α-2,6-sialyltransferase, i.e., transferring sialic acid from the activated sugar CMP-Neu5Ac to the terminal galactose (acceptor) of LNnT to form LST-c.

[0447] Table 4 provides the genotypes of the background strain (MDO), the LNnT strain, and the α-2,6-sialyltransferase expressing strain capable of producing LST-c.

[0448] Table 4. Genotypes of strains capable of producing LST-c used in this example

[0449]

[0450] *2,6ST is the abbreviation for α-2,6-sialyltransferase, and the sequence is inserted into the genome of the host strain.

[0451] Deep hole measurement

[0452] Use 4 days scheme to screen strains in 96 deep well plates. Within the first 24 hours, pre-cultures grow to high density, and are subsequently transferred to the culture medium that allows inducible gene expression and product formation. More specifically, on the 1st day, fresh pre-cultures are prepared using the basic minimal culture medium supplemented with magnesium sulfate, thiamine and glucose. Pre-cultures are incubated for 24 hours at 34 ℃ and 1000rpm vibration, and then further transferred to new basic minimal culture medium (BMM, pH 7.5) to start main cultivation. Magnesium sulfate, thiamine, 20% glucose solution (50 microliters per 100 milliliters) and 20% lactose solution (5 milliliters per 100 milliliters) are added in the new BMM. In addition, 50% sucrose solution is also provided as a carbon source, while sucrose hydrolase (invertase) is added, so as to release glucose at a rate suitable for C restricted growth. Add IPTG (50mg / ml) inducible gene expression and ampicillin antibiotic (100mg / ml). Main culture was cultivated for 72 hours at 28 ℃ and 1000rpm vibration.

[0453] Fermentation

[0454] Escherichia coli (E. coli) strains were cultured in 250 mL fermenters (Ambr 250 bioreactor system, Sartorius) in duplicate starting with 100 mL mineral medium consisting of 25 g / L glucose, lactose, (NH4)2HPO4, KH2PO4, MgSO4 x 7H2O, KOH, NaOH, citric acid, trace element solution, defoamer and thiamine. The dissolved oxygen level was maintained at 20% by first stirring and then by airflow starting at 700 rpm (up to 4500 rpm) and 1 VVM (up to 3 VVM). The pH was maintained at 6.8 by titration with 8.5% NH4OH solution. At the start of the culture, 2% (v / v) inoculum was used from a pre-culture grown in a similar glucose-containing medium with an optical density of 2.5-5 measured at 600 nm. After glucose was depleted in the batch medium, a feed solution containing 49% (w / w) glucose, MgSO4 x 7H2O, trace metals and defoamers was continuously fed using a feed curve with an initial feed rate of 0.224 g glucose / hour, linearly increased to 0.447 g / hour over 5 hours, and then fed at a constant rate of 0.447 g / hour. The temperature was maintained at 34°C throughout the process. Additional lactose was added as a bolus 20 hours after the start of feeding 25% lactose monohydrate solution, and then added every 19 hours to prevent lactose limitation. The growth and metabolic activity and state of the cells were tracked by online measurement of reflectance and CO2 evolution rate.

[0455] Throughout the fermentation process, samples were taken to determine the concentration of HMOs, byproducts, lactose and other minor byproducts using HPLC. The total fermentation broth samples were diluted three times in deionized water and boiled for 20 minutes. They were then centrifuged at 17000 g for 3 minutes and the resulting supernatant was analyzed by HPLC.

[0456] Example 1 - In vivo LST-c synthesis

[0457] Genetically modified cells expressing individual α-2,6-sialyltransferases were screened for their ability to produce sialylated HMOLST-c.

[0458] A panel of 19 enzymes (Table 3) was compiled and tested for their ability to synthesize LST-c when introduced into genetically modified cells producing LNnT and activated sialic acid (CMP-Neu5Ac).

[0459] Genetically modified strains expressing 19 individual α-2,6-sialyltransferases (Table 3) were generated as described in the Methods section. Cells were screened in a fed-batch deep-well assay as described in the Methods section. The molar content of the individual HMOs produced by the strains was measured by HPLC. In addition, NMR analysis was performed on the LST-c fraction to confirm that it was indeed LST-c.

[0460] Table 4 lists the genotypes of the 12 strains found to produce LST-c (albeit in very small amounts), the remaining 7 strains tested did not produce any LST-c at all.

[0461] Table 7 shows the results of LST-c producing cells, ie the fraction (percent, %) of the total HMO content produced by each strain.

