Production of oligosaccharides by microbial cell fermentation using glycerol

Abstract

Disclosed are a methylglyoxal-resistant genetically engineered microbial cell for fermentative production of an oligosaccharide of interest, and a method for fermentative production of an oligosaccharide of interest using the methylglyoxal-resistant microbial cell.

Description

[0001] The present invention relates to producing oligosaccharides by fermentation of microbial cells. More specifically, the present invention relates to producing oligosaccharides by fermentation of microbial cells using glycerol as a carbon source and an energy source. Background Art

[0002] Among the oligosaccharide group, human milk oligosaccharides (HMO) have attracted a lot of attention in recent years due to their beneficial health effects. For example, HMO can prevent pathogenic microorganisms from attaching to gastrointestinal mucosal cells, thus preventing the emergence of diseases. In addition, HMO also plays a prebiotic role, that is, it promotes the proliferation of non-pathogenic intestinal bacteria. Beneficial effects on the mature immune system and nervous system have also been described.

[0003] Due to the beneficial effects of HMOs, their industrial production has also attracted much attention. The most economically viable HMO production appears to be fermentative production by genetically engineered microbial cells such as Escherichia coli cells, Bacillus subtilis cells, Corynebacterium glutamicum cells or Saccharomyces cerevisiae cells.

[0004] In general, industrial-scale bioprocesses (such as the production of oligosaccharides by / in microbial cells by fermentation) involve a production phase, during which biosynthesis of the target oligosaccharide occurs. The production phase is followed by a product recovery phase, during which the target oligosaccharide is recovered from the culture fluid and / or the microbial cells. Different substrates such as glucose, sucrose or glycerol are known to serve as carbon and energy sources for the microbial cells in the production phase.

[0005] Glycerol is a carbon source for microbial cells and has recently become economically attractive for fermentation production processes. As glycerol is a byproduct of biodiesel production, the supply of glycerol in the market has increased with the increase in the production of biodiesel as a substitute for gasoline-based fuels.

[0006] One microbial cell that has received particular attention in the fermentative production of oligosaccharides, especially human milk oligosaccharides, is the Gram-negative bacterium Escherichia coli. Escherichia coli is a microorganism that is able to metabolize glycerol as a carbon and energy source. Catabolism in E. coli can occur under aerobic or anaerobic conditions. The catabolism pathways of glycerol in E. coli are as follows: Figure 1As shown. The main aerobic pathway involves glycerol internalization through the glycerol absorption promoter "GlpF" (encoded by the glpF gene), glycerol kinase (encoded by the glpK gene) and glycerol-3-phosphate dehydrogenase (encoded by the glpD gene). The glycerol decomposition pathway adopted by Escherichia coli under anaerobic conditions requires glycerol-3-phosphate dehydrogenase (encoded by the glpABC gene) instead of glycerol-3-phosphate dehydrogenase to convert glycerol-3-phosphate into dihydroxyacetone phosphate. After glycerol is absorbed by the glycerol absorption promoter, the alternative anaerobic pathway of glycerol contains glycerol dehydrogenase (encoded by the gldA gene) and dihydroxyacetone kinase (encoded by the dhaKLM gene). In all these metabolic pathways, dihydroxyacetone phosphate is produced as a key metabolite and enters the central metabolism through the Embden-Meyerhof-Parnas (EMP) pathway.

[0007] Due to the location of the gldA gene in the E. coli genome, i.e., close to the ptsA gene encoding polyphosphoryl transporter 2 and fsaB encoding fructose-6-phosphate aldolase, and because the glycerol dehydrogenase GldA has a higher affinity for dihydroxyacetone than for glycerol, it has been suggested that the main role of GldA is to convert dihydroxyacetone to glycerol to protect E. coli cells from dihydroxyacetone toxicity. In addition, dihydroxyacetone can also be spontaneously converted to methylglyoxal, a highly toxic metabolite of E. coli, which can inhibit the growth of E. coli at concentrations as low as 0.3 mM.

[0008] Therefore, it is desirable that the microbial cells used to produce the target oligosaccharides are less susceptible to the toxicity of metabolites from the glycerol decomposition pathway when the microbial cells are cultured in the presence of glycerol as a carbon source and energy source. Summary of the invention

[0009] Surprisingly, it was found that when E. coli cells capable of synthesizing human milk oligosaccharides intracellularly possess certain variants of their glycerol kinase, they are able to increase the production of human milk oligosaccharides and are not affected by the toxicity of methylglyoxal.

[0010] Therefore, in the first aspect, a genetically engineered microbial cell for producing a target oligosaccharide is provided, wherein the genetically engineered microbial cell is capable of synthesizing the target oligosaccharide intracellularly when cultured in the presence of glycerol as the sole carbon source. The microbial cell has a glycerol permease (also known as a glycerol absorption promoter) for internalizing exogenous glycerol. The microbial cell further has a functional variant of the native Escherichia coli glycerol kinase GlpK, wherein the amino acid sequence of the native Escherichia coli GlpK is as shown in SEQ ID NO: 1, wherein the functional variant has an amino acid residue containing a non-ionized but polar acting side chain at a position corresponding to amino acid position 55 in the amino acid sequence of the native Escherichia coli GlpK, and / or wherein the functional variant has an amino acid containing an anionic side chain at a position corresponding to amino acid position 231 in the amino acid sequence of the native Escherichia coli GlpK. The genetically engineered microbial cell further has a metabolic pathway for intracellular biosynthesis of nucleotide-activated monosaccharides, the nucleotide-activated monosaccharides comprising a nucleotide portion and a monosaccharide portion, wherein the nucleotide-activated monosaccharide is a donor substrate for transferring its monosaccharide portion to an acceptor molecule. The genetically engineered microbial cells further have a glycosyltransferase for transferring the monosaccharide moiety from the nucleotide-activated monosaccharide to an acceptor molecule for intracellular biosynthesis of the target oligosaccharide.

[0011] In the second aspect, the use of the genetically engineered microbial cell according to the first aspect for producing the target oligosaccharide is provided. Therefore, the use includes the use of the genetically engineered microbial cell, wherein the genetically engineered microbial cell is capable of synthesizing the target oligosaccharide intracellularly when cultured in the presence of glycerol as the sole carbon source, wherein the genetically engineered microbial cell is capable of synthesizing the target oligosaccharide intracellularly when cultured in the presence of glycerol as the sole carbon source. The microbial cell has a glycerol permease (also known as a glycerol absorption promoter) to internalize exogenous glycerol. The microbial cell further has a functional variant of the native Escherichia coli glycerol kinase GlpK, wherein the amino acid sequence of the native Escherichia coli GlpK is as shown in SEQ ID NO: 1, wherein the functional variant has an amino acid residue containing a non-ionized but polar acting side chain at the position corresponding to amino acid position 55 in the amino acid sequence of the native Escherichia coli GlpK in its amino acid sequence, and / or wherein the functional variant has an amino acid containing an anionic side chain at the position corresponding to amino acid position 231 in the amino acid sequence of the native Escherichia coli GlpK in its amino acid sequence. The genetically engineered microbial cell further comprises a metabolic pathway for intracellular biosynthesis of a nucleotide-activated monosaccharide as a donor substrate for the monosaccharide moiety, and the genetically engineered microbial cell further comprises a glycosyltransferase for transferring the monosaccharide moiety from the nucleotide-activated monosaccharide to an acceptor molecule.

[0012] In a third aspect, a method for producing oligosaccharides of interest by fermentation is provided, wherein the method comprises:

[0013] - providing a genetically engineered microbial cell for producing a target oligosaccharide according to the first aspect, i.e., a genetically engineered microbial cell capable of synthesizing a target oligosaccharide intracellularly when cultured in the presence of glycerol as the sole carbon source, wherein the genetically engineered bacterium comprises a glycerol permease and a functional variant of a native Escherichia coli glycerol kinase GlpK, a metabolic pathway for intracellular biosynthesis of a nucleotide-activated monosaccharide as a donor substrate for its monosaccharide moiety, and a glycosyltransferase for transferring a monosaccharide moiety from the nucleotide-activated monosaccharide to an acceptor molecule, wherein the amino acid sequence of the native Escherichia coli GlpK is as shown in SEQ ID NO: 1, wherein the functional variant has an amino acid residue containing a non-ionized but polar acting side chain at the position in its amino acid sequence corresponding to amino acid position 55 in the amino acid sequence of the native Escherichia coli GlpK, and / or wherein the functional variant has an amino acid containing an anionic side chain at the position in its amino acid sequence corresponding to amino acid position 231 in the amino acid sequence of the native Escherichia coli GlpK;

[0014] - culturing the genetically engineered microbial cells in a culture medium and under conditions that allow the genetically engineered microbial cells to synthesize the target oligosaccharide within the cells, wherein the culture medium contains glycerol as a carbon source for the genetically engineered microbial cells; and

[0015] - Optionally, recovering the oligosaccharides of interest from the microbial cells and / or the culture medium.

[0016] In a further aspect, a method for reducing the toxicity of methylglyoxal to microbial cells when the microbial cells are cultured in the presence of glycerol as a carbon source is provided, the method comprising transforming the microbial cells to have a functional variant of the native Escherichia coli glycerol kinase GlpK, wherein the amino acid sequence of the native Escherichia coli GlpK is as shown in SEQ ID NO: 1, wherein the functional variant has an amino acid residue comprising a non-ionized but polar acting side chain at the position in its amino acid sequence corresponding to amino acid position 55 in the amino acid sequence of the native Escherichia coli GlpK, and / or wherein the functional variant has an amino acid comprising an anionic side chain at the position in its amino acid sequence corresponding to amino acid position 231 in the amino acid sequence of the native Escherichia coli GlpK.

