Genetically modified UDP-N-acetylglucosamine producing cells

Abstract

The present invention relates to a genetically modified cell capable of producing UDP-N-acetylglucosamine (UDP-GlcNAc), wherein the mRNA stability of native glucosamine-6-phosphate synthase (glmS) is enhanced by modifying the cell such that its RNase adapter protein RapZ (yhbJ) function is reduced or completely lost. In particular, the present invention relates to said cells which are also capable of producing one or more N-acetylated and / or sialylated molecules, such as N-acetylated and / or sialylated human milk oligosaccharides (HMOs). The invention also relates to a method for producing one or more N-acetylated-and / or sialylated human milk oligosaccharides (HMO) using such cells.

Description

Technical Field

[0001] The present invention relates to a genetically modified cell capable of producing UDP-N-acetylglucosamine (UDP-GlcNAc), wherein the mRNA stability of native glucosamine-6-phosphate synthase (glmS) is enhanced by modifying the cell to reduce or completely abolish the function of its RNase adaptor protein RapZ (yhbJ). In particular, the present invention relates to such cells further modified to produce one or more N-acetylated- and / or sialylated molecules, such as N-acetylated- and / or sialylated human milk oligosaccharides (HMOs). The present invention also relates to a method for producing one or more N-acetylated- and / or sialylated molecules, such as human milk oligosaccharides (HMOs), using such cells. Background Art

[0002] Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is an acetylated amino sugar nucleotide that naturally serves as a precursor for bacterial cell wall synthesis and is involved in prokaryotic and eukaryotic ribosylation reactions. UDP-GlcNAc has applications in multiple fields, including the production of oligosaccharides and glycoproteins with therapeutic benefits.

[0003] The physiological level of UDP-GlcNAc in cells plays an important role in the biotechnological production of various compounds. In particular, the level of UDP-GlcNAc in cells can affect the yield of a desired compound during its biotechnological production, since UDP-GlcNAc plays a key role in a series of biosynthetic pathways of desired compounds, especially in its function as a glycosyl donor.

[0004] For example, it is known that the cell-based biotechnological production of human milk oligosaccharides (HMOs) and the resulting HMO yield are affected by the physiological level of UDP-GlcNAc, since the compound is involved in the biosynthetic pathway leading to the formation of certain human milk oligosaccharides.

[0005] Therefore, there is a desire to provide cells for the biotechnological production of certain human milk oligosaccharides that exhibit high levels of UDP-GlcNAc to increase the HMO yield. However, finding suitable cell modifications that are practically feasible and separately providing a sufficiently high increase in UDP-GlcNAc concentration and HMO yield is challenging.

[0006] Regarding the production of HMOs, examples of increasing the expression of phosphoglucosamine mutase (GlmM), N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU), and / or glucosamine-6-phosphate synthase (GlmS) are described, for example, in WO2014 / 153253 and Zhu et al. 2021 J Agric Food Chem 69:3702-3711.

[0007] Sugita and Koketsu described a slight increase in LNT-II in cells containing a yhbJ deletion, but did not disclose an increase in the UDP-GlcNAc pool in the cells (Sugita and Koketsu 2022 J. Agric. Food Chem. 70:5106-5114).

[0008] In addition, during the biotechnological production of human milk oligosaccharides, a particular cell typically produces more than one human milk oligosaccharide. However, in some cases, only one or two specific human milk oligosaccharides are actually desired in the produced human milk oligosaccharide mixture. Thus, in addition to merely optimizing the overall HMO yield during the biotechnological production process, it is desirable to provide modifications to the cells to allow for the significant formation of specific human milk oligosaccharides in the produced human milk oligosaccharide mixture, i.e., to increase the proportion of certain human milk oligosaccharides in the human milk oligosaccharide mixture. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Schematic diagram of the metabolic pathway responsible for UDP-GlcNAc biosynthesis. Proteins are shown in bold.

[0010] Figure 2 UDP-GlcNAc concentrations for various modified strains and the control strain MDO.

[0011] Figure 3 Shows the total and individual HMO concentrations produced by the LNnT-producing strains M12 and M13 with two copies of the β-1,3-N-acetylglucosaminyltransferase LgtA gene. Figure 3 A shows the total and individual HMO concentrations (mM) relative to the total HMO amount produced by the control strain (MP12). Figure 3 B shows the individual HMO concentrations (mM) relative to the specific HMO concentration in the control strain (MP12).

[0012] Figure 4 Shows the total and individual HMO concentrations produced by the LNnT-producing strains M14 and M15 with one copy of the β-1,3-N-acetylglucosaminyltransferase LgtA gene. Figure 4 A shows the total and individual HMO concentrations (mM) relative to the total HMO amount produced by the control strain (MP14). Figure 4 B shows the individual HMO concentrations (mM) relative to the specific HMO concentration in the control strain (MP14).

[0013] Figure 5 Shows the total and individual HMO concentrations produced by the LNT-producing strains M16 and M17 with two copies of the β-1,3-N-acetylglucosaminyltransferase LgtA gene. Figure 5A shows the total and individual HMO concentrations (mM) relative to the total amount of HMOs produced by the control strain (MP16). Figure 5 B shows the individual HMO concentrations (mM) relative to the specific HMO concentration in the control strain (MP16).

[0014] Figure 6 Shows the total and individual HMO concentrations produced by the LNT-producing strains MP18 and M19 with one copy of the β-1,3-N-acetylglucosaminyltransferase LgtA gene. Figure 6 A shows the total and individual HMO concentrations (mM) relative to the total amount of HMOs produced by the control strain (MP18). Figure 6 B shows the concentration (mM) of individual HMOs in MP19 relative to the specific HMO concentration in the control strain (MP18). The control strain was set to 100% and not shown.

[0015] Figure 7 Is the relative concentration of 3'SL in the test strains MP21 (ΔyhbJ), MP22 (ΔyhbJ+glmS), and MP23 (ΔyhbJ+glmU) compared to the control strain MP20.

[0016] Figure 8 Is the relative concentration of LNT-II in the test strain MP25 (ΔyhbJ+glmU+glmM) compared to the control strain MP24. Summary of the Invention

[0017] The present invention relates to a genetically modified cell capable of producing UDP-N-acetylglucosamine (UDP-GlcNAc), wherein the mRNA stability of the native glucosamine-6-phosphate synthase (glmS) is enhanced by modifying the cell to reduce or completely abolish the function of its RNase adaptor protein RapZ (yhbJ).

[0018] In one embodiment, the genetically modified cell is further modified to overexpress phosphoglucosamine mutase (glmM) and / or N-acetylglucosamine-1-phosphate uridylyltransferase (glmU), and / or overexpress glucosamine-6-phosphate synthase (glmS). Preferably, such a cell is capable of producing N-acetylated- and / or sialylated molecules, such as oligosaccharides, lipids, or proteins containing N-acetylglucosamine, or sialylated oligosaccharides, lipids, or proteins.

[0019] In one embodiment, the genetically modified cell contains a yhbJ deletion and expresses an α-2,3-sialyltransferase or an α-2,6-sialyltransferase and a biosynthetic pathway for preparing sialic acid sugar nucleotides. Preferably, such a cell is capable of producing sialylated oligosaccharides.

[0020] In a particular embodiment, the genetically modified cell according to the invention is capable of producing one or more human milk oligosaccharides (HMOs), in particular N-acetylated- and / or sialylated HMOs.

[0021] Another aspect of the invention relates to a method for producing one or more N-acetylated- and / or sialylated molecules, which comprises the steps of:

[0022] a) providing a genetically modified cell according to the invention, which is capable of producing one or more N-acetylated- and / or sialylated molecules; and

[0023] b) culturing the cell according to (a) in a suitable cell culture medium to produce said molecules.

[0024] Another aspect of the invention relates to human milk oligosaccharides (HMOs) produced by the method according to the invention.

[0025] Another aspect of the invention relates to the use of a genetically modified cell according to the invention for the production of human milk oligosaccharides (HMOs), said genetically modified cell being capable of producing one or more human milk oligosaccharides. Detailed Description

[0026] Cells capable of producing UDP-N-acetylglucosamine (UDP-GlcNAc)

[0027] The invention relates to a genetically modified cell capable of producing UDP-N-acetylglucosamine (UDP-GlcNAc), wherein the mRNA stability of the native glucosamine-6-phosphate synthase (glmS) is enhanced by modifying the cell to reduce or completely abolish the function of its RNase adaptor protein RapZ (yhbJ).

[0028] In this context, the terms "genetically modified cell" and "genetically engineered cell" can be used interchangeably. As used herein, a "genetically modified cell" refers to a host cell whose genetic material has been altered by human intervention using genetic engineering techniques such as, but not limited to, transformation or transfection, e.g., with a heterologous polynucleotide sequence, Crispr / Cas editing, and / or random mutagenesis. In one embodiment, the genetically engineered cell has been transformed or transfected with a recombinant nucleic acid sequence.

[0029] The genetically modified cell according to the invention is characterized in that it is capable of producing UDP-N-acetylglucosamine (UDP-GlcNAc).

[0030] Thus, the modified cell according to the invention has the genetic ability to produce UDP-N-acetylglucosamine (UDP-GlcNAc).

[0031] In one embodiment, the genetically modified cell is capable of producing UDP-N-acetylglucosamine (UDP-GlcNAc) via the de novo pathway. In this regard, UDP-GlcNAc is prepared by the cell from simple carbon sources such as glycerol, sucrose, fructose or glucose in a stepwise reaction sequence under the action of enzymes involved in the de novo biosynthetic pathway.

[0032] The enzymes involved in the de novo biosynthesis pathway of UDP-GlcNAc may be naturally present in the cell or introduced into the cell by genetic engineering or recombinant DNA techniques, all of which are part of the general knowledge of those skilled in the art. Figure 1 Show the de novo biosynthesis pathway of UDP-GlcNAc.

[0033] UDP-GlcNAc is a nucleotide-activated sugar that can serve as a glycosyl donor in various biosynthetic pathways, where the glycosylation reaction is mediated by the corresponding glycosyltransferase.

[0034] A further feature of the genetically modified cell according to the invention is that by modifying the cell to reduce or completely abolish the function of its RNase adaptor protein RapZ (yhbJ), the mRNA stability of the native glucosamine-6-phosphate synthase (glmS) is enhanced.

[0035] The "native" glucosamine-6-phosphate synthase (glmS) refers to the glmS gene that is naturally present in the cell, not introduced into the cell by genetic engineering techniques or otherwise manipulated to increase its expression, such as by recombinantly introducing a stronger promoter or other regulatory sequences.

[0036] The yhbJ gene encodes the RNase adaptor protein RapZ (RapZ), which has an effect on the stability of the messenger RNA encoding the GlmS enzyme, which may be a rate-limiting factor for UDP-GlcNAc biosynthesis in the cell. The inventors of the present invention have now found that cell modification resulting in reduced or complete loss of function of the RNase adaptor protein RapZ is beneficial for increasing the level of UDP-N-acetylglucosamine (UDP-GlcNAc) in the cell. In Escherichia coli, the ybhJ gene contains the nucleic acid sequence according to SEQ ID NO:5 or consists of it, which encodes the RapZ protein, which contains the amino acid sequence according to SEQ ID NO:4 or consists of it.