[0462] Table 7: Mole % of individual HMO content produced by each strain relative to the total HMO content (mM)

[0463] LST-c 6'SL LqCy HkDJ Poral_2 2.4 12.7 84.9 0.0 phot 3.3 8.5 77.8 10.4 Pd2 4.9 23.3 64.6 7.3 plst6_145 8.2 15.6 41.1 35.1 ppho1 8.4 13.1 69.9 8.6 pdam 8.9 16.3 66.3 8.5 plst6_119 10.2 16.1 66.4 7.3 pmult 10.5 8.1 76.1 5.3 valg2 11.7 17.2 64.5 6.6 HAC1268 17.9 4.8 71.5 5.8 Shal 21.2 28.5 46.4 3.9

[0464] In deep well assays with the indicated strains, no HMOs other than those shown in Table 7 were identified.

[0465] From the data in Table 7, it can be seen that five enzymes (Shal, HAC1268, Valg2, pmult and plst6_119) are able to transfer sialic acid units to the terminal galactose of the LNnT molecule to form LST-c at levels greater than 10% of the total molar content of HMO produced by each modified cell, which is greater than the amount of LST-c produced by Pdam in this experiment. Pdam is known in the prior art and has been proposed to be active against LNnT, although WO 2021 / 123113 does not provide any evidence of the level of LST-c produced. Figure 1 The mole % of LST-c produced by these five strains and the Pdam strain are shown.

[0466] Furthermore, the levels of LST-c formed by Shal, HAC1268, and Valg2 were greater than 10.5% of the total molar HMO content produced, which was higher than the level formed by plst6_119, which had been previously used to form 6'SLs in genetically modified strains but was believed to have never been shown to be able to transfer a sialic acid unit to the terminal galactose of the LNnT molecule.

[0467] HAC1268 produced almost 4 times more LST-c than 6'SL (produced by sialylation of lactose). This suggests that this enzyme has a higher activity towards LNnT as a substrate than towards lactose as a substrate. Furthermore, the HAC1268 enzyme was described as a bifunctional α-2,3 / -2,8-sialyltransferase, rather than an α-2,6-sialyltransferase, which makes it even more surprising since it is very efficient in forming LST-c.

[0468] Expression of HAC1268 in a strain producing LNnT and sialic acid resulted in 17.9% LST-c. Similarly, expression of the Shal enzyme in a strain producing LNnT and sialic acid resulted in 21.2% LST-c, but the 6'SL ratio was significantly higher. If purification of LST-c is desired, a lower 6'SL ratio is preferred because separation of two acidic HMOs (LST-c and 6'SL) is more challenging than separation of a single acidic HMO (LST-c) from a neutral HMO (LNnT and pLNnH).

[0469] Example 2 - Fermentation using Shal and HAC1268 α-2,6-sialyltransferase strains

[0470] To confirm the high levels of LST-c observed in the deep well assays of the Sha1 and HAC1268 strains of Example 1, fermentations were performed with these four strains as described in the Methods section above.

[0471] The results are shown in Table 8.

[0472] Table 8: Mole % of individual HMO content produced by each strain relative to the total HMO content

[0473] strain ref. HkDJ LNT-II LqCy 6'SL LST-c Shal 3.8 0.0 33.9 26.6 26.7 HAC1268 16.8 0.0 67.2 0.0 15.4

[0474] Apart from the HMOs shown in Table 8, no other HMOs were identified during the fermentation.

[0475] As can be seen from the data in Table 8, the LST-c fraction of the strain expressing Shal was higher when cultivated in the fermenter compared to the deep well assay presented in Example 1, indicating that the cells expressing Shal were able to produce LST-c at levels greater than 26% of the total HMO produced by the strain. During the fermentation, the cells expressing HAC1268 provided LST-c levels very similar to the deep well assay, but a big benefit was the surprising absence of 6'SL in the final mixture, which made it much easier to purify LST-c since the only charged HMO was LST-c.

[0476] Example 3

[0477] This example investigated whether changes in the CMP-Neu5Ac production system in the strains and additional copies of the α-2,6-sialyltransferase of the Sha1, HAC1268 and Valg2 strains would improve LST-c production and alter the ratio of LST-c:6'SL.

[0478] The strain was constructed by inserting the neuA (SEQ ID NO:52), neuB (SEQ ID NO:53) and neuC (SEQ ID NO:54) genes into separate genomic integration sites in the genome under the control of the PglpF promoter (SEQ ID NO:40) to generate a strain capable of producing CMP-Neu5Ac from the genome rather than from a plasmid.

[0479] The genotypes of the tested strains are shown in Table 9. MP0 was used as the background strain for this example, and MP1 to MP8 all had the same genetic background as MP0, but with additional modifications as shown in the table.

[0480] Table 9: Genotypes of strains used in this example

[0481]

[0482]

[0483] 2x indicates that there are two copies of the nucleic acid sequence encoding the indicated enzyme in the genome

[0484] Deep well assay for product formation

[0485] The strains were screened in 96-well deep well plates using a 3-day protocol to evaluate HMO production. Fresh pre-cultures were grown to high density within the first 24 hours. More specifically, fresh pre-cultures were prepared using minimal basal medium supplemented with magnesium sulfate, thiamine, and glucose. The pH was adjusted to pH 7.0 with NaOH. The pre-cultures were incubated at 34°C and 1000 rpm shaking for 24 hours.

[0486] To produce HMO, aliquots of the pre-culture were transferred to new deep-well plates with basal minimal medium (BMM7, pH 7.5) to start the main culture. The new BMM was supplemented with thiamine and magnesium sulfate, 0.01% glucose, 2% lactose, 1.75% maltodextrin, thiamine and glucoamylase to optimize the hydrolysis of maltodextrin. The HMO main culture was incubated at 28°C and 1000rpm shaking for 48 hours. The assay was usually repeated three times.

[0487] After incubation, the main culture was boiled and centrifuged. The samples were analyzed by HPLC.

[0488] result

[0489] The strains in Table 9 were tested in the deep well assay described in connection with this example. Table 10 shows the average levels of the following HMOs for at least 3 replicates in each strain: LNT-II, LNnT, pLNnH, 6'SL, LST-c. In addition, an LST-c isomer was identified that is essentially a LNnT molecule with the sialic acid in a different part than the terminal galactose, suggesting that some α-2,6-sialyltransferases have some affinity for other parts of LNnT.

[0490] Table 10 shows the results of LST-c producing cells, ie, the total HMO + LST-c isomer content (percent, %) produced by each strain.

[0491] Table 10: Mole % of the content of individual HMOs / isomers relative to the total content of the listed molecules produced by each strain

[0492]

[0493] From these data it can be seen that switching CMP-Neu5Ac formation from a plasmid-based system (Example 1) to a genomically integrated system (this Example) still allows for efficient sialylation of LNnT for all three enzymes. As the analysis was performed using a modified deep well assay compared to Example 1, the data cannot be directly compared.

[0494] From Table 10, it can be observed that for all three enzymes, when introduced into strains containing a genomically integrated CMP-Neu5Ac production pathway and a CMP-Neu5Ac production pathway on a plasmid, the formation of LST-c was increased, as MP2 produced 4% more LST-c than MP1, MP5 produced 5% more LST-c than MP4, and MP8 produced 3% more LST-c than MP7. However, for Sha1 and Valg2, the increase in CMP-Neu5Ac formation also significantly increased the amount of 6'SL formed, resulting in LST-c:6'SL ratios of 0.44 and 0.40, respectively, while for HAC1268, the LST-c:6'SL ratio was 17.2. If pure LST-c is desired, a high LST-c:6'SL ratio is a significant benefit, as it is more difficult to separate 6'SL from LST-c than to separate any neutral HMO from LST-c.

[0495] Another benefit of HAC1268 appears to be that this enzyme produces very little of the LST-c isomer compared to Shal and Valg2. Of particular note, even when the number of copies of the nucleic acid sequence encoding the enzyme was increased to two (MP6), the production of the isomer did not increase in the HAC1268 strain, whereas for the Shal strain, the production of the LST-c isomer increased when additional copies of the nucleic acid sequence encoding the enzyme were added to the high-producing CMP-Neu5Ac strain (MP3 compared to MP2).

[0496] Insertion of an additional copy of the nucleic acid sequence encoding α-2,6-sialyltransferase further increased LST-c production in the HAC1268 strain by another 10% to a total of 39% (MP6), while for the Shal strain the increase in enzyme activity appeared to result in increased 6'SL formation and decreased LST-c (MP3). This clearly demonstrates that HAC1268 has a very high specificity for the terminal galactose moiety on LNnT, and low specificity for both lactose and other moieties in LNnT, making it an ideal enzyme for producing pure LST-c.

[0497] If a mixture of 6'SL and LST-c is desired, Shal and valg2 appear to be very suitable enzymes, where the ratio of 6'SL to LST-c can be adjusted by regulating the enzyme activity (e.g. by increasing the number of copies of the nucleic acid sequence encoding the enzyme) or by regulating the formation of CMP-Neu5Ac. By testing three Shal strains and two valg2 strains, the ratio of 6'SL:LST-c can be in the range of 1.5:1 to 3:1.

Claims

1. A genetically modified cell comprising a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the cell is capable of producing one or more HMOs, and wherein at least 10% of the total molar HMO content produced by the cell is LST-c.

2. The genetically modified cell according to claim 1, wherein the α-2,6-sialyltransferase is selected from: a. HAC1268, which comprises or consists of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 80% identity with SEQ ID NO: 2, b. Shal, which comprises or consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80% identity with SEQ ID NO: 1, and c. Valg2 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80% identity with SEQ ID NO:

3.

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

4. The genetically modified cell according to claim 3, wherein the genetically modified cell further comprises a recombinant nucleic acid sequence encoding β-1,3-N-acetyl-glucosaminyltransferase.

5. The genetically modified cell of any one of the preceding claims, wherein the cell comprises a biosynthetic pathway for producing a sialic acid sugar nucleotide such as CMP-Neu5Ac.

6. A genetically modified cell according to any one of the preceding claims, wherein the modified cell is a microorganism, such as a bacterium or a fungus.

7. The genetically modified cell according to claim 6, wherein the fungus is selected from a yeast cell of the genera Komagataella, Kluyveromyces, Yarrowia, Pichia, Saccaromyces, Schizosaccharomyces or Hansenula, or a filamentous fungus selected from the genera Aspargillus, Fusarium or Thricoderma, or the bacterium is selected from the genera Escherichia, Bacillus, Lactobacillus, Corynebacterium and Campylobacter.

8. The genetically modified cell according to claim 7, wherein the bacterium is Escherichia coli (E. coli).

9. A method for producing one or more sialylated human milk oligosaccharides (HMOs), the method comprising culturing a genetically modified cell, the genetically modified cell comprising, a. A recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the enzyme is selected from: i. HAC1268, which comprises or consists of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 80% identity with SEQ ID NO: 2, ii. Shal, which comprises or consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80% identity with SEQ ID NO: 1, iii. Valg2, which comprises or consists of the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80% identity with SEQ ID NO: 3; iv. Pmult comprising or consisting of the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 80% identity with SEQ ID NO: 4; and v. Plst6_119, which comprises or consists of the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 80% identity with SEQ ID NO:5, and b. a recombinant nucleic acid sequence encoding an enzyme having β-1,4-galactosyltransferase activity; and c. Optionally, a recombinant nucleic acid sequence encoding an enzyme having β-1,3-N-acetyl-glucosaminyltransferase activity; and Wherein at least one sialylated HMO is LST-c.

10. The method of claim 9, wherein the genetically modified cell is a microorganism according to any one of claims 6 to 8, and the genetically modified cell optionally comprises at least one additional modification according to any one of claims 4 or 5.

11. A process according to claim 9 or 10, wherein at least 10% of the total molar HMO content produced by the process is LST-c.

12. The method according to any one of claims 9 to 11, wherein the sialylated human milk oligosaccharides (HMOs) produced are LST-c and optionally 6'SL.

13. The method according to any one of claims 9 to 12, wherein lactose or LNT-II is provided as substrate for the formation of HMOs during the cultivation of the genetically engineered cells.

14. The method of any one of claims 9 to 13, wherein the sialylated human milk oligosaccharides (HMOs) are recovered from the culture medium and / or the genetically modified cells.

15. The method of claim 14, wherein LST-c is purified to produce at least 75% pure LST-c.

16. A nucleic acid construct comprising a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity, wherein the recombinant nucleic acid sequence is selected from: a. HAC1268, which comprises or consists of the nucleic acid sequence of SEQ ID NO: 14 or a nucleic acid sequence having at least 80% identity with SEQ ID NO: 14, b. Shal, which comprises or consists of the nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence having at least 80% identity with SEQ ID NO: 13, and / or c. Valg2, which comprises or consists of the nucleic acid sequence of SEQ ID NO: 15 or a nucleic acid sequence having at least 80% identity with SEQ ID NO: 15; and The enzyme coding sequence is controlled by a promoter sequence selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 40, 49, 37, 38 and 39, respectively) and variants thereof.

17. Use of a nucleic acid construct comprising a recombinant nucleic acid sequence encoding an enzyme having α-2,6-sialyltransferase activity according to claim 16 for producing sialylated HMOs in a host cell, wherein at least 10% of the total molar HMO content produced by the method is LST-c.

18. Use of the genetically modified cell according to any one of claims 1 to 8 in the production of sialylated HMOs.

19. An HMO mixture consisting essentially of LST-c, LNnT, 6'SL and pLNnH.

20. The HMO mixture of claim 19, wherein a. LST-c is in the range of 10-45 mol % of the mixture, b. LNnT in the range of 25-70 mol % of the mixture, c. 6'SL is in the range of 0-65 mol % of the mixture, and d. pLNnH is in the range of 1-20 mol % of the mixture.

21. The HMO mixture of claim 19 or 20, wherein the mixture is produced according to the method of claims 9 to 15, and wherein the HMO mixture is purified such that it contains less than 15% (w / w) lactose.

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