[0017] In a further aspect, a method for reducing the responsiveness of microbial cells to methylglyoxal toxicity during production of oligosaccharides of interest by fermentation of microbial cells is provided, when the microbial cells are cultured in the presence of exogenous glycerol as a carbon source to produce oligosaccharides of interest, the method comprising transforming the microbial cells to have a functional variant of native Escherichia coli glycerol kinase GlpK, wherein the amino acid sequence of the native Escherichia coli GlpK is as shown in SEQ ID NO: 1, wherein the functional variant has an amino acid residue containing a non-ionized but polar acting side chain at the position in its amino acid sequence corresponding to amino acid position 55 in the amino acid sequence of the native Escherichia coli GlpK, and / or wherein the functional variant has an amino acid containing an anionic side chain at the position in its amino acid sequence corresponding to amino acid position 231 in the amino acid sequence of the native Escherichia coli GlpK. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The catabolic pathway of glycerol in E. coli is shown.

[0019] Figure 2 Shown are the nucleotide sequence of the protein coding region of the native E. coli glpK gene and the deduced amino acid sequence of the native E. coli glycerol kinase GlpK.

[0020] Figure 3 Shown are graphs comparing growth (A) and 3-FL production (B) of a 3-FL-producing E. coli strain containing the native E. coli (K12) GlpK enzyme (control) and progeny of the control strain containing the functional variant GlpK(A55T) instead of the native E. coli (K12) GlpK.

[0021] Figure 4 Shown are graphs comparing growth (A) and 2'-FL production (B) of a 2'-FL-producing E. coli strain containing the native E. coli GlpK enzyme (control), an offspring of the control strain containing a functional variant of the native E. coli GlpK, GlpK(A55T), instead of the native E. coli GlpK, and another offspring of the control strain containing a functional variant of the native E. coli GlpK, GlpK(G231D), instead of the native E. coli GlpK.

[0022] Figure 5 Shown are graphs comparing growth (A) and LNT production (B) of a LNT-producing E. coli strain containing the native E. coli GlpK enzyme (control) and progeny of the control strain containing a functional variant of the native E. coli GlpK, GlpK(A55T), instead of the native E. coli GlpK.

[0023] Figure 6 Shown is a graph comparing the growth profiles of E. coli strains producing 2'-FL (A) or LNT (B), wherein the growth profiles of these strains carrying their endogenous mgsA gene are compared to the growth profiles of the corresponding progeny strains lacking their endogenous mgsA gene. DETAILED DESCRIPTION

[0024] According to a first aspect, a genetically engineered microbial cell for producing oligosaccharides of interest by fermentation is provided.

[0025] As used herein, the term "microbial cell" refers to a unicellular organism. The microbial cell may be a prokaryotic cell or may be a eukaryotic cell. Suitable prokaryotic cells include bacterial cells and archaeal cells. Suitable eukaryotic cells include yeast cells and fungal cells.

[0026] In some embodiments, prokaryotic cell is bacterial cell.Bacterial cell is selected from the bacterial cell of the following genus: Bacillus, Bifidobacterium, Citrobacter, Clostridium, Corynebacterium, Enterococcus, Erwinia, Escherichia coli, Lactobacillus, Lactococcus, Micrococcus, Micromonospora, Pantoea, Pectobacterium, Propionibacterium, Pseudomonas, Rhodococcus, Sporolactobacillus, Streptococcus and Xanthomonas.Suitable bacterial species in the genus are Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus brevis, Bacillus lentus, Bacillus cereus, Bacillus circulans, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Citrobacter freundii, Clostridium cellulolyticum, Clostridium yongdalii (Clostridium In another embodiment, the microbial cell is an Escherichia coli cell.

[0027] In some embodiments, the eukaryotic cell is a yeast cell. Suitable yeast cells may be selected from the genera: Saccharomycopsis sp., Saccharomycopsis sp., Pichia sp., Hansenula sp., Kluyveromyces sp., Yarrowia sp., Rhodotorula sp., and Schizosaccharomyces sp. Additionally and / or alternatively, the yeast cell is a Saccharomyces cerevisiae cell, a Pichia sp. cell, or a Hansenula polymorpha cell.

[0028] The microbial cells are microbial cells for producing target oligosaccharides. The term "for production" used herein for producing target oligosaccharides means that when cultured in a culture medium and under conditions that allow the microbial cells to synthesize target oligosaccharides in the cells, the microbial cells are able to synthesize target oligosaccharides in the cells.

[0029] The term "oligosaccharide" as used herein refers to a sugar molecule consisting of at least three monosaccharide moieties but not more than twenty monosaccharide moieties (preferably not more than 12 monosaccharide moieties, and more preferably not more than 10 monosaccharide moieties), wherein each of the monosaccharide moieties is bound to at least one other monosaccharide moiety by a glycosidic bond. The oligosaccharide of interest may consist of a linear chain of monosaccharide moieties, or may consist of a branched chain of monosaccharide moieties.

[0030] The monosaccharide portion of the oligosaccharide of interest can be selected from aldoses (e.g., arabinose, xylose, ribose, deoxyribose, lyxose, glucose, idose, galactose, talose, allose, altrose, mannose), ketoses (e.g., ribulose, xylulose, fructose, sorbose, tagatose), deoxysugars (e.g., rhamnose, fucose, quinovose), deoxyaminosugars (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine), uronic acids (e.g., galacturonic acid, glucuronic acid) and ketouronic acids (e.g., N-acetylneuraminic acid).

[0031] The term "target oligosaccharide" used herein refers to the oligosaccharide expected to be produced by genetically engineered microbial cells. Usually, microbial cells are selected and / or genetically engineered to synthesize specific oligosaccharides, i.e., target oligosaccharides, in cells. Except target oligosaccharides, any other oligosaccharides that microbial cells may synthesize are considered to be by-products, whether these other oligosaccharides are the product of another metabolic pathway outside the biosynthetic metabolic pathways of the target oligosaccharide, the reaction product of an intermediate reaction, the metabolic pathways that constitute the target oligosaccharide, or the undesirable by-products produced due to the heterogeneity of the enzymes that participate in the metabolic pathways of the synthetic target oligosaccharide.

[0032] Typically, the oligosaccharide of interest is not naturally present in the native cells of the microbial species selected for genetic engineering to produce the oligosaccharide of interest. Thus, a genetically engineered microbial cell is a microbial cell that has been genetically engineered to produce the oligosaccharide of interest.

[0033] In some embodiments, the oligosaccharide of interest is a human milk oligosaccharide (HMO), i.e., an oligosaccharide selected from the group of oligosaccharides present in human milk. Human milk oligosaccharides are composed of a diverse mixture of oligosaccharides that cannot be digested by humans. The oligosaccharide composition and quantity of human milk are unique. To date, more than 150 structurally different HMOs have been discovered. The vast majority of HMOs are characterized by a lactose moiety (Gal-β1,4-Glc) at the reducing end. HMOs contain at least one fucose moiety, at least one sialic acid moiety, and / or at least one N-acetylglucosamine moiety. More generally, the monosaccharides that make up HMOs are selected from D-glucose, D-galactose, N-acetyl-D-glucosamine, L-fucose, and N-acetylneuraminic acid.

[0034] Additionally and / or alternatively, the oligosaccharide of interest is an HMO selected from the group consisting of 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 2',3-difucosyllactose (DFL), lacto-N-triose II, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP-I), lacto-N-neofucopentaose I (LNnFP-I), lacto-N-fucopentaose II (LNFP-II), Lactose-N-fucopentaose III (LNFP-III), lactose-N-fucopentaose V (LNFP-V), lactose-N-neofucopentaose V (LNnFP-V), lactose-N-hexaose (LNH), lactose-N-neohexaose (LNnH), para-lactose-N-hexaose (paraLNH), para-lactose-N-neohexaose (paraLNnH), difucosyl-lactose-N-neohexaose (DF-LNnH), lactose-N-difucosylhexaose I, lactose-N-di Fucosylhexaose II, para-lactose-N-fucosylhexaose (paraLNH), fucosyl-lactose-N-sialylpentaose a (F-LST-a), fucosyl-lactose-N-sialylpentaose b (F-LST-b), fucosyl-lactose-N-sialylpentaose c (F-LST-c), fucosyl-lactose-N-sialylpentaose c, disialyl-lactose-N-fucopentaose, 3-fucosyl-3'-sialyl lactose (3F-3'-SL), 3 -fucosyl-6'-sialyl lactose (3F-6'-SL), lactose-N-neodifucohexaose I, 3'-sialyl lactose (3-SL), 6'-sialyl lactose (6-SL), sialyl lactose-N-tetraose a (LST-a), sialyl lactose-N-tetraose b (LST-b), sialyl lactose-N-tetraose c (LST-c), disialyl lactose-N-tetraose (DS-LNT), disialyl-lactose-N-fucopentaose (DS-LNFP V), lactose-N-neodifucohexaose I (LNnDFH I), 3'-galactosyl lactose (3'-GL), 6'-galactosyl lactose (6'-GL).

[0035] Genetically engineered microbial cells are microbial cells that have been genetically engineered to have metabolic pathways for intracellular biosynthesis of target oligosaccharides. Therefore, genetically engineered microbial cells are capable of intracellular synthesis of target oligosaccharides.

[0036] As used herein, the terms "capable" and "may" with respect to biosynthesis of target oligosaccharides by genetically engineered microbial cells mean that when the microbial cells are cultured in a culture medium and under conditions (e.g., in terms of temperature, pH, osmotic pressure, nutrient composition) that allow intracellular biosynthesis of the target oligosaccharides, the genetically engineered microbial cells synthesize the target oligosaccharides intracellularly.

[0037] When genetically engineered microbial cells are cultured in the presence of exogenous glycerol as the sole carbon / energy source, they are able to synthesize the desired oligosaccharides within the cells. Therefore, genetically engineered microbial cells have a glycerol permease for internalizing exogenous glycerol. Glycerol permease, also known as a glycerol uptake enhancer, is able to internalize exogenously supplied glycerol.

