Glycerol-based fermentative production of oligosaccharides by microbial cells

Abstract

Disclosed are methylglyoxal-resistant genetically engineered microbial cells for the fermentative production of oligosaccharides of interest, and methods for the fermentative production of oligosaccharides of interest using the methylglyoxal-resistant microbial cells.

Description

[Technical Field]

[0001] The present invention relates to the fermentative production of target oligosaccharides by microbial cells. More specifically, the present invention relates to the fermentative production of target oligosaccharides by microbial cells using glycerol as a carbon and energy source. [Background technology]

[0002] Among the oligosaccharides, human milk oligosaccharides (HMOs) have attracted considerable attention in recent years due to their beneficial health effects. For example, HMOs can prevent the adhesion of pathogenic microorganisms to mucosal cells of the gastrointestinal tract, thereby preventing the development of diseases. Furthermore, HMOs exert a prebiotic effect, i.e., promote the growth of non-pathogenic intestinal bacteria. Beneficial effects on the maturing immune and nervous systems have also been described.

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

[0004] Generally, industrial-scale biological processes, such as the fermentative production of oligosaccharides by / in microbial cells, include a production phase during which biosynthesis of the desired oligosaccharide occurs. The production phase is followed by a product recovery phase in which the desired oligosaccharide is recovered from the culture broth and / or the microbial cells. It is known that different substrates, such as glucose, sucrose, or glycerol, can be used as carbon and energy sources for the microbial cells in the production phase.

[0005] Glycerol, a carbon source for microbial cells, has recently become economically attractive for fermentation production processes. Because glycerol is a by-product of biodiesel production, the increase in biodiesel production as an alternative to petroleum-based fuels has coincided with an increase in the supply of glycerol on the market.

[0006] A microbial cell of particular interest for the fermentative production of oligosaccharides, particularly human milk oligosaccharides, is the Gram-negative bacterium Escherichia coli. Escherichia coli is a microorganism capable of metabolizing glycerol as a carbon and energy source. Catabolism in E. coli can occur aerobically or anaerobically. The glycerol catabolic pathway in E. coli is shown schematically in Figure 1. The primary aerobic pathway involves the internalization of glycerol by the glycerol uptake facilitator 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 catabolic pathway used by Escherichia coli (E. coli) in the absence of oxygen requires glycerol-3-phosphate dehydrogenase (encoded by the glpABC genes) in place of the aforementioned glycerol-3-phosphate dehydrogenase for the conversion of glycerol-3-phosphate to dihydroxyacetone phosphate. The alternative anaerobic glycerol pathway involves glycerol dehydrogenase (encoded by the gldA gene) and dihydroxyacetone kinase (encoded by the dhaKLM gene) after glycerol uptake by a glycerol uptake enhancer. In all of these metabolic pathways, dihydroxyacetone phosphate is produced as a key metabolite that enters central metabolism via the Embden-Meyerhof-Parnas (EMP) pathway.

[0007] Due to the location of the gldA gene in the E. coli genome, i.e., near the ptsA gene encoding multiphosphoryl transfer protein 2 and the fsaB gene encoding fructose-6-phosphate aldolase, and the higher affinity of the glycerol dehydrogenase GldA for dihydroxyacetone compared to glycerol, it has been suggested that the primary role of GldA is to convert dihydroxyacetone to glycerol to prevent E. coli cells from dihydroxyacetone toxicity. Furthermore, dihydroxyacetone can spontaneously convert to methylglyoxal, a highly toxic metabolite for E. coli that inhibits its growth at concentrations as low as 0.3 mM.

[0008] Therefore, it is desirable to make microbial cells for producing desired oligosaccharides less susceptible to the toxicity of metabolic products from the glycerol catabolic pathway when cultured in the presence of glycerol as a carbon and energy source. Summary of the Invention [Problem to be solved by the invention]

[0009] Surprisingly, it was found that Escherichia coli (E. coli) cells, which are capable of synthesizing human milk oligosaccharides intracellularly, have improved production of human milk oligosaccharides without being affected by methylglyoxal toxicity when they harbor specific mutants of glycerol kinase. [Means for solving the problem]

[0010] Thus, in a first aspect, there is provided a genetically engineered microbial cell for producing an oligosaccharide of interest, the genetically engineered microbial cell being capable of intracellularly synthesizing the oligosaccharide of interest when cultured in the presence of glycerol as a sole carbon source, the microbial cell possessing a glycerol permease (also known as a glycerol uptake facilitator) for internalization of exogenous glycerol. The microbial cell further possesses a functional variant of a naturally occurring Escherichia coli (E. coli) glycerol kinase GlpK, the amino acid sequence of which is set forth in SEQ ID NO: 1, wherein the functional variant has an amino acid residue at a position in its amino acid sequence corresponding to amino acid position 55 in the naturally occurring E. coli (E. coli) GlpK, the amino acid residue comprising a non-ionized but polar-acting side chain, and / or the functional variant possesses an amino acid comprising an anionic side chain at a position in its amino acid sequence corresponding to amino acid position 231 in the naturally occurring E. coli (E. coli) GlpK. The genetically engineered microbial cell further possesses a metabolic pathway for the intracellular biosynthesis of a nucleotide-activated monosaccharide comprising a nucleotide moiety and a monosaccharide moiety, the nucleotide-activated monosaccharide being a donor substrate for transfer of the monosaccharide moiety to an acceptor molecule. The genetically engineered microbial cells further possess glycosyltransferases for the transfer of monosaccharide moieties from nucleotide-activated monosaccharides to acceptor molecules for intracellular biosynthesis of the desired oligosaccharides.

[0011] In a second aspect, there is provided a use of the genetically engineered microbial cell according to the first aspect for producing an oligosaccharide of interest. This use thus includes the use of a genetically engineered microbial cell capable of intracellularly synthesizing the oligosaccharide of interest when cultured in the presence of glycerol as a sole carbon source, wherein the microbial cell possesses a glycerol permease (also known as a glycerol uptake facilitator) for internalization of exogenous glycerol. The microbial cell further comprises a functional variant of a naturally occurring Escherichia coli (E. coli) glycerol kinase GlpK, the amino acid sequence of which is set forth in SEQ ID NO: 1, wherein the functional variant comprises an amino acid residue at a position in the amino acid sequence corresponding to amino acid position 55 in the naturally occurring E. coli (E. coli) GlpK, the amino acid residue comprising a non-ionized but polar-acting side chain, and / or the functional variant comprises an amino acid comprising an anionic side chain at a position in the amino acid sequence corresponding to amino acid position 231 in the naturally occurring E. coli (E. coli) GlpK. The genetically engineered microbial cell further comprises a metabolic pathway for the 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 the transfer of the monosaccharide moiety from the nucleotide-activated monosaccharide to an acceptor molecule.

[0012] In a third aspect, there is provided a method for the fermentative production of an oligosaccharide of interest, the method comprising: A genetically engineered microbial cell according to a first aspect for producing a desired oligosaccharide, i.e., a genetically engineered microbial cell capable of intracellularly synthesizing a desired oligosaccharide when cultured in the presence of glycerol as a sole carbon source, comprising a glycerol permease, a functional variant of native Escherichia coli (E. coli) glycerol kinase GlpK, the amino acid sequence of which is set forth in SEQ ID NO: 1 (this functional variant has an amino acid sequence corresponding to amino acid position 55 in the amino acid sequence of native Escherichia coli (E. coli) GlpK). a genetically engineered bacterium having a nucleotide-activated monosaccharide moiety (wherein the nucleotide-activated monosaccharide moiety is a donor substrate for the monosaccharide moiety), a metabolic pathway for the intracellular biosynthesis of a nucleotide-activated monosaccharide, and a glycosyltransferase for the transfer of the monosaccharide moiety from the nucleotide-activated monosaccharide to an acceptor molecule; Culturing the genetically engineered microbial cells in a culture medium (containing glycerol as a carbon source for the genetically engineered microbial cells) and under conditions that allow the genetically engineered microbial cells to synthesize the desired oligosaccharides intracellularly; and Optionally, recovering the oligosaccharides of interest from the microbial cells and / or culture medium.

[0013] In a further aspect, there is provided a method for mitigating the toxicity of methylglyoxal to microbial cells when the microbial cells are cultured in the presence of glycerol as a carbon source, comprising transforming the microbial cells so that they harbor a functional variant of a naturally occurring Escherichia coli (E. coli) glycerol kinase GlpK, the amino acid sequence of which is set forth in SEQ ID NO: 1, wherein the functional variant has an amino acid residue at a position in its amino acid sequence corresponding to amino acid position 55 in the amino acid sequence of naturally occurring E. coli (E. coli) GlpK, wherein the amino acid residue comprises a non-ionized but polar-acting side chain, and / or the functional variant has an amino acid comprising an anionic side chain at a position in its amino acid sequence corresponding to amino acid position 231 in the amino acid sequence of naturally occurring E. coli (E. coli) GlpK.

[0014] In a further aspect, there is provided a method for reducing the sensitivity of a microbial cell to methylglyoxal toxicity during the fermentative production of an oligosaccharide of interest by the microbial cell when cultured in the presence of exogenous glycerol as a carbon source to produce the oligosaccharide of interest, the method comprising transforming the microbial cell so that it harbors a functional variant of a naturally occurring Escherichia coli (E. coli) glycerol kinase GlpK, the amino acid sequence of which is set forth in SEQ ID NO: 1, wherein the functional variant has an amino acid residue at a position in its amino acid sequence corresponding to amino acid position 55 in the amino acid sequence of naturally occurring E. coli GlpK, the amino acid residue comprising a non-ionized but polar-acting side chain, and / or the functional variant has an amino acid comprising an anionic side chain at a position in its amino acid sequence corresponding to amino acid position 231 in the amino acid sequence of naturally occurring E. coli GlpK. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 shows the catabolic pathway of glycerol in Escherichia coli (E. coli). [Figure 2-1]FIG. 2 shows 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. [Figure 2-2] FIG. 2 shows 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. [Figure 3] Figure 3 shows graphs comparing the 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 the progeny of this control strain containing the functional mutant GlpK(A55T) instead of the native E. coli (K12) GlpK. [Figure 4] Figure 4 shows graphs comparing the growth (A) and 2'-FL production (B) of a 2'-FL-producing E. coli strain containing the native E. coli GlpK enzyme (control), a progeny of this control strain containing the functional variant GlpK(A55T) of native E. coli GlpK instead of native E. coli GlpK, and another progeny of the control strain containing the functional variant GlpK(G231D) of native E. coli GlpK instead of native E. coli GlpK. [Figure 5] Figure 5 shows graphs comparing the growth (A) and LNT production (B) of an LNT-producing E. coli strain containing the native E. coli GlpK enzyme (control) and the progeny of this control strain containing the native E. coli functional mutant GlpK(A55T) instead of the native E. coli GlpK. [Figure 6]Figure 6 shows graphs comparing the growth profiles of Escherichia coli (E. coli) strains producing 2'-FL (A) or LNT (B), comparing the growth profiles of these strains with the endogenous mgsA gene with the growth profiles of their respective progeny strains lacking the endogenous mgsA gene. DETAILED DESCRIPTION OF THE INVENTION

[0016] In a first aspect, genetically engineered microbial cells are provided for the fermentative production of oligosaccharides of interest.

[0017] As used herein, the term "microbial cell" refers to a unicellular organism. Microbial cells may be prokaryotic or eukaryotic. Suitable prokaryotic cells include bacterial and archaeal cells. Suitable eukaryotic cells include yeast and fungal cells.

[0018] In some embodiments, the prokaryotic cell is a bacterial cell, such as a Bacillus, Bifidobacterium, Citrobacter, Clostridium, Corynebacterium, Enterococcus, Erwinia, Escherichia, Lactobacillus, Lactococcus, Micrococcus, or any of the following: The bacterial cell is a bacterial cell of a genus selected from the group consisting of Micromonospora, Pantoea, Pectobacterium, Proprionibacterium, Pseudomonas, Rhodococcus, Sporolactobacillus, Streptococcus, and Xanthomonas. Suitable bacterial species within the genera include Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, Bacillus circulans, Bifidobacterium longum, Bacillus spp. ... longum, Bifidobacterium infantis, Bifidobacterium bifidumbifidum, Citrobacter freundii, Clostridium cellulolyticum, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium acetobutylicum, Corynebacterium glutamicum, Enterococcus faecium, Enterococcus thermophiles, Erwinia herbicola (Pantoea agglomerans), Escherichia coli coli), Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri (Lactobacillus casei), Lactobacillus reuteri reuteri, Lactobacillus jensenii, Lactococcus lactis, Pantoea citreacitrea, Pectobacterium carotovorum, Proprionibacterium freudenreichii, Pseudomonas fluorescens, Pseudomonas aeruginosa, Streptococcus thermophiles, and Xanthomonas campestris. In another embodiment, the microbial cell is an Escherichia coli cell.

[0019] In some embodiments, the eukaryotic cell is a yeast cell. Suitable yeast cells can be selected from a genera selected from the group consisting of Saccharomyces sp., Saccharomycopsis sp., Pichia sp., Hanensula sp., Kluyveromyces sp., Yarrowia sp., Rhodotorula sp., and Schizosaccharomyces sp. Additionally and / or alternatively, the yeast cell is a Saccharomyces cerevisiae cell, a Pichia pastoris cell, or a Hanensula polymorpha cell.

[0020] The microbial cell is a microbial cell for producing an oligosaccharide of interest. As used herein with respect to producing an oligosaccharide of interest, the term "for producing" means that the microbial cell is capable of synthesizing the oligosaccharide of interest intracellularly when the microbial cell is cultured in a medium and under conditions that allow for the intracellular synthesis of the oligosaccharide of interest.

[0021] The term "oligosaccharide," as used herein, refers to a sugar molecule consisting of at least three monosaccharide moieties, but not more than 20 monosaccharide moieties, preferably not more than 12 monosaccharide moieties, and more preferably not more than 10 monosaccharide moieties, each of which is linked to at least one other monosaccharide moiety by a glycosidic bond. Oligosaccharides of interest may consist of linear chains of monosaccharide moieties or branched chains of monosaccharide moieties.

[0022] The monosaccharide portion of the oligosaccharide of interest can be selected from the group consisting of aldoses (e.g., arabinose, xylose, ribose, desoxyribose, lyxose, glucose, idose, galactose, talose, allose, altrose, mannose), ketoses (e.g., ribulose, xylulose, fructose, sorbose, tagatose), deoxysugars (e.g., rhamnose, fucose, quinovose), deoxy-aminosugars (e.g., N-acetylglucosamine, N-acetyl-mannosamine, N-acetyl-galactosamine), uronic acids (e.g., galacturonic acid, glucuronic acid), and ketoaldonic acids (e.g., N-acetylneuraminic acid).

[0023] As used herein, the term "oligosaccharide of interest" refers to an oligosaccharide that is expected to be produced by a genetically engineered microbial cell. Typically, the microbial cell has been selected and / or genetically engineered to synthesize a specific oligosaccharide, i.e., the oligosaccharide of interest, intracellularly. Any other oligosaccharide other than the oligosaccharide of interest that may be further synthesized by the microbial cell is also considered to be a by-product, regardless of whether the other oligosaccharide is a reaction product of an intermediate reaction in the biosynthesis of the oligosaccharide of interest, constitutes a product of another metabolic pathway other than the metabolic pathway for the biosynthesis of the oligosaccharide of interest, or is an unwanted by-product due to the promiscuity of the specificity of the enzymes involved in the metabolic pathway for the synthesis of the oligosaccharide of interest.

[0024] Typically, the oligosaccharide of interest does not naturally occur in the native cells of the microbial species selected to be engineered to produce the oligosaccharide of interest. Thus, an engineered microbial cell is a microbial cell that has been engineered to be capable of producing the oligosaccharide of interest.

[0025] 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 constitute a diverse mixture of oligosaccharides that are not digested by humans. Human milk is unique in terms of the composition and amount of its oligosaccharides. To date, over 150 structurally distinct HMOs have been identified. Most HMOs are characterized by a lactose moiety (Gal-β1,4-Glc) at their reducing end. HMOs contain at least one fucose moiety, at least one sialic acid moiety, and / or at least one N-acetylglucosaminyl moiety. More generally, the monosaccharides that make up HMOs are selected from the group consisting of D-glucose, D-galactose, N-acetyl-D-glucosamine, L-fucose, and N-acetylneuraminic acid.

[0026] Additionally and / or alternatively, the oligosaccharide of interest may be 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 ... Copentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP-V), lacto-N-neofucopentaose V (LNnFP-V), lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para-lacto-N-hexaose (paraLNH), para-lacto-N-neohexaose (paraLNnH), difucosyl-lacto-N-neohexaose (DF-LNnH), lacto-N-difucosylhexaose I, la Fucosyl-lacto-N-difucosylhexaose II, para-lacto-N-fucosylhexaose (paraLNH), fucosyl-lacto-N-sialylpentaose a (F-LST-a), fucosyl-lacto-N-sialylpentaose b (F-LST-b), fucosyl-lacto-N-sialylpentaose c (F-LST-c), fucosyl-lacto-N-sialylpentaose c, disialyl-lacto-N-fucopentaose, 3-fucosyl-3'-sialyllactose (3F-3'-SL) ), 3-fucosyl-6'-sialyllactose (3F-6'-SL), lacto-N-neodifucohexaose I, 3'-sialyllactose (3-SL), 6'-sialyllactose (6-SL), sialyllactose-N-tetraose a (LST-a), sialyllactose-N-tetraose b (LST-b), sialyllactose-N-tetraose c (LST-c), disialyl-lacto-N-tetraose (DS-LNT), disialyl-lacto-N-fucopentaose (DS-LNFP V), lacto-N-neodifucohexaose(hxaose) I (LNnDFH I), 3'-galactosyllactose (3'-GL), and 6'-galactosyllactose (6'-GL).

[0027] Thus, a genetically engineered microbial cell is a microbial cell that has been genetically engineered to possess metabolic pathway(s) for the intracellular biosynthesis of an oligosaccharide of interest, and thus is capable of synthesizing the oligosaccharide of interest intracellularly.

[0028] As used herein, the terms "capable" and "capable" with respect to biosynthesis of an oligosaccharide of interest by a genetically engineered microbial cell mean that the genetically engineered microbial cell synthesizes the oligosaccharide of interest intracellularly when the microbial cell is cultured in a medium and under conditions (e.g., with respect to temperature, pH, osmolarity, nutrient composition) that permit the intracellular biosynthesis of the oligosaccharide of interest.

[0029] When genetically engineered microbial cells are cultured in the presence of exogenous glycerol as the sole carbon / energy source, they can synthesize desired oligosaccharides intracellularly. Therefore, the genetically engineered microbial cells possess a glycerol permease for the internalization of exogenous glycerol. Glycerol permease, also known as a glycerol uptake facilitator, internalizes exogenously supplied glycerol.

[0030] In some embodiments, the glycerol permease is an endogenous glycerol permease of the microbial cell, i.e., a glycerol permease that is naturally present in naturally occurring cells of the same species to which the engineered microbial cell belongs. In further and / or alternative embodiments, the glycerol permease is a heterologous glycerol permease, i.e., a glycerol permease that is not naturally present in naturally occurring cells of the microbial species to which the engineered microbial cell belongs.

[0031] Additionally and / or alternatively, the genetically engineered microbial cell is genetically engineered to possess a glycerol permease or to possess increased glycerol permease activity compared to a progenitor cell that has not been genetically engineered to possess increased glycerol permease activity. When the microbial cell is genetically engineered to possess a glycerol permease or increased glycerol permease activity, the glycerol permease is expressed from an exogenous glycerol permease-encoding gene inserted into the microbial cell and / or by genetic modification of one or more endogenous glycerol permease-encoding genes of the microbial cell. In some of these embodiments, a progenitor cell of the genetically engineered microbial cell contains an exogenous glycerol permease-encoding gene and is genetically engineered to express the glycerol permease. In some embodiments, the exogenous glycerol permease-encoding gene is integrated into one or more chromosomes of the microbial cell or into at least one of the chromosomes. Additionally and / or alternatively, the exogenous glycerol permease-encoding gene is present on an episomal nucleic acid molecule within the microbial cell. In some embodiments, the glycerol permease-encoding gene is a recombinant gene.

[0032] One example of a suitable glycerol permease is the glycerol uptake facilitator protein GlpF of Escherichia coli (K-12 strain), encoded by the E. coli glpF gene. The deduced amino acid sequence of E. coli K-12 GlpF is disclosed in the UniProt Knowledge Base (www.uniprot.org), Release 2022_01, entry number P0AER0, as of February 23, 2022. Thus, in some embodiments, the engineered microbial cell contains E. coli GlpF or a functional variant thereof. Additionally and / or alternatively, the microbial cell is engineered to contain the E. coli K-12 glycerol permease GlpF or a functional variant thereof. Thus, in some embodiments, the microbial cell is a cell that has been genetically engineered to harbor a recombinant gene that includes and expresses a nucleotide sequence encoding Escherichia coli (E. coli) GlpF or a functional variant thereof.

[0033] As used herein, the term "variant(s)" refers to a polynucleotide or polypeptide whose nucleotide or amino acid sequence differs from that of a reference polynucleotide or polypeptide, respectively, but retains essential properties (e.g., catalysis) of the reference polynucleotide or polypeptide. A typical variant of a polynucleotide differs in nucleotide sequence from another polynucleotide, the reference polynucleotide. Differences in the nucleotide sequence of a variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide deviations may result in amino acid substitutions, additions, deletions, fusions, and / or truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, or deletions, in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0034] Within the scope of the present invention, the terms also include nucleic acid / polynucleotide and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that possess a nucleotide or amino acid sequence with greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference sequence, where the sequence identity covers a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides or amino acids compared to the reference sequence.

[0035] A "variant" of any gene / polypeptide is meant to refer to a sequence variant of the gene / protein that retains the same activity, lesser or greater activity, or a different activity with respect to the substrate specificity of the reference polypeptide, or a different activity with respect to the reaction specificity of the reference polypeptide, as the gene, polypeptide, and / or polypeptide encoded by the gene.

[0036] As used herein, the term "operon" refers to a nucleotide sequence that includes two or more protein-coding sequences that are transcribed from the same regulatory elements to mediate and / or control expression in a microbial cell.

[0037] As used herein, the term "functional gene" refers to a nucleic acid molecule that includes a nucleotide sequence that encodes a protein or polypeptide and also contains regulatory sequences operably linked to the protein-encoding nucleotide sequence (open reading frame) such that the nucleotide sequence encoding the protein or polypeptide can be expressed in / by a microbial cell harboring the functional gene. Thus, when cultured under conditions that allow expression of the functional gene, the gene is expressed, and microbial cells that express the functional gene typically contain the protein or polypeptide encoded by the protein-coding region of the functional gene. As used herein, the terms "nucleic acid" and "polynucleotide" refer to deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form and, unless otherwise limited, encompass known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.

[0038] The term "deregulated" as used herein with respect to expression of an endogenous gene refers to altered expression of the protein coding region of an endogenous gene(s) compared to the expression of the gene(s) in a natural precursor cell of a genetically engineered microbial cell that has not been genetically engineered to alter expression of the gene(s). The term "deregulated" includes increased expression of a gene as well as decreased or impaired expression of a gene. Gene expression can be deregulated by different means known to those skilled in the art. Examples of deregulated expression of a gene include altering the native nucleotide sequence of the gene's promoter or altering the native nucleotide sequence of the gene's ribosome binding site.

[0039] As used herein, the term "operably linked" refers to a functional linkage between an expression-controlling nucleotide sequence (such as a promoter, signal sequence, or transcription factor binding site array) and a second nucleotide sequence, where the expression-controlling nucleotide sequence affects the transcription and / or translation of the second nucleotide sequence. Thus, the term "promoter" refers to a nucleotide sequence that typically "precedes" a protein-coding nucleotide sequence in a DNA polynucleotide and provides a site for initiation of transcription into mRNA. "Regulatory" DNA sequences are also typically "upstream" (i.e., preceding) the protein-coding nucleotide sequence of a gene in a given DNA polymer and bind proteins that determine the frequency (or rate) of transcription initiation. These sequences preceding a selected gene (or set of genes) in a functional DNA polymer, collectively referred to as "promoter / regulator" or "control" DNA sequences, cooperate to determine whether transcription (and ultimately expression) of the gene occurs. Nucleotide sequences that "follow" a protein-coding nucleotide sequence in a DNA polymer and provide a signal for termination of transcription into mRNA are referred to as transcription "terminator" sequences.

[0040] The term "recombinant" as used herein indicates that a polynucleotide, such as a gene or operon, has been produced by genetic engineering. Thus, a recombinant gene, operon, or polynucleotide does not naturally occur in microbial cells of the same species as the microbial cell used to produce the desired oligosaccharide. A recombinant gene or operon contains nucleotide sequences derived from at least two different ancestors, e.g., different genetic loci or different species. An example is the protein coding region of a glycerol permease-encoding gene operably linked to the promoter of a different gene. A recombinant polynucleotide may contain a heterologous nucleotide sequence or express a polypeptide (i.e., a nucleotide sequence foreign to the microbial cell) encoded by a heterologous nucