Genetically engineered strain for reducing formation of by-products

Abstract

The present disclosure relates to the production of oligosaccharides, in particular human milk oligosaccharides (HMO), using genetically engineered cells of phosphoglycerotransferases I and II and / or phosphoethanolamine transferases and / or dextran biosynthetic protein C with reduced or completely lost function to reduce oligosaccharide byproducts and / or increase oligosaccharide yield.

Description

Technical Field

[0001] The present disclosure relates to the production of oligosaccharides, in particular human milk oligosaccharides (HMOs), and the genetic engineering of suitable cells for producing oligosaccharides by modifying the osmo-regulatory periplasmic glucan biosynthetic machinery of the cells, e.g., so that the formation of phosphoglycerol and / or phosphoethanolamine and / or succinyl conjugation byproducts is reduced. Background Art

[0002] The design and construction of bacterial cell factories for the production of oligosaccharides, such as human milk oligosaccharides (HMOs), are constantly evolving to facilitate and improve production, including main product yield, by-product profile, lactose consumption, energy efficiency, strain stability, etc. Some of this work is summarized in Petschacher and Nidetzkya 2016 Journal of Biotechnology 235:61–83, Bych et al. 2019 Current Opinion in Biotechnology 56:130–137, and Deng et al. 2019 ACSSynth.Biol. 8:2418-2427.

[0003] Gram-negative bacteria, such as the Enterobacteriaceae, possess an osmoregulatory periplasmic glucan (OPG) biosynthetic machinery that produces periplasmic oligosaccharides. The OPG structures found in different bacterial species share several common features: (i) D-glucose is the only constituent sugar; (ii) the glucose units are at least partially linked by β-glycosidic bonds; (iii) the number of glucose units is limited (5 to 24); and (iv) in most cases, but with a few exceptions, the OPG concentration in the periplasm increases with decreasing environmental osmotic pressure. In many, but not all, species, the OPG backbone can be modified to varying degrees with a variety of substituents. These substituents appear to belong to two categories: (i) residues derived from membrane phospholipids, such as phosphoglycerol, phosphoethanolamine, or phosphocholine; and (ii) residues derived from intermediary metabolism, such as acetyl, succinyl, and methylmalonyl groups (Bontemps-Gallo 2017 EcoSalPlus 72) ESP-0001-2017, the author of this article Figure 2 Included in this article as Figure 1 Jacson et al. (Journal of Bacteriology, 160:976-981) completed the characterization of phosphoglycerol transferases I and II in 1984 by deleting opgB (mdoB) in Escherichia coli, resulting in the synthesis of endogenous membrane-derived oligosaccharides without phosphoglycerol residues.

[0004] In the MDO background strain used in the examples herein, the mdoH (opgH) gene has been deleted (see the methods section herein) to prevent the presence of large amounts of unwanted oligosaccharides (byproducts) in the HMO products produced by the cells, as well as to divert metabolic energy to the formation of other sugar nucleotide precursors required for HMO synthesis. WO 2021 / 160830 describes glycominimized bacterial host cells, characterized by the deletion of the genes opgG and opgH, which are responsible for the formation of the OPG core structure, and are therefore also referred to as OPG backbone enzymes. Summary of the invention

[0005] In a first aspect, the present disclosure relates to a genetically engineered bacterial cell having reduced or completely lost function of phosphoglycerol transferases I and II and / or phosphoethanolamine transferase and / or OPG biosynthetic protein C. The cell is suitable for the production of hetero-oligosaccharides and therefore comprises at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase.

[0006] Preferably, the genes encoding phosphoglycerol transferases I and II (opgB) and / or the genes encoding phosphoethanolamine transferases (opgE) and / or the genes encoding OPG biosynthesis protein C (opgC), or their functional equivalents / homologous genes are deleted or become dysfunctional. In an embodiment, such genetically engineered bacterial cells are capable of producing or produce one or more oligosaccharides, such as one or more human milk oligosaccharides (HMOs) having a lacto-N-triose II (LNT-II) core structure. Preferably, such HMOs are selected from LNT, LNnT, LNT-II, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, pLNH, F-pLNH I, pLNnH, LST a, LST b, LST c, DSLNT.

[0007] Of particular interest is that the genetically engineered bacterial cells produce lower amounts of phosphoglycerol-oligosaccharides and / or phosphoethanolamine-oligosaccharides and / or succinyl-oligosaccharides when compared to cells in which phosphoglyceroltransferase I and II and / or phosphoethanolaminetransferase and / or OPG biosynthesis protein C (opgC) function is not reduced or completely lost.

[0008] Additionally, at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase in the genetically engineered bacterial cell may comprise one or more heterologous nucleic acids encoding one or more heterologous glycosyltransferases selected from the group consisting of β-1,3-N-acetyl-glucosaminyltransferase, β-1,3-galactosyltransferase, and β-1,4-galactosyltransferase.

[0009] In additional embodiments, the genetically engineered bacterial cell is a Gram-negative bacterium.

[0010] In a specific embodiment, the engineered bacterial cell is an Escherichia sp., Klebsiella sp., Salmonella sp., or Shigella sp., such as Escherichia coli.

[0011] In a second aspect, the present disclosure relates to a method for producing an oligosaccharide product, wherein the method comprises,

[0012] a. providing a genetically engineered cell as described herein,

[0013] b. culturing the genetically engineered cells in a culture medium under conditions that allow production of the oligosaccharide product; and optionally

[0014] c. recovering the oligosaccharide product from the culture.

[0015] Preferably, the production of the desired oligosaccharide is reduced by no more than 15%, preferably by no more than 10%, and preferably is not substantially reduced, when compared to cells in which the function of phosphoglycerol transferases I and II and / or phosphoethanolamine transferase and / or OPG biosynthetic protein C is not reduced or completely lost.

[0016] In addition, compared to methods using cells containing phosphoglycerol transferases I and II and / or phosphoethanolamine transferases or their functional equivalents / homologous genes, it is expected that the method produces reduced amounts of byproduct oligosaccharides containing phosphoethanolamine and / or glycerophosphate moieties. Such byproducts can, for example, be byproduct phosphoglycerol-LNT and / or phosphoethanolamine-LNT, or phosphoglycerol-LNnT and / or phosphoethanolamine-LNnT. Thus, in a specific embodiment, the desired oligosaccharide is one or more human milk oligosaccharides (HMOs) selected from the group consisting of LNT, LNnT, LNT-II, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, pLNH, F-pLNH I, pLNnH, LST a, LST b, LST c, DSLNT, such as LNT or LNnT.

[0017] Preferably, when the desired oligosaccharide is LNT, the ratio of LNT:phosphoglycerol-LNT (w / w) produced in the culturing step according to the method is at least 500: 1, such as at least 750: 1, 1000: 1, 2000: 1 or at least 5000: 1. Along the same line, when the desired oligosaccharide is LNT, preferably, the ratio of LNT:phosphoethanolamine-LNT (w / w) produced in the culturing step according to the method is at least 50: 1, such as at least 100: 1, 300: 1, 500: 1 or such as at least 1000: 1.

[0018] A third aspect of the present disclosure relates to the use of the genetically engineered cells described herein in the production of oligosaccharide products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 An overview of the key enzymes in the osmoregulated periplasmic glucan biosynthetic machinery associated with osmoregulated periplasmic glucan (OPG) synthesis in Escherichia coli (from Bontemps-Gallo 2017 EcoSal Plus 72) ESP-0001-2017).

[0020] Figure 2 An overview of the synthesis of LNT and LNnT is given, and the byproducts phosphoglycerol-LNT (GP-LNT), phosphoethanolamine-LNT (EP-LNT), galactose-LNT, p-LNT, phosphoglycerol-LNnT (GP-LNnT), and phosphoethanolamine-LNnT (EP-LNnT), as well as GlcNAc-LNnT and pLNnH are shown.

[0021] Figure 3 Conformational structures of A) E. coli OPG with 9 glucose residues connected by β-1,2 and β-1,6 bonds and B) phosphoglycerol-LNT.

[0022] Figure 4 Chromatograms of fermentation samples of MP0 (control, top line) and MP1 (ΔopgB and ΔopgE, bottom line). Peak 11 eluted at 6.041 is LNT, peak 12 eluted at 6.716 is GP-LNT (marked with an arrow), peak 13 eluted at 8.047 is Gal-LNT, peak 14 eluted at 8.857 is pLNnH, peak 15 eluted at 9.125 is EP-LNT (marked with an arrow), and peak 16 eluted at 10.102 is galpLNH2. MP1 (bottom line) shows that MP1 does not produce byproducts EP-LNT and GP-LNT compared to MP0, which has a phosphoglycerol-LNT peak (peak 12) eluted at 6.716 minutes and a phosphoethanolamine-LNT peak (peak 15) eluted at 9.152 minutes, as indicated by arrows.

[0023] Figure 5 is the relative yield of LNT after MP0 and MP1 fermentation. The yield is relative to the maximum LNT yield obtained by MP0 fermentation.

[0024] Figure 6 is the relative EP-LNT yield (ratio of EP-LNT:LNT (w / w%)) during MP0 and MP1 fermentation.

[0025] Figure 7 is the relative yield of GP-LNT (ratio of GP-LNT:LNT (w / w%)) during the fermentation of MP0 and MP1.

[0026] Figure 8 Relative LNnT, pLNnH and total HMO production of ΔopgE (DopgE) and ΔopgB (DopgB) (MP3) and the control strain (MP2). DETAILED DESCRIPTION

[0027] The purpose of the present application is to be able to reduce the formation of unwanted oligosaccharide by-products in oligosaccharide production, while also optimizing the energy consumption of the production strain, which in turn may cause the output of the desired oligosaccharide to increase. The production of oligosaccharides is preferably the production of one or more heterologous oligosaccharides that the host cell does not naturally produce. In a preferred embodiment, the heterologous oligosaccharides produced by the genetically engineered cells comprise at least one galactose (Gal) and / or N-acetylglucosamine (GlcNAc) moiety, preferably at least one Gal and at least one GlcNAc moiety. The ratio of Gal:GlcNAc moiety is preferably between 1.5-2:1.

[0028] The genetically engineered cells described herein are preferably modified to be able to produce at least one desired heterologous oligosaccharide, such as at least one HMO. Such modification can be, for example, the expression of at least one recombinant or heterologous glycosyltransferase or the engineering of a nucleotide activated sugar pathway required to produce the desired oligosaccharide. The genetically engineered cells are preferably suitable for industrial production of the desired oligosaccharide.

[0029] A first aspect of the present invention relates to a genetically engineered Gram-negative bacterial cell having reduced or completely absent function of phosphoglycerol transferases I and II and / or phosphoethanolamine transferase, wherein the cell comprises at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase.

[0030] The present inventors have recognized that compromising the portion of the osmoregulatory periplasmic glucan biosynthetic machinery responsible for modifying the OPG backbone can increase the amount of desired oligosaccharide products and prevent the formation of undesirable oligosaccharide byproducts, particularly undesirable modifications of the primary oligosaccharide products (e.g., LNT, LNnT or other HMO products). In particular, the deletion of the opgB gene encoding phosphoglycerol transferases I and II (responsible for phosphoglycerol modification) and the opgE gene encoding phosphoethanolamine transferase (responsible for phosphoethanolamine modification) from the OPG biosynthetic machinery prevents the formation of phosphoglycerol and phosphoethanolamine conjugated byproducts, e.g., phosphoglycerol and phosphoethanolamine modifications, particularly of desired oligosaccharide products such as HMOs. Since such byproducts are similar in size to the desired product, removal of these byproducts would require additional purification steps, which may also have an impact on overall yield, e.g., due to conversion of the product HMOs into undesirable byproducts, thereby affecting overall production costs.

[0031] Therefore, when aiming to increase the yield of heterologous products in industrial-scale biological systems, avoiding side processes (e.g. leading to the formation of by-products during the production process) is of vital importance.

[0032] In the biosynthetic production of oligosaccharides, the formation of by-products is largely unavoidable due to the complexity of the cellular processes required to maintain cell viability and the excessive processes performed by cells when producing the desired oligosaccharide products. In particular, glycosyltransferases catalyze the transfer of sugar moieties from activated sugar donors to sugar or non-sugar acceptors. These glycosyltransferases are key to the biosynthesis of biological products (e.g., disaccharides, oligosaccharides and polysaccharides, glycolipids and glycoproteins). A strategy to reduce the formation of by-products is to reduce the presence of non-product oligosaccharides, which is accomplished, for example, by utilizing specific glycosyltransferases, which are highly specific to the expected acceptor oligosaccharides, see, for example, WO2024 / 013399.

[0033] In addition to non-product oligosaccharides produced by glycosyltransferases, other cellular machinery, such as oligosaccharide-modifying enzymes, are also responsible for the production of non-product oligosaccharides in certain circumstances. For example, enzymes of the osmo-regulated periplasmic glucan (OPG) biosynthetic machinery are responsible for the production of OPG backbones, such as opgG and opgH.

[0034] In Gram-negative bacteria, glycerol phosphate and ethanolamine phosphate are primarily found in phosphatidylglycerol or ethanolamine phosphate lipids in the membrane and in osmoregulatory periplasmic glucans (OPGs), where they play a role in maintaining the membrane and periplasmic charge of the bacteria and in overall resilience and virulence.

[0035] To our knowledge, the enzymes responsible for modifying the OPG backbone in the osmoregulatory periplasmic glucan (OPG) biosynthetic machinery have never been implicated in the formation of unwanted oligosaccharide byproducts.

[0036] Surprisingly, the inventors of the present application have recognized that oligosaccharides having a lacto-N-triose II (LNT-II) core structure are substrates for modifying enzymes in the OPG biosynthetic machinery. Figure 3 The conformational structures of OPG, which has nine glucose residues linked by β-1,2 and β-1,6 bonds (Bontemps-Gallo et al., Osmoregulated Periplasmic Glucans, EcoSal Plus. 2017 Jun; 7(2)), and LNT, in which the glycosyl moieties are linked by β-1,3 and β-1,4 bonds, are illustrated.

[0037] Specifically, during the production of LNT from an opgH-deleted E. coli strain, two byproducts were identified on the HPLC chromatogram of the fermentation broth: glycerophosphodiester-LNT (GP-LNT) and ethanolaminephosphodiester-LNT (EP-LNT) ( Figure 4 ). The pathways leading to the formation of these byproducts are unclear, as the production strain E. coli produces many transferases that could be responsible for the production of these two species. However, deletion of opgB and opgE resulted in the elimination of these byproducts.

[0038] Furthermore, it was observed that the deletion of opgB and opgE also had a beneficial effect on the overall production of the desired oligosaccharides, especially LNnT ( Figure 8 ), indicating that the energetic optimization obtained by deleting opgB and opgE favors the formation of additional products.

[0039] Osmoregulatory periplasmic glucan

[0040] Osmoregulatory periplasmic glucans (OPGs), formerly known as "membrane-derived oligosaccharides" (MDOs), are oligosaccharides that accumulate in the envelope of many Gram-negative bacteria. OPGs are essential for cellular adaptation to osmotic flux. OPGs contain 6 to 12 glucose residues linked by α-1,2 and α-1,6 bonds, with the major species containing 8 to 9 glucose units. OPGs are osmoregulatory in that their synthesis and accumulation are inversely proportional to the osmotic strength of the environment. OPGs contain a glucose backbone that can be substituted (modified) with various substituents such as succinyl, phosphoglycerol, and phosphoethanolamine moieties ( Figure 1 In E. coli, OPG consists of the opg gene, opgB, opgC, opgD, opgE, opgG and opgH (see Figure 1 ) and acpP (in Figure 1The OPG backbone structure is synthesized by an enzyme encoded by two gene products, OpgH and OpgG. The backbone is further modified with a phosphoglycerol moiety by two gene products, OpgB and OpgB' (phosphoglycerol transferases I and II), with a phosphoethanolamine moiety by OpgE (phosphoethanolamine transferase), and with a succinyl moiety by OpgC (biosynthetic protein C). Figure 1 ).

[0041] In the context of the present disclosure, it has been recognized that oligosaccharides with an LNT-II backbone, particularly LNT, can be modified by OpgB / B' and opgE and possibly OpgC, as described in more detail below. The modification may not occur in all strains that produce oligosaccharides with an LNT-II backbone, because for the modification to occur, the oligosaccharide must be present in the periplasm of the production cell at a sufficient concentration. Therefore, the deletion of ogpB, opgC, and opqE is most likely to be beneficial to high-yielding strains.

[0042] In one embodiment, the strain with reduced or completely lost function of phosphoglycerol transferase I and II and / or phosphoethanolamine transferase and / or OPG biosynthesis protein C produces more than 50 g / L of the desired oligosaccharides, such as more than 60 g / L of the desired oligosaccharides, such as more than 70 g / L of the desired oligosaccharides in fermentation, preferably in large-scale fermentation.

[0043] Furthermore, in strains that efficiently export the desired oligosaccharides from the periplasm to the culture medium, modification by OpgB / B', opgC and / or opqE may not be observed. Nevertheless, deletion of the opgB, opgC and opqE genes may still be beneficial in such strains, as they can lead to increased product formation, as observed for LNnT production strains (Example 2).

[0044] In one embodiment, a strain in which the function of phosphoglyceroltransferase I and II and / or phosphoethanolamine transferase and / or OPG biosynthesis protein C is reduced or completely lost produces at least 5%, such as at least 10%, such as at least 15%, such as at least 20% more desired oligosaccharides than a comparison strain in which phosphoglyceroltransferase I and II and / or phosphoethanolamine transferase and / or OPG biosynthesis protein C are not reduced (i.e., having opgB, opgC and / or opqE genes with similar or higher activity than wild-type opgB, opgC and opqE genes.

[0045] OgB

[0046] The opgB gene of E. coli encodes two phosphoglycerol transferases (OpgB and OpgB') that are responsible for transferring the phosphoglycerol moiety from the phosphoglycerol containing phospholipids to the glucose backbone of OPG. Figure 1 OpgB in the inner membrane cleaves phosphoglycerol from phospholipid-containing phosphoglycerol, and the soluble periplasmic phosphoglycerol transferase I, also known as phosphoglycerol transferase II ( Figure 1 OpgB' in E. coli transfers the phosphoglycerol residue from the phosphatidylglycerol to the OPG molecule. Thus, it is well known that the phosphoglycerol transferase I and phosphoglycerol transferase II encoded by the opgB gene in E. coli can act as transferases that recognize the glucose part in the complex glucose oligosaccharide as a suitable substrate for the transferase reaction, and these transferases transfer the phosphoglycerol part to the glucose part.

[0047] Surprisingly, as disclosed herein, the phosphoglycerol transferase encoded by the opgB gene can recognize non-glucose moieties in oligosaccharides and modify the N-acetylglucosamine (GlcNAc) moiety in LNT with the phosphoglycerol moiety (confirmed by NMR and MS analysis of the eluate eluted from the HPLC column at about 6.7 minutes). Generally speaking, when aiming to produce pure oligosaccharides (e.g., HMO LNT), phosphoglycerol-conjugated oligosaccharides are unwanted byproducts, wherein phosphoglycerol-LNT is an unwanted byproduct. The generation of such byproducts in fermentation requires additional downstream purification to remove the byproducts.

[0048] In embodiments, the gene encoding phosphoglycerol transferases I and II (opgB) is deleted or rendered dysfunctional in the genetically engineered cells described herein.

[0049] In E. coli, an example of an opgB gene comprises a nucleic acid sequence according to SEQ ID NO: 1, it being understood that nucleic acid sequences encoding phosphoglycerol transferases I and II may have homology between species and strains. In an example, the phosphoglycerol transferase I encoded by the opgB gene in E. coli comprises or consists of an amino acid sequence according to SEQ ID NO: 3. The phosphoglycerol transferase II encoded by the opgB gene in E. coli is obtained by a native N-terminal truncation of SEQ ID NO: 3 (removal of the transmembrane domain of phosphoglycerol transferase I), i.e., the phosphoglycerol transferase II comprises or consists of an amino acid sequence approximately located between positions 130 to 763 and 165 to 763 of SEQ ID NO: 3.

[0050] Thus, in embodiments, a gene functionally equivalent / homologous to the opgB gene of E. coli is deleted or rendered dysfunctional in a genetically engineered cell. The introduction of nonsense mutations that result in premature termination codons can render the transcribed polypeptide dysfunctional and can be used to generate host cells that completely lose the function of phosphoglycerol transferase I and / or phosphoglycerol transferase II. In addition, mutations in the active sites of phosphoglycerol transferases I and II can also reduce or eliminate the function of the enzymes.

[0051] In an embodiment, the gene product responsible for the production of oligosaccharides having a phosphoglycerol moiety is deleted or rendered dysfunctional in the genetically modified bacterial cell.

[0052] In an embodiment, the opgB gene is deleted from the genome of the genetically modified bacterial cell.

[0053] In an embodiment, a gene functionally equivalent / homologous to the opgB gene of E. coli is deleted or rendered dysfunctional.

[0054] In an embodiment, a functional equivalent / homolog of phosphoglycerol transferase I (OpgB) is functionally dysregulated in the genetically modified bacterial cell, and the amino acid sequence of the functional equivalent / homolog is at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identical to SEQ ID NO:3.

[0055] In an embodiment, the functional equivalent / homolog of phosphoglycerol transferase I (OpgB) is phosphoglycerol transferase II (OpgB').

[0056] As in Example 1 and Figure 6 and 7 As shown, deletion of the opgB and opgE genes in the LNT-producing strain resulted in abrogation of both phosphoglycerol-LNT and phosphoethanolamine-LNT production.

[0057] Og

[0058] The opgE gene encodes phosphoethanolamine transferase (OpgE), which catalyzes the phosphoethanolamine modification of OPG. OpgE catalyzes the transfer of a phosphoethanolamine group from phosphatidylethanolamine lipids in the membrane to OPG in the periplasm.

[0059] Surprisingly, as disclosed herein, the phosphoethanolamine transferase encoded by the opgE gene can recognize non-glucose moieties in oligosaccharides and modify the N-acetylglucosamine (GlcNAc) moieties in LNT with phosphoethanolamine moieties (confirmed by NMR and MS analysis of the eluate eluted from the HPLC column at about 9.15 minutes). Generally speaking, when aiming to produce pure oligosaccharides (e.g., HMO LNT), phosphoethanolamine-conjugated oligosaccharides are unwanted byproducts, wherein phosphoethanolamine-LNT is an unwanted byproduct. The generation of such byproducts in fermentation requires additional downstream purification to remove the byproducts.

[0060] In an embodiment, the gene encoding phosphoethanolamine transferase (opgE) is deleted or becomes dysfunctional in the genetically engineered cells described herein. The introduction of nonsense mutations that result in premature termination codons can cause dysfunctional transcribed polypeptides and can be used to generate host cells that completely lose the function of phosphoglycerol transferase I and / or phosphoglycerol transferase II. In addition, mutations in the active site of phosphoethanolamine transferase can also reduce or eliminate the function of the enzyme.

[0061] In some E. coli strains, the opgE gene comprises the nucleic acid sequence according to SEQ ID NO: 2. It will be appreciated that nucleic acid sequences encoding phosphoethanolamine transferases may have homology between species and strains. For example, the phosphoethanolamine transferase encoded by the opgE gene comprises or consists of the amino acid sequence according to SEQ ID NO: 4.

[0062] In embodiments, the gene product responsible for the production of oligosaccharides having a phosphoethanolamine moiety is deleted or rendered dysfunctional in the genetically modified bacterial cell.

[0063] In an embodiment, the opgE gene is deleted in the genetically modified bacterial cell.

[0064] In an embodiment, a gene functionally equivalent / homologous to the opgE gene of E. coli is deleted or rendered dysfunctional.

[0065] In an embodiment, a functional equivalent / homolog of phosphoethanolamine transferase OpgE is functionally dysregulated in the genetically modified bacterial cell, and the amino acid sequence of the functional equivalent / homolog is at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identical to SEQ ID NO:4.

[0066] OgC

[0067] The opgC gene encodes the OPG biosynthetic protein C (OpgC), which transfers a succinyl residue from the cytoplasmic side of the membrane to the nascent glucan backbone on the periplasmic side of the membrane and catalyzes the conjugation of the succinyl residue to OPG.

[0068] Generally speaking, when aiming to produce pure oligosaccharides (e.g. HMOs having an LNT-II core structure), succinyl-conjugated oligosaccharides are unwanted byproducts. The production of such byproducts in fermentation requires additional downstream purification to remove the byproducts.

[0069] In an embodiment, the gene encoding OPG biosynthesis protein C (opgC) is deleted or becomes dysfunctional in the genetically engineered cells described herein. The introduction of nonsense mutations that result in premature termination codons can render the transcribed polypeptide dysfunctional and can be used to generate host cells that completely lose the function of OPG biosynthesis protein C (OpgC). In addition, mutations in the active site of OPG biosynthesis protein C can also reduce or eliminate the function of the enzyme.

[0070] In some E. coli strains, the opgC gene comprises a nucleic acid sequence corresponding to positions 1,107,784 to 1,108,941 of GenBank: CP032667.1 (E. coli str. K-12 substr. MG1655 DNA, complete genome). It should be understood that the nucleic acid sequence encoding OpgC may have homology between species and strains. For example, the OPG biosynthetic protein C encoded by the opgC gene comprises or consists of the amino acids corresponding to UniProt accession number P75920 or RefSeq ID. NP_415565.1.

[0071] In an embodiment, the gene product responsible for the production of oligosaccharides having a succinyl moiety is deleted or rendered dysfunctional in the genetically modified bacterial cell.

[0072] In an embodiment, the opgC gene is deleted in the genetically modified bacterial cell.

[0073] In an embodiment, a gene functionally equivalent / homologous to the opgC gene of E. coli is deleted or rendered dysfunctional.

[0074] In an embodiment, a functional equivalent / homolog of biosynthetic protein C (OpgC) is functionally dysregulated in a genetically modified bacterial cell, and the amino acid sequence of the functional equivalent / homolog is at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identical to UniProt Accession No. P759204.

[0075] Due to the nature of OPG and the complex cellular pathways involved in its synthesis, it is foreseeable that deletion of the opgB and opgE genes will affect the production of desired oligosaccharides in unforeseen ways, such as by negatively affecting the production of desired oligosaccharides. This is not the case for the production of LNT, as deletion of the opgB and opgE genes in LNT production strains does not largely affect the yield of LNT produced.

[0076] "Deleted" means that the coding region is completely or partially removed so that no (functional) gene product is produced. Inactivation or "dysfunctionality" means that the polypeptide encoding nucleic acid sequence has been altered so that the resulting gene product is functionally inactive or encodes a gene product with less than 100% activity of the native, naturally occurring, endogenous gene product, for example, 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20%. A "non-mutated" gene or protein does not differ from the native, naturally occurring or endogenous coding sequence by 1, 2, at most 5, at most 10, at most 20, at most 50, at most 100, at most 200 or at most 500 codons or more, or from the corresponding encoded amino acid sequence.

[0077] Oligosaccharides

[0078] In this article, the term "oligosaccharide" refers to a sugar polymer containing at least three monosaccharide units, i.e., tri-oligosaccharides, tetra-oligosaccharides, penta-oligosaccharides, hexa-oligosaccharides or higher oligosaccharides. Oligosaccharides may have a straight or branched structure containing monosaccharide units connected to each other by interglycosidic bonds. Specifically, oligosaccharides are included in the lactose residue at the reducing end and one or more naturally occurring monosaccharides selected from the following 5-9 carbon atoms: aldose (e.g., glucose, galactose, ribose, arabinose, xylose, etc.), ketose (e.g., fructose, sorbose, tagatose, etc.), deoxy sugar (e.g., rhamnose, fucose, etc.), deoxy amino sugar (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acid and keto-uronic acid (e.g., N-acetylneuraminic acid). Therefore, one or more oligosaccharides can be produced. Preferably, one or more oligosaccharides are one or more human milk oligosaccharides (HMO).

[0079] Human Milk Oligosaccharides (HMO)

[0080] Preferred oligosaccharides of the present disclosure are human milk oligosaccharides (HMOs).

[0081] The term "human milk oligosaccharide" or "HMO" herein refers to a complex carbohydrate found in human breast milk. HMO has a core structure comprising a lactose unit at the reducing end, which may be extended by one or more β-N-acetyl-lactosamine and / or one or more β-lactosyl-N-disaccharide units, and the core structure may be partially substituted by α-L-pyranosyl and / or α-N-acetyl-neuraminic acid (fucosyl). The HMO structure is disclosed, for example, by Xi Chen in "Advances in Carbohydrate Chemistry and Biochemistry" 2015, Vol. 72, Chapter 4.

[0082] HMOs are either neutral or acidic. In this regard, non-acidic (or neutral) HMOs do not contain sialic acid residues, whereas acidic HMOs have at least one sialic acid residue in their structure. Non-acidic (or neutral) HMOs may be fucosylated or non-fucosylated.

[0083] Examples of such neutral non-fucosylated HMOs include lacto-N-triose II (LNT-II), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), p-lacto-N-neohexaose (pLNnH), p-lacto-N-hexaose (pLNH), and lacto-N-hexaose (LNH).

[0084] Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2'FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3'FL), difucosyllactose (DFL or LDFT), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucohexaose I (LNDFH-I), lacto-N-difucohexaose II (LNFP-II), lacto-N-difucohexaose III (LNFP-III), lacto-N-difucohexaose II ... Algae hexaose III (LNDFH-III), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-p-lacto-N-hexaose I (FpLNH-I), fucosyl-p-lacto-N-neohexose II (F-pLNnH II) and fucosyl-lacto-N-neohexose (FLNnH).

[0085] 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'-O-sialyllacto-N-tetraose b (LST b), fucosyl-LST b (FLST b), 6'-O-sialyllacto-N-neotetraose (LST c), fucosyl-LST c (FLST c), 3'-O-sialyllacto-N-neotetraose (LST d), fucosyl-LST d (FLST d). d), sialic acid-lacto-N-hexaose (SLNH), sialic acid-lacto-N-neohexaose I (SLNH-I), sialic acid-lacto-N-neohexaose II (SLNH-II) and disialyl-lacto-N-tetraose (DSLNT).

[0086] In the context described herein, the desired oligosaccharide, such as HMO, comprises a Gal and / or GlcNAc sugar unit, preferably both, such as HMO LNT-II, LNT and / or LNnT. Preferably, the ratio of Gal:GlcNAc is 1.5-2:1. In addition, the desired oligosaccharide, such as HMO, comprises a lacto-N-triose II (LNT-II, GlcNAc (β1-3) Gal (β1-4) Glc) core structure. Preferably, the oligosaccharide with the LNT-II core structure has a glucose moiety at the reducing end. Examples of oligosaccharides with the LNT-II core structure are Gal (β1-4) GlcNAc (β1-3) Gal (β1-4) Glc or Gal (β1-3) GlcNAc (β1-3) Gal (β1-4) Glc structures. Examples of HMOs comprising an LNT-II core structure (or backbone) are, for example, LNT, LNnT, LNT-II, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, pLNH, F-pLNH I, pLNnH, LST a, LST b, LST c, and DSLNT.

[0087] In one embodiment, the desired human milk oligosaccharide (HMO) is a HMO selected from the group consisting of LNT, LNnT, LNT-II, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, pLNH, F-pLNH I, pLNnH, LST a, LST b, LST c and DSLNT.

[0088] In a further embodiment, the desired human milk oligosaccharide (HMO) is a HMO selected from the group consisting of LNT-II, LNT, LNnT and LNFP-I.

[0089] By-products

[0090] In the production of one or more specific desired HMOs, the synthesis may also produce undesirable oligosaccharides, such as HMO species (e.g., pLNH2) or HMO-derived species (e.g., gal-LNT, EP-LNT, or GP-LNT), described herein as by-products.

[0091] Examples of by-products are oligosaccharides containing, for example, phosphoglycerol- and / or phosphoethanolamine- and / or succinyl-moieties. In particular, HMO products (HMO-derived species) with phosphoglycerol- and / or phosphoethanolamine- and / or succinyl moieties (see Figure 1 and 2 ).

[0092] Deletion of the transferase responsible for the production of oligosaccharides containing glycerol phosphate and / or ethanolamine phosphate and / or succinyl enables the production of oligosaccharides without the production of byproducts containing glycerol phosphate and / or ethanolamine phosphate and / or succinyl.

[0093] Thus, cells with reduced or complete loss of the function of phosphoglyceroltransferases I and II (OpgB and OpgB') and / or phosphoethanolaminetransferase (OpgE) and / or OPG biosynthesis protein C (OpgC) as presented herein produce reduced amounts of byproduct oligosaccharides containing phosphoethanolamine- and / or glycerophosphate- and / or succinyl-moieties, compared to cells containing functional phosphoglyceroltransferases I (OpgB) and II (OpgB') and / or functional phosphoethanolaminetransferase (OpgE) and / or OPG biosynthesis protein C (OpgC).

[0094] In an embodiment, in a genetically modified cell suitable for producing oligosaccharides, in particular heterologous oligosaccharides, the function of phosphoglycerol transferase I (OpgB) and / or II (OpgB') is reduced or completely eliminated. In an embodiment, in a genetically modified cell suitable for producing oligosaccharides, the function of phosphoethanolamine transferase (OpgE) is reduced or completely eliminated. In an embodiment, in a genetically modified cell suitable for producing oligosaccharides, in particular heterologous oligosaccharides, the function of OPG biosynthetic protein C (OpgC) is reduced or completely eliminated. In an embodiment, in a genetically modified cell suitable for producing oligosaccharides, in particular heterologous oligosaccharides, the function of phosphoglycerol transferase I (OpgB) and / or II (OpgB') and the function of phosphoethanolamine transferase (OpgE) are reduced or completely eliminated. In an embodiment, the function of phosphoglycerol transferase I (OpgB) and / or II (OpgB') and the function of phosphoglycerol transferase I (OpgB) and / or II (OpgB') and the function of OPG biosynthesis protein C (OpgC) are reduced or completely eliminated in a genetically modified cell suitable for producing oligosaccharides, especially heterologous oligosaccharides. In an embodiment, the function of phosphoglycerol transferase I (OpgB) and / or II (OpgB') and the function of phosphoethanolamine transferase (OpgE) and the function of OPG biosynthesis protein C (OpgC) are reduced or completely eliminated in a genetically modified cell suitable for producing oligosaccharides, especially heterologous oligosaccharides. In an embodiment, in a genetically modified cell suitable for producing oligosaccharides, in particular heterologous oligosaccharides, the function of phosphoglycerol transferase I (OpgB) and / or II (OpgB') and the function of phosphoglycerol transferase I (OpgB) and / or II (OpgB'), phosphoethanolamine transferase (OpgE) and the function of OPG biosynthesis protein C (OpgC) are reduced or completely eliminated. In an embodiment, in a genetically modified cell suitable for producing oligosaccharides, in particular heterologous oligosaccharides, only the function of phosphoglycerol transferase I (OpgB) and / or II (OpgB') and the function of phosphoethanolamine transferase (OpgE) are reduced or completely eliminated.

[0095] like Figure 2 As shown, in the production of LNT and LNnT, phosphoethanolamine and / or glycerophosphate moieties are attached to the GlcNAc moieties of LNT or LNnT. Therefore, it is preferred that the reduction or loss of phosphoglycerol transferase I and II and / or functional phosphoethanolamine transferase function reduces the amount of phosphoglycerol-modified oligosaccharides produced that carry phosphoethanolamine and / or GlcNAc moieties.

[0096] Depending on the oligosaccharide produced by the cell (e.g., HMO having an LNT-II core structure), the level of oligosaccharide byproducts containing phosphoethanolamine and / or glycerophosphate will also vary. The LNT production strain MP0 presented in Example 1 produces about 0.21% (mass %) of phosphoethanolamine-LNT relative to the amount of LNT produced and about 2.6% (mass %) of phosphoglycerol-LNT relative to the amount of LNT produced.

[0097] Preferably, the ratio (w / w) of desired oligosaccharide: phosphoglycerol-oligosaccharide is at least 500:1, such as at least 750:1, 1000:1, 2000:1 or at least 5000:1.

[0098] Preferably, the ratio (w / w) of desired oligosaccharide:phosphoethanolamine-oligosaccharide is at least 50:1, such as at least 100:1, 300:1, 500:1 or at least 1000:1.

[0099] Therefore, for LNT-producing strains, it is highly preferred that genetically engineered bacterial cells in which the functions of phosphoglycerol transferases I and II and / or phosphoethanolamine transferase are reduced or completely lost produce less than 0.2% (mass %) of phosphoethanolamine-LNT relative to the amount of LNT produced, and less than 2.6% (mass %) of phosphoglycerol-LNT relative to the amount of LNT produced.

[0100] Thus, preferably, the cells with reduced or complete loss of phosphoglycerol transferase I and II and / or phosphoethanolamine function as presented herein produce lower amounts of phosphoglycerol-LNT and / or phosphoethanolamine-LNT or phosphoglycerol-LNnT and / or phosphoethanolamine-LNnT compared to cells with no reduction or complete loss of phosphoglycerol transferase I and II and / or phosphoethanolamine transferase function. Preferably, the ratio (w / w) of LNT: phosphoglycerol-LNT is at least 500:1, such as at least 750:1, 1000:1, 2000:1 or at least 5000:1.

[0101] Additionally, preferably the ratio of LNT:phosphoethanolamine-LNT (w / w) is at least 50:1, such as at least 100:1, 300:1, 500:1, or at least 1000:1.

[0102] Receptor / precursor molecule

[0103] Genetically engineered cells according to the present disclosure comprise one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases capable of producing a desired oligosaccharide, such as a HMO, from a precursor molecule, such as a disaccharide or oligosaccharide, such as lactose or LNT-II.

[0104] As described herein, an acceptor oligosaccharide is a molecule that can serve as a substrate for a glycosyltransferase, and is preferably a disaccharide or trisaccharide, which is capable of transferring a glycosyl moiety from a glycosyl donor to a precursor molecule. The glycosyl donor is preferably a nucleotide activated sugar, as described in the "Glycosyl Donor-Nucleotide Activated Sugar Pathway" section. Preferably, the acceptor sugar is a precursor for preparing a more complex oligosaccharide such as an HMO, and may also be referred to as a precursor molecule.

[0105] The acceptor oligosaccharide may be an intermediate product of the fermentation process of the invention, the end product of a separate fermentation process employing separate genetically engineered cells, or an enzymatically or chemically produced molecule.

[0106] In this context, the acceptor oligosaccharide for producing the desired oligosaccharide, e.g. the desired HMO, is preferably lactose and / or LNT-II, which is directly input from the production medium or, in the case of LNT-II, is produced from the initial precursor molecule lactose (e.g. the acceptor of β-1,3-N-acetylglucosaminyltransferase) (see Figure 2 ). Preferably, the initial precursor molecule is supplied to the genetically engineered cell, which is capable of producing, for example, LNT-II, LNT, LNnT or a more complex HMO from the precursor molecule. Most commonly, the initial precursor is lactose, and the genetically engineered cell is capable of producing the intermediate precursors (acceptor oligosaccharides, such as LNT-II and LNnT) intracellularly. Alternatively, if the cell is capable of importing LNT-II, the initial precursor may also be LNT-II.

[0107] As an alternative to supplying a precursor (e.g., lactose) to genetically engineered cells, cells can be engineered to produce their own lactose (see, e.g., Parschat et al. 2020 ACS Synth. Biol. 9:2784-27969).

[0108] Glycosyltransferase

[0109] Preferably, the genetically engineered cell according to the present disclosure comprises at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase.

[0110] Genetically engineered cells according to the present disclosure may comprise one or more additional recombinant nucleic acids encoding one or more recombinant and / or heterologous glycosyltransferases capable of transferring glycosyl residues from a glycosyl donor to an acceptor oligosaccharide. Preferably, the additional glycosyltransferases enable the genetically engineered cells to synthesize LNT from precursor molecules such as lactose or LNT-II. In embodiments, the genetically engineered cells described herein comprise one or more additional recombinant nucleic acids encoding one or more recombinant and / or heterologous glycosyltransferases.

[0111] The glycosyltransferase may be selected from the group consisting of galactosyltransferase, glucosaminyltransferase, N-acetylglucosaminyltransferase and N-acetylglucosaminyltransferase. The glycosyltransferase is preferably selected from the group consisting of galactosyltransferase and glucosaminyltransferase.

[0112] In one aspect, the glycosyltransferase is selected from the β-1,4-galactosyltransferase or β-1,3-galactosyltransferase listed herein. In an embodiment, the one or more glycosyltransferases are selected from β-1,3-N-acetylglucosaminyltransferase, β-1,4-galactosyltransferase and β-1,3-galactosyltransferase.

[0113] In one embodiment, the cell is capable of producing or produces LNT and comprises a heterologous β-1,3-N-acetyl-glucosaminyltransferase and a heterologous β-1,3-galactosyltransferase.

[0114] In another embodiment, the cell is capable of producing or produces LNnT and comprises a heterologous β-1,3-N-acetyl-glucosaminyltransferase and a heterologous β-1,4-galactosyltransferase.

[0115] In addition, the genetically engineered cells may further comprise one or more heterologous nucleic acids encoding one or more heterologous glycosyltransferases selected from the group consisting of α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-3,4-fucosyltransferase, α-2,3-sialyltransferase and α-2,6-sialyltransferase. Such additional fucosyltransferases allow for the production of, for example, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, pLNH, F-pLNH I, pLNnH, LST a, LST b, LST c and DSLNT.

[0116] In an embodiment, the β-1,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis. In a further embodiment, the β-1,3-galactosyltransferase and / or the β-1,4-galactosyltransferase are each from Helicobacter pylori.

[0117] Typically, the glycosyl donor is a nucleotide activating sugar or oligosaccharide, for example selected from glucose-UDP-GlcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc) and CMP-N-acetylneuraminic acid (CMP-Neu5Ac), preferably UDP-Gal and / or UDP-GlcNAc.

[0118] The glycosyl donor is synthesized by one or more genetically engineered cells and / or added exogenously to the culture medium. Preferably, the glycosyl donor is a nucleotide activating sugar, which is synthesized by the host cell using existing pathways that can be modified to increase the pool of relevant nucleotide activating sugars, or synthesized by introducing a nucleotide sequence encoding an enzyme required to produce the relevant nucleotide activating sugar in the cell.

[0119] In an embodiment, at least one functional enzyme capable of transferring a sugar moiety from a glycosyl donor to an acceptor oligosaccharide can be selected from β-1,4-galactosyltransferases, such as galT and β-1,3-galactosyltransferases, such as galTK. For example, these enzymes can be used to generate LNnT or LNT, respectively, starting from LNT-II as an acceptor oligosaccharide.

[0120] In a preferred embodiment, the genetically modified cell according to the present disclosure comprises at least one recombinant nucleic acid sequence encoding a glycosyltransferase capable of transferring a glycosyl residue from a glycosyl donor to the acceptor oligosaccharide to synthesize a desired oligosaccharide, preferably a heterologous oligosaccharide.

[0121] In order to enable a cell to produce oligosaccharides having a lacto-N-triose II (LNT-II) core structure, such as HMO having a lacto-N-triose II (LNT-II) core structure, one or more recombinant nucleic acid sequences encoding glycosyltransferases may be required, for example, the synthesis of LNT-II from lactose requires β-1,3-N-acetyl-glucosaminyl-transferase, the synthesis of LNT from LNT-II requires β-1,3-galactosyltransferase, and the synthesis of LNT from lactose requires both β-1,3-N-acetyl-glucosaminyl-transferase and β-1,3-galactosyltransferase. Similarly, the synthesis of LNnT from LNT-II requires β-1,4-galactosyltransferase, and the synthesis of LNnT from lactose requires both β-1,3-N-acetyl-glucosaminyl-transferase and β-1,4-galactosyltransferase.

[0122] Such glycosyltransferase activity is preferably selected from the following activities.

[0123] Heterologous β-1,3-N-acetyl-glucosaminyl-transferase

[0124] β-1,3-N-acetyl-glucosaminyl-transferase is any protein that has the ability to transfer the N-acetyl-glucosamine of UDP-N-acetyl-glucosamine to lactose or another acceptor molecule in a β-1,3-linked manner (see Figure 2). Preferably, the β-1,3-N-acetyl-glucosaminyl-transferase used herein does not originate from the species of the genetically engineered cell, ie, the gene encoding the β-1,3-N-acetyl-glucosaminyl-transferase is of heterologous origin.

[0125] Thus, in an embodiment, the genetically engineered cell comprises one or more recombinant nucleic acid sequences encoding a β-1,3-N-acetyl-glucosaminyltransferase.

[0126] Non-limiting examples of β-1,3-N-acetyl-glucosaminyltransferases are given in Table 1. β-1,3-N-acetyl-glucosaminyltransferase variants may also be useful, preferably such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to the amino acid sequence of any β-1,3-N-acetyl-glucosaminyltransferase in Table 1.

[0127] Table 1 - List of β-1,3-N-acetyl-glucosaminyltransferases

[0128]

[0129] In an embodiment, the genetically engineered cell comprises a recombinant nucleic acid sequence encoding a β-1,3-N-acetyl-glucosaminyl transferase. In one embodiment, the recombinant nucleic acid sequence encodes a β-1,3-N-acetyl glucosaminyl transferase comprising or consisting of the amino acid sequence shown in SEQ ID NO:6 (LgtA from Neisseria meningitidis) or a functional homologue thereof, wherein the amino acid sequence of the functional homologue 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:6.

[0130] To produce LNnT or LNT using lactose as a substrate, a β-1,3-N-acetylglucosaminyltransferase is used to form the LNT-II precursor. In an embodiment, the genetically engineered cell comprises a β-1,3-N-acetylglucosaminyltransferase gene or a functional homolog or fragment thereof to produce the intermediate LNT-II from lactose.

[0131] Some of the examples below use a heterologous β-1,3-N-acetyl-glucosaminyl-transferase named LgtA from Neisseria meningitidis or a variant thereof.

[0132] Heterologous β-1,3-galactosyltransferase

[0133] β-1,3-Galactosyltransferase is any protein that has the ability to transfer the galactose of UDP-galactose to the N-acetyl-glucosaminyl portion of an acceptor molecule in a β-1,3-linked manner (see Figure 2). Preferably, the β-1,3-galactosyltransferase used herein does not originate from the species of the genetically engineered cell, ie, the gene encoding the β-1,3-galactosyltransferase is of heterologous origin.

[0134] In embodiments, cells of the present disclosure comprise a β-1,3-glycosyltransferase and optionally a β-1,3-N-acetylglucosaminyltransferase.

[0135] As described herein, the acceptor molecule is an acceptor sugar, such as LNT-II or a more complex HMO structure.

[0136] The following examples use a heterologous β-1,3-galactosyltransferase named GalTK or a variant thereof to produce LNT.

[0137] The β-1,3-galactosyltransferase can be obtained from any of a variety of sources, for example, the galTK gene from Helicobacter pylori (homologous to GenBank protein accession number BD182026.1) or the WbgO gene from Escherichia coli 055:H7 (GenBank accession number WP_000582563.1) or the jhp0563 gene from Helicobacter pylori (GenBank accession number AEZ55696.1) as described.

[0138] In one embodiment, the β-1,3-galactosyltransferase encoded by the recombinant nucleic acid sequence comprises or consists of the amino acid sequence shown in SEQ ID NO:7 (galTK from Helicobacter pylori), or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% identical to SEQ ID NO:7.

[0139] To produce LNT from the LNT-II precursor, a β-1,3-galactosyltransferase is required. In one embodiment, the genetically modified cell comprises a β-1,3-galactosyltransferase gene or a functional homologue or fragment thereof.

[0140] In a further embodiment, the β-1,3-N-acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO:6, or a functional homolog thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO:6, and the β-1,3-galactosyltransferase has an amino acid sequence according to SEQ ID NO:7, or a functional homolog thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO:7.

[0141] The following are examples of genetically modified strains described herein that have specific combinations of glycosyltransferases that will result in the production of LNT using either lactose or LNT-II as the initial substrate.

[0142] In one example, LgtA from Neisseria meningitidis was used in combination with galTK from Helicobacter pylori to produce LNT using lactose as the initial substrate.

[0143] In one example, galTK from Helicobacter pylori was used to produce LNT starting from LNT-II as the initial substrate.

[0144] Heterologous β-1,4-galactosyltransferase

[0145] β-1,4-Galactosyltransferase is any protein that has the ability to transfer the galactose of UDP-galactose to the N-acetyl-glucosaminyl portion of an acceptor molecule in a β-1,4-linked manner (see Figure 2 ). Preferably, the β-1,4-galactosyltransferase used herein does not originate from a species of genetically engineered cells, ie, the gene encoding the β-1,4-galactosyltransferase is of heterologous origin. In the context described herein, the acceptor molecule is an acceptor sugar, such as LNT-II or a more complex HMO structure.

[0146] The following examples use a heterologous β-1,4-galactosyltransferase GalT or a variant thereof to produce LNnT. Thus, in an embodiment, the genetically engineered cell comprises one or more recombinant nucleic acid sequences encoding a β-1,4-galactosyltransferase.

[0147] Non-limiting examples of β-1,4-galactosyltransferases are provided in Table 2. β-1,4-galactosyltransferase variants may also be useful, preferably such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% identity to the amino acid sequence of any β-1,4-galactosyltransferase in Table 2.

[0148] Table 2- List of β-1,4-glycosyltransferases

[0149]

[0150] In one embodiment, the β-1,4-galactosyltransferase encoded by the recombinant nucleic acid sequence comprises or consists of the amino acid sequence shown in SEQ ID NO:8 (galT from Helicobacter pylori), or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% identical to SEQ ID NO:8.

[0151] To produce LNnT from the LNT-II precursor, a β-1,4-galactosyltransferase is required. In one embodiment, the genetically engineered cell comprises a β-1,4-galactosyltransferase gene or a functional homologue or fragment thereof.

[0152] In an embodiment, the β-1,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis and the β-1,4-galactosyltransferase is from Helicobacter pylori. In a further embodiment, the β1,3-N-acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO: 6, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 6, and the β-1,4-galactosyltransferase has an amino acid sequence according to SEQ ID NO: 8, or a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 8.

[0153] In one example, LgtA from Neisseria meningitidis was used in combination with galT from Helicobacter pylori to produce LNnT starting from lactose as the initial substrate.

[0154] In one example, galT from Helicobacter pylori was used to produce LNnT starting from LNT-II as the initial substrate.

[0155] In one example, LgtA from Neisseria meningitidis is used in combination with LgtB from Neisseria meningitidis to produce LNnT starting from lactose as the initial substrate.

[0156] In one example, LgtB from Neisseria meningitidis was used to produce LNnT starting from LNT-II as the initial substrate.

[0157] In order to obtain more complex oligosaccharides having a lacto-N-triose II (LNT-II) core structure, such as fucosylated and / or sialylated derivatives of LNT or LNnT, additional glycosyltransferase activities may be required in the synthesis in addition to the glycosyltransferase activity required for the production of LNT or LNnT.

[0158] Thus, the genetically engineered cells may further comprise one or more heterologous nucleic acids encoding one or more heterologous glycosyltransferases selected from the group consisting of α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3 / 4-fucosyltransferase, α-2,3-sialyltransferase, and α-2,6-sialyltransferase.

[0159] In embodiments where the complex oligosaccharide having a lacto-N-triose II (LNT-II) core structure is fucosylated, the genetically engineered cells may further comprise one or more heterologous nucleic acids encoding one or more heterologous glycosyltransferases selected from the group consisting of α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, and α-1,3 / 4-fucosyltransferase.

[0160] In embodiments where the complex oligosaccharide having a lacto-N-triose II (LNT-II) core structure is sialylated, the genetically engineered cells may further comprise one or more heterologous nucleic acids encoding one or more heterologous glycosyltransferases selected from the group consisting of α-2,3-sialyltransferase and α-2,6-sialyltransferase.

[0161] Heterologous α-1,2-fucosyltransferase

[0162] A heterologous α-1,2-fucosyltransferase is a protein having the ability to catalyze the transfer of fucose from a donor substrate, such as GDP-fucose, to an acceptor molecule in an α-1,2-linkage. Preferably, the α-1,2-fucosyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding the α-1,2-fucosyltransferase is of heterologous origin. In the context of the present disclosure, the acceptor molecule is an acceptor oligosaccharide of at least three or four monosaccharide units, such as 2'-fucosyllactose, 3-fucosyllactose, LNT, LNFP-1 or a more complex HMO structure.