[0038] In some embodiments, the glycerol permease is an endogenous glycerol permease of the microbial cell, i.e., a glycerol permease that naturally occurs in native cells of the same species to which the genetically engineered microbial cell belongs. In additional and / or alternative embodiments, the glycerol permease is a heterologous glycerol permease, i.e., a glycerol permease that does not naturally occur in native cells of the microbial species to which the genetically engineered microbial cell belongs.

[0039] Additionally and / or alternatively, the genetically engineered microbial cell has been genetically engineered to have glycerol permease or has increased glycerol permease activity compared to the ancestral cell that has not yet been genetically engineered to have increased glycerol permease activity. After genetically engineering the microbial cell to have glycerol permease or the glycerol permease activity that improves, the exogenous glycerol permease encoding gene is inserted into the microbial cell, and / or by genetically modifying the endogenous glycerol permease encoding gene of the microbial cell, the glycerol permease is expressed. In some of these embodiments, the ancestral cell of the genetically engineered microbial cell has been genetically engineered to contain the exogenous glycerol permease encoding gene, and expresses the glycerol permease. In some embodiments, the exogenous glycerol permease encoding gene is integrated into the chromosome of the microbial cell or at least into one of the chromosomes of the microbial cell. Additionally and / or alternatively, the exogenous glycerol permease encoding gene is present on the free nucleic acid molecule in the microbial cell. In some embodiments, the glycerol permease encoding gene is a recombinant gene.

[0040] An example of a suitable glycerol permease is the glycerol uptake promoting protein GlpF of Escherichia coli (strain K-12), encoded by the Escherichia coli glpF gene. The deduced amino acid sequence of Escherichia coli K-12 GlpF has been published in the UniProt knowledge base (www.uniprot.org), version 2022_01 published from February 23, 2022: entry number P0AER0. Therefore, in some embodiments, the genetically engineered microbial cell has Escherichia coli GlpF or a functional variant thereof. Additionally and / or alternatively, the microbial cell has been genetically engineered to have the Escherichia coli K-12 glycerol permease GlpF or a functional variant thereof. Therefore, in some embodiments, the microbial cell is a cell genetically engineered to have a recombinant gene that contains and expresses a nucleotide sequence encoding Escherichia coli GlpF or a functional variant thereof.

[0041] The term "variant" as used herein refers to a polynucleotide or polypeptide whose nucleotide sequence or amino acid sequence is different from the sequence of a reference polynucleotide or polypeptide, respectively, but retains the basic properties of the reference polynucleotide or polypeptide (e.g., catalyticity). A typical variant of a polynucleotide is different from another polynucleotide as a reference polynucleotide in nucleotide sequence. The difference in the nucleotide sequence of the variant may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. As described below, nucleotide deviations may cause amino acid substitutions, additions, deletions, fusions, and / or truncations in the polypeptide encoded by the reference sequence. A typical variant of a polypeptide is different from another reference polypeptide in amino acid sequence. In general, the difference is limited, so that the sequences of the reference polypeptide and the variant are generally very similar and identical in many regions. The difference between the variant and the reference polypeptide in amino acid sequence may be any combination of one or more substitutions, additions, and deletions. The substituted or inserted amino acid residues may or may not be encoded by the genetic code. The variant of a polynucleotide or polypeptide may be naturally occurring, such as an allele variant, or may be an unknown naturally occurring variant. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, direct synthesis, and other recombinant methods known to those skilled in the art.

[0042] Within the scope of the present invention, these terms also include nucleic acid / polynucleotide and polypeptide polymorphic variants, alleles, mutants and interspecies homologs having nucleotide sequences or amino acid sequences having greater than about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater sequence identity to a reference sequence. The sequence identity extends over a region of at least about 25, 50, 100, 200, 500, 1000 or more nucleotides or amino acids compared to the reference sequence.

[0043] A "variant" of any gene / polypeptide refers to a sequence variant of a gene / protein that retains the same, lower or higher activity of the gene, polypeptide and / or polypeptide encoded by the gene, or a different activity in substrate specificity relative to a reference polypeptide, or a different activity in reaction specificity relative to a reference polypeptide.

[0044] As used herein, the term "operon" refers to a nucleotide sequence comprising two or more protein coding sequences that are transcribed by the same regulatory elements that are used to mediate and / or control expression in a microbial cell.

[0045] The term "functional gene" used herein refers to a nucleic acid molecule that includes a nucleotide sequence encoding a protein or polypeptide, and also contains a regulatory sequence that is operably linked to the protein-encoding nucleotide sequence (open reading frame), so that the nucleotide sequence encoding the protein or polypeptide can be expressed in a microbial cell carrying the functional gene or expressed by the microbial cell. Therefore, when cultured under conditions that allow expression of the functional gene, the gene is expressed, and the microbial cell expressing the functional gene generally includes a protein or polypeptide encoded by the protein coding region of the functional gene. The terms "nucleic acid" and "polynucleotide" used herein refer to deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, encompass known analogs of natural nucleotides that hybridize with nucleic acids in a manner similar to naturally occurring nucleotides.

[0046] As used herein, the term "deregulated" with respect to endogenous gene expression refers to an altered expression of a protein coding region of an endogenous gene compared to the expression of the gene in the native progenitor of a genetically engineered microbial cell, wherein the progenitor has not been genetically engineered to alter the expression of the gene. The term "deregulated" includes increased gene expression as well as decreased or impaired gene expression. Regulation of gene expression can be deregulated by various methods known to those skilled in the art. Examples of deregulated gene expression include altering the native nucleotide sequence of a gene promoter, altering the native nucleotide sequence of a gene ribosome binding site.

[0047] The term "operably connected" as used herein refers to a functional connection between a nucleotide sequence (such as a promoter, a signal sequence or an array of transcription factor binding sites) and a second nucleotide sequence that controls expression, wherein the nucleotide sequence that controls expression affects the transcription and / or translation of the second nucleotide sequence. Therefore, the term "promoter" refers to a nucleotide sequence that is usually located "before" the protein encoding nucleotide sequence of a DNA polynucleotide and provides a start site for transcription into mRNA. A "regulator" DNA sequence is also usually located "upstream" (i.e., before) of the protein encoding nucleotide sequence of a gene in a given DNA polymer, in conjunction with a protein that determines the frequency (or rate) of transcription initiation. These sequences are collectively referred to as "promoter / regulator" or "control" DNA sequences, which are located before a selected gene (or a series of genes) in a functional DNA polymer, and jointly determine whether transcription (and final expression) of a gene occurs. The nucleotide sequence that "follows" a protein encoding nucleotide sequence in a DNA polymer and provides a termination signal for transcription into mRNA is called a transcription "terminator" sequence.

[0048] The term "recombination" as used herein means a polynucleotide produced by a genetic engineering method, such as a gene or an operon. Therefore, a recombinant gene, an operon or a polynucleotide is not naturally present in a microbial cell of the same species as the microbial cell for producing the oligosaccharide of interest. A recombinant gene or an operon comprises a nucleotide sequence derived from at least two different ancestors (e.g., derived from different loci or different species). An example is the protein coding region of a glycerol permease encoding gene, which is operably connected to the promoter of different genes. A recombinant polynucleotide may contain a heterologous nucleotide sequence, or express a polypeptide encoded by a heterologous nucleotide sequence (i.e., a nucleotide sequence that is foreign to the microbial cell). A recombinant microbial cell may contain genes not found in its native (non-recombinant) ancestor. Recombinant cells may also contain gene variants found in the native ancestors of recombinant cells, wherein the gene is modified and reintroduced into cells by technical means. The term "recombination" also encompasses microbial cells containing endogenous nucleotide sequences of bacterial cells, which have been modified, but the nucleic acid molecules carrying the nucleotide sequence have not been removed from the bacterial cell. Such modifications include modifications obtained by gene replacement, site-specific mutations, and related techniques. Thus, a "recombinant polypeptide" is a polypeptide produced by a recombinant cell.

[0049] As used herein, a "heterologous nucleotide sequence" or "heterologous nucleic acid" is a sequence derived from a source foreign to a particular host cell (e.g., from a different species), or, if derived from the same source, a sequence that has been modified from its original form. Thus, a heterologous nucleotide sequence may be a nucleotide sequence in which a heterologous protein encoding nucleotide sequence is operably linked to a promoter, wherein the protein encoding nucleotide sequence and the nucleotide sequence of the promoter are obtained from different source organisms, or, if derived from the same source organism, the protein encoding nucleotide sequence or the promoter has been modified from its original form. The heterologous nucleotide sequence may be stably introduced into the genome of the bacterial cell, for example, by transposition, transfection, transformation, conjugation, or transduction. The techniques that may be applied will depend on the specificity of the microbial cell and the nucleic acid molecule to be introduced into the microbial cell. Various techniques are known to those skilled in the art and are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Therefore, "genetically engineered microbial cells" should be understood as microbial cells that have been transformed or transfected, or can be transformed or transfected by exogenous polynucleotide sequences. Therefore, the nucleotide sequence used in the present invention can, for example, be included in a vector to be stably transformed / transfected or otherwise introduced into a host microbial cell. A variety of vectors can be used to synthesize polypeptides in microbial cells. Such vectors especially include chromosomal vectors, episomal gene vectors and viral-derived vectors, for example, vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, and vectors derived from their combinations, such as vectors derived from plasmids and bacteriophage genetic elements (such as clay and phagemids). The expression system construct can contain a control region that regulates and causes expression. Generally speaking, any system or vector suitable for maintaining, breeding or expressing polynucleotides and synthetic polypeptides in bacterial host cells can be used for expression in this regard. Appropriate nucleotide sequences can be inserted into an expression system by any of a variety of well-known conventional techniques, such as the technology described in, for example, Sambrook et al. (the same). Preferably, the polynucleotide containing the recombinant nucleotide sequence is stably introduced into the genome of the microbial cell. Genomic integration can be achieved by recombination or transposition.