[0037] The potential mechanism by which the RNase adaptor protein RapZ affects the stability of the messenger RNA encoding the GlmS enzyme is as follows: Two small RNAs, glmY and glmZ, are involved in the regulation of GlmS expression, as glmZ stabilizes the glmS mRNA and permits the expression of GlmS. RapZ (encoded by yhbJ) is capable of binding to both glmY and glmZ. When the concentration of glucosamine-6-phosphate (GlcN-6-P) in the cell is high, the content of the small RNA glmY is low. Under these conditions, the RNase adaptor protein RapZ can freely bind to another small RNA, glmZ, and recruit it via protein interaction to the processing by RNase E. As a result, glmZ is inactivated and thus unable to stabilize the glmS mRNA, leading to the degradation of GlmS. In contrast, when the GlcN-6-P level decreases, glmY accumulates and binds and sequesters the RNase adaptor protein RapZ. Consequently, glmZ remains unbound and cannot be processed by RNase E. As a result, the glmZ base pairs with glmS in an Hfq-dependent manner and activates the synthesis of GlmS, thereby resynthesizing GlcN-6-P. In other words, the RNase adaptor protein RapZ is involved in a feedback coupling mechanism that controls the synthesis of GlmS.

[0038] The inventors of the present invention have now found that a decrease or complete loss of the function of the RNase adaptor protein RapZ results in the stabilization of the messenger RNA encoding the GlmS enzyme, which in turn leads to an increase in the synthesis of the GlmS enzyme. Since glucosamine-6-phosphate synthase (GlmS) is the enzyme that mediates the biosynthesis of GlcN-6-P from Fru-6-P (fructose-6-phosphate) in the biosynthetic pathway leading to UDP-N-acetylglucosamine (UDP-GlcNAc), the inventors have found that the level of UDP-N-acetylglucosamine (UDP-GlcNAc) in the cell can thus be effectively increased.

[0039] In a preferred embodiment, a decrease or complete loss of the function of the RNase adaptor protein RapZ (yhbJ) is achieved by deleting or rendering dysfunctional the gene encoding the RNase adaptor protein RapZ (yhbJ).

[0040] The terms "deleting" and "rendering dysfunctional" refer to the native yhbJ gene and may include 1) partial or complete removal of the coding sequence of the gene, 2) introduction of a stop codon in its coding sequence, 3) inactivation or significant attenuation of the gene promoter or Shine-Dalgarno sequence, 4) deletion of the gene encoding the direct activator of the native promoter of the yhbJ (rapZ) gene or enhanced expression of the gene encoding its direct repressor, 5) inactivation of the gene by insertion of an expression cassette encoding another gene product (such as, but not limited to, a drug or antibiotic marker or a glycosyltransferase).

[0041] The genetically engineered cell can be any cell capable of producing UDP-N-acetylglucosamine (UDP-GlcNAc). Preferably, the host cell is a unicellular microorganism of eukaryotic or prokaryotic origin. Suitable microbial cells that can serve as host cells include yeast cells, bacterial cells, archaeal cells, algal cells, and fungal cells.

[0042] The genetically engineered cell (host cell) can be, for example, a bacterial or yeast cell. In a preferred embodiment, the genetically engineered cell is a bacterial cell.

[0043] In principle, there is no restriction on the bacterial host cell; it can be a eubacterium (Gram-positive or Gram-negative) or an archaeon, provided that they allow genetic manipulation for inserting the gene of interest and can be cultured on a production scale. Preferably, the host cell has the property of allowing culturing to a high cell density. Non-limiting examples of suitable bacterial host cells are Escherichia coli, Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Campylobacter sp., Corynebacterium sp., Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillus can also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans.Similarly, bacteria of the genus Lactobacillus and Lactococcus can be used, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles, Streptomyces lividans, and Proprionibacterium freudenreichii are also suitable bacterial species of the present invention. As part of the present invention, it also includes strains from the genus Enterococcus (such as Enterococcus faecium and Enterococcus thermophiles), the genus Bifidobacterium (such as Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and the genus Pseudomonas (such as Pseudomonas fluorescens and Pseudomonas aeruginosa).

[0044] Non-limiting examples of fungal host cells are yeast cells such as Komagataella phaffii, Kluyveromyces lactis, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae, or filamentous fungi such as Aspargillus sp, Fusarium sp, or Thricoderma sp. Exemplary species are Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Fusarium solani, Fusarium graminearum, and Trichoderma reesei.

[0045] In one or more exemplary embodiments, the genetically engineered cell is selected from Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

[0046] In one embodiment, the genetically engineered cell is Pichia pastoris.

[0047] In one embodiment, the genetically engineered cell is selected from bacteria of the genus Escherichia sp., Bacillus sp., Lactobacillus sp, Corynebacterium sp., and Campylobacter sp.

[0048] In one embodiment, the genetically engineered cell is selected from Escherichia coli, Corynebacterium glutamicum, Lactobacillus lactis, Bacillus subtilis, and Streptomyces lividans.

[0049] In one embodiment, the genetically engineered cell is Bacillus subtilis.

[0050] In one embodiment, the genetically engineered cell is Corynebacterium glutamicum.

[0051] In a preferred embodiment, the genetically modified cell is a microbial cell, such as a cell selected from Escherichia coli, Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum, Campylobacter sp., Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

[0052] In a preferred embodiment, the genetically engineered cell is Escherichia coli, preferably Escherichia coli K-12 strain or Escherichia coli DE3, and most preferably Escherichia coli K-12.

[0053] As Figure 1 shown, the UDP-GlcNAc de novo pathway includes three glucosamine modifying enzymes (Glm), starting from glucosamine-6-phosphate synthase (glmS), which promotes the conversion of Fru-6-P to GlcN-6-P. Then, GlcN-6-P is converted to GlcN-1-P by glucosamine-6-phosphate synthase (GlmS), and finally N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU) converts GlcN-1-P to GlcNAc-1-P, which is then converted to UDP-GlcNAc in a second reaction. Once a limiting factor in UDP-GlcNAc formation is eliminated by increasing GlmS through deletion of the yhbJ gene, a new limiting factor may appear downstream of GlmS, which can be alleviated by overexpressing one or more Glm enzymes.

[0054] In an embodiment, the cell further comprises one or more of the following modifications:

[0055] a) Overexpression of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU), and / or

[0056] b) Overexpression of glucosamine-6-phosphate synthase (GlmS), and / or

[0057] c) Overexpression of phosphoglucosamine mutase (GlmM).

[0058] In the biosynthetic pathway of UDP-N-acetylglucosamine (UDP-GlcNAc), phosphoglucosamine mutase (GlmM) mediates the formation of GlcN-1-P (glucosamine-1-phosphate) from GlcN-6-P. The inventors of the present invention surprisingly found that cells showing reduced or complete loss of function of the RNase adaptor protein RapZ (yhbJ) and overexpression of phosphoglucosamine mutase (GlmM) produce more UDP-N-acetylglucosamine (UDP-GlcNAc) than expected based solely on the additive effect of the above cell modifications. So far, the inventors of the present invention have found that there is a synergistic effect between these two modifications.

[0059] In a preferred embodiment, the cell is further modified to overexpress phosphoglucosamine mutase (GlmM).

[0060] In a further embodiment, the cell further comprises at least one of the following modifications:

[0061] a) Overexpression of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU), and / or

[0062] b) Overexpression of glucosamine-6-phosphate synthase (GlmS).

[0063] Although the loss of RapZ function stabilizes glmS mRNA, thereby increasing the level of GlmS, further increasing the expression by genetic modification of the glmS gene may be beneficial.

[0064] In certain embodiments, the cell comprises the following modifications:

[0065] a) A deleted or dysfunctional yhbJ gene, and

[0066] b) Overexpression of glucosamine-6-phosphate synthase (GlmS).

[0067] In certain embodiments, the cell comprises the following modifications:

[0068] a) A deleted or dysfunctional yhbJ gene, and

[0069] b) Overexpression of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU).

[0070] In certain embodiments, the cell comprises the following modifications:

[0071] a) A deleted or dysfunctional yhbJ gene, and

[0072] b) Overexpression of phosphoglucosamine mutase (GlmM).

[0073] In certain embodiments, the cell comprises the following modifications:

[0074] a) A deleted or dysfunctional yhbJ gene, and

[0075] b) Overexpression of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU), and

[0076] c) Overexpression of glucosamine-6-phosphate synthase (GlmS).

[0077] In certain embodiments, the cell comprises the following modifications:

[0078] a) A deleted or dysfunctional yhbJ gene, and

[0079] b) Overexpression of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU), and

[0080] c) Overexpression of phosphoglucosamine mutase (GlmM).

[0081] In certain embodiments, the cell comprises the following modifications:

[0082] a) a missing or dysfunctional yhbJ gene, and

[0083] b) overexpression of glucosamine-6-phosphate synthase (GlmS), and

[0084] c) overexpression of phosphoglucosamine mutase (GlmM).

[0085] In certain embodiments, the cell comprises the following modifications:

[0086] a) a missing or dysfunctional yhbJ gene, and

[0087] b) overexpression of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU), and

[0088] c) overexpression of glucosamine-6-phosphate synthase (GlmS), and

[0089] d) overexpression of phosphoglucosamine mutase (GlmM).

[0090] The use of the term "overexpression", such as "overexpression" of one or more of the GlmS, GlmM, and GlmU proteins, refers to an elevated level of the expressed protein as compared to the normal protein expression level of the native protein in the cell. In the context of the present invention, overexpression is preferably achieved directly by manipulating the target gene. As described herein, the level of GlmS can be increased, for example, by loss of function of the RapZ protein that indirectly stabilizes glmS mRNA. The level of GlmS can also be increased by directly manipulating the expression of the glmS gene, such as by modifying the gene copy number, controlling the expression of any copy of the gene at the transcriptional or translational level, such as by replacing the native promoter with a strong promoter, deleting regulatory elements in the gene that inhibit gene expression, or introducing episomal elements, such as plasmids, that carry and express the coding sequence of the gene of interest.

[0091] In embodiments, glmS, glmM, and / or glmU genes (target genes) are overexpressed by directly manipulating the genes, such as i) increasing the promoter strength regulating the expression of the target gene, ii) modifying the native Shine-Dalgarno sequence of the target gene with a stronger sequence to facilitate ribosome binding, iii) increasing the chromosomal copy number of the target gene, i.e., by inserting one or more additional copies of the target gene at genomic loci outside the native locus, and iv) expressing the target gene from a low-copy number (5-10 copies per cell) episome to a high-copy number plasmid (300-500 copies per cell).

[0092] In a particular embodiment, overexpression is obtained by

[0093] a) Replace the native promoter encoding the following genes with a stronger promoter

[0094] i. Phosphoglucosamine mutase (glmM), and / or

[0095] ii. N-acetylglucosamine-1-phosphate uridyltransferase (glmU), and / or

[0096] iii. Glucosamine-6-phosphate synthase (glmS); and / or

[0097] b) Insert into said cell a recombinant nucleic acid encoding

[0098] i. Phosphoglucosamine mutase (glmM) that comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1, or consists of the same

[0099] ii. N-acetylglucosamine-1-phosphate uridyltransferase (glmU) that comprises the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:2, or consists of the same

[0100] iii. Glucosamine-6-phosphate synthase that comprises the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:3, or consists of the same

[0101] In this context, the terms "recombinant nucleic acid sequence", "recombinant gene / nucleic acid / nucleotide sequence / DNA encoding" or "encoding nucleic acid sequence" are used interchangeably and are intended to refer to an artificial nucleic acid sequence (i.e., a nucleic acid sequence prepared in vitro using standard laboratory methods) that comprises a set of contiguous, non-overlapping triplets (codons) that are transcribed into mRNA and translated into a protein under the control of appropriate control sequences (i.e., promoter sequences). The recombinant nucleic acid can be an endogenous gene, for example, by introducing a stronger promoter to manipulate its expression level.

[0102] The boundaries of the coding sequence are typically determined by a ribosome binding site located upstream of the open reading frame at the 5' end of the mRNA, a transcription start codon (AUG, GUG or UUG), and a translation stop codon (UAA, UGA or UAG). The coding sequence can include, but is not limited to, genomic DNA, cDNA, synthetic and recombinant nucleic acid sequences.

[0103] The term "nucleic acid" includes RNA, DNA and cDNA molecules. It should be understood that due to the degeneracy of the genetic code, a large number of nucleic acid sequences can be generated that encode a given protein.