[0163] Non-limiting examples of α-1,2-fucosyltransferases are given in the following table. α-1,2-fucosyltransferase variants may also be useful. Preferably, such variants comprise at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with the α-1,2-fucosyltransferases shown in Table 8.

[0164] Table 8 - List of α-1,2-fucosyltransferases

[0165]

[0166] A heterologous α-1,2-fucosyltransferase can, for example, be used to produce DFL or LNFP-I or LNDFH-I.

[0167] Heterologous α-1,3-fucosyltransferase

[0168] A heterologous α-1,3-fucosyltransferase refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate, such as GDP-fucose, to an acceptor molecule in an α-1,3-linkage. Preferably, the α-1,3-fucosyltransferase used herein does not originate from a species of genetically engineered cells, i.e., the gene encoding the α-1,3-fucosyltransferase has a heterologous origin. In the context of the present disclosure, the acceptor molecule is an acceptor oligosaccharide of at least three or four monosaccharide units, such as 2'-fucosyllactose, 3-fucosyllactose, LNT, LNFP-I or a more complex HMO structure.

[0169] Non-limiting examples of α-1,3-fucosyltransferases are given in the following table. α-1,3-fucosyltransferase variants may also be useful. Preferably, such variants comprise at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with the α-1,3-fucosyltransferases shown in Table 9.

[0170] Table 9 - List of α-1,3-fucosyltransferases

[0171]

[0172] A heterologous α-1,3-fucosyltransferase can, for example, be used to produce 3FL, DFL, LNFP-III, LNFP-V, LNDFH-II, or LNDFH-III.

[0173] Heterologous α-1,3 / 4-fucosyltransferase

[0174] A heterologous α-1,3 / 4-fucosyltransferase refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate such as GDP-fucose to an acceptor molecule in an α-1,3- or α1,4-linkage. Preferably, the α-1,3 / 4-fucosyltransferase used herein does not originate from a species of genetically engineered cells, i.e., the gene encoding the α-1,3 / 4-fucosyltransferase has a heterologous origin. In the context of the present disclosure, the acceptor molecule is an acceptor oligosaccharide of at least three or four monosaccharide units, such as 2'-fucosyllactose, 3-fucosyllactose, LNT, LNFP-I or a more complex HMO structure.

[0175] Non-limiting examples of α-1,3 / 4-fucosyltransferases are given in the table below. α-1,3 / 4-fucosyltransferase variants may also be useful. Preferably, such variants comprise at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with the α-1,3 / 4-fucosyltransferases shown in Table 10.

[0176] Table 10 - List of α-1,3 / 4-fucosyltransferases

[0177]

[0178]

[0179] A heterologous α-1,3 / 4-fucosyltransferase can, for example, be used to produce 3FL, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, or LNDFH-III, or a mixture thereof.

[0180] Heterologous α-2,3-sialyltransferase

[0181] Heterologous α-2,3-sialyltransferase refers to a glycosyltransferase that catalyzes sialic acid from a donor substrate such as CMP-N-acetylneuraminic acid to transfer to an acceptor molecule with an α-2,3-linked connection. Preferably, the α-2,3-sialyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding 2,3-sialyltransferase has a heterologous source. In the context of the present disclosure, the acceptor molecule is an acceptor oligosaccharide of at least three or four monosaccharide units, such as 3-fucosyllactose, LNT, LNnT or a more complex HMO structure. Non-limiting examples of α-2,3-sialyltransferases are given in the following table. α-2,3-sialyltransferase variants may also be useful. Preferably, such variants comprise and have at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with the α-2,3-sialyltransferase shown in Table 11.

[0182] Table 11 - List of α-2,3-sialyltransferases

[0183]

[0184] If combined with the expression of a fucosyltransferase, a heterologous 2,3-sialyltransferase can be used to produce, for example, 3'SL or LST-a, or fucosylated variants thereof.

[0185] Heterologous α-2,6-sialyltransferase

[0186] Heterologous α-2,6-sialyltransferase refers to a glycosyltransferase that catalyzes the transfer of sialic acid from a donor substrate, such as 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 2,6-sialyltransferase has a heterologous origin. In the context of the present disclosure, the acceptor molecule is an acceptor oligosaccharide of at least three or four monosaccharide units, such as 3-fucosyllactose, LNT, LNnT or a more complex HMO structure.

[0187] Non-limiting examples of α-2,6-sialyltransferases are given in the following table. α-2,6-sialyltransferase variants may also be useful. Preferably, such variants comprise at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with the α-2,6-sialyltransferases shown in Table 12.

[0188] Table 12- List of α-2,6-sialyltransferases

[0189]

[0190] If combined with the expression of a fucosyltransferase, a heterologous 2,6-sialyltransferase can be used to produce, for example, 6'SL or Lst-c, or fucosylated variants thereof.

[0191] Glycosyl donor-nucleotide activated sugar pathway

[0192] The production of oligosaccharides is mainly mediated by glycosyltransferases, which utilize activated sugar nucleotides as donors for the glycosylation reaction.

[0193] Activated sugar nucleotides usually have a phosphorylated glycosyl residue connected to a 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-acetylgalactosamine (GlcNAc) and CMP-N-acetylneuraminic acid.

[0194] The genetically engineered cells described herein may comprise one or more pathways for producing a nucleotide activated sugar selected from the group consisting of glucose-UDP-GlcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, and CMP-N-acetylneuraminic acid.

[0195] In one embodiment of the present disclosure, the genetically engineered cells are capable of producing one or more of the above-mentioned activated sugar nucleotides by a de novo approach. In this regard, activated sugar nucleotides are produced by cells in a stepwise reaction sequence starting from a simple carbon source (such as glycerol, sucrose, fructose or glucose) under the action of enzymes participating in the de novo biosynthetic pathway of the corresponding sugar nucleotides (for a review of monosaccharide metabolism, see, for example, HH Freeze and AD Elbein: Chapter 4 Glycosylation precursors, in: Essentials of Glycobiology, 2nd edition (Eds. A. Varki et al.), Cold Spring Harbour Laboratory Press (2009))

[0196] The enzymes involved in activating the de novo sugar nucleotide biosynthetic pathway 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 skilled person.

[0197] In another embodiment, genetically engineered cells can utilize salvaged monosaccharides for sugar nucleotides. In the salvage synthesis pathway, monosaccharides derived from degraded oligosaccharides are phosphorylated by kinases and converted into nucleotide sugars by pyrophosphorylases. The enzymes involved in this process can be heterologous enzymes or native enzymes of the host cell.

[0198] β-Galactosidase

[0199] Host cells suitable for HMO production, such as E. coli, may contain an endogenous β-galactosidase gene or an exogenous β-galactosidase gene, such as E. coli containing an endogenous lacZ gene (e.g., GenBank Accession No. V00296 (GI:41901)). For the purposes of the present disclosure, when producing HMOs, if lactose is used as the initial substrate for producing the desired HMO, it is preferred that the genetically engineered cells do not express a functional β-galactosidase to avoid degradation of lactose. In embodiments, the lacZ gene may be inactivated by completely or partially deleting the corresponding nucleic acid sequence from the bacterial genome, or the gene sequence may be mutated in a manner that it is not transcribed, or if transcribed, the transcript is not translated, or if translated into a protein (i.e., β-galactosidase), the protein does not have the corresponding enzymatic activity. In this way, the HMO-producing bacteria accumulate a larger intracellular lactose pool, which is beneficial to the production of HMOs.

[0200] Lactose permease

[0201] Lactose permease is a membrane protein, a member of the major facilitator superfamily, and can be classified as a symporter, which utilizes the proton gradient toward the cell to transport β-galactosides (e.g., lactose) into the cell in the same direction. In oligosaccharide production, particularly in the production of human milk oligosaccharides (HMOs), lactose is often the initial substrate that is modified to produce any HMO of interest in the biotransformation that occurs inside the cell. Therefore, in the production of HMOs, it is necessary to be able to import lactose into the cell, for example by expression and / or overexpression of a lactose permease such as lacY of E. coli K.-12.

[0202] In an embodiment, the lactose permease is as shown in SEQ ID NO:9, or a functional homolog thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO:9, such as at least 85%, 90%, 95%, 98% or 99% identical.

[0203] In an embodiment, expression of the lactose permease is regulated by a promoter according to the present disclosure.

[0204] Transporter

[0205] Oligosaccharide products, such as HMOs produced by cells, can accumulate in the cell and in the extracellular matrix. The product can be transported to the supernatant in a passive manner, that is, it diffuses to the outside through the cell membrane. More complex HMO products may be retained in the cell, which may ultimately damage cell growth, thereby affecting the possible total yield of a single fermentation product. HMO transport can be promoted by major facilitator superfamily transport proteins, which promote the efflux of sugar derivatives from cells to the supernatant. Major facilitator superfamily transport proteins can exist exogenously or endogenously and are overexpressed under fermentation conditions to enhance the output of the produced oligosaccharide derivatives (HMOs). The specificity for the sugar portion of the product to be secreted can be changed by mutations known in the art of recombinant DNA technology.

[0206] Thus, the genetically engineered cells according to the present disclosure may further comprise a nucleic acid sequence encoding a major facilitator superfamily transporter protein capable of exporting a fucosylated human milk oligosaccharide product, such a transporter protein may for example be a member of the major facilitator superfamily transporter protein.

[0207] In recent years, several new, potent major facilitator superfamily transporters have been identified, each specific for a different recombinantly produced HMO, and the development of recombinant cells expressing the proteins has facilitated large-scale industrial HMO manufacturing.

[0208] Therefore, in one or more exemplary embodiments, the genetically engineered cell according to the present disclosure also comprises a gene product that serves as an oligosaccharide transporter. The gene product that serves as an oligosaccharide transporter can be encoded by a recombinant nucleic acid sequence expressed in a genetically engineered cell. The recombinant nucleic acid sequence encoding the oligosaccharide transporter can be integrated into the genome of the genetically engineered cell, or expressed using a plasmid.

[0209] In one embodiment, the genetically engineered cells of the present disclosure comprise a nucleic acid sequence encoding a major facilitator superfamily transporter capable of exporting product oligosaccharides into the extracellular medium, in particular a transporter specific for exporting HMOs, preferably having a lacto-N-triose II (LNT-II) core structure, such as LNT, LNnT, and derivatives thereof.

[0210] Nec

[0211] In an embodiment, the genetically engineered cells described herein comprise a nucleic acid sequence encoding an efflux transporter capable of exporting the produced oligosaccharides (e.g., LNT and / or LNnT) to the extracellular medium. In this context, the efflux transporter is preferably a heterologous gene encoding a putative MFS (major facilitator superfamily) transporter derived from the bacterium Rosenbergiella nectarea. More specifically, the present disclosure relates to a genetically engineered cell optimized to produce oligosaccharides, in particular oligosaccharides having a lacto-N-triose II (LNT-II) core structure, such as LNT, comprising a recombinant nucleic acid encoding a protein having at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 5 and GenBank Accession No. WP_092672081.1.

[0212] In addition, the MFS transporter of SEQ ID NO:5 and GenBank accession number WP_092672081.1 is further described in WO2021 / 148615 and is interchangeably identified herein as "Nec protein" or "Nec transporter" or "Nec"; the nucleic acid sequence encoding the Nec protein is identified herein as "nec encoding nucleic acid / DNA" or "nec gene" or "nec".

[0213] Nec is expected to promote an increase in the efflux of oligosaccharides (eg, LNT) produced in the genetically engineered cells of the present disclosure.

[0214] Vag

[0215] An alternative MFS transporter is identified herein as a "Vag protein" or "Vag transporter" or "Vag", interchangeably, having an amino acid sequence corresponding to GenBank Accession No. WP_048785139.1.

[0216] In one or more embodiments of the present invention, the genetically engineered cells express the heterologous MFS transporter vag or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the amino acid sequence of GenBank Accession No. WP_048785139.1.

[0217] Vag is expected to promote increased efflux of oligosaccharides (eg, LNnT) produced in the genetically engineered cells of the present disclosure.

[0218] YberC

[0219] Another alternative MFS transporter is identified herein as "YberC protein" or "YberC transporter" or "YberC", interchangeably, having an amino acid sequence corresponding to GenBank Accession No. EEQ08298.1.

[0220] In one or more embodiments of the present invention, the genetically engineered cells express the heterologous MFS transporter YberC or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the amino acid sequence of GenBank Accession No. EEQ08298.1.

[0221] Vag is expected to contribute to increased efflux of oligosaccharides (eg, LNT or LNnT) produced in the genetically engineered cells of the present disclosure.

[0222] Genetically engineered cells

[0223] As used herein, a "genetically engineered cell" is a cell 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 a heterologous polynucleotide sequence, Crisper / Cas editing, and / or random mutagenesis. In one embodiment, the genetically engineered cell has been transformed or transfected with a recombinant nucleic acid sequence.

[0224] The genetically engineered cell is preferably a prokaryotic cell, such as a microbial cell, preferably a bacterial cell. In a preferred embodiment, the genetically engineered cell is a bacterial cell, preferably a Gram-negative bacterium.

[0225] Host cells

[0226] In an embodiment, the engineered cell is a microorganism. The genetically engineered cell is preferably a microbial cell, such as a prokaryotic cell, such as a bacterial cell.

[0227] The genetically engineered cell may be, for example, a bacterial cell.

[0228] For bacterial host cells, there is no restriction in principle; but preferably, the bacterial host cell is a Gram-negative cell, provided that such cell allows genetic manipulation, for example, by inserting a target gene, and the cell can be cultured on a manufacturing scale. Preferably, the host cell has a property that allows cultivation to a high cell density. Non-limiting examples of bacterial host cells suitable for recombinant industrial production of oligosaccharides according to the present disclosure can be members of the Enterobacterales order, preferably Escherichia sp., Klebsiella sp., Salmonella sp. or Shigella sp.

[0229] Other examples of suitable Gram-negative host cells are Bdellovibrio sp., Campylobacter sp., Citrobacter sp. (e.g., Citrobacter freundii), Erwinia sp. (e.g., Erwinia herbicola), Haemophilus sp., Helicobacter sp., Legionella sp., Moraxella sp., Neisseria sp., Pantoea sp. (e.g., Pantoea agglomerans), Pantoea sp. (e.g., Pantoea citrea), Pectobacterium sp. sp.) (e.g., Pectobacterium carotovorum), Pseudomonas sp., Stenotrophomonas sp., Xanthomonas sp. (e.g., Xanthomonas campestris).

[0230] In one or more exemplary embodiments, the genetically engineered cell is selected from Escherichia coli.

[0231] Preferably, the genetically engineered cell is a Gram-negative bacterium.

[0232] In one or more exemplary embodiments, the genetically engineered cell is Escherichia coli.

[0233] In one or more exemplary embodiments, the present disclosure relates to a genetically engineered cell, wherein the cell is derived from E. coli K-12 strain or DE3.

[0234] Recombinant nucleic acid sequence

[0235] As used herein, the terms "recombinant nucleic acid sequence", "recombinant gene / nucleic acid / nucleotide sequence / DNA encoding..." or "coding nucleic acid sequence" are used interchangeably and are intended to mean an artificial nucleic acid sequence (i.e., produced in vitro using standard laboratory methods for preparing nucleic acid sequences) that comprises a set of consecutive, non-overlapping triplets (codons) that are transcribed into mRNA and translated into protein when under the control of appropriate control sequences (i.e., promoter sequences).

[0236] 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.

[0237] The term "nucleic acid" includes RNA, DNA and cDNA molecules. It will be appreciated that due to the degeneracy of the genetic code, a large number of nucleic acid sequences can be generated that encode a given protein.

[0238] The recombinant nucleic acid sequence can be a coding DNA sequence, such as a gene, or a non-coding DNA sequence, such as regulatory DNA, such as a promoter sequence or other non-coding regulatory sequences.

[0239] In addition, the recombinant nucleic acid sequence can 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, amino acid sequence, nucleic acid molecule or nucleotide sequence that is not naturally present in the cell or organism.

[0240] The present disclosure also includes nucleic acid constructs, which include a coding nucleic acid sequence, i.e., a recombinant DNA sequence of a target gene (e.g., a glycosyltransferase), and a non-coding regulatory DNA sequence, such as a promoter DNA sequence, such as a recombinant promoter sequence derived from a promoter of a lac operon or a glp operon, or a promoter sequence derived from another genome, a promoter DNA sequence, or a synthetic promoter sequence, wherein the coding sequence and the promoter sequence are operably connected. Examples of such constructs are constructs such as those comprising PglpF-galTK, PglpF-galT, or PglpF-lgtA.

[0241] The term "operably linked" refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. It refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter sequence is operably linked to a coding sequence if the promoter sequence stimulates or regulates transcription of the coding sequence in an appropriate host cell or other expression system.

[0242] Generally, promoter sequences operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, ie, they are cis-acting.

[0243] In one exemplary embodiment, the nucleic acid construct of the present disclosure 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.

[0244] Thus, 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 contained in the construct.

[0245] 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 specific sites on the chromosome, as described for the attTn7 site (Waddell CS and Craig NL, Genes Dev. (1988) Feb; 2(2): 137-49.); methods for 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 Rec 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. (2005), 12(3):349-56.; Vetcher et al., Appl Environ Microbiol. (2005); 71(4):1829-35); Positive clones, ie clones carrying the expression cassette, can be selected, for example, by loss or gain of marker gene, or gene function.

[0246] Preferably, the heterologous element, such as a glycosyltransferase or transporter encoding sequence, is under the control of a promoter sequence selected from the promoter sequences having the nucleic acid sequence identified in Table 3.

[0247] Table 3 – Selected promoter sequences

[0248]

[0249] *Promoter activity was assessed in the LacZ assay described below and the PglpF promoter was run as a positive reference in the same assay. To compare activities from different assays, activity relative to the PglpF promoter was calculated and ranges represent results from multiple assays.

[0250] The promoter may be of heterologous origin, native to the genetically engineered cell, or it may be a recombinant promoter combining heterologous and / or native elements.

[0251] One approach to increasing product yields may be to modulate the production of a desired enzyme activity for production of the product, such as a glycosyltransferase or an enzyme involved in a glycosyl donor biosynthetic pathway.

[0252] Increasing the promoter strength that drives 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, in which the β-galactosidase activity is determined as previously described (see, e.g., Miller J.H. Experiments in molecular genetics, Cold spring Harbor Laboratory Press, NY, 1972). In short, cells are diluted in Z buffer (Z-buffer) and permeabilized with sodium dodecyl sulfate (0.1%) and chloroform. The LacZ assay is performed at 30°C. The sample is preheated, the assay is started by adding 200 μl of o-nitrophenyl-β-galactosidase (4 mg / ml), and the assay is stopped by adding 500 μl of 1M Na2CO3 when the sample becomes slightly yellow. The release of o-nitrophenol is subsequently determined as a change in optical density at 420 nm. The specific activity is reported in Miller units (MU) [A420 / (min*ml*A600)]. The regulatory elements with an activity higher than 10,000MU are considered to be strong regulatory elements, while the regulatory elements with an activity lower than 3,000MU are considered to be weak regulatory elements, and the regulatory elements between the two have medium strength. An example of a strong regulatory element is the PglpF promoter with an activity of about 14000MU, and an example of a weak promoter is Plac, which has an activity of about 2300MU when induced with IPTG. In a preferred embodiment, the expression of the nucleic acid sequence is controlled by a strong promoter selected from SEQ ID NO 10, 11, 12, 13, 14, 15, 16, 18 and 19.

[0253] In an embodiment, the expression of the nucleic acid sequence described herein is controlled by: PglpF (SEQ ID NO:22) or Plac (SEQ ID NO:31) promoter or PmglB_UTR70 (SEQ ID NO:19) or PglpA_70UTR (SEQ ID NO:20) or PglpT_70UTR (SEQ ID NO:21), or a variant thereof, such as the promoter shown in Table 3, in particular the PglpF_SD4 variant of SEQ ID NO:17, or the Plac_70UTR variant of SEQ ID NO:13, or the PmglB_70UTR variant of SEQ ID NO:10, 11, 12, 13, 14, 15, 16, 18 and 19. Other suitable variants of the PglpF, PglpA_70UTR, PglpT_70UTR and PmglB_70UTR promoter sequences are described in WO2019 / 123324 and WO2020 / 255054, respectively (incorporated herein by reference).

[0254] In a preferred embodiment, the recombinant nucleic acid sequences are each under the control of one or more promoters selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 22, 31, 19, 20 and 21, respectively), and variants thereof.

[0255] Sequence identity

[0256] The term "sequence identity" as used herein describes the correlation between two amino acid sequences or between two nucleotide sequences (i.e., between a candidate sequence (e.g., sequence disclosed herein) and a reference sequence (e.g., prior art sequence)) based on their pairwise alignment. For purposes disclosed herein, 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 the 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 (see https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). The parameters used are gap opening penalty 10, gap extension penalty 0.5, -endopen 10.0, -endextend 0.5 and EBLOSUM62 (EMBOSS version of 30BLOSUM62) substitution matrix. The output (obtained using -nobrief option) marked as "identity" by Needle is used as percentage identity. In general, sequence identity can be calculated as follows: (same residue × 100) / (alignment region).

[0257] For purposes disclosed herein, 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 open penalty 10, gap extension penalty 0.5, -endopen 10.0, -endextend 0.5 and DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output (obtained using the -nobrief option) marked as "identity" by Needle is used as percentage identity. In general, sequence identity can be calculated as follows: (identical nucleotide residues × 100) / (alignment region).

[0258] Functional homologs

[0259] Functional homologues or functional variants of the 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 to the functionality of the protein / nucleic acid sequence, the functional homologue should have at least 50% residual functionality, such as at least 60%, 70%, 80%, 90% or at least 100% or more.

[0260] With regard to the modification of the osmotic regulation periplasmic glucan (OPG) biosynthetic machinery in cells as described herein, the complete loss of function is understood to be the complete loss of enzymatic activity, for example by deleting the nucleic acid encoding the enzyme, or by introducing a stop codon (which terminates the transcription of the nucleic acid encoding the enzyme at a position where a functional enzyme cannot be formed). Functional reduction is understood to be a reduction in enzyme functionality after genetic modification, wherein the functional reduction is sufficient to avoid the significant formation of byproduct oligosaccharides in genetically modified cells capable of producing one or more desired oligosaccharides (e.g., HMOs). The functional reduction of the OPG enzyme can be assessed by measuring the ability of the enzyme to modify LNT or LNnT compared to an unmodified enzyme. Preferably, the ability to modify LNT and / or LNnT is reduced by at least 50%, for example at least 60%, 70%, 80%, 90% or at least 95%, compared to an unmodified enzyme.

[0261] Uses of genetically engineered cells

[0262] The present disclosure also relates to the use of the genetically engineered cells described herein in the production of oligosaccharide products, particularly heterologous oligosaccharide products isolated / purified from their genetically engineered production cells.

[0263] Preferably, the oligosaccharide product is one or more human milk oligosaccharides (HMOs) having a lacto-N-triose II (LNT-II) core structure, such as HMOs selected from the group consisting of LNT, LNnT, LNT-II, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, pLNH, F-pLNH I, pLNnH, LST a, LST b, LST c, DSLNT. The use of such cells should result in lower production of by-products such as phosphoglycerol-oligosaccharides and / or phosphoethanolamine-oligosaccharides.

[0264] Method for producing one or more oligosaccharides

[0265] The present disclosure also relates to a method of producing one or more desired oligosaccharides, in particular one or more heterologous oligosaccharides, comprising culturing a genetically engineered cell according to the present disclosure.

[0266] A second aspect of the present disclosure is a method for producing an oligosaccharide product, in particular a heterologous oligosaccharide product, wherein the method comprises,

[0267] a) providing a genetically engineered cell according to the present disclosure,

[0268] b) culturing the genetically engineered cell in a culture medium under conditions that allow production of said oligosaccharide product; and optionally

[0269] c) recovering the oligosaccharide product from the culture.

[0270] "Desired oligosaccharide" or "product oligosaccharide" is understood to be an oligosaccharide that is intentionally produced by the process, i.e. the main product to be obtained from the process. The desired oligosaccharide may be a single oligosaccharide, such as HMO LNT, or it may be a mixture of oligosaccharides, such as a mixture comprising HMO LNT-II, LNT and LNFP-I. A by-product may be considered as an unwanted product produced by the process, such as a phosphoethanolamine-modified oligosaccharide and / or a glycerophosphate-modified oligosaccharide and / or a succinyl-modified oligosaccharide, or it may be an intermediate oligosaccharide product, such as LNT-II, or an extended oligosaccharide product, such as pLNH-II.

[0271] One of the benefits of using the methods disclosed herein to produce one or more oligosaccharides is the absence of phosphoethanolamine and / or glycerophosphate and / or succinyl modified oligosaccharides, which allows for cleaner production of the desired oligosaccharides. This is exemplified in Example 1, where the desired oligosaccharide is LNT, and the phosphoethanolamine and / or glycerophosphate modified oligosaccharides are the LNT derivatives phosphoethanolamine-LNT (EP-LNT) and glycerophosphate-LNT (GP-LNT). Upon culturing the cells of the present disclosure, it was shown that the cells were able to produce LNT without producing the byproducts EP-LNT and GP-LNT. In addition, it was found that the cultivation of the cells produced almost the same LNT yield compared to strains with functional opgB and opgE genes.

[0272] Therefore, the present application also relates to a method for producing a desired oligosaccharide product, wherein the method comprises: providing a genetically engineered cell capable of producing or producing the desired oligosaccharide product and having reduced or completely lost phosphoglycerol transferase I and II and / or phosphoethanolamine transferase and / or OPG biosynthesis protein C function, culturing the genetically engineered cell in a culture medium under conditions that allow the production of the oligosaccharide product; and optionally, recovering the oligosaccharide product. Preferably, phosphoglycerol transferase I and II are encoded by the opgB gene. Preferably, phosphoethanolamine transferase is encoded by the opgE gene. Preferably, OPG biosynthesis protein C is encoded by the opgC gene.

[0273] Preferably, the production of the desired oligosaccharides is reduced by no more than 15%, preferably no more than 10%, and preferably is substantially unaffected, when compared to a method comprising culturing cells which do not have reduced or completely lost function of phosphoglycerol transferases I and II and / or phosphoethanolamine transferase and / or OPG biosynthetic protein C.

[0274] As also shown in Example 2, the production yield of the desired oligosaccharide LNnT can also be increased upon deletion of key enzymes of the osmoregulatory periplasmic glucan biosynthetic machinery (eg, OpgB and OpgE), probably due to a reduction in cellular energy consumption.

[0275] In a preferred embodiment, the yield of the desired oligosaccharides according to the methods disclosed herein can even be increased by at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, compared to a method comprising culturing cells that do not have reduced or completely lost function of phosphoglycerol transferases I and II and / or phosphoethanolamine transferase and / or OPG biosynthetic protein C.

[0276] In addition, compared to the method using cells containing phosphoglycerol transferases I and II and / or phosphoethanolamine transferase and / or OPG biosynthetic protein C, smaller amounts of by-product oligosaccharides containing phosphoethanolamine- and / or glycerophospho- and / or succinyl- moieties are produced by the present method, for example, the by-product is phosphoglycerol-LNT and / or phosphoethanolamine-LNT and / or succinyl-LNT, or phosphoglycerol-LNnT and / or phosphoethanolamine-LNnT and / or succinyl-LNnT.

[0277] In an embodiment, the desired oligosaccharide is one or more human milk oligosaccharides (HMOs) selected from the group consisting of LNT, LNnT, LNT-II, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, pLNH, F-pLNH I, pLNnH, LST a, LST b, LST c, DSLNT. Preferably, the HMO is LNT, LNnT or LNFP-I.

[0278] The benefit of the methods disclosed herein is that the amount of phosphoethanolamine and / or glycerophosphate-modified oligosaccharides produced by the cell is reduced. Therefore, in an embodiment, the ratio (w / w) of the desired oligosaccharide produced in the culturing step according to the method: phosphoglycerol-oligosaccharide is at least 500: 1, such as at least 750: 1, 1000: 1, 2000: 1 or such as at least 5000: 1 and / or the ratio (w / w) of the desired oligosaccharide: phosphoethanolamine-oligosaccharide is at least 50: 1, such as at least 100: 1, 300: 1, 500: 1 or such as at least 1000: 1.

[0279] In a preferred embodiment, the ratio (w / w) of LNT: phosphoglycerol-LNT produced in the culturing step according to the method is at least 500: 1, such as at least 750: 1, 1000: 1, 2000: 1 or such as at least 5000: 1. In a further embodiment, the ratio (w / w) of LNT: phosphoethanolamine-LNT is at least 50: 1, such as at least 100: 1, 300: 1, 500: 1 or such as at least 1000: 1.

[0280] Therefore, in the method for producing LNT, it is highly preferred that less than 0.2% of phosphoethanolamine-LNT is produced relative to the amount of LNT, such as less than 0.1%, 0.05%, 0.01%, 0.005% or less than 0.001% (mass %). In addition, it is also highly preferred that less than 2.6% of glycerophosphophosphate-LNT is produced relative to the amount of LNT, such as less than 2%, 1.5%, 1%, 0.5%, 0.1%, 0.05% or less than 0.01% (mass %).

[0281] Preferably, glycerophospho-LNT and / or phosphoethanolamine-LNT are not produced.

[0282] Further genetic modifications may be selected from, for example, the inclusion of additional glycosyltransferases and / or metabolic pathway engineering, and the inclusion of transporters, such as substrate importers or product exporters, such as MFS transporters, as described in the above sections, and the skilled person will know how to combine these modifications into a genetically engineered cell capable of producing one or more oligosaccharides.

[0283] The method comprises culturing a genetically engineered cell, and further comprises culturing the genetically engineered cell in the presence of an energy source (carbon source) selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.

[0284] On the one hand, the method of the present disclosure includes providing an acceptor sugar as an initial substrate for oligosaccharide such as HMO formation. In one embodiment, the acceptor sugar comprises at least two monosaccharide units, which are added to the culture medium by an exogenous source and / or produced by independent microbial fermentation. As an alternative to adding the initial substrate to the fermentation medium, genetically modified cells can be further transformed to produce the initial substrate in the cell (see, for example, WO2015 / 150328). Preferably, the acceptor sugar is lactose, or LNT-II.

[0285] In an embodiment, the substrate for HMO formation is lactose, which is supplied to the culture during fermentation of the genetically engineered cells. Preferably, the initial substrate is lactose and / or LNT-II, and the substrate for HMO formation is added during the culture of the genetically engineered cells.

[0286] The desired oligosaccharide, such as the desired HMO, can be recovered from the culture, medium and / or genetically engineered cells.

[0287] Cultivation / fermentation

[0288] Cultivation, cultivation or fermentation (fermenting) or fermentation (fermentation) in a controlled bioreactor (used interchangeably herein) generally includes (a) a first phase of exponential cell growth in a medium in which a carbon source is ensured, and (b) a second phase of cell growth in a medium operated under carbon limitation, wherein a carbon source is continuously added together 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).

[0289] The terms "manufacturing" or "manufacturing scale" or "large-scale production" or "large-scale fermentation" are used interchangeably and define, in the meaning of the present disclosure, 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. Typically, a "manufacturing scale" process is defined as being able to process large production quantities of a desired oligosaccharide product, such as an HMO product, which, for example, in the case of a therapeutic compound or composition, 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 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 cultures. To a large extent, the behavior of the expression system in a laboratory scale process, such as the shake flask, benchtop bioreactor or deep well format described in the embodiments of the present disclosure, does allow prediction of the behavior of the system in the complex environment of a bioreactor.

[0290] There is no restriction on the suitable cell culture medium used in the fermentation process. The culture medium can be semi-restricted, i.e. containing complex culture medium compounds (e.g., yeast extract, soy peptone, casamino acids, etc.), or it can be chemically restricted, without any complex compounds. The carbon source can be selected from glucose, sucrose, fructose, galactose, maltose, xylose and glycerol or alternative carbon source. In one or more exemplary embodiments, the culture medium is supplemented with one or more energy sources and carbon sources selected from glycerol, sucrose and glucose. In other embodiments, lactose is added as the substrate formed by oligosaccharides during the cultivation of genetically engineered cells.

[0291] In one or more embodiments, the culture medium contains sucrose as the sole carbon and energy source. In further embodiments, at least one energy source is added to the culture medium. In embodiments, at least one energy source is preferably selected from glucose, sucrose, fructose, galactose, maltose, xylose, glycerol, and combinations thereof. In one or more exemplary embodiments, the genetically engineered cell comprises 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 cell.

[0292] In an embodiment, the precursor molecule (initial substrate) for synthesizing the desired oligosaccharide is added as the substrate for oligosaccharide formation during the cultivation of genetically engineered cells. In a further embodiment, lactose and / or LNT-II are added during the cultivation of genetically engineered cells as the substrate for forming the desired oligosaccharide.

[0293] In one or more 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).

[0294] The method according to the present disclosure comprises culturing genetically engineered bacterial cells in a culture medium designed to support microbial growth and containing one or more carbohydrate sources, or only a carbon source, such as one or more carbon sources selected from glucose, sucrose, fructose, galactose, maltose, xylose and glycerol, or an alternative carbon source.

[0295] Recycling / Harvesting

[0296] In this article, the term "retrieving" is used interchangeably with the term "harvesting". "Recovery" and "harvesting" in the context all refer to collecting the oligosaccharides produced from the culture / fermentation broth after the fermentation is terminated. In one or more exemplary embodiments, it can include collecting the oligosaccharides 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, i.e., before the removal of the biomass at the end of the fermentation, the cells are lysed to release the oligosaccharide content into the fermentation broth. In other embodiments, the oligosaccharides produced 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).

[0297] The separation of cells from the culture medium can be carried out by any method well known to those skilled in the art, such as any suitable type of centrifugation or filtration. The separation of cells from the culture medium can be carried out immediately after the fermentation broth is harvested, or at a later stage after the fermentation broth is stored under appropriate conditions. Recovering the produced oligosaccharides from the remaining biomass (or total fermentation broth) includes extracting them from the biomass (i.e., the production cells).

[0298] After recovery from fermentation, the oligosaccharides are available for further processing and purification.

[0299] Oligosaccharides can be purified according to procedures known in the art, such as those described in WO2017 / 182965. Purified oligosaccharides can be used as nutraceuticals, pharmaceuticals, or for any other purpose, such as for research.

[0300] manufactures

[0301] The term "finished product" refers to one or more oligosaccharides intended as one or more oligosaccharide products, such as HMOs with an LNT-II core, or a composition of an oligosaccharide mixture. Preferably, the product oligosaccharide is produced by the methods described herein using the genetically engineered cells described herein. In a preferred embodiment, the finished product is LNT or LNnT or LNFP-I or LNT and LNFP-I. Therefore, the present disclosure also relates to oligosaccharides produced by the methods described herein. For example, Example 1 shows that when LNT is produced as described in the present disclosure, the levels of byproducts phosphoglycerol-LNT and phosphoethanolamine-LNT can be eliminated.

[0302] Thus, oligosaccharides (eg, LNT) produced according to the present method can be distinguished from oligosaccharides (eg, LNT) produced by another method in which phosphoglycerol-oligosaccharides (eg, phosphoglycerol-LNT) and phosphoethanolamine-oligosaccharides (eg, phosphoethanolamine-LNT) may be present.

[0303] The finished product may be a powder, composition, liquid, suspension or gel comprising one or more oligosaccharides.

[0304] sequence

[0305] This application contains a Sequence Listing in both text and electronic formats, which is incorporated herein by reference.

[0306] An overview of the sequences described in this application can be found in Table 4, and additional promoter sequences used in this application can be found in Table 3 (promoter sequences SEQ ID NO: 10-34).

[0307] Table 4 – Sequences

[0308]

[0309]

[0310] Example

[0311] method

[0312] 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).

[0313] The examples described below were chosen to illustrate the invention and are not intended to limit the invention in any way.

[0314] strains

[0315] 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 an MDO strain with the following modifications: lacZ: 1.5 kbp deleted, lacA: 0.5 kbp deleted, nanKETA: 3.3 kbp deleted, melA: 0.9 kbp deleted, wcaJ: 0.5 kbp deleted, mdoH: 0.5 kbp deleted, and the Plac promoter was inserted upstream of the gmd gene. This strain was used as the MDO background strain.

[0316] Methods for inserting or deleting a gene of interest in 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).

[0317] To obtain LNT production strains, the MDO strain was further modified by chromosomal integration of β-1,3-GlcNAc transferase (LgtA from Neisseria meningitidis, homologous to NCBI accession number WP_033911473.1, and as shown in SEQ ID NO: 6) and β-1,3-galactosyltransferase (GalTK from Helicobacter pylori, homologous to GenBank accession number BD182026.1, and as shown in SEQ ID NO: 7), both under the control of the PglpF promoter (SEQ ID NO: 22). In addition, the strain was supplemented with MFS transporter to export LNT to the outside of the cell, and the strain was named LNT strain (MP0 in Table 5 below).

[0318] To obtain a LNnT production strain, β-1,3-GlcNAc transferase (LgtA from Neisseria meningitidis, homologous to NCBI accession number WP_033911473.1 and shown in SEQ ID NO:6) and β-1,4-galactosyltransferase (GalT from Helicobacter pylori, homologous to GenBank ID WP_001262061.1 and shown in SEQ ID NO:8) were integrated by chromosome, both under the control of the PglpF promoter (SEQ ID NO:22). To increase LNnT production and export from cells, the strain was supplemented with the MFS transporter, which was named LNnT strain (MP2 in Table 5 below).

[0319] The LNT and LNnT strains were further engineered by deleting the endogenous genes opgE (SEQ ID NO: 1) and opgB (SEQ ID NO: 2) (MP1 and MP3, respectively, in Table 5 below).

[0320] Table 5 provides the genotypes of the background strain (MDO) and the LNT and LNnT production strains MP0, MP1, MP2 and MP3.

[0321] Table 5 - Genotypes of strains used in fermentation

[0322]

[0323] 1 lgtA-PglpF - three genomic inserted copies of the gene encoding β-1,3-N-acetyl-glucosaminyltransferase (SEQ ID NO: 6) under the control of the PglpF promoter (SEQ ID NO: 22).

[0324] 2 galTK-PglpF - A genomic insert encoding β-1,3-galactosyltransferase (SEQ ID NO:7) under the control of the PglpF promoter (SEQ ID NO:22).

[0325] 3 PglpF-SD1-Pscr-scrYA - one genomic insertion copy of the scrY and scrA gene cluster (WO2015 / 197082) under the control of the double PglpF-SD1 (SEQ ID NO: 33) Pscr promoter (SEQ ID NO: 34).

[0326] 4 Pscr-scrBR - a genomic insertion copy of the scrB and scrR gene cluster (WO2015 / 197082) under the control of the Pscr (SEQ ID NO: 34) promoter.

[0327] 5 PglpF-LacY - A genomic insert encoding the lactose permease LacY (SEQ ID NO: 9) under the control of the PglpF promoter (SEQ ID NO: 22).

[0328] 6 PglpF-Nec - a genomic insert encoding the MFS transporter Nec (SEQ ID NO: 5) under the control of the PglpF promoter (SEQ ID NO: 22).

[0329] 7 ΔopgE deletes the gene encoding phosphoethanolamine transferase OpgE (SEQ ID NO: 2).

[0330] 8 ΔopgB deletes the genes encoding phosphoglycerol transferases I and II (SEQ ID NO: 1).

[0331] 9PglpF-galT - A genomic insert encoding beta-1,4-galactosyltransferase (SEQ ID NO: 8) under the control of the PglpF promoter.

[0332] 10 2xPglpF - Exporter - Encodes two different genomic insertion genes for a transporter capable of exporting LNnT from cells. Such transporters are described, for example, in WO2021 / 148611, WO2021 / 148610 and PCT / EP2024 / 054663.

[0333] Deep hole measurement

[0334] The deep well assay in the present example was performed as originally described by Lv et al. (Bioprocess Biosyst Eng 20 (2016) 39: 1737-1747) and optimized for the purposes of the present disclosure. More specifically, the strains disclosed in this example were screened in 96 deep well plates using a 4-day protocol. Within the first 24 hours, the preculture was grown to high density (OD600 up to 5) and then transferred to a medium capable of inducing gene expression and product formation.

[0335] More specifically, on day 1, a fresh preculture was prepared using basal minimal medium (BMM) (pH 7.0) supplemented with magnesium sulfate (0.12 g / L), thiamine (0.004 g / L) and glucose (5.5 g / L). The composition of the basal minimal medium was as follows: NaOH (1 g / L), KOH (2.5 g / L), KH2PO4 (7 g / L), NH4H2PO4 (7 g / L), citric acid (0.5 g / l), trace mineral solution (5 mL / L). The trace mineral stock solution contained ZnSO4*7H2O 0.82 g / L, citric acid 20 g / L, MnSO4*H2O 0.98 g / L, FeSO4*7H2O 3.925 g / L, CuSO4*5H2O 0.2 g / L. The pH of the basal minimal medium was adjusted to 7.0 with 5N NaOH and autoclaved. Pre-culture was incubated for 24 hours at 34°C and 1000rpm vibration, then further transferred to 0.75mL of new BMM (pH 7.5) to start main culture. New BMM has been supplemented with magnesium sulfate (0.12g / L), thiamine (0.02g / L), a group of glucose solution (0.1-0.15g / L) and a group of lactose solution (5-20g / L). In addition, 20% sucrose (40-45g / L) or maltodextrin (19-20g / L) storage solution is provided as carbon source, specific hydrolase, sucrose hydrolase or saccharifying enzyme are added respectively simultaneously, therefore, glucose is released at a rate suitable for carbon-restricted growth, and is similar to typical batch fed fermentation process. Main culture was incubated for 72 hours at 28°C and 1000rpm vibration. In order to analyze total fermentation broth, 96 orifice plates were boiled at 100°C, centrifuged subsequently, and finally supernatant was analyzed by HPLC.

[0336] Fermentation

[0337] Two LNT-producing E. coli strains, MP0 (control) and MP1 (opgB / opgE double knockout) were cultured in duplicate in 250 mL disposable fermenters (AMBR250 automated microbioreactor system, Sartorius, Germany), starting with 100 mL mineral medium containing 31.2 g / kg sucrose as carbon source, MgSO4 x 7H2O, KOH, H3PO4, citric acid, trace element solution (containing Fe, Mn, Zn, Cu, Mo and Se), defoamer and thiamine. The dissolved oxygen level was maintained at 23% by a series of operations: first stirring, then starting the airflow at 700 rpm and 1 VVM, respectively. The pH was maintained at 6.8 by titration with 8.5% NH4OH solution, which also served as a nitrogen source. The culture was started with an inoculum of 2% (v / v) from a preculture grown in a similar sucrose-containing medium. After depletion of sucrose contained in the batch medium, a feed solution containing 49% (w / w) sucrose, MgSO4 x 7H2O, KOH, H3PO4, citric acid, trace element solution (elements as described above), defoamer and thiamine was continuously fed using a predetermined feed profile to ensure carbon-limited growth conditions, starting at an initial feed rate of 3.14 g sucrose / kg fermentation broth / h. In addition, a (NH4)2SO4 stock solution (400 g / kg) was added daily to maintain adequate ammonium levels in the fermentation broth. The temperature was initially controlled at 33°C and decreased to 27°C in a 5-hour gradient after 20 hours of feeding. Acceptor lactose was added in the form of a bolus, starting with a 25% (w / w) lactose monohydrate solution, and then added every 24 hours to prevent lactose from being limited. The growth and metabolic activity and state of the cells were tracked by online measurement of reflectance and CO2 release rate and offline measurement of biological wet mass concentration (BWM). Daily fermentation samples were analyzed by HPLC for product (LNT), acceptor (lactose) and major byproducts such as trisaccharide LNT II, ​​hexasaccharide pLNH2, phosphoethanolamine-LNT (EP-LNT) and phosphoglycerol-LNT (GP-LNT). Fermentation was stopped after approximately 115 hours.

[0338] Example 1 - Production of LNT by fermentation

[0339] Genetically engineered cells capable of producing LNT carrying the ΔopgE and ΔopgB deletions were fermented and evaluated for their ability to reduce byproduct production while maintaining acceptable levels of LNT production.

[0340] To evaluate the effects of the deletions of the opgE and opgB genes described herein, the strains were tested in a fermentation process, wherein the strains were fermented as described above in the "Methods" section.

[0341] After the fermentation of the strain was completed, the contents of the desired product (LNT), acceptor (lactose), and major byproducts (such as trisaccharide LNT II, ​​hexasaccharide pLNH2, phosphoethanolamine-LNT (EP-LNT), and phosphoglycerol-LNT (GP-LNT)) were analyzed by HPLC. Figure 4 shown.

[0342] exist Figure 4 In the chromatogram of MP0 (top chromatogram), GP-LNT and EP-LNT were eluted from the HPLC column at about 6.7 minutes and about 9.15 minutes, respectively. For MP1, there were no peaks at about 6.7 minutes (GP-LNT) and about 9.15 minutes (EP-LNT), clearly indicating that the strain carrying the deletion of opgE and opgB did not produce GP-LNT or EP-LNT.

[0343] The total LNT content from fermentations of LNT-producing strains MP0 and MP1 was analyzed and the results are shown in Tables 6 and Figure 5 (LNT titers normalized to maximum MP0 LNT production, expressed in %), Figure 6 (EP-LNT / LNT ratio (mass %)) and Figure 7 (GP-LNT / LNT ratio (mass %)).

[0344] Table 6 - Average LNT production of the ΔopgE, ΔopgB strain (MP1) relative to the average LNT production of the strain (MP0) set as 100% (fermentations were performed in duplicate).

[0345]

[0346] From Table 6 and Figure 5 As can be seen in the graph, the total production of LNT in the MP1 strain was comparable to the amount of LNT produced by the strain MP0 (99.8%), indicating that the deletion of the ΔopgE and ΔopgB genes did not have a significant effect on LNT production.

[0347] From Table 4 and Figure 6 It can also be seen that MP0 produced about 0.21% (mass %) EP-LNT relative to the amount of LNT produced during the fermentation process. Therefore, for the MP0 strain, the mass ratio of LNT:EP-LNT is about 475:1. In addition, from Tables 4 and Figure 7 It can also be seen that MP0 produced about 2.6% (mass %) GP-LNT relative to the amount of LNT produced during fermentation. Therefore, for the MP0 strain, the mass ratio of LNT:EP-LNT is about 38:1.

[0348] In contrast, from Table 4 and Figure 6 and 7It can be seen that MP1 did not produce detectable amounts of byproducts GP-LNT and EP-LNT.

[0349] Thus, deletion of the ΔopgE and ΔopgB genes abolished the formation of phosphoglycerol-LNT and phosphoethanolamine-LNT without significantly affecting the overall production of LNT.

[0350] Deletion of the opgE and opgB genes is beneficial in reducing the levels of byproducts carrying phosphoethanolamine (EP) and phosphoglycerol (GP) moieties, because these two genes express the transferases responsible for transferring the EP and GP moieties to oligosaccharides (see Figure 1 ). Therefore, deletion of these genes allows the production of LNT without the unwanted byproducts glycerophospho-LNT (GP-LNT) and ethanolamine-phospho-LNT (EP-LNT) (see Figure 2 Importantly, deletion of the OpgE and OpgB genes should not negatively affect the production of the major product to a large extent, as this would reduce the commercial value of the strain, as a large decrease in yield would have a significant impact on production costs.

[0351] Example 2 - Production of LNnT by fermentation

[0352] As described in the Methods section above, the ability to produce LNnT in genetically engineered cells with or without the opgE and opgB genes was assessed by deep well assay to evaluate the effect of deletion of the opgE and opgB genes on LNnT production.

[0353] The formation of HMO LNnT and pLNnH was detected using HPLC. In the deep well assay, no phosphoglycerol and phosphoethanolamine substituted byproducts were observed in the HPLC of the control strain (MP2) or the ΔopgE and ΔopgB strains (MP3). Table 7 and Figure 8 Comparative data are shown, where the total amount of individual HMO or total HMO in the control strain was set to 100%.

[0354] Table 7 - Average LNnT, pLNnH and total HMO production of the ΔopgE, ΔopgB strains (MP3) relative to the respective average LNnT, pLNnH and total HMO production of the control strain (MP2).

[0355] strains describe LqCy HkDJ Total HMO MP2 Control, opgB+, opgE+ 100 100 100 MP3 ΔopgE and ΔopgB 122 134 123

[0356] From Table 7 (also Figure 8 As shown in Figure 2, the deletion of opgE and opgB in the LNnT production strain increased the total HMO formation by more than 20% compared to the control strain. The formation of LNnT and pLNnH was also increased by more than 20% relative to the formation of the same products in the control strain.

[0357] The ΔopgE, ΔopgB strain (MP3) produced 88% LNnT and 12% pLNnH of the total HMO, which was similar to the control strain (MP2) which produced 89% LNnT and 11% pLNnH.

[0358] The absence of phosphoglycerol and phosphoethanolamine substituted byproducts in the control strain may be due to the efficient export system that exports LNnT out of the cell.

Claims

1. A genetically engineered Gram-negative bacterial cell having reduced or completely lost function of phosphoglycerol transferase I and II and / or phosphoethanolamine transferase, wherein the cell comprises at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase.

2. The genetically engineered bacterial cell according to claim 1, wherein the function of the OPG biosynthesis protein C of the cell is reduced or completely lost.

3. A genetically engineered bacterial cell according to claim 1 or 2, wherein the gene encoding the phosphoglycerol transferases I and II (opgB) and / or the gene encoding the phosphoethanolamine transferase (opgE) and / or the gene encoding the biosynthetic protein C (opgC), or their functional equivalents / homologous genes, are deleted or become dysfunctional.

4. The genetically engineered bacterial cell of any one of the preceding claims, wherein the gene encoding the phosphoglycerol transferases I and II (opgB) and the gene encoding the phosphoethanolamine transferase (opgE) are deleted or rendered dysfunctional.

5. A genetically engineered bacterial cell according to any one of the preceding claims, wherein the cell is capable of producing one or more hetero-oligosaccharides.

6. The genetically engineered bacterial cell of any one of the preceding claims, wherein the cell is capable of producing an HMO selected from the group consisting of LNT, LNnT, LNT-II, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, pLNH, F-pLNHI, pLNnH, LSTa, LSTb, LSTc and DSLNT.

7. The genetically engineered bacterial cell of any of the preceding claims, wherein the genetically engineered cell comprises one or more heterologous nucleic acids encoding one or more heterologous glycosyltransferases selected from the group consisting of β-1,3-N-acetyl-glucosaminyltransferase, β-1,3-galactosyltransferase and β-1,4-galactosyltransferase.

8. The genetically engineered bacterial cell of any one of the preceding claims, wherein the genetically engineered Gram-negative bacterium is Escherichia coli.

9. A method for producing an oligosaccharide product, wherein the method comprises, a. providing a genetically engineered cell according to any one of claims 1 to 8, b. culturing the genetically engineered cells in a culture medium under conditions that allow production of the oligosaccharide product; and optionally c. recovering the oligosaccharide product.

10. A method according to claim 9, wherein the production of the desired oligosaccharide is reduced by no more than 15%, preferably by no more than 10%, and preferably is not substantially reduced, when compared to cells in which the function of phosphoglycerol transferases I and II and / or phosphoethanolamine transferase and / or OPG biosynthetic protein C is not reduced or completely lost.

11. The method according to claim 9 or 10, wherein the method produces reduced amounts of by-product oligosaccharides comprising phosphoethanolamine-moieties and / or glycerophosphate-moieties and / or succinyl-moieties compared to a method using cells containing phosphoglyceroltransferases I and II and / or phosphoethanolaminetransferases and / or OPG biosynthetic protein C, or functionally equivalent / homologous genes thereof.

12. The method according to claim 11, wherein the byproduct is phosphoglycerol-LNT and / or phosphoethanolamine-LNT or phosphoglycerol-LNnT and / or phosphoethanolamine-LNnT.

13. The method of any one of claims 9 to 12, wherein the desired oligosaccharide is one or more human milk oligosaccharides (HMOs) selected from the group consisting of LNT, LNnT, LNT-II, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, pLNH, F-pLNHI, pLNnH, LSTa, LSTb, LSTc and DSLNT.

14. The method of claim 13, wherein the HMO is LNT or LNnT.

15. Use of a genetically engineered cell according to any one of claims 1 to 8 in the production of an oligosaccharide product.

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