[0050] The genetically engineered microbial cells contain glycerol kinase. Glycerol kinase is an enzyme that uses adenosine triphosphate (ATP) to catalyze the phosphorylation of glycerol to generate sn-glycerol 3-phosphate. More specifically, the microbial cells have glycerol kinase, which is a functional variant of Escherichia coli K12 glycerol kinase GlpK.

[0051] The glycerol kinase of Escherichia coli (strain K12) is encoded by the protein-coding nucleotide sequence of the Escherichia coli (K12) glpK gene. The nucleotide sequence of the protein-coding region of the Escherichia coli (K12) glpK gene is shown in SEQ ID NO: 2 in the attached sequence listing. The deduced amino acid sequence of Escherichia coli K-12 GlpK is disclosed in the UniProt knowledge base (www.uniprot.org), version 2022_01 published from February 23, 2022: entry number P0A6F3. Additionally or alternatively, the amino acid sequence of the native Escherichia coli K12 glycerol kinase GlpK is also shown in SEQ ID NO: 1 of the sequence listing. Figure 2 Shown are the nucleotide sequence of the protein coding region of Escherichia coli (K12) glpK and the amino acid sequence of Escherichia coli (K12) GlpK deduced from the nucleotide sequence.

[0052] In certain embodiments, the functional variant of the Escherichia coli (K12) glycerol kinase GlpK comprises an amino acid residue at a position within its amino acid sequence corresponding to amino acid position 55 in the amino acid sequence of Escherichia coli (K12) GlpK shown in SEQ ID NO:1, wherein the amino acid residue comprises a non-ionized but polar acting side chain, and / or the variant comprises an amino acid residue at a position within its amino acid sequence corresponding to amino acid position 231 in the amino acid sequence of Escherichia coli (K12) GlpK shown in SEQ ID NO:1, wherein the amino acid residue comprises an anionic side chain.

[0053] Thus, the microbial cell comprises a glycerol kinase, wherein the glycerol kinase is a functional variant of the Escherichia coli glycerol kinase GlpK, wherein the functional variant has an amino acid residue at a position corresponding to amino acid position 55 in the amino acid sequence of the Escherichia coli GlpK shown in SEQ ID NO: 1, wherein the amino acid residue comprises a non-ionized (also called uncharged) but polar acting side chain.

[0054] Amino acids with non-ionizing but polar acting side chains belong to the group of amino acids having side chains with a functional group comprising at least one atom with an electron pair which can form hydrogen bonds with water.

[0055] In some embodiments, the amino acid residues containing non-ionizing but polar acting side chains are hydrophilic amino acids.

[0056] Additionally and / or alternatively, the amino acid comprising a non-ionizing but polar acting side chain is an amide amino acid. The amide amino acid is preferably selected from L-asparagine and L-glutamine.

[0057] Additionally and / or alternatively, the amino acid comprising a non-ionizing but polar acting side chain is a sulfur-containing amino acid. The sulfur-containing amino acid is preferably selected from L-serine, L-threonine, L-cysteine ​​and L-methionine.

[0058] Additionally and / or alternatively, the amino acid residue comprising a non-ionizing but polar acting side chain is an amino acid selected from the group consisting of L-serine, L-threonine, L-cysteine, L-methionine, L-asparagine and L-glutamine.

[0059] Additionally and / or alternatively, the microbial cell comprises a glycerol kinase, wherein the glycerol kinase is a functional variant of the Escherichia coli (K12) glycerol kinase GlpK, wherein the functional variant has an amino acid residue at the position corresponding to amino acid position 231 in the amino acid sequence of the Escherichia coli GlpK shown in SEQ ID NO: 1, wherein the amino acid residue comprises an anionic side chain. The amino acid residue comprising an anionic side chain is preferably selected from the amino acids L-aspartic acid and L-glutamic acid.

[0060] In order to have any of the functional variants of Escherichia coli (K12) glycerol kinase GlpK, the progenitor cell of the genetically engineered microbial cell is genetically engineered to contain a nucleic acid molecule that encodes and expresses a nucleotide sequence encoding at least one of the functional variants. Therefore, the genetically engineered microbial cell contains an exogenous functional gene that encodes and expresses a functional variant of Escherichia coli (K12) glycerol kinase GlpK.

[0061] In some embodiments, the exogenous gene encoding the functional variant of Escherichia coli (K12) GlpK is integrated into the chromosome of the microbial cell or into at least one of the chromosomes of the microbial cell. Additionally and / or alternatively, the recombinant functional gene encoding the functional variant of Escherichia coli (K12) glycerol kinase GlpK is included in the free nucleic acid molecule present in the genetically engineered microbial cell.

[0062] In some embodiments, the microbial cell has been genetically engineered in that the nucleotide sequence of the endogenous glycerol kinase gene of the microbial cell has been altered such that the resulting glycerol kinase encoding gene encodes a functional variant of E. coli (K12) GlpK.

[0063] The genetically engineered microbial cell has a glycerol kinase that is one of the functional variants of the E. coli (K12) glycerol kinase GlpK described herein. The microbial cell does not have a native or endogenous glycerol kinase that is not one of the functional variants of the E. coli (K12) glycerol kinase GlpK and has an enzyme activity higher than any of the functional variants of the E. coli (K12) glycerol kinase GlpK described herein. Therefore, in some embodiments, the genetically engineered microbial cell comprises a deletion or functional inactivation of its endogenous gene encoding glycerol kinase.

[0064] Functional inactivation can be the deletion and / or inactivation of one or more expression control sequences of the native glycerol kinase gene of the microbial cell, so that the expression of the native glycerol kinase gene of the microbial cell is eliminated. Additionally or alternatively, the functional inactivation can be the deletion or alteration of the protein coding region of the native glycerol kinase gene of the microbial cell, such as, for example, by introducing a frameshift mutation, a mutation that impairs translation initiation, a stop codon or a mutation that is introduced into the amino acid sequence of the polypeptide encoded by the altered nucleotide sequence, so that the resulting polypeptide does not have glycerol kinase activity.

[0065] In some embodiments, in addition to the deletion or functional inactivation of the endogenous glycerol kinase gene of the microbial cell, the genetically engineered microbial cell further comprises a functional exogenous gene encoding a functional variant of the E. coli (K12) glycerol kinase GlpK disclosed herein.

[0066] In some embodiments, the genetically engineered microbial cell is genetically engineered so that the protein coding region of its native glycerol kinase gene is altered to encode a functional variant of E. coli (K12) GlpK. When the genetically engineered microbial cell is an E. coli cell, altering the protein coding region of the native or endogenous glycerol kinase gene of the microbial cell is an option.

[0067] In some embodiments, the microbial cell is a genetically engineered microbial cell that has been genetically engineered because an exogenous gene encoding a functional variant of the E. coli (K12) glycerol kinase replaces the native / endogenous glycerol kinase gene of the microbial cell. When the genetically engineered microbial cell is an E. coli cell, replacing the protein coding region of the native or endogenous glycerol kinase gene of the microbial cell is an alternative option to changing the protein coding region of the native or endogenous glycerol kinase encoding gene of the microbial cell.

[0068] The genetically engineered microbial cell further comprises a metabolic pathway for intracellular biosynthesis of oligosaccharides of interest. Thus, the genetically engineered microbial cell comprises: a metabolic pathway for intracellular biosynthesis of nucleotide-activated monosaccharides, which serve as donor substrates for their monosaccharide moieties transferred to acceptor molecules; all enzymes and transporters necessary to provide acceptor molecules for obtaining the monosaccharide moieties; and a glycosyltransferase for transferring the monosaccharide moieties from the nucleotide-activated monosaccharides to the acceptor molecules.

[0069] The genetically engineered microbial cell comprises a metabolic pathway for intracellular synthesis of nucleotide-activated monosaccharides, wherein the nucleotide-activated monosaccharides serve as donor substrates for their monosaccharide moieties, wherein glycosyltransferases transfer the monosaccharide moieties from the donor substrates to acceptor molecules.

[0070] In some embodiments, the nucleotide activated monosaccharide is selected from guanosine-5'-diphospho-β-L-fucose (GDP-Fuc), cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NeuNAc), uridine-5'-diphospho-α-D-galactose (UDP-Gal), uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) and uridine-5'-diphospho-α-D-glucose (UDP-Glc).

[0071] The nucleotide activated monosaccharide can be synthesized by genetically engineered microbial cells through a de novo pathway. Additionally and / or alternatively, the microbial cell can use a salvage pathway to provide the nucleotide activated monosaccharide.

[0072] In embodiments where nucleotide-activated monosaccharides are synthesized by a de novo pathway, the microbial cell has the enzymes necessary for the de novo biosynthetic pathway of nucleotide-activated monosaccharides. In some embodiments, the microbial cell is genetically engineered to have at least one exogenous gene encoding an enzyme necessary for the de novo biosynthetic pathway of nucleotide-activated monosaccharides.

[0073] At least one gene encoding an enzyme necessary for the de novo biosynthetic pathway of nucleotide-activated monosaccharides may be endogenous to the microbial cell, or they may be introduced into the microbial cell from an exogenous source for expression. The expression of proteins encoded by endogenous genes, i.e., functional transcription and translation, may be altered by genetically engineering microbial cells. For example, the change in endogenous gene expression may be achieved by modifying the transcription promoter of the gene, modifying the ribosome binding site of the gene, and / or modifying the codon usage of the protein encoding nucleotide sequence of the gene. In addition, the protein encoding nucleotide sequence of the gene may be modified so that the activity and / or specificity of the enzyme encoded by the gene becomes conducive to the desired biosynthetic pathway. Those skilled in the art know which genes need to be expressed in genetically modified organisms in order to synthesize de novo nucleotide-activated sugar donor molecules.

[0074] In embodiments where the salvage pathway is used to biosynthesize a nucleotide-activated monosaccharide as a donor substrate, the microbial cell comprises an enzyme that catalyzes the attachment of a nucleotide and a monosaccharide. An example of such an enzyme is a bifunctional fucokinase / L-fucose-1-phosphate-guanylate transferase FKP from Bacteroides fragilis, which catalyzes a kinase reaction and a pyrophosphatase reaction to form GDP-L-fucose. Another example of an enzyme that catalyzes the attachment of a nucleotide and a monosaccharide is an N-acetylneuraminic acid cytidylic acid transferase NeuA from Neisseria meningitidis, which forms CMP-neuraminic acid.

[0075] In some embodiments, a microbial cell having a salvage pathway that provides a nucleotide-activated monosaccharide as a donor substrate contains an exogenous gene that encodes and expresses an enzyme that catalyzes the ligation of a nucleotide and a monosaccharide.

[0076] Monosaccharides used as substrates for forming nucleotide-activated monosaccharides in the salvage pathway can be internalized by microbial cells utilizing monosaccharide import proteins. Examples of such monosaccharide import proteins are fucose permeases, such as FucP, and sialic acid importers, such as NanT from Escherichia coli. Monosaccharide import proteins can be encoded and expressed by endogenous genes, or can be encoded and expressed by exogenous genes.

[0077] For the biosynthesis of target oligosaccharides, the acceptor molecule is a carbohydrate, i.e., an acceptor saccharide. The acceptor molecule can be selected from monosaccharides, disaccharides, and oligosaccharides. In some embodiments, the acceptor saccharide is a disaccharide that is converted into a trisaccharide. In other embodiments, the acceptor saccharide is a trisaccharide that is converted into a tetrasaccharide. In other embodiments, the acceptor saccharide is a tetrasaccharide that is converted into a pentasaccharide. In some embodiments, the acceptor saccharide is a pentasaccharide that is converted into a hexasaccharide.

[0078] In some embodiments, the acceptor sugar or the precursor of the acceptor sugar is internalized by the microbial cell, preferably by utilizing a specific transporter present in the cell membrane of the microbial cell. In the process of using the genetically engineered microbial cell to produce the target oligosaccharide, the genetically engineered microbial cell is cultured in the presence of the acceptor sugar or the precursor of the acceptor sugar. The acceptor sugar or its precursor present in the culture medium is internalized by the genetically engineered microbial cell and used for the biosynthesis of the target oligosaccharide.

[0079] Additionally and / or alternatively, the acceptor sugar is synthesized intracellularly by the genetically engineered microbial cell.

[0080] In some embodiments, the acceptor carbohydrate is synthesized by microbial cells in the cell from the monosaccharide that has been internalized by the microbial cells, and is extended by the monosaccharide moiety to obtain a disaccharide, wherein the disaccharide can be further extended by adding a further monosaccharide moiety, so that the oligosaccharide composed of three, four, five, six or more monosaccharide moieties is synthesized in the cell, and then it constitutes the acceptor carbohydrate for the biosynthesis purpose oligosaccharide. For example, the microbial cells internalize glucose, and it is converted into lactose by adding the galactose moiety. Then, the lactose can be converted into oligosaccharides, such as for example 2'-FL, 3-FL, LNT-II, LNT, LNnT, 3'-SL, 6'-SL. Each of the oligosaccharides can be a purpose oligosaccharide, or can constitute the acceptor carbohydrate.

[0081] In some embodiments, the microbial cell is genetically engineered to have an enzyme that catalyzes the conversion of an acceptor saccharide (i.e., a disaccharide or oligosaccharide) into a desired oligosaccharide by transferring a monosaccharide moiety from a donor substrate to an acceptor saccharide. Thus, the microbial cell contains and expresses at least one exogenous gene, homologous or heterologous gene, encoding an enzyme that catalyzes the conversion of an acceptor saccharide into a desired oligosaccharide.

[0082] The gene or equivalent functional nucleotide sequence encoding the enzymatic ability of a microbial cell to catalyze the conversion of an acceptor sugar into a target oligosaccharide (i.e., adding a monosaccharide moiety from a donor substrate to the acceptor sugar) can be a homologous nucleotide sequence or a heterologous nucleotide sequence. The term "homologous" used herein with respect to nucleotide sequences refers to a nucleotide sequence native to the species to which the microbial cell used to produce the target oligosaccharide belongs. The term "heterologous" used herein with respect to nucleotide sequences refers to a nucleotide sequence artificially produced or derived from a species different from the species to which the microbial cell used to produce the target oligosaccharide belongs. Heterologous nucleotide sequences derived from species different from the species of the microbial cell used to produce the target oligosaccharide can be derived from plants, animals (including humans), bacteria, archaea, fungi or viruses.

[0083] The enzyme that catalyzes the transfer of a monosaccharide moiety to an acceptor molecule is a glycosyltransferase or transglycosidase. While transglycosidase catalyzes the transfer of a monosaccharide moiety from one sugar to another, glycosyltransferase catalyzes the transfer of a monosaccharide moiety from a nucleotide activated sugar as a donor substrate to an acceptor carbohydrate. The glycosyltransferase can be selected from galactosyltransferase, glucosyltransferase, fucosyltransferase, sialyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, glucuronyltransferase, mannosyltransferase and xylosyltransferase. Additionally and / or alternatively, the glycosyltransferase is selected from the group consisting of β-1,3-galactosyltransferase, β-1,4-galactosyltransferase, β-1,6-galactosyltransferase, α-1,3-glucosyltransferase, α-1,4-glucosyltransferase, α-1,2-fucosyltransferase and α-1,3-fucosyltransferase, α1,4-fucosyltransferase, α-2,3-sialyltransferase, α-2 ,6-sialyltransferase, α-2,8-sialyltransferase, β-1,3-N-acetylglucosaminyltransferase, β-1,4-N-acetylglucosaminyltransferase, α-1,3-N-acetylgalactosaminyltransferase, β-1,3-N-acetylgalactosaminyltransferase, β-1,4-N-acetylgalactosaminyltransferase, α-1,2-mannosyltransferase, β-1,4-xylosyltransferase.

[0084] It is understood that a specific glycosyltransferase catalyzes the transfer of the corresponding monosaccharide moiety from the nucleotide activated monosaccharide to the acceptor molecule.

[0085] That is, some genetically engineered microbial cells have a metabolic pathway for providing GDP-Fuc, and further have a fucosyltransferase for biosynthesis of fucosylated oligosaccharides of interest, such as, for example, 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 2',3-difucosyllactose (DFL), lacto-N-fucopentaose I (LNFP-I), lacto-N-neofucopentaose I (LNnFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP-V), lacto-N-neofucopentaose V (LNnFP-V), difucosyl-lactose-N-neohexose (DF -LNnH), lactose-N-difucosyllhexaose I, lactose-N-difucosyllhexaose II, para-lactose-N-fucosyllhexaose (paraLNH), fucosyl-lactose-N-sialylpentaose a (F-LST-a), fucosyl-lactose-N-sialylpentaose b (F-LST-b), fucosyl-lactose-N-sialylpentaose c (F-LST-c), fucosyl-lactose-N-sialylpentaose c, disialyl-lactose-N-fucopentaose, 3-fucosyl-3'-sialyl lactose (3F-3'-SL), 3-fucosyl-6'-sialyl lactose (3F-6'-SL), lactose-N-neodifucohexaose I, disialyl-lactose-N-fucopentaose (DS-LNFP V) or lactose-N-neodifucohexaose I (LNnDFH I).

[0086] Some genetically engineered microbial cells have a metabolic pathway that provides CMP-NeuNAc, and further have a sialyltransferase for the biosynthesis of sialylated oligosaccharides of interest, such as, for example, 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), sialyl lactose-N-tetraose a (LST-a), sialyl lactose-N-tetraose b (LST-b), sialyl lactose-N-tetraose c (LST-c), disialyl lactose-N-tetraose (DS-LNT), fucosyl-lactose-N- Sialylpentasaccharide a (F-LST-a), fucosyl-lactose-N-sialylpentasaccharide b (F-LST-b), fucosyl-lactose-N-sialylpentasaccharide c (F-LST-c), fucosyl-lactose-N-sialylpentasaccharide c, disialyl-lactose-N-fucopentasaccharide, 3-fucosyl-3'-sialyl lactose (3F-3'-SL), 3-fucosyl-6'-sialyl lactose (3F-6'-SL), lactose-N-neodifucohexaose I, disialyl-lactose-N-fucopentasaccharide (DS-LNFP V) or lactose-N-neodifucohexaose I (LNnDFH I).

[0087] Some genetically engineered microbial cells have a metabolic pathway for providing UDP-GlcNAc and further have an N-acetylglucosaminyltransferase for the biosynthesis of oligosaccharides containing N-acetylglucosamine, such as, for example, lactose-N-triose II, lactose-N-tetraose (LNT), lactose-N-neotetraose (LNnT), lactose-N-fucopentaose I (LNFP-I), lactose-N-neofucopentaose I (LNnFP-I), lactose-N-fucopentaose II (LNFP-II), lactose-N-fucopentaose III (LNFP-III), lactose-N-fucopentaose V (LNFP-V), lactose-N-neofucopentaose V (LNnFP-V), lactose-N-hexaose (LNH), lactose-N-neohexaose (LNnH), para-lactose-N-hexaose (paraLNH), para-lactose-N-neohexaose (paraLNnH), difucosyl-lactose-N-neohexaose (DF-LNnH), lactose- N-difucosylated hexaose I, lactose-N-difucosylated hexaose II, para-lactose-N-fucosylated hexaose (paraLNH), fucosyl-lactose-N-sialylpentasaccharide a (F-LST-a), fucosyl-lactose-N-sialylpentasaccharide b (F-LST-b), fucosyl-lactose-N-sialylpentasaccharide c (F-LST-c), fucosyl-lactose-N-sialylpentasaccharide c, disialyl-lactose-N-fucosidopentaose, 3-LST-a Fucosyl-3'-sialyl lactose (3F-3'-SL), 3-fucosyl-6'-sialyl lactose (3F-6'-SL), lactose-N-neodifucohexaose I, sialyl lactose-N-tetraose a (LST-a), sialyl lactose-N-tetraose b (LST-b), sialyl lactose-N-tetraose c (LST-c), disialyl lactose-N-tetraose (DS-LNT), disialyl-lactose-N-fucopentaose (DS-LNFP V) or lactose-N-neodifucohexaose I (LNnDFH I).

[0088] Some genetically engineered microbial cells have a metabolic pathway for providing UDP-Gal and further have a galactosyltransferase for the biosynthesis of oligosaccharides containing galactose moieties, such as, for example, 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 2',3-difucosyllactose (DFL), lacto-N-triose II, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP-I), lacto-N-neofucopentaose I (LNFP-II). (LNnFP-I), lactose-N-fucopentaose II (LNFP-II), lactose-N-fucopentaose III (LNFP-III), lactose-N-fucopentaose V (LNFP-V), lactose-N-neofucopentaose V (LNnFP-V), lactose-N-hexaose (LNH), lactose-N-neohexose (LNnH), para-lactose-N-hexaose (paraLNH), para-lactose-N-neohexose (paraLNnH), difucosyl-lactose-N-neohexose (DF-LNnH), Lactose-N-difucosylated hexaose I, lactose-N-difucosylated hexaose II, para-lactose-N-fucosylated hexaose (paraLNH), fucosyl-lactose-N-sialylpentasaccharide a (F-LST-a), fucosyl-lactose-N-sialylpentasaccharide b (F-LST-b), fucosyl-lactose-N-sialylpentasaccharide c (F-LST-c), fucosyl-lactose-N-sialylpentasaccharide c, disialyl-lactose-N-fucopentasaccharide, 3-fucosyl-3'-sialyl lactose (3F-LST-c) -3'-SL), 3-fucosyl-6'-sialyl lactose (3F-6'-SL), lactose-N-neodifucohexaose I, 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), sialyl lactose-N-tetraose a (LST-a), sialyl lactose-N-tetraose b (LST-b), sialyl lactose-N-tetraose c (LST-c), disialyl lactose-N-tetraose (DS-LNT), disialyl-lactose-N-fucopentaose (DS-LNFP V), lactose-N-neodifucohexaose I (LNnDFH I), 3'-galactosyl lactose (3'-GL), 6'-galactosyl lactose (6'-GL).

[0089] In some embodiments, the genetically engineered microbial cell has a sugar transporter in its cell membrane. The sugar transporter transfers the target oligosaccharide from the cytoplasm of the microbial cell across the cell membrane into the culture medium, or for Gram-negative bacteria, into the periplasmic space.

[0090] A carbohydrate transporter that mediates the translocation of a carbohydrate (e.g., a target oligosaccharide) may consist of a single polypeptide, or may consist of multiple polypeptides (each polypeptide being considered as a subunit) that form homomeric or heteromeric complexes that can translocate the target oligosaccharide across the cell membrane.

[0091] In some embodiments, the carbohydrate transporter is a member selected from the major facilitator superfamily (MFS), the sugar:cation symporter family, the nucleoside-specific transporter family, the ATP-binding cassette superfamily, and the phosphotransferase system family.

[0092] In certain embodiments, the sugar transporter is encoded and expressed by an exogenous gene. Thus, in such embodiments, the microbial cell comprises an exogenous gene encoding a sugar transporter or at least one subunit of a sugar transporter complex.

[0093] The exogenous gene encoding the transporter or transporter complex that facilitates the transport of the oligosaccharide of interest across the cell membrane may be endogenous to the genetically engineered microbial cell, or may be exogenous.

[0094] In some embodiments, the sugar transporter gene is endogenous for genetically engineered bacterial cells.The functional element that affects the protein synthesis of the open reading frame encoding of the sugar transporter gene may be modified so that the transcription level of the open reading frame or the translation level that causes the synthetic mRNA of transporter are different from the level of natural discovery in the predecessor cell.Compared with unmodified state, the modification of functional element can increase transcription and / or translation level, or reduce transcription and / or translation level.Modification can be the exchange of mononucleotide modification or complete functional element.In any case, for the transportation of intracellular oligosaccharide across inner membrane, optimized the non-natural level of transcription and / or translation.

[0095] In another embodiment, one or more genes encoding a carbohydrate transporter or transporter complex are exogenous to a genetically engineered microbial cell. In such embodiments, the protein coding region is operably linked to a naturally occurring or artificial functional element, such as a promoter or ribosome binding site, in a manner that the transcription and translation levels of the transporter are optimized for the transport of oligosaccharides across the inner membrane of the target.

[0096] The transcription of the gene encoding the sugar transporter or the sugar transporter subunit can be constitutive, or can be regulated. Those skilled in the art will know how to select and use promoters and / or promoters in combination with further transcriptional regulators to achieve optimal expression of the gene encoding the sugar transporter.

[0097] In embodiments where the gene encoding a sugar transporter is foreign to the microbial cell used to produce the oligosaccharide of interest, the nucleotide sequence of the gene may be modified compared to the naturally occurring nucleotide sequence but may encode a polypeptide having an unchanged amino acid sequence.

[0098] The sugar transporter for translocating the oligosaccharide across the cell membrane of the target can have its native amino acid sequence, i.e., a naturally occurring amino acid sequence. Alternatively, the amino acid sequence of the sugar transporter can be modified compared to the native amino acid sequence so that the resulting variant has enhanced activity and / or may have other beneficial properties in the translocation of the oligosaccharide of the target, such as enhanced protein stability or more stably integrated into the inner membrane.

[0099] In a specific embodiment, the nucleotide sequence encoding the carbohydrate transporter is artificial, and thus, the amino acid sequence of the resulting protein that functions as a transporter to export the oligosaccharide of interest from the cytoplasm into the periplasm is not itself found in nature.

[0100] In some embodiments, the genetically engineered microbial cell further comprises a deletion or functional inactivation of its endogenous methylglyoxal synthase gene. Methylglyoxal synthase catalyzes the conversion of dihydroxyacetone phosphate into methylglyoxal and phosphoric acid. Surprisingly, the deletion or functional inactivation of the endogenous gene of the microbial cell encoding methylglyoxal synthase further improves the fermentation production of target oligosaccharides by the microbial cell, and the microbial cell has a functional variant of the native Escherichia coli glycerol kinase GlpK, wherein the amino acid sequence of the native Escherichia coli GlpK is as shown in SEQ ID NO: 1, wherein the functional variant has an amino acid residue containing a non-ionized but polar acting side chain at the position corresponding to amino acid position 55 in the amino acid sequence of the native Escherichia coli GlpK in its amino acid sequence, and / or wherein the functional variant has an amino acid containing an anionic side chain at the position corresponding to amino acid position 231 in the amino acid sequence of the native Escherichia coli GlpK in its amino acid sequence.

[0101] The genetically engineered microbial cells described above can be used to produce target oligosaccharides by fermentation.

[0102] Therefore, a method for producing oligosaccharides of interest by fermentation is also disclosed, wherein the method comprises:

[0103] - providing a genetically engineered microbial cell for producing a target oligosaccharide according to the first aspect, i.e. a genetically engineered microbial cell capable of synthesizing a target oligosaccharide intracellularly when cultured in the presence of glycerol as a sole carbon source, wherein the genetically engineered microbial cell comprises a glycerol permease and a functional variant of a native Escherichia coli glycerol kinase GlpK, a metabolic pathway for intracellular biosynthesis of a nucleotide-activated monosaccharide as a donor substrate for its monosaccharide moiety, and a glycosyltransferase for transferring a monosaccharide moiety from the nucleotide-activated monosaccharide to an acceptor molecule, wherein the amino acid sequence of the native Escherichia coli GlpK is as shown in SEQ ID NO: 1, wherein the functional variant has an amino acid residue containing a non-ionized but polar acting side chain at the position in its amino acid sequence corresponding to amino acid position 55 in the amino acid sequence of the native Escherichia coli GlpK, and / or wherein the functional variant has an amino acid containing an anionic side chain at the position in its amino acid sequence corresponding to amino acid position 231 in the amino acid sequence of the native Escherichia coli GlpK;

[0104] - culturing the genetically engineered microbial cells in a culture medium and under conditions that allow the genetically engineered microbial cells to synthesize the target oligosaccharide intracellularly, wherein the culture medium contains glycerol as a carbon source for the genetically engineered microbial cells; and

[0105] - Optionally, recovering the desired oligosaccharides from the microbial cells and / or culture medium.

[0106] Also disclosed are methods for reducing the toxicity of methylglyoxal to microbial cells when the microbial cells are cultured in the presence of glycerol as a carbon source, and methods for reducing the responsiveness of microbial cells to methylglyoxal toxicity during the fermentation production of target oligosaccharides. These methods include providing genetically engineered microbial cells that synthesize target oligosaccharides in cells; deleting or functionally inactivating the endogenous glycerol kinase gene of the microbial cells; and transforming the microbial cells to contain and express a functional variant of the Escherichia coli glycerol kinase GlpK, wherein the functional variant has an amino acid residue at a position corresponding to amino acid position 55 in the amino acid sequence of the Escherichia coli GlpK as shown in SEQ ID NO: 1, wherein the amino acid residue contains a non-ionized but polar acting side chain, and / or has an amino acid residue at a position corresponding to amino acid position 231 in the amino acid sequence of the Escherichia coli GlpK as shown in SEQ ID NO: 1, wherein the amino acid residue contains an anionic side chain.

[0107] The present invention will be described in conjunction with specific embodiments and with reference to the accompanying drawings, but the invention is not limited thereto and is limited only by the claims. In addition, the terms "first", "second", etc. in the specification and claims are used to distinguish similar elements and are not necessarily used to describe the order in time, space, ranking or any other manner. It should be understood that the terms so used are interchangeable where appropriate, and that the embodiments of the invention described herein are capable of operating in a different order than described or shown herein.

[0108] It is worth noting that the term "comprising" used in the claims should not be interpreted as being limited to the means listed thereafter; it does not exclude other elements or steps. Therefore, it should be interpreted as specifying the presence of the mentioned features, integers, steps or components, but does not exclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Therefore, the scope of the expression "a device comprising means A and B" should not be limited to a device consisting only of components A and B. This means that, with respect to the present invention, the only relevant components of the device are A and B.

[0109] Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in conjunction with that embodiment is included in at least one embodiment of the present invention. Thus, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment. Furthermore, it will be apparent to one of ordinary skill in the art from this disclosure that the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0110] Similarly, it should be understood that in the description of exemplary embodiments of the present invention, various features of the present invention are sometimes grouped together in a single embodiment, figure, or description thereof in order to simplify the disclosure and aid in understanding one or more of the various inventive aspects. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than those expressly recited in each claim. On the contrary, as reflected in the following claims, inventive aspects lie in less than all the features of a single preceding disclosed embodiment. Therefore, the claims following the detailed description are hereby expressly incorporated into this detailed description, and each claim itself serves as a separate embodiment of the present invention.

[0111] In addition, although some embodiments described herein include some features included in other embodiments but do not include other features, the combination of features of different embodiments is intended to fall within the scope of the present invention and form different embodiments, as understood by those skilled in the art. For example, in the following claims, any claimed embodiment can be used in any combination.

[0112] In the description and drawings provided herein, many specific details are set forth. However, it should be understood that embodiments of the present invention may be practiced without these specific details. In other cases, well-known methods, structures, and techniques are not shown in detail to avoid obscuring the understanding of this description.

[0113] The present invention will now be described by detailed description of its multiple embodiments. Other embodiments of the present invention may be configured according to the knowledge of those skilled in the art without departing from the true spirit or technical teaching of the present invention, and the present invention is limited only by the terms of the appended claims.

[0114] Example

[0115] Example 1: Adaptive laboratory evolution of 3-FL, ΔmgsA, GlpK(A55T)-Escherichia coli

[0116] Adaptive laboratory evolution experiments were conducted on an E. coli strain capable of producing 3-FL to investigate the effects of cured methylglyoxal toxicity on growth and 3-FL production using glycerol as the sole carbon and energy source.

[0117] The E. coli strain capable of producing 3-FL lacks the lacZ gene, expresses a heterologous α-1,3-fucosyltransferase gene, and lacks the mgsA gene. All molecular biology procedures were performed according to Sambrook, J. and Russell, D., Molecular Cloning: A laboratory Manual, 3 rdEdition, Cold Spring Harbor Laboratory Press, New York, 2001. In order to delete the mgsA gene in the Escherichia coli strain that can produce 3-FL, the upstream and downstream regions near the mgsA open reading frame were amplified by polymerase chain reaction (PCR), and two DNA fragments were produced. Primers 1 and 2 (Table 1) were used to generate the upstream fragment, and primers 3 and 4 (Table 1) were used to amplify the downstream region of the mgsA flank. These two DNA fragments were used as templates in the third PCR, and the third PCR used primers 1 and 4 (Table 1) to generate a DNA construct, and then according to Datsenko, KA and Wanner, BL (One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc. Natl. Acid. Sci 97 (2000) 6640-6645) The modified version of the method described, the mgsA gene was deleted using this DNA construct. Single clones were analyzed by colony PCR using primers 1 and 4 (Table 1). The selected strain, designated 3-FL-2, was exposed to N-methyl-N'-nitro-N-nitrosoguanidine for chemical mutagenesis and then cultured in two consecutive fed-batch cycles in a bioreactor. The culture was carried out as described in WO 2018 / 077892A1.

[0118] The cells obtained from the first fed-batch culture were used as inoculum for the second fed-batch culture. Cell samples were collected during the second culture, diluted in 0.9% (w / v) NaCl, and 100 μL aliquots were inoculated into plates containing 2YT medium, which consisted of 16 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl, and 18 g / L agar. After incubation at 30 ° C for 48 hours, the isolated colonies that increased 3-FL production were screened in deep-well plates using the same batch culture medium as in the fed-batch culture described previously herein. For the pre-culture used in this experiment, the plate was incubated at 30 ° C, 800 rpm, 80% humidity (Microtron, Infors AG, Switzerland) for 24 hours. In the main deep-well plate culture, the culture medium was supplemented with 10 mM lactose, and the culture conditions were the same as those used for the pre-culture. Sample preparation and HPLC analysis were performed as described in WO 2018 / 077892A1.

[0119] A selected E. coli isolate (3-FL-3) obtained in the adaptive laboratory evolution experiment was cultivated in a bioreactor in fed-batch mode to evaluate its growth characteristics and 3-FL production and compare them with those of the ancestral strain that was not subjected to the laboratory evolution experiment.

[0120] The results are shown in Figure 3 All data points are relative to the control strain.

[0121]

[0122] Table 1. Primers

[0123] The optical density (OD) of the culture medium was analyzed at a wavelength of 600 nm. 600 ), the selected E. coli isolate 3-FL-3 (■) showed a 44% reduction in time to complete the batch phase compared to the ancestral strain (▲) Figure 3 A). In addition, the selected E. coli isolate 3-FL-3 (■) showed an increase of approximately 18% in 3-FL in the culture supernatant after nearly 100 hours of cultivation, compared to the amount of 3-FL found in the culture supernatant of the ancestral strain (▲) ( Figure 3 B).

[0124] The selected E. coli isolate 3-FL-3, which showed faster growth and increased 3-FL production compared to the ancestral strain, was found to contain a mutated glpK gene encoding a variant of GlpK in which the alanine residue at position 55 was replaced by a threonine residue. This variant of E. coli GlpK was designated GlpK (A55T).

[0125] Example 2: Production of 2'-FL or LNT by GlpK mutant E. coli strains

[0126] To investigate the effect of GlpK(A55T) on the production of HMOs other than 3-FL, E. coli strains capable of producing 2'-FL or LNT were genetically engineered so that their endogenous glpK gene was replaced by a variant encoding E. coli (K12) GlpK(A55T). In 2'-FL-producing E. coli strains and LNT-producing E. coli strains, the endogenous glpK gene was replaced by a variant encoding E. coli (K12) GlpK(A55T). rdAll molecular biological procedures used to modify the endogenous glpK gene into a mutant glpK gene were performed according to the methods described in Glucan®, Glucan®, Glucan®, Glucan® Edition, Cold Spring Harbor Laboratory Press, New York, 2001. To change the nucleotide sequence of the native glpK gene, primer 5 (Table 1) was used for the recombination event.

[0127] In order to identify clones containing mutant glpK genes, colony PCR was performed using a combination of primers 6 and 8 (Table 1) to confirm the presence of the wild-type glpK gene, while a combination of primers 7 and 8 (Table 1) was used to confirm the presence of the mutant version of glpK. Nucleotide sequence analysis was performed on bacterial clones identified as carrying mutant versions of the glpK gene using primers 8 and 9 (Table 1) to confirm that they carried the glpK gene variant encoding GlpK (A55T). The E. coli strain producing 2'-FL thus constructed was named E. coli 2'-FL-2 (Table 2). The E. coli strain producing LNT thus constructed was based on a metabolically engineered E. coli BL21 (DE3) similar to the E. coli LNT-1 strain (Table 2), and was named E. coli LNT-2 (Table 2). Both E. coli strains 2'-FL2 and LNT-2 were cultured in a fed-batch process to compare growth characteristics and HMO production profiles. Fed-batch cultivation and HPLC analysis were performed under similar conditions as described in WO 2018 / 077892A1.

[0128] Despite the presence of endogenous mgsA gene in E. coli 2'-FL-2 and E. coli LNT-2 strains, they showed improved growth ( Figure 4 A and Figure 5 A) and HMO production ( Figure 4 B and Figure 5 B). For E. coli 2'-FL-2 strain (Table 2) (■), Figure 4 A shows the time required to complete the batch phase is reduced, and Figure 4 B shows the increase in 2'-FL titer. The time to complete the batch phase was reduced by approximately 35%, while the 2'-FL titer increased by approximately 36%. Figure 5 In Figure 5 As shown in A, the time to complete the batch process of E. coli LNT-2 strain (Table 2) (■) was reduced by 41% compared to the control strain (▲). Furthermore, the LNT-2 titer in the culture supernatant of E. coli 2'-FL-2 (■) increased by about 14% (see Figure 5 B).

[0129]

[0130] Table 2. Escherichia coli strains

[0131] Example 3: Overcoming growth inhibition in 2'-FL and LNT production strains containing GlpK(A55T) by mgsA inactivation

[0132] To ensure that methylglyoxal toxicity would not be an issue during HMO production using E. coli strains carrying GlpK(A55T), the growth kinetics of different E. coli strains carrying GlpK(A55T) for 2'-FL or LNT production were recorded under different production conditions. The growth kinetics were compared between E. coli strains carrying the native mgsA gene and the progeny strains in which the mgsA gene was deleted.

[0133] Sudden exposure of E. coli strains containing feedback-resistant variants of GlpK to glycerol may result in inhibition of cell growth. In the fermentative production of HMOs, a bacterial preculture obtained from a seed fermenter is usually used to inoculate the main fermenter while the bacterial cells are still in the exponential growth phase. However, it may happen that the bacterial cell culture in the seed fermenter has reached the stationary growth phase before the main fermenter is inoculated. This results in the bacterial cells being exposed to the high glycerol concentration in the main fermenter at the time of inoculation, which in turn can lead to methylglyoxal toxicity.

[0134] To investigate whether this situation would lead to growth problems and affect the production of HMOs, E. coli strains carrying GlpK(A55T) and capable of synthesizing 2'-FL or LNT were tested using the following method. Fed-batch precultures were prepared to obtain the inoculum to be used for the production of HMOs in fed-batch operation mode, as described in Example 2. Figure 6 As shown, when the preculture originated from fed-batch culture, two E. coli strains 2′-FL-2 (▲) (Table 2; Figure 6 A) and LNT-2 (▲) ( Table 2 ; Figure 6 B) were unable to grow in the primary fermenter.

[0135] Subsequently, the experiment was repeated using the E. coli strains producing 2'-FL or producing LNT that additionally deleted the offspring of the mgsA gene. In order to inactivate the mgsA gene in the E. coli 2'-FL-2 strain and the E. coli LNT-2 strain, the same single-stranded DNA-based method described in Example 2 was used. The mgsA gene was functionally inactivated by inserting a stop codon and frameshift at position 43 using primer 10 (Table 1). The functional inactivation of the mgsA gene was confirmed using a combination of primers 11 and 13 (Table 1). The presence of the endogenous mgsA gene was confirmed using a combination of primers 12 and 13 (Table 1). After the colony PCR of mgsA verified the functional inactivation, single bacterial clones were selected and further characterized by nucleotide sequence analysis using primers 13 and 14 (Table 1).

[0136] Regarding the E. coli ΔmgsA variant of the 2′-FL-producing E. coli strain 2′-FL-2 (designated 2′-FL-3) ( Table 2 ; Figure 6 A ) and the E. coli ΔmgsA variant of E. coli strain LNT-2 (designated LNT-3) ( Table 2 ; Figure 6 B), When challenged with glycerol after inoculation from the stationary growth phase, cell growth was observed for both strains (■).

[0137] These results demonstrate the importance of the ΔmgsA genotype as a preventive measure and / or to further improve the robustness of bacterial HMO production strains to be able to cope with plant operation variability.

[0138] Example 4. Comparison of 2'-FL production in E. coli carrying GlpK(A55T) or GlpK(G231D)

[0139] The results shown in Examples 1 and 2 prompted us to investigate whether other variants of GlpK could also improve HMO production. To test this hypothesis, the 2'-FL productivity of E. coli strain 2'-FL-2 (Table 2) was compared with that of E. coli strain 2'-FL-4 (Table 2), which contains the glpK gene encoding the previously reported feedback-resistant version of GlpK (G231D) (Honisch, C. et al., (2004) Genome Res. 14: 2495-2502). E. coli strain 2'-FL-4 (Table 2) was constructed by replacing the native glpK gene with the glpK (G692A, C693T) gene in the same E. coli precursor strain used to construct E. coli strain 2'-FL-2 (Table 2). In order to improve the efficiency of the genetic engineering procedures adopted, the glpK gene mutations G692A and C693T were used in combination instead of using only the mutation G692A described in the literature. Both the nucleotide mutation G692A or the G692A mutation combined with C693T encode the target GlpK enzyme with the amino acid substitution G231D. The construction of the E. coli 2'-FL-4 strain (Table 2) followed the same method used to create the E. coli 2'-FL-2 strain (Table 2) in Example 2. Primer 15 (Table 1) was used for glpK gene modification. For clone selection, primer combinations 16 and 18 (Table 1) or primer pairs 17 and 18 (Table 1) were used to identify mutant glpK variants and native glpK genes, respectively. In order to confirm the desired modifications, primers 18 and 19 were used to sequence the glpK gene range (Table 1). Similar to E. coli 2'-FL-2 strain (Table 2), E. coli 2'-FL-4 (Table 2) was also cultured in a fed-batch to produce 2'-FL using the same culture conditions and analytical procedures as described in Example 2. The G231D mutation in the GlpK enzyme of E. coli strain 2'-FL-4 (·) (Table 2) had a higher positive effect on 2'-FL production ( Figure 4 B). The 2'-FL titer of E. coli 2'-FL-4 (·) was increased by 53% compared to the 2'-FL titer of the control strain (▲). In addition, a 34% reduction in batch processing time was achieved compared to the control strain (▲).

Claims

1. A genetically engineered microbial cell for intracellular biosynthesis of target oligosaccharides, wherein: When cultured in the presence of glycerol as the sole carbon source and energy source, the genetically engineered microbial cell synthesizes the target oligosaccharide within the cell, wherein the genetically engineered microbial cell comprises: - Glycerol permease for internalization of exogenous glycerol; - glycerol kinase, wherein the glycerol kinase is a functional variant of the Escherichia coli glycerol kinase GlpK, wherein the functional variant has an amino acid residue at the position corresponding to amino acid position 55 in the amino acid sequence of the Escherichia coli GlpK shown in SEQ ID NO: 1, wherein the amino acid residue comprises a non-ionizing but polar acting side chain, and / or has an amino acid residue at the position corresponding to amino acid position 231 in the amino acid sequence of the Escherichia coli GlpK shown in SEQ ID NO: 1, wherein the anionic amino acid residue comprises an anionic side chain; - metabolic pathways for the intracellular biosynthesis of nucleotide-activated monosaccharides as donor substrates for the monosaccharide moiety; and - a glycosyltransferase for transferring the monosaccharide moiety from the nucleotide activated monosaccharide to an acceptor substrate.

2. The genetically engineered microbial cell according to claim 1, wherein The amino acid residues having non-ionizing but polar acting side chains are selected from the group consisting of L-serine, L-threonine, L-cysteine, L-methionine, L-asparagine and L-glutamine.

3. The genetically engineered bacterial cell according to claim 1 or 2, wherein The anionic amino acid residue is selected from L-aspartic acid and L-glutamic acid.

4. The genetically engineered microbial cell according to any one of claims 1 to 3, wherein The functional variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with E. coli (K12) GlpK.

5. The genetically engineered microbial cell according to any one of claims 1 to 4, wherein The genetically engineered microbial cell has been genetically engineered to contain and express a recombinant gene encoding the functional variant of the E. coli GlpK.

6. The genetically engineered microbial cell according to claim 5, wherein (i) the endogenous glycerol kinase gene of the microbial cell is deleted or functionally inactivated, and wherein the microbial cell has been transformed to contain and express a recombinant gene encoding the functional variant of the E. coli GlpK; or (ii) the endogenous glycerol kinase gene of the microbial cell has been edited to contain and express a recombinant gene encoding the functional variant of the E. coli GlpK.

7. The genetically engineered microbial cell according to any one of claims 1 to 6, wherein The endogenous methylglyoxal synthase gene is deleted or functionally inactivated.

8. The genetically engineered microbial cell according to any one of claims 1 to 7, wherein The target oligosaccharide is an oligosaccharide selected from human milk oligosaccharides.

9. Use of the genetically engineered microbial cell according to any one of claims 1 to 8 for producing target oligosaccharides.

10. The use according to claim 9, wherein The target oligosaccharide is an oligosaccharide selected from human milk oligosaccharides.

11. A method for producing oligosaccharides of interest by fermentation, the method comprising: - Providing a genetically engineered microbial cell for intracellular biosynthesis of the target oligosaccharide according to any one of claims 1 to 8; - culturing the genetically engineered microbial cells in a culture solution containing glycerol as a carbon source and energy source for the genetically engineered microbial cells; as well as - Recovering the target oligosaccharide from the microbial cells and / or the culture solution.

12. The method according to claim 11, wherein: The glycerol is the only carbon source and energy source added exogenously to the culture medium.

13. The method according to claim 11 or 12, wherein: The target oligosaccharide is an oligosaccharide selected from human milk oligosaccharides.

14. A method for reducing the toxicity of methylglyoxal in microbial cells, wherein: The method comprises: -Providing genetically engineered microbial cells for fermentation production of oligosaccharides of interest; - Deletion or functional inactivation of the endogenous glycerol kinase gene of the microbial cell; - Transforming the microbial cell to contain and express a gene encoding a functional variant of the Escherichia coli (K12) glycerol kinase GlpK, wherein the functional variant has an amino acid residue at the position corresponding to amino acid position 55 in the amino acid sequence of the Escherichia coli GlpK shown in SEQ ID NO: 1, wherein the amino acid residue comprises a non-ionizing but polar acting side chain, and / or has an amino acid residue at the position corresponding to amino acid position 231 in the amino acid sequence of the Escherichia coli GlpK shown in SEQ ID NO: 1, wherein the anionic amino acid residue comprises an anionic side chain.

15. A method for reducing the responsiveness of microbial cells to methylglyoxal toxicity during the fermentation process for producing oligosaccharides of interest, wherein: The method comprises: -Providing genetically engineered microbial cells for fermentation production of oligosaccharides of interest; - Deletion or functional inactivation of the endogenous glycerol kinase gene of the microbial cell; - transforming the microbial cell to contain and express a gene encoding a functional variant of the Escherichia coli glycerol kinase GlpK, wherein the functional variant has an amino acid residue at the position corresponding to amino acid position 55 in the amino acid sequence of the Escherichia coli GlpK shown in SEQ ID NO: 1, wherein the amino acid residue comprises a non-ionizing but polar acting side chain, and / or has an amino acid residue at the position corresponding to amino acid position 231 in the amino acid sequence of the Escherichia coli GlpK shown in SEQ ID NO: 1, wherein the anionic amino acid residue comprises an anionic side chain.

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