[0104] The recombinant nucleic acid sequence can be heterologous. As used herein, "heterologous" refers to a nucleic acid sequence or nucleotide sequence that is exogenous to a cell or organism, i.e., a nucleic acid sequence that does not naturally occur in the cell.

[0105] Increasing the promoter strength driving the expression of a desired target gene can be a way to achieve overexpression. The strength of a promoter can be evaluated using the lacZ enzyme assay, in which β-galactosidase activity is assayed as described previously (see, for example, Miller J.H. Experiments in molecular genetics, Cold Spring Harbor Laboratory Press, New York, 1972). Briefly, cells are diluted in Z-buffer and permeabilized with sodium dodecyl sulfate (0.1%) and chloroform. The LacZ assay is carried out at 30 °C. The samples are pre-warmed, the assay is initiated by adding 200 μl of o-nitrophenyl-β-galactoside (4 mg / ml), and the assay is stopped by adding 500 μl of 1 M Na2CO3 when the sample turns slightly yellow. The release of o-nitrophenol is then assayed as the change in optical density at 420 nm. The specific activity is reported in Miller units (MU) [A420 / (min*ml*A600)]. A regulatory element with an activity higher than 10,000 MU is considered strong, a regulatory element with an activity lower than 3,000 MU is considered weak, and a regulatory element in between has medium strength. An example of a strong regulatory element is the PglpF promoter, which has an activity of approximately 14,000 MU, and an example of a weak promoter is Plac, which has an activity of approximately 2,300 MU when induced with IPTG.

[0106] Alternatively, if it is desired to balance the expression levels of one or more proteins to optimize production, it may be beneficial to use a promoter with a desired strength (e.g., medium or low strength). Table 0 below lists a series of wild-type and recombinant promoters according to their strength relative to the PglpF promoter.

[0107] Table 0 - Promoter sequences according to strength

[0108]

[0109] * Promoter activity was evaluated in the LacZ assay described below, and the PglpF promoter was used as a positive reference in the same assay. To compare between assays, the activity relative to the PglpF promoter was calculated, and a range represents the results of multiple detections.

[0110] The promoter can be of heterologous origin, the native promoter of a genetically modified cell, or can be a recombinant promoter combining heterologous and / or native elements.

[0111] In one or more exemplary embodiments, the promoter sequence is selected from the group consisting of Plac, PglpF, PglpA, PglpT or PmglB and their variants.

[0112] In one or more exemplary embodiments, the promoter is selected from PglpF (SEQ ID NO:23), PglpT_70UTR (SEQ ID NO:21), PgatY_70UTR (SEQ ID NO:22), Plac_70UTR (SEQ ID NO:13), PmglB_54UTR (SEQ ID NO:12), PmglB_70UTR (SEQ ID NO:19), PglpA_70UTR (SEQ ID NO:20), or variants thereof. Specifically, the variants disclosed in Table 0 are preferred.

[0113] In the currently preferred embodiment, the promoter sequence is selected from the strong promoters PmglB_70UTR_SD8 (SEQ ID NO:10), PmglB_70UTR_SD10 (SEQ ID NO:11), PmglB_54UTR (SEQ ID NO:12), Plac_70UTR (SEQ ID NO:13), PmglB_70UTR_SD9 (SEQ ID NO:14), PmglB_70UTR_SD4 (SEQ ID NO:15), PmglB_70UTR_SD5 (SEQ ID NO:16), PglpF_SD4 (SEQ ID NO:17), PmglB_70UTR_SD7 (SEQ ID NO:18), PmglB_70UTR (SEQ ID NO:19), PglpA_70UTR (SEQ ID NO:20), PglpT_70UTR (SEQ ID NO:21), PgatY_70UTR (SEQ ID NO:22), PglpF (SEQ ID NO:23), PglpF_SD10 (SEQ ID NO:24), PglpF_SD5 (SEQ ID NO:25), PglpF_SD8 (SEQ ID NO:26), PmglB_16UTR (SEQ ID NO:27).

[0114] In a preferred embodiment, the overexpression of the nucleic acid sequence encoding the enzymes of the UPD-GlcNAc pathway is controlled by the PglpF (SEQ ID NO:23) promoter or another strong promoter selected from Table 0.

[0115] In one embodiment, the nucleic acid encoding GlmM is under the control of the PglpF (SEQ ID NO:23) promoter or another strong promoter selected from Table 0. Preferably, by replacing the native promoter with a stronger promoter.

[0116] In one embodiment, the nucleic acid encoding GlmU is under the control of the PglpF (SEQ ID NO:23) promoter or another strong promoter selected from Table 0. Preferably, by replacing the native promoter with a stronger promoter.

[0117] In one embodiment, the nucleic acid encoding GlmS is under the control of the PglpF (SEQ ID NO:23) promoter or another strong promoter selected from Table 0. Preferably, by replacing the native promoter with a stronger promoter.

[0118] The term "sequence identity" as used herein describes the relatedness between two amino acid sequences or between two nucleotide sequences (i.e., a candidate sequence (e.g., a sequence of the present invention) and a reference sequence (e.g., a prior art sequence)) based on their pairwise comparison. For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), which is implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or higher (available at https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of 30BLOSUM62) substitution matrix. The output of Needle labeled "identity" (obtained using the -nobrief option) is used as the percent identity. Generally, sequence identity can be calculated as follows: (identical residues x 100) / (length of the alignment region).

[0119] For the purposes of the present invention, 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), preferably version 5.0.0 or higher. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "identity" (obtained using the -nobrief option) is used as the percentage identity. Generally, sequence identity can be calculated as follows: (number of identical deoxyribonucleotides x 100) / (length of the alignment region).

[0120] Cells capable of producing N-acetylated- and / or sialylated molecules

[0121] In an embodiment, the cells according to the invention are capable of producing one or more oligosaccharides, glycoproteins or glycolipids, in particular one or more N-acetylated- and / or sialylated oligosaccharides, or one or more N-acetylated- and / or sialylated glycoproteins, or one or more N-acetylated- and / or sialylated glycolipids.

[0122] The expressions "N-acetylated oligosaccharide" or "N-acetylated protein" or "N-acetylated lipid" refer to an oligosaccharide, protein or lipid containing at least one N-acetylglucosamine moiety, also referred to as an N-acetylglucosamine-containing molecule (e.g., an oligosaccharide, lipid or protein). Suitable examples of N-acetylated oligosaccharides are lacto-N-triose II (LNT-II), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), para-lacto-N-neohexose (para-LNnH), disialyl lacto-N-tetraose (DSLNT) or any combination thereof. Other examples are lacto-N-hexose, lacto-N-neohexose, para-lacto-N-hexose, para-lacto-N-neohexose, lacto-N-octose, lacto-N-neooctose, iso-lacto-N-octose, para-lacto-N-octose and lacto-N-decose or other HMOs containing an LNT-II backbone listed below. Other examples of N-acetylated oligosaccharides are polysaccharides such as chitin, chitosan, hyaluronic acid, chondroitin sulfate or heparan sulfate.

[0123] The terms "sialylated oligosaccharide" or "sialylated protein" or "sialylated lipid" refer to an oligosaccharide, protein, or lipid that contains at least one N-acetylneuraminic acid (Neu5Ac) or at least one O-acetylneuraminic acid (Neu9Ac) moiety. Suitable examples of N-acetylated oligosaccharides include 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), and 3-fucosyl-3'-sialyllactose (FSL), sialyl-Lewis-X, 6'-sialyl-lacto-N-tetraose b (LST-b), 6'-sialyl-lacto-N-neotetraose (LST-c), 3'-sialyl-lacto-N-neotetraose (LST-d), or other HMOs containing sialic acid moieties listed below.

[0124] Examples of N-acetylated and / or sialylated glycoproteins are, for example, mucins. The therapeutic potential of lectins has also been increasingly explored, such as monoclonal antibodies designed to contain specific glycosylation patterns, including GlcNAc residues, which are crucial for the targeting and efficacy of monoclonal antibodies.

[0125] Examples of N-acetylated and / or sialylated glycolipids are, for example, sialylated liposomes or gangliosides or glycosphingolipids, all of which are modified with varying amounts of sialic acid and N-acetylglucosamine.

[0126] In an embodiment, the N-acetylated- and / or sialylated molecule is selected from an oligosaccharide, a glycoprotein, and a glycolipid. If the molecule is N-acetylated, then preferably, at least one N-acetylation in the molecule is an N-acetylglucosamine moiety.

[0127] In an embodiment, a cell according to the present invention is capable of producing one or more oligosaccharides selected from: LNT-II, LNT, LNnT, LNH, LNnH, pLNH, pLNnH, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, F-pLNnH, DF-LNH a, DF-LNH b, DF-LNH c, DF-pLNnH, TF-pLNnH, FDS-LNH, TF-LNH, DS-LNH, F-LNH-I, F-LNH-II, F-LNH-III, FLST-a (S-LNFP-II), FLST-b, pLNnH, FLST-c, FLST-d, FSL, sialyl-Lewis-X, 3'SL, 6'SL, LST-a, LST-b, LST-c, LST-d, DSLNT, SLNH, SLNH-II, and any mixture thereof, or a polysaccharide selected from chitin, chitosan, hyaluronic acid, chondroitin sulfate, or heparan sulfate.

[0128] In a preferred embodiment, the cells according to the invention are capable of producing one or more human milk oligosaccharides (HMOs), in particular one or more N-acetylated- and / or sialylated HMOs.

[0129] The term "human milk oligosaccharide" or "HMO" in this context refers to complex carbohydrates found in human breast milk. HMOs have a core structure containing a lactose unit at the reducing end, which can be extended by one or more β-N-acetyl-lactosamine groups and / or one or more β-lacto-N-biose units, and the core structure can be partially substituted by α-L-fucopyranosyl and / or α-N-acetyl-neuraminyl (sialic acid) moieties. HMO structures are disclosed, for example, by Xi Chen in Chapter 4 of Advances in Carbohydrate Chemistry and Biochemistry 2015 vol 72.

[0130] HMOs can be non-acidic (or neutral) or acidic. Neutral HMOs do not have sialic acid residues, and acidic HMOs have at least one sialic acid residue in their structure.

[0131] Non-acidic (or neutral) HMOs can be fucosylated or non-fucosylated.

[0132] In an embodiment, the HMOs produced by the genetically modified cells are HMOs containing an N-acetylglucosamine (GlcNAc) moiety or a sialic acid (Neu5Ac) moiety. The production of oligosaccharides with these moieties depends on a functional UDP-GlcNAc pathway.

[0133] Examples of GlcNAc-containing neutral non-fucosylated HMOs include lacto-N-triose II (LNT-II), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexose (LNnH), para-lacto-N-neohexose (pLNnH), para-lacto-N-hexose (pLNH), and lacto-N-hexose (LNH).

[0134] Examples of GlcNAc-containing neutral fucosylated HMOs include lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexose I (LNDFH-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucohexose III (LNDFH-III), fucosyl-lacto-N-hexose II (FLNH-II), lacto-N-pentaose V (LNFP-V), lacto-N-difucohexose II (LNDFH-II), fucosyl-lacto-N-hexose I (FLNH-I), fucosyl-para-lacto-N-hexose I (FpLNH-I), fucosyl-para-lacto-N-neohexose II (F-pLNnH II), and fucosyl-lacto-N-neohexose (FLNnH).

[0135] Examples of acidic HMOs containing sialic acid moieties include 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), and 3-fucosyl-3'-sialyllactose (FSL).

[0136] Examples of acidic HMOs containing sialic acid moieties and GlcNAc moieties include 3'-O-sialyllacto-N-tetraose a (LST-a), fucosyl-LSTa (FLST-a), 6'-O-sialyllacto-N-tetraose b (LST-b), fucosyl-LSTb (FLST-b), 6'-O-sialyllacto-N-neotetraose (LST-c), fucosyl-LSTc (FLST-c), 3'-O-sialyllacto-N-neotetraose (LST-d), fucosyl-LST d (FLST-d), sialyl-lacto-N-hexose (SLNH), sialyl-lacto-N-neohexose I (SLNH-I), sialyl-lacto-N-neohexose II (SLNH-II), and disialyl-lacto-N-tetraose (DSLNT).

[0137] In a particular embodiment, the cell is capable of producing one or more human milk oligosaccharides (HMOs) selected from the group consisting of: LNT, LNT-II, LNnT, LNH, LNnH, pLNH, pLNnH, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, F-pLNnH, DF-LNH a, DF-LNH b, DF-LNH c, DF-pLNnH, TF-pLNnH, FDS-LNH, TF-LNH, DS-LNH, F-LNH-I, F-LNH-II, F-LNH-III, FLST-a (S-LNFP-II), FLST-b, pLNnH, FLST-c, FLST-d, FSL, 3’SL, 6’SL, LST-a, LST-b, LST-c, LST-d, DSLNT, SLNH, SLNH-II, and any mixture thereof.

[0138] According to the invention, a genetically modified cell capable of producing one or more human milk oligosaccharides (HMOs) comprises at least one recombinant nucleic acid sequence encoding a glycosyltransferase activity capable of transferring a glycosyl moiety from an activated sugar to a receptor oligosaccharide.

[0139] In the context of the present invention, a receptor oligosaccharide is an oligosaccharide that can serve as a substrate for a glycosyltransferase that is capable of transferring a glycosyl moiety from a glycosyl donor to the receptor oligosaccharide. The glycosyl donor can be a nucleotide-activated sugar, such as UDP-N-acetylglucosamine (UDP-GlcNAc), optionally in combination with other glycosyl donors as described hereinafter. The receptor oligosaccharide can be a precursor for the preparation of more complex HMOs (composed of 4 monosaccharides or more). Thus, the receptor oligosaccharide can also be referred to as a precursor molecule.

[0140] In this context, the receptor oligosaccharide for the glycosyltransferase can be lactose, lacto-N-triose II (LNT-II), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 2’-fucosyllactose (2’FL), 3-fucosyllactose (3FL), 3’-sialyllactose (3’SL) or 6’-sialyllactose (6’SL). In a preferred embodiment, the receptor molecule is lactose.

[0141] Glycosyltransferase

[0142] As described above, the genetically modified cells according to the present invention are capable of producing one or more oligosaccharides, such as human milk oligosaccharides (HMOs) having GlcNAc or sialic acid moieties, and comprise at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase capable of transferring a glycosyl residue from a glycosyl donor to a recipient oligosaccharide to synthesize human milk oligosaccharides. The nucleic acid sequence encoding one or more expressed glycosyltransferases can be integrated into the genome of the genetically engineered cells (by chromosomal integration), or alternatively, it can be contained in a plasmid and expressed in a plasmid-borne form.

[0143] The genetically modified cells according to the present invention can comprise at least two recombinant nucleic acid sequences encoding two different glycosyltransferases capable of transferring glycosyl residues from glycosyl donors to recipient oligosaccharides.

[0144] One or more glycosyltransferases are preferably selected from enzymes having the following enzymatic activities: α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3 / 4-fucosyltransferase, α-1,4-fucosyltransferase, α-2,3-sialyltransferase, α-2,6-sialyltransferase, β-1,3-N-acetylglucosaminyltransferase, β-1,6-N-acetylglucosaminyltransferase, β-1,3-galactosyltransferase, and β-1,4-galactosyltransferase, which will be described in detail below.

[0145] For the production of chito-oligosaccharides such as chitin and chitosan, the glycosyltransferase can be chitin synthase (NodC) (see, for example, CN114990174), and for the production of hyaluronic acid, hyaluronic acid synthase, which also has glycosyltransferase activity, can be used (see, for example, Widner et al. 2005 Appl Environ Microbiol. 71(7):3747–3752 and Sze et al. 2016, 3 Biotech. 6(1):67).

[0146] β-1,3-N-acetyl-glucosaminyltransferase

[0147] A β-1,3-N-acetyl-glucosaminyltransferase is any protein that includes the ability to transfer the N-acetyl-glucosamine of UDP-N-acetyl-glucosamine to lactose or another acceptor molecule via a β-1,3-bond. Preferably, the β-1,3-N-acetyl-glucosaminyltransferase used herein is not a species derived from a genetically engineered cell, i.e., the gene encoding the β-1,3-N-acetyl-glucosaminyltransferase is of heterologous origin. Non-limiting examples of β-1,3-N-acetyl-glucosaminyltransferases are given in Table 1. Variants of the β-1,3-N-acetyl-glucosaminyltransferase may also be useful. Preferably, these variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to one of the β-1,3-N-acetyl-glucosaminyltransferases given in Table 1.

[0148] Table 1. List of β-1,3-N-acetyl-glucosaminyltransferases

[0149]

[0150]

[0151] In an embodiment, the genetically modified cell is capable of producing HMO and includes i) a deleted or dysfunctional yhbJ gene and ii) a β-1,3-N-acetyl-glucosaminyltransferase.

[0152] heterologous β-1,6-N-acetylglucosaminyltransferase

[0153] A heterologous β-1,6-N-acetyl-glucosaminyltransferase is any protein that includes the ability to transfer the N-acetyl-glucosamine of UDP-N-acetyl-glucosamine to an acceptor molecule via a β-1,6-bond. The β-1,6-N-acetyl-glucosaminyltransferase used herein is not a species derived from a genetically engineered cell, i.e., the gene encoding the β-1,6-galactosyltransferase is of heterologous origin. An example of a β-1,6-N-acetyl-glucosaminyltransferase is Csp2 (NCBI accession number WP_22844786.1) from Chryseobacterium sp. KBW03 or a variant thereof, such as one that can produce LNH or LNnH.

[0154] β-1,3-galactosyltransferase

[0155] β-1,3-galactosyltransferase is any protein that includes the ability to transfer the galactose of UDP-galactose to the N-acetyl-glucosaminyl moiety of a receptor molecule in a β-1,3-linkage. Preferably, the β-1,3-galactosyltransferase used herein is not a species derived from a genetically engineered cell, i.e., the gene encoding the β-1,3-galactosyltransferase is of heterologous origin. Non-limiting examples of β-1,3-galactosyltransferase are given in Table 2. Variants of β-1,3-galactosyltransferase may also be useful. Preferably, these variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to one of the β-1,3-galactosyltransferases given in Table 2.

[0156] Table 2. List of β-1,3-glycosyltransferases

[0157]

[0158] In an embodiment, the genetically modified cell is capable of producing HMO and comprises i) a deleted or dysfunctional yhbJ gene, ii) a β-1,3-N-acetyl-glucosaminyltransferase, and iii) a β-1,3-galactosyltransferase.

[0159] In a further embodiment, the HMO produced by the cell is selected from LNT, LNFP-I, LNFP-II, LNFP-V, LNDFH-I, LNDFH-II, LST-a, FLST-a, LNH, pLNH, pLNH2, FLNH-I, FLNH-II, F-LNH-III.

[0160] β-1,4-galactosyltransferase

[0161] β-1,4-galactosyltransferase is any protein that includes the ability to transfer the galactose of UDP-galactose to the N-acetyl-glucosaminyl moiety of a receptor molecule in a β-1,4-linkage. Preferably, the β-1,4-galactosyltransferase used herein is not a species derived from a genetically engineered cell, i.e., the gene encoding the β-1,4-galactosyltransferase is of heterologous origin. Non-limiting examples of β-1,4-galactosyltransferase are given in Table 3. Variants of β-1,4-galactosyltransferase may also be useful. Preferably, these variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to one of the β-1,4-galactosyltransferases in Table 3.

[0162] Table 3. List of β-1,4-glycosyltransferases

[0163]

[0164] In an embodiment, the genetically modified cell is capable of producing HMOs and comprises i) a deleted or dysfunctional yhbJ gene, ii) a β-1,3-N-acetyl-glucosaminyltransferase, and iii) a β-1,4-galactosyltransferase.

[0165] In a further embodiment, the HMOs produced by the cell are selected from LNnT, LNFP-III, LNFP-VI, LNDFH-III, DF-LNH a, DF-LNH b, DF-LNH c, DF-pLNnH, TF-pLNnH, FDS-LNH, TF-LNH, DS-LNH, F-LNH-I, F-LNH-II, F-LNH-III, LST-c, FLST-c, LNnH or pLNnH.

[0166] α-1,2-fucosyltransferase

[0167] An α-1,2-fucosyltransferase is a protein that comprises the ability to catalyze the transfer of fucose from a donor substrate (e.g., GDP-fucose) to an acceptor molecule in an α-1,2-linkage. Preferably, the α-1,2-fucosyltransferase used herein is not from a species of genetically engineered cell, i.e., the gene encoding the α-1,2-fucosyltransferase is of heterologous origin. Non-limiting examples of α-1,2-fucosyltransferases are given in Table 4. Variants of the α-1,2-fucosyltransferase may also be useful, preferably, these variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to one of the α-1,2-fucosyltransferases in Table 4.

[0168] Table 4. List of α-1,2-fucosyltransferases

[0169]

[0170] α-1,3-fucosyltransferase

[0171] An α-1,3-fucosyltransferase refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate (e.g., GDP-fucose) to an acceptor molecule in an α-1,3-linkage. Preferably, the α-1,3-fucosyltransferase used herein is not from a species of genetically engineered cell, i.e., the gene encoding the α-1,3-fucosyltransferase is of heterologous origin. Non-limiting examples of α-1,3-fucosyltransferases are given in Table 5. Variants of the α-1,3-fucosyltransferase may also be useful, preferably, these variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to one of the α-1,3-fucosyltransferases in Table 5.

[0172] Table 5. List of α-1,3-fucosyltransferases

[0173]

[0174] α-1,3 / 4-fucosyltransferase

[0175] 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 via an α-1,3- or α-1,4-bond. Preferably, the α-1,3 / 4-fucosyltransferase used herein is not a species derived from genetically engineered cells, that is, the gene encoding the α-1,3 / 4-fucosyltransferase is of heterologous origin. Non-limiting examples of α-1,3 / 4-fucosyltransferases are given in Table 6. Variants of α-1,3 / 4-fucosyltransferases may also be useful. Preferably, these variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity with one of the α-1,3 / 4-fucosyltransferases in Table 6.

[0176] Table 6. List of α-1,3 / 4-fucosyltransferases

[0177] Protein name GenBank ID Source FucTIII AY450598.1 Helicobacter pylori strain DSM 6709 FutA WP_000487428.1 Helicobacter pylori ATCC 26695 Med1 WP_087337236.1 Mediterranea sp. An20

[0178] α-2,3-sialyltransferase

[0179] α-2,3-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 via an α-2,3-bond. Preferably, the α-2,3-sialyltransferase used herein is not a species derived from genetically engineered cells, that is, the gene encoding the α-2,3-sialyltransferase is of heterologous origin. Non-limiting examples of α-2,3-sialyltransferases are given in Table 7. Variants of α-2,3-sialyltransferases may also be useful. Preferably, these variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity with one of the α-2,3-sialyltransferases in Table 7.

[0180] Table 7. List of α-2,3-sialyltransferases

[0181]

[0182] In an embodiment, the genetically modified cell is capable of producing HMO and comprises i) a deleted or dysfunctional yhbJ gene, and ii) an α-2,3-sialyltransferase.

[0183] In a further embodiment, the HMO produced by the cell is selected from 3'SL, FSL, LST-a, DS-LNT, and FLST-a.

[0184] α-2,6-sialyltransferase

[0185] An 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 the glycosyl group of a receptor molecule via an α-2,6-linkage. Preferably, the α-2,6-sialyltransferase used herein is not a species derived from a genetically engineered cell, i.e., the gene encoding the α-2,6-sialyltransferase is of heterologous origin. Non-limiting examples of α-2,6-sialyltransferases are given in Table 8. Variants of the α-2,6-sialyltransferase may also be useful. Preferably, these variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to one of the α-2,6-sialyltransferases in Table 8.

[0186] Table 8. List of α-2,6-sialyltransferases

[0187]

[0188]

[0189] In an embodiment, the genetically modified cell is capable of producing HMO and comprises i) a deleted or dysfunctional yhbJ gene, and ii) an α-2,6-sialyltransferase.

[0190] In a further embodiment, the HMO produced by the cell is selected from 6’SL, LST-c, or FLST-c.

[0191] Preferred embodiments of cells capable of producing human milk oligosaccharides (HMO)

[0192] In a preferred embodiment, the genetically modified cell is capable of producing an HMO mixture comprising LNT-II, LNnT, and pLNnH and includes the following modifications:

[0193] a) deletion or dysfunction of the yhbJ gene;

[0194] b) recombinant β-1,3-N-acetyl-glucosaminyltransferase;

[0195] c) recombinant β-1,4-galactosyltransferase; and

[0196] d) optionally, overexpression of phosphoglucomutase (GlmM);

[0197] e) optionally, overexpression of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU), and / or overexpression of glucosamine-6-phosphate synthase (GlmS).

[0198] In the above-described embodiments, preferably, at least 15%, such as at least 20%, such as at least 25% of the total molar content of HMOs produced by the cells is pLNnH.

[0199] In a preferred embodiment, the genetically modified cell is capable of producing an HMO mixture comprising LNT-II, LNT, and pLNH2, and comprises the following modifications:

[0200] a) Deletion or dysfunction of the yhbJ gene;

[0201] b) Overexpression of phosphoglucosamine mutase (GlmM);

[0202] c) Recombinant β-1,3-N-acetyl-glucosaminyltransferase;

[0203] d) Recombinant β-1,4-galactosyltransferase; and

[0204] e) Optionally, overexpression of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU), and / or overexpression of glucosamine-6-phosphate synthase (GlmS).

[0205] In the above-described embodiments, preferably, compared to cells without the modifications of a), b), and e), the formation of LNT is increased by at least 50%.

[0206] In a preferred embodiment, the genetically modified cell is capable of producing sialylated oligosaccharides, such as human milk oligosaccharides, and comprises the following modifications:

[0207] a) Deletion or dysfunction of the yhbJ gene;

[0208] b) α-2,3-sialyltransferase or α-2,6-sialyltransferase, and

[0209] c) A biosynthetic pathway for the production of sialyl sugar nucleotides, and

[0210] d) Optionally, overexpression of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU).

[0211] In the above-described embodiments, the genetically modified cell produces sialylated human milk oligosaccharides selected from 3'SL, 6'SL, LST-a, LST-b, LST-c, and DS-LST, preferably the sialylated HMO is 3'SL or 6'SL. Even more preferably, the modified cell comprises α-2,3-sialyltransferase and produces at least 1.5-fold, such as 1.75-fold, such as more than 2-fold of 3'SL compared to cells without the modifications of a) and d).

[0212] In a preferred embodiment, the genetically modified cell is capable of producing LNT-II, and comprises the following modifications:

[0213] a) Deletion or dysfunction of the yhbJ gene;

[0214] b) Overexpression of phosphoglucosamine mutase (GlmM);

[0215] c) Recombinant β-1,3-N-acetyl-glucosaminyltransferase; and

[0216] d) Optionally, overexpression of N-acetylglucosamine-1-phosphate uridyltransferase (GlmU), and / or overexpression of glucosamine-6-phosphate synthase (GlmS).

[0217] In the above embodiments, preferably, compared with modified cells without a), b) and e), the formation of LNT-II is increased by at least 75%, for example 100%.

[0218] Other glycosyl donors and nucleotide-activated sugars

[0219] In addition to the ability to produce UDP-N-acetylglucosamine (UDP-GlcNAc), the cells according to the invention can also produce other glycosyl donor compounds for the production of, for example, fucosylated or sialylated HMOs. Thus, the modified cells according to the invention can have the genetic ability to produce other glycosyl donor compounds such as nucleotide-activated sugars.

[0220] Other glycosyl donor compounds can be: UDP-galactose, GDP-fucose, CMP-N-acetylneuraminic acid (CMP-Neu5Ac), or any combination thereof. In Table 9 below, the HMOs that can be produced with the said glycosyl donors are given:

[0221] Table 9. List of glycosyl donor HMO products

[0222]

[0223]

[0224] In one embodiment, the genetically modified cells are capable of producing further activated sugar nucleotides via the de novo pathway. In this regard, the further activated sugar nucleotides can be prepared by the cells in a stepwise reaction sequence from simple carbon sources such as glycerol, sucrose, fructose or glucose under the action of enzymes involved in the de novo biosynthetic pathway.

[0225] The enzymes involved in the de novo biosynthetic pathway can be naturally present in the cells or introduced into the cells by genetic engineering or recombinant DNA technology, all of which are part of the common knowledge of those skilled in the art.

[0226] Sialic acid sugar nucleotide synthesis pathway

[0227] To produce sialylated molecules, such as sialylated HMOs, the genetically modified cells contain the ability to synthesize sialyl sugar nucleotides, i.e., the genetically modified cells contain a biosynthetic pathway for making sialyl sugar nucleotides (such as CMP-N-acetylneuraminic acid (CMP-Neu5Ac), which is a glycosyl donor for sialyltransferases). For example, the genetically modified cells are provided with exogenous UDP-GlcNAc 2-epimerase (such as neuC of Campylobacter jejuni (GenBank AAK91727.1) or an equivalent (such as, GenBank CAR04561.1)), Neu5Ac synthase (such as, neuB of Campylobacter jejuni (GenBank AARK91726.1) or an equivalent (such as sialic acid synthase of Flavobacterium limnosediminis, GenBank WP_023580510.1)), and / or CMP-Neu5Ac synthase (such as, neuA of Campylobacter jejuni (GenBank AAK91728.1) or an equivalent (such as CMP-sialic acid synthase of Vibrio brasiliensis, GenBank WP_006881452.1)).

[0228] UDP-GlcNAc is a substrate for UDP-GlcNAc 2-epimerase (neuC), and thus, as described herein, an increase in the ability to produce UDP-GlcNAc is also expected to increase the formation of CMP-Neu5Ac.

[0229] In one or more instances, the UDP-GlcNAc 2-epimerase, CMP-Neu5Ac synthase, and Neu5Ac synthase from Campylobacter jejuni, also referred to as neuBCA from Campylobacter jejuni or simply the neuBCA operon, can be plasmid-borne or integrated into the genome of the genetically modified cells. Preferably, the sialyl sugar nucleotide pathway is encoded by a nucleic acid sequence encoding neuBCA of Campylobacter jejuni (SEQ ID NO:35) or a functional variant thereof, which has an amino acid sequence having at least 80% identity, such as at least 85%, at least 90%, or at least 99% identity with SEQ ID NO:35.

[0230] In addition, genetically modified cells may lack sialic acid catabolic pathways. A "sialic acid catabolic pathway" refers to a series of reactions, typically controlled and catalyzed by enzymes, that result in the degradation of sialic acid. The exemplary sialic acid catabolic pathway described below is the E. coli pathway. In this pathway, sialic acid (Neu5Ac; N-acetylneuraminic acid) is degraded by the enzymes NanA (N-acetylneuraminate lyase), NanK (N-acetylmannosamine kinase), and NanE (N-acetylmannosamine-6-phosphate epimerase), all of which are encoded by the nanATEK-yhcH operon and repressed by NanR (http: / / ecocyc.org / ECOLI).

[0231] Colanic acid gene cluster

[0232] For the production of fucosylated HMOs, the colanic acid gene cluster is important for ensuring the presence of sufficient GDP-fucose. In E. coli, GDP-fucose is an intermediate in the production of the extracellular polysaccharide colanic acid (which is the major oligosaccharide of the bacterial cell wall). In the context of the present invention, the colanic acid gene cluster encodes most of the enzymes (gmd, wcaG, wcaH, wcaI, manB, manC) involved in the de novo synthesis of GDP-fucose, while one or more genes downstream of GDP-L-fucose, such as wcaJ, may be deleted to prevent the conversion of GDP-fucose to colanic acid.

[0233] The de novo GDP-fucose pathway genes responsible for forming GDP-fucose include or consist of the following genes:

[0234] i) manA, which encodes the protein mannose-6-phosphate isomerase (EC 5.3.1.8, UniProt accession number P00946), which facilitates the interconversion of fructose 6-phosphate (F6P) and mannose-6-phosphate;

[0235] ii) manB, which encodes the protein phosphomannomutase (EC 5.4.2.8, UniProt accession number P24175), which is involved in the biosynthesis of GDP-mannose by catalyzing the conversion of mannose-6-phosphate into mannose-1-phosphate;

[0236] iii) manC, which encodes the protein mannose-1-phosphate guanylyltransferase (EC:2.7.7.13, UniProt accession number P24174), which is involved in the biosynthesis of GDP-mannose by synthesizing GDP-mannose from GTP and α-D-mannose-1-phosphate;

[0237] iv) gmd, which encodes the protein GDP-mannose-4,6-dehydratase (UniProt accession number P0AC88), which catalyzes the conversion of GDP-mannose to GDP-4-dehydro-6-deoxy-D-mannose;

[0238] v) wcaG (fcl), which encodes the protein GDP-L-fucose synthase (EC 1.1.1.271, UniProt accession number P32055), which catalyzes the two-step NADP-dependent conversion of GDP-4-dehydro-6-deoxy-D-mannose to GDP-fucose.

[0239] Thus, preferably, when producing one or more fucosylated heterologous products, the genetically engineered cells overexpress the entire colonic acid gene cluster and / or one or more genes of the de novo GDP-fucose pathway selected from manA, manB, manC, gmd, and wcaG.

[0240] In one or more exemplary embodiments, the colonic acid gene cluster responsible for forming GDP-fucose can be expressed from its native genomic locus. The expression can be positively regulated to increase the formation of GDP-fucose. The expression can be regulated by exchanging the native promoter with a promoter of interest, and / or by expressing the gene cluster from another genomic locus outside the native locus to increase the copy number of the colonic acid genes encoding the proteins, or by epigenetically expressing the colonic acid gene cluster or its specific genes.

[0241] For the purposes of the present invention, the term "native genomic locus" in relation to the colonic acid gene cluster refers to the original and natural position of the gene cluster in the genome of the genetically engineered cell.

[0242] A method for producing N-acetylated- and / or sialylated molecules

[0243] The present invention also relates to a method for producing one or more N-acetylated- and / or sialylated molecules, comprising the steps of:

[0244] a) providing a genetically modified cell according to the present invention; and

[0245] b) culturing the cell according to (a) in a suitable cell culture medium to produce the N-acetylated- and / or sialylated molecules.

[0246] Preferably, the N-acetylated- and / or sialylated molecules are selected from oligosaccharides, glycoproteins, and glycolipids. If the molecule is N-acetylated, then preferably, at least one N-acetylation in the molecule is an N-acetylglucosamine moiety.

[0247] One embodiment of the present invention relates to a method for producing one or more oligosaccharides, such as human milk oligosaccharides (HMOs), comprising the steps of:

[0248] a) providing a genetically modified cell according to the present invention; and

[0249] b) culturing the cell according to (a) in a suitable cell culture medium to produce said HMO.

[0250] The above-described embodiments of the genetically modified cell according to the present invention are also applicable to the method according to the present invention.

[0251] In an embodiment, the one or more oligosaccharides are selected from: LNT-II, LNT, LNnT, LNH, LNnH, pLNH, pLNnH, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, F-pLNnH, DF-LNH a, DF-LNH b, DF-LNH c, DF-pLNnH, TF-pLNnH, FDS-LNH, TF-LNH, DS-LNH, F-LNH-I, F-LNH-II, F-LNH-III, FLST-a (S-LNFP-II), FLST-b, pLNnH, FLST-c, FLST-d, FSL, sialyl-Lewis-X, 3'SL, 6'SL, LST-a, LST-b, LST-c, LST-d, DSLNT, SLNH, SLNH-II, and any mixture thereof, or a polysaccharide selected from chitin, chitosan, hyaluronic acid, chondroitin sulfate, or heparan sulfate.

[0252] In a particular embodiment, the human milk oligosaccharides produced are selected from: LNT, LNT-II, LNnT, LNH, LNnH, pLNH, pLNnH, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, F-pLNH, pLNnH, FLST-a, FLST-b, FLST-c, FSL, 3’SL, 6’SL, LST-a, LST-b, LST-c, DSLNT, SLNH, SLNH-II, DF-LNH a, DF-LNH b, DF-LNH c, DF-pLNnH, TF-pLNnH, pLNnH, FDS-LNH, TF-LNH, DS-LNH, F-LNH-I, F-LNH-II, F-LNH-III and any mixtures thereof. Preferably, the human milk oligosaccharides to be produced are a mixture of LNT-II, LNnT and pLNnH, or a mixture of LNT-II, LNT and pLNH2 or sialylated human milk oligosaccharides such as 3’SL or 6’SL.

[0253] In one embodiment, step b) of the cultivation is carried out in the presence of an energy source (carbon source) selected from glucose, sucrose, fructose, xylose and glycerol. Preferably, the carbon source is a carbon source that causes overflow metabolism when present in excess during fermentation. Preferably, the carbon source is glucose or sucrose, including hydrolyzed sucrose.

[0254] In one embodiment, the cultivation is fed-batch fermentation or continuous fed-batch fermentation (fed-batch discharge fermentation), wherein the carbon source is continuously supplied to the fermentation broth during fermentation.

[0255] In one embodiment, the method of the present invention further comprises providing a receptor sugar as a substrate for HMO formation, the receptor sugar comprising at least two monosaccharide units, which are exogenously added to the culture medium and / or produced by separate microbial fermentation. In a preferred embodiment, the substrate for HMO formation is lactose, which is provided (e.g., fed) to the culture during the fermentation of the genetically engineered cells. For example, alternative receptor sugars can be, for example, LNT-II, 2’FL, 3FL, 3’SL, 6’SL, LNnT and LNT, which can be exogenously added to the culture medium and / or produced by separate microbial fermentation. If the fermentation is fed-batch fermentation or continuous fed-batch fermentation, the receptor sugar is preferably provided at the end of the batch phase.

[0256] Culturing or fermenting (used interchangeably herein) in a controlled bioreactor generally includes (i) a first stage of exponential cell growth in a culture medium ensured by a carbon source, and (ii) a second stage of cell growth in a culture medium operating under carbon limitation, where the carbon source is continuously added together with a receptor oligosaccharide (such as lactose), allowing the formation of HMO products in this stage. Carbon (sugar) limitation refers to a stage in the fermentation process where the growth rate is dynamically controlled by the concentration of the carbon source (sugar) in the culture broth, and the concentration of the carbon source in the culture broth is in turn determined by the rate of carbon addition (sugar feed rate) to the fermenter.

[0257] There is no limitation on the suitable cell culture medium used in the fermentation process. The culture medium can be semi-defined, i.e., containing complex culture medium compounds (such as yeast extract, soy peptone, casein amino acids, etc.), or can be chemically defined without any complex compounds. The carbon source can be selected from glucose, sucrose, fructose, xylose, and glycerol. In one or more exemplary embodiments, the culture medium is supplemented with one or more energy sources and carbon sources selected from glycerol, sucrose, and glucose. Preferably, the carbon source is glucose or sucrose.

[0258] In one or more exemplary embodiments, the culture medium contains sucrose as the sole carbon and energy source. In one or more exemplary embodiments, the genetically engineered cells contain one or more heterologous nucleic acid sequences encoding one or more heterologous polypeptides capable of utilizing sucrose as the sole carbon source and energy source of the genetically engineered cells, such as a PTS-dependent sucrose utilization system, further comprising the scrYA and scrBR operons as described in WO2015 / 197082.

[0259] In an embodiment, it is preferred to recover HMO from the culture broth and / or biomass at the end of the culturing / fermentation process. Suitable methods for recovering the produced HMO are known to those skilled in the art, such as those described in WO2016095924, WO2015188834, WO2017152918, WO2017182965, or US20190119314.

[0260] Use of genetically modified cells

[0261] The present invention also relates to any commercial use of the genetically modified cells disclosed herein, such as but not limited to in a method for producing one or more human milk oligosaccharides (HMO).

[0262] In an embodiment of the present invention, the genetically modified cells and / or nucleic acid constructs according to the present invention are used for the manufacture of HMOs. Preferably, the genetically modified cells are used for the large-scale manufacture of one or more HMOs. In one embodiment of the present invention, large-scale fermentation reaches a final fermentation volume of greater than 1,000 L, such as greater than 10,000 L, such as greater than 50,000 L, preferably greater than 100,000 L.

[0263] N-acetylated- and / or sialylated molecules prepared by the method according to the present invention

[0264] Another aspect of the present invention relates to N-acetylated- and / or sialylated molecules prepared by the method according to the present invention.

[0265] In an embodiment, one or more N-acetylated- and / or sialylated molecules are selected from oligosaccharides, glycoproteins and glycolipids. If the molecule is N-acetylated, then preferably at least one N-acetylation in the molecule is an N-acetylglucosamine moiety.

[0266] In a preferred embodiment, the N-acetylated- and / or sialylated molecule is an oligosaccharide, such as human milk oligosaccharide (HMO).

[0267] The resulting N-acetylated- and / or sialylated molecules, such as HMOs, can be in the form of a powder, composition, suspension or gel.

[0268] Sequence

[0269] The present invention includes a sequence listing in text format and electronic format, which is incorporated herein by reference.

[0270] The following table is a summary of the sequences listed in the present invention.

[0271]

[0272] Examples

[0273] Methods

[0274] Strain engineering

[0275] The strains (genetically engineered cells) constructed in the present invention are based on Escherichia coli K-12 DH1, with the genotype: Fˉ, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. Additional modifications were made to Escherichia coli K-12 DH1 strain to generate an MDO parental / strain platform strain with the following modifications: lacZ: deletion of 1.5 kbp, lacA: deletion of 0.5 kbp, nanKETA: deletion of 3.3 kbp, melA: deletion of 0.9 kbp, wcaJ: deletion of 0.5 kbp, mdoH: deletion of 0.5 kbp, and insertion of Plac promoter upstream of the gmd gene. Unless otherwise stated, standard techniques, vectors, control sequence elements, and other expression system elements known in the field of molecular biology were used for nucleic acid manipulation, transformation, and expression. These standard techniques, vectors, and elements can be found, for example, in: 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, New York); 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, New York); Miller, J.H. Experiments in molecular genetics (1972.) (Cold Spring Harbor Laboratory Press, New York).

[0276] Deep well assay

[0277] For strain characterization, the following deep well assay protocol was employed:

[0278] Determination of UDP-GlcNAc level in MDO parent strain variant

[0279] The strains used in the following examples were screened in 96-well deep-well plates using a 4-day protocol. During the initial 24 hours, the precultures were grown to high density and subsequently transferred to a medium capable of inducing gene expression and UDP-GlcNAc formation. More specifically, on Day 1, fresh precultures were prepared using minimal basal medium supplemented with magnesium sulfate, thiamine, and glucose. The precultures were incubated at 34 °C with shaking at 1000 rpm for 24 hours and then further transferred to fresh minimal basal medium (BMM, pH 7.5) to initiate the main culture. The fresh BMM was supplemented with magnesium sulfate, thiamine, a 20% glucose solution (0.1 - 0.15 g / L) as a carbon source, and a 50% sucrose solution (40 - 45 g / L), and invertase (sucrose hydrolase) was added to release glucose at a rate suitable for carbon-limited growth. The main culture was incubated at 28 °C with shaking at 1000 rpm for 72 hours.

[0280] Determination of HMO level produced by the strain

[0281] The strains used in the following examples were screened in 96-well deep-well plates using a 4-day protocol. During the initial 24 hours, the precultures were grown to high density and subsequently transferred to a medium capable of inducing gene expression and product formation. More specifically, on Day 1, fresh precultures were prepared using minimal basal medium supplemented with magnesium sulfate, thiamine, and glucose. The precultures were incubated at 34 °C with shaking at 1000 rpm for 24 hours and then further transferred to fresh minimal basal medium (BMM, pH 7.5) to initiate the main culture. The fresh BMM was supplemented with magnesium sulfate, thiamine, a 20% glucose solution (0.1 - 0.15 g / L), and a lactose solution (5 - 20 g / L). In addition, a 50% sucrose solution (40 - 45 g / L) was provided as a carbon source, and invertase (sucrose hydrolase) was added to release glucose at a rate suitable for carbon-limited growth. The main culture was incubated at 28 °C with shaking at 1000 rpm for 72 hours.

[0282] Example 1 - Increasing the UDP-GlcNAc Pool in Escherichia coli Strains Using Genetic Engineering Tools

[0283] Based on the platform strain (MDO), various further modifications were made to evaluate their potential impact on the UDP-GlcNAc pool in the cells, as they are all related to the metabolic pathway for producing UDP-GlcNAc ( Figure 1 ). The modifications are summarized in Table 10 below to obtain the whole-chromosome strains MP1, MP2, MP3, MP4, MP5, MP6, MP7, MP8, MP9, MP10, and MP11.

[0284] Table 10. Genotypes of strains carrying gene modifications involved in UDP-GlcNAc pathway biosynthesis or regulation

[0285]

[0286] As can be observed from Table 10, the strains lacked the genes yhbJ or nagB (strains MP1 and MP2) that are closely related to the UDP-GlcNAc pathway, or carried an additional PglpF-driven copy of one or all of the key biosynthetic genes glmM, glmU, glmS (strains MP3 to MP6), or a combination of the above modifications (strains MP7 to MP11).

[0287] The strains in Table 10 were screened using the UDP-GlcNAc deep-well assay described in the strain usage section, repeated twice. UDP-GlcNAc levels were evaluated using HPLC, and the identity of the UDP-GlcNAc HPLC-peak was confirmed by LC-MS.

[0288] The results are shown in Table 11 and Figure 2 as follows. Table 11 and Figure 2 present the measured concentrations [g / L] of UDP-GlcNAc for all the strains listed in Table 10.

[0289] Table 11. Measured concentrations [g / L] of UDP-GlcNAc for all the strains listed in Table 10

[0290] Strain Mutation UDP-GlcNAc [g / L] OD MDO Control 0.02 11.55 MP2 ΔnagB 0.03 12.40 MP3 PglpF-glmS 0.02 11.90 MP4 PglpF-glmU 0.02 12.35 MP5 PglpF-glmM 0.02 11.35 MP6 PglpF-glmS + PglpF-glmU + PglpF-glmM 0.02 9.70 MP1 ΔyhbJ 0.05 11.85 MP7 ΔyhbJ + PglpF-glmS 0.06 9.00 MP8 ΔyhbJ + PglpF-glmU 0.06 12.75 MP9 ΔyhbJ + PglpF-glmM 0.20 8.40 MP10 ΔyhbJ + PglpF-glmM + PglpF-glmU 0.59 9.35 MP11 ΔyhbJ PglpF-glmS + PglpF-glmU + PglpF-glmM 0.61 10.05

[0291] As can be observed from Table 11 and Figure 2 it can be seen that compared with the control strain (MDO), the deletion of the yhbJ gene (MP1) led to an increase in the UDP-GlcNAc concentration. It can also be observed that this increase was more significant compared with the deletion of the nagB gene (MP2), which is also involved in UDP-GlcNAc biosynthesis, for example.

[0292] It can also be observed that although the individual introduction of PglpF-driven glmU or glmS copies did not result in an increase in the UDP-GlcNAc concentration (see strains MP3 and MP4 respectively), the individual introduction of PglpF-driven glmU or glmS copies, combined with the deletion of the yhbJ gene (see strains MP8 / MP7), led to a slight increase in the UDP-GlcNAc concentration compared with the individual deletion of the yhbJ gene (MP1). Therefore, this effect is greater than additive.

[0293] For the single PglpF-driven glmM copy, this also did not result in an increase in UDP-GlcNAc concentration (see strain MP5). However, the combination of the deletion of the yhbJ gene and the introduction of the PglpF-driven glmM copy (see strain MP9) led to an increase in UDP-GlcNAc concentration that exceeded a mere additive effect. Thus, a synergistic effect of the said modifications was demonstrated.

[0294] It could further be observed that the introduction of a PglpF-driven glmU copy and the deletion of the glmM and yhbJ genes (see strain MP10) led to an increase in UDP-GlcNAc concentration that was more than 3-fold higher than the concentration achieved by the synergistic combination of glmM overexpression and yhbJ deletion (MP9).

[0295] Similarly, it could be observed that the introduction of PglpF-driven glmU, glmS, and glmM copies, in combination with the deletion of the yhbJ gene (see strain MP11), led to an increase in UDP-GlcNAc concentration that exceeded the expected mere additive effect based on individual modifications (see strains MP1, MP5, MP3, and MP4) and the combination of PglpF-driven glmU, glmS, and glmM copies (without deletion of the yhbJ gene, MP6) (which did not result in an improvement over individual modifications (strain MP6 compared to MP5 / MP4 / MP3)).

[0296] The said examples demonstrate that the single deletion of the yhbJ gene or in combination with overexpression of one or more of glmU, glmS, and / or glmM is an effective tool for increasing the UDP-GlcNAc concentration in Escherichia coli strains. In particular, the combination with glmM overexpression was found to be very beneficial.

[0297] Example 2 - Escherichia coli strains for producing LNnT

[0298] Since UDP-GlcNAc is used as a donor substrate in the formation of LNT-II, and LNT-II is further used as an acceptor in the formation of LNnT, the effect of the increased UDP-GlcNAc pool on LNnT formation has been evaluated.

[0299] Based on the platform strain (MDO), further modifications were made as summarized in Table 12 below to obtain the whole-chromosome LNnT strains MP12, MP13, MP14, and MP15. Each of these strains contains one or two PglpF-driven copies of the β-1,3-N-acetylglucosaminyltransferase LgtA gene (SEQ ID NO:6) from Neisseria meningitidis and a single PglpF-driven copy of the β-1,4-galactosyltransferase GalT gene (SEQ ID NO:8) from Helicobacter pylori 26695. The only differences between the two pairs of strains are (a) the deletion of the yhbJ gene in the MP12-MP13 strain pair, and (b) the deletion of the yhbJ gene and the overexpression of the PglpF-driven genes glmM and glmU in the MP14-MP15 strain pair. All four strains can produce the tetrasaccharide HMOLNnT as the main product. In addition to LNnT, LNT-II and pLNnH can also be formed.

[0300] Table 12. Genotypes of strains expressing glycosyltransferases (genomic integration) that are capable of achieving the biosynthesis of LNnT, with or without modification of genes encoding proteins related to the UDP-GlcNAc biosynthesis pathway in Escherichia coli.

[0301] Strain ID Genotype MP12 MDO, x2 PglpF-lgtA, x1 PglpF-galT MP13 MDO, x2 PglpF-lgtA, x1 PglpF-galT, ΔyhbJ MP14 MDO, x1 PglpF-lgtA, x1 PglpF-galT MP15 MDO, x1 PglpF-lgtA, x1 PglpF-galT, ΔyhbJ, x1 PglpF-glmM, x1 PglpF-glmU

[0302] The strains in Table 12 were screened using the HMO deep-well assay described in the Methods section, repeated four times, and the levels of all HMOs produced by the strains were evaluated using HPLC.

[0303] Figure 3 (MP12-MP13) and Figure 4 (MP14-MP15) show the results for the two strain pairs. In Figure 3 A and Figure 4 A, the amount of HMO (mM) produced by each test strain (MP13 and MP15) is shown relative to the total amount of HMO produced by the control strain (MP12 or MP14, respectively). Figure 3 B and Figure 4 B show the relative concentration (mM) of each individual oligosaccharide (sugar) relative to the amount of the same individual oligosaccharide (sugar) produced by the control strain (MP12 or MP14, respectively) (set to 100%).

[0304] From Figure 3 A, it can be observed that the deletion of the yhbJ gene led to a 20% increase in the total HMO content, while the combination of three modifications in strain MP15 (i.e., the deletion of the yhbJ gene and the overexpression of the glmU and glmM genes) led to a slight decrease of approximately 13% in the total amount of HMO formed compared to the control strain MP14 ( Figure 4A).

[0305] In addition, as can be observed from Figure 3 B, the deletion of the yhbJ gene leads to a slight decrease in the amount of the main product LNnT, which is beneficial for HMO LNT-II and pLNnH. This indicates that the deletion of the yhbJ gene increases the proportion of the minor HMO products LNT-II and pLNnH, while decreasing the proportion of the main product LNnT. This is surprising and shows that not only the total oligosaccharide content can be affected by the deletion of the yhbJ gene, but also the individual proportions of the HMOs produced are affected. In particular, if the production of the hexasaccharide pLNnH is desired, the deletion of yhbJ seems to be advantageous.

[0306] Although the total HMO content of strain MP15 with additional genomic copies of the glmU and glmM genes and the deletion of the yhbJ gene is slightly reduced compared to the control strain MP14 and even the modified strain MP13, the previously observed increase in the proportion of the minor HMO product pLNnH mediated by ΔyhbJ ( Figure 3 B) is significantly amplified with the additional overexpression of the glmM and glmU genes ( Figure 4 B). Specifically, compared to the control strain MP14, the amount of pLNnH formed by strain MP15 is more than three times greater and far higher than the pLNnH concentration observed for strain MP13 ( Figure 4 B).

[0307] The individual proportions of the HMOs formed are also as shown in Figure 3 A and 4A. In Figure 3 A, it can be seen that the deletion of the yhbJ gene leads to a relative decrease in the proportion of the main product (LNnT) from 73% to 62%. In addition, it can be observed that LNT-II increases from 12% relatively to 23%. In addition, it can be observed that pLNnH increases from 15% relatively to 35%. Similarly, in Figure 4 A, we observe that the deletion of the yhbJ gene and the overexpression of the glmU and glmM genes lead to a relative decrease in the proportion of the main product (LNnT) from 92% to 64%, an increase in LNT-II from 3% relatively to 4%, and finally an increase in pLNnH from 5% relatively to 19%.

[0308] These results show that the proportion of individual HMOs in the HMO mixture produced by the strain can be altered when the yhbJ gene is deleted alone or in combination with the overexpression of the glmU and glmM genes, and this alteration is more pronounced for the strain with all three modifications.

[0309] Example 3 - Escherichia coli strain for producing LNT

[0310] Based on the platform strain (MDO), further modifications were carried out as summarized in Table 13 below to obtain the whole-chromosome LNT-producing strains MP16 - MP19. Each of these strains contains one or two PglpF-driven copies of the β-1,3-N-acetylglucosaminyltransferase lgtA gene (SEQ ID NO:6) from Neisseria meningitidis (strains pairs MP16 - MP17 or MP18 - MP19, respectively) and a single PglpF-driven copy of the β-1,3-galactosyltransferase galTK gene (SEQ ID NO:7) from Helicobacter pylori 43504. The only differences between these two strain pairs are (a) the deletion of the yhbJ gene in the strain pair MP16 - MP17, and (b) the deletion of the yhbJ gene and the overexpression of the PglpF-driven genes glmM and glmU in the strain pair MP18 - MP19. All four of these strains can produce the tetrasaccharide HMO LNT as the main product. In addition to LNT, LNT-II and pLNH2 can also be formed.

[0311] Table 13. Genotypes of strains expressing glycosyltransferases that are capable of biosynthesizing LNT from genomic copies of glycosyltransferase classes, with and without modification of genes encoding proteins related to the UDP-GlcNAc biosynthetic pathway in Escherichia coli.

[0312] Strain ID Genotype MP16 MDO, x2 PglpF-lgtA, x1 PglpF-galTK MP17 MDO, x2 PglpF-lgtA, x1 PglpF-galTK, ΔyhbJ MP18 MDO, x1 PglpF-lgtA, x1 PglpF-galTK MP19 MDO, x1 PglpF-lgtA, x1 PglpF-galTK, ΔyhbJ, x1 PglpF-glmM, x1 PglpF-glmU

[0313] The strains in Table 13 were screened using the HMO deep-well assay described in the Methods section, repeated four times, and the levels of all HMOs produced by the strains were evaluated using HPLC.

[0314] Figure 5 (MP16 - MP17) and Figure 6 (MP18 - MP19) show the results for the two strain pairs. In Figure 5 A and Figure 6 A, the amount of HMO (mM) produced by each test strain (M17 and MP19) is shown relative to the total amount of HMO produced by the control strain (MP16 or MP18, respectively). Figure 5 B and Figure 6 B show the relative concentration (mM) of each individual oligosaccharide (sugar) relative to the amount of the same individual oligosaccharide (sugar) produced by the control strain (MP16 or MP18, respectively) (set to 100%). Figure 6 B does not show the oligosaccharide levels of the control strain because these levels are default set to 100%.

[0315] From Figure 5It can be observed that compared with the control strain MP18, the deletion of the yhbJ gene only led to a slight increase in the total HMO content, by about 5%, while the combination of the three modifications in strain MP19 (i.e., the deletion of the yhbJ gene and the overexpression of the glmU and glmM genes) led to a total HMO formation more than twice as much ( Figure 6 A).

[0316] In addition, from Figure 5 It can be observed from B that the deletion of the yhbJ gene led to a slight decrease in the amount of the main product LNT and a slight increase in the by-product pLNH2, and a significant increase in the HMO LNT-II, whose concentration almost doubled in the final HMO profile obtained. This indicates that the deletion of the yhbJ gene led to an increase in the proportion of the minor HMO product LNT-II, while the final titer of the main product LNT decreased slightly.

[0317] As described above, the total HMO content of strain MP19 carrying extra genomic copies of the glmU and glmM genes and the deletion of the yhbJ gene was significantly higher than that of the control strain MP18 and even the modified strain MP17 ( Figure 6 A). Contrary to the significant increase in the proportion of the minor HMO product LNT-II mediated by ΔyhbJ alone ( Figure 5 B), the additional overexpression of the glmM and glmU genes not only led to a significant increase in the concentration of LNT-II, but also led to significant increases in the concentrations of LNT and pLNH2 (1.5-fold and 4-fold higher, respectively) ( Figure 6 B).

[0318] The respective proportions of the HMOs formed are also as shown in Figure 5 A and 6A. In Figure 5 A, it can be observed that the deletion of the yhbJ gene led to a relative decrease in the proportion of the main product (LNT) from 85% to 77%. In addition, it can be observed that LNT-II increased relatively from 14% to 27%. Similarly, in Figure 6 A, we observed that the combination of the deletion of the yhbJ gene and the overexpression of the glmU and glmM genes led to a relative increase in the proportion of the main product (LNT) from 96% to 159%, LNT-II from 3% to 54%, and finally pLNH2 from 0.5% to 2.1%.

[0319] These results indicate that when the yhbJ gene is deleted alone or in combination with the overexpression of the glmU and glmM genes, the proportion of individual HMOs in the HMO mixture produced by the strain can be changed, and this change is more pronounced for the strain with all three modifications. This is surprising and indicates that not only the total sugar content can be affected by modifying these three genes in the manner proposed in the present invention, but also the individual proportions of the HMOs produced are affected.

[0320] Example 4 - Escherichia coli Strain for Producing 3'SL

[0321] The formation of 3'SL uses CMP - Neu5Ac as a donor and is catalyzed by a suitable α - 2,3 - sialyltransferase. In the Escherichia coli cells used in this example, CMP - Neu5Ac is formed from UDP - GlcNAc by inserting genes in the sialic acid sugar nucleotide pathway, which includes exogenous UDP - GlcNAc 2 - epimerase (neuC), Neu5Ac synthase (neuB), and CMP - Neu5Ac synthetase (neuA).

[0322] Based on the platform strain (MDO), further modifications were made as summarized in Table 14 below to obtain strain MP20 as a control strain that can produce HMO 3'SL. In addition, test strains MP21 - MP23 were prepared, which differed from MP20 only in that the yhbJ gene was individually deleted from the chromosome of the MP20 strain (to create strain MP21), or the deletion of the yhbJ gene was combined with the overexpression of the glmS gene (to create strain MP22), or the deletion of the yhbJ gene was combined with the overexpression of the glmM gene (to create strain MP23).

[0323] All four strains express the genomically integrated α - 2,3 - sialyltransferase Poral gene, which encodes the wild - type enzyme of SEQ ID NO:9 from Pasteurella oralis. In addition, the strain contains the high - copy plasmid pBS - neuBCA - amp, carrying the neuBCA operon of Campylobacter jejuni (SEQ ID NO:35).

[0324] Table 14. Genotypes of strains carrying gene modifications involved in 3'SL biosynthesis, with genes encoding proteins related to the UDP - GlcNAc biosynthesis pathway modified and unmodified in Escherichia coli simultaneously.

[0325] Strain ID Genotype MP20 MDO, PglpF-Poral, pBS-neuBCA(Plac) MP21 MDO, PglpF-Poral, pBS-neuBCA(Plac), ΔyhbJ MP22 MDO, PglpF-Poral, pBS-neuBCA(Plac), ΔyhbJ, x1 PglpF-glmS MP23 MDO, PglpF-Poral, pBS-neuBCA(Plac), ΔyhbJ, x1 PglpF-glmU

[0326] The strains in Table 14 were screened using the HMO deep - well assay described in the method section, repeated four times, and the 3'SL levels were evaluated using HPLC.

[0327] The results are as Figure 7 shown, as the relative concentration (mM) of 3'SL, where the amount of 3'SL found in the control strain MP20 (without deletion of the yhbJ gene or overexpression of the glmU and / or glmS genes) was set to 100%.

[0328] From Figure 7It was observed that the deletion of the yhbJ gene led to a significant increase in the concentration of 3’SL by approximately 2-fold (strain MP21 relative to strain MP20). When the deletion of the yhbJ gene was accompanied by the overexpression of the glmU gene (strain MP23 relative to strain MP21), slightly better results were obtained, while the deletion of the yhbJ gene accompanied by the overexpression of glmS seemed to have no effect beyond that of the yhbJ deletion alone observed in the HMO deep-well assay used.

[0329] The above observations indicate that the deletion of the yhbJ gene alone or especially in combination with the overexpression of the glmU gene can significantly increase the production of 3’SL in Escherichia coli strains that can produce 3’SL.

[0330] Example 5 - Escherichia coli Strains Producing LNT-II

[0331] The formation of LNT-II uses UDP-GlcNAc as a donor and is catalyzed by a suitable β-1,3-N-acetylglucosaminyltransferase reaction. In the Escherichia coli cells used in this example, UDP-GlcNAc is formed in the native biosynthetic pathway of Escherichia coli cells involving the genes glmM, glmS, and glmU.

[0332] Based on the platform strain (MDO), further modifications were made as summarized in Table 15 below to obtain strain MP24 as a control strain that can produce the HMO LNT-II. In addition, a test strain MP25 was prepared, which differed from MP24 only in the deletion of the yhbJ gene from the chromosome of the MP24 strain and the overexpression of the glmM and glmU genes. Both MP24 and MP25 carry a single PglpF-driven genomic copy of the β-1,3-N-acetylglucosaminyltransferase lgtA gene (SEQ ID NO:6) from Neisseria meningitidis, while the MP25 strain also carries additional genomic copies of the glmU and glmM genes under the control of the PglpF promoter.

[0333] Table 15. Genotypes of strains carrying gene modifications involved in LNT-II biosynthesis, with simultaneous modification and non-modification of genes encoding proteins related to the UDP-GlcNAc biosynthetic pathway in Escherichia coli.

[0334] Strain ID Genotype MP24 MDO, x1 PglpF-lgtA MP25 MDO, x1 PglpF-lgtA, ΔyhbJ, x1 PglpF-glmM, x1 PglpF-glmU

[0335] The strains in Table 15 were screened using the HMO deep-well assay described in the Methods section, repeated four times, and the LNT-II levels were evaluated using HPLC.

[0336] The results are as Figure 8Shown is the relative concentration (mM) of LNT-II, where the amount of LNT-II found in the control strain MP24 (where the yhbJ gene was not deleted or the glmU and glmS genes were not overexpressed) was set to 100%.

[0337] From Figure 8 It can be observed that the combination of deletion of the yhbJ gene and overexpression of the glmM and glmU genes results in a more than two-fold greater concentration of LNT-II present in the resulting final HMO profile.

[0338] The above observations indicate that the combination of deletion of the yhbJ gene and overexpression of the glmM and glmU genes can significantly increase the production of LNT-II in E. coli strains that can produce LNT-II, which is highly relevant to the data observed in Example 1, where the UDP-GlcNAc pool was significantly increased in MDO strains with this specific modification (which do not express HMO).

Claims

1. A genetically modified cell capable of producing UDP-N-acetylglucosamine (UDP-GlcNAc), wherein the mRNA stability of native glucosamine-6-phosphate synthase (glmS) is enhanced by modifying the cell to reduce or completely abolish the function of its RNase adaptor protein RapZ (yhbJ).

2. The genetically modified cell according to claim 1, wherein the gene encoding the RNase adaptor protein RapZ (yhbJ) is deleted or rendered dysfunctional.

3. The genetically modified cell according to claim 1 or 2, wherein the cell is further modified to overexpress phosphoglucosamine mutase (GlmM).

4. The genetically modified cell according to any one of claims 1 to 3, wherein the cell further comprises at least one of the following modifications: a) overexpression of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU), and / or b) overexpression of glucosamine-6-phosphate synthase (GlmS).

5. The genetically modified cell according to claim 3 or 4, wherein the overexpression is obtained by: a) replacing the native promoter of the gene encoding i. phosphoglucosamine mutase (glmM), and / or ii. N-acetylglucosamine-1-phosphate uridylyltransferase (glmU), and / or iii. glucosamine-6-phosphate synthase (glmS); with a stronger promoter; and / or b) inserting into the cell a recombinant nucleic acid encoding i. phosphoglucosamine mutase (glmM) comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1, or consisting thereof, ii. N-acetylglucosamine-1-phosphate uridylyltransferase (glmU) comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:2, or consisting thereof, iii. glucosamine-6-phosphate synthase (glmS) comprising the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:3, or consisting thereof.

6. The genetically modified cell according to any one of the preceding claims, wherein the cell is capable of producing one or more N-acetylated molecules and / or sialylated molecules selected from oligosaccharides, glycoproteins, and glycolipids.

7. The genetically modified cell according to claim 6, wherein the cell is capable of producing one or more oligosaccharides selected from the following: LNT-II, LNT, LNnT, LNH, LNnH, pLNH, pLNnH, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, F-pLNnH, DF-LNH a, DF-LNH b, DF-LNH c, DF-pLNnH, TF-pLNnH, FDS-LNH, TF-LNH, DS-LNH, F-LNH-I, F-LNH-II, F-LNH-III, FLST-a(S-LNFP-II), FLST-b, pLNnH, FLST-c, FLST-d, FSL, sialyl-Lewis-X, 3’SL, 6’SL, LST-a, LST-b, LST-c, LST-d, DSLNT, SLNH, SLNH-II, and any mixture thereof, or a polysaccharide selected from chitin, chitosan, hyaluronic acid, chondroitin sulfate or heparan sulfate.

8. The genetically modified cell according to any one of the preceding claims, wherein the cell further comprises the following modifications: a) a recombinant β-1,3-N-acetylglucosaminyltransferase, and wherein the cell is capable of producing LNT-II.

9. The genetically modified cell according to any one of claims 1 to 7, wherein the cell further comprises the following modifications: a) a recombinant β-1,3-N-acetylglucosaminyltransferase, and b) a recombinant β-1,4-galactosyltransferase, and wherein the cell is capable of producing an HMO mixture comprising LNT-II, LNnT and pLNnH.

10. The genetically modified cell according to any one of claims 1 to 7, wherein the cell further comprises the following modifications: a) a recombinant β-1,3-N-acetylglucosaminyltransferase, and b) a recombinant β-1,3-galactosyltransferase, and wherein the cell is capable of producing an HMO mixture comprising LNT-II, LNT and pLNH2.

11. The genetically modified cell according to any one of claims 1 to 7, wherein the cell further comprises the following modifications: a) an α-2,3-sialyltransferase or an α-2,6-sialyltransferase, and b) a biosynthetic pathway for producing sialyl sugar nucleotides, and wherein the cell is capable of producing sialylated human milk oligosaccharides.

12. The genetically modified cell according to claim 11, wherein the sialylated human milk oligosaccharides are selected from 3’SL, 6’SL, LST-a, LST-b, LST-c and DS-LST.

13. A genetically modified cell according to any one of the preceding claims, wherein the modified cell is a microbial cell, such as a cell selected from Escherichia coli, Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum, Campylobacter sp., Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

14. A method for producing one or more N-acetylated molecules and / or sialylated molecules, comprising the following steps: a) providing a genetically modified cell according to any one of claims 6 to 13; and b) culturing the cell according to (a) in a suitable cell culture medium to produce the N-acetylated molecule and / or sialylated molecule.

15. The method according to claim 14, wherein the molecule is an oligosaccharide selected from the following: LNT, LNT-II, LNnT, LNH, LNnH, pLNH, pLNnH, LNFP-I, LNFP-II, LNFP-III, LNFP-IV, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, F-pLNnH, DF-LNH a, DF-LNH b, DF-LNH c, DF-pLNnH, TF-pLNnH, FDS-LNH, TF-LNH, DS-LNH, F-LNH-I, F-LNH-II, F-LNH-III, FLST-a (S-LNFP-II), FLST-b, pLNnH, FLST-c, FLST-d, FSL, sialyl-Lewis X, 3’SL, 6’SL, LST-a, LST-b, LST-c, LST-d, DSLNT, SLNH, SLNH-II, and any mixture thereof, or a polysaccharide selected from chitin, chitosan, hyaluronic acid, chondroitin sulfate, or heparan sulfate.

16. The method according to claim 15, wherein the oligosaccharide is a human milk oligosaccharide selected from the following: a) a mixture of LNT-II, LNnT, and pLNnH, or b) a mixture of LNT-II, LNT, and pLNH2, or c) a sialylated human milk oligosaccharide, such as 3’SL or 6’SL.

17. The method according to any one of claims 14 to 16, wherein the N-acetylated molecule and / or sialylated molecule is recovered from the culture broth and / or biomass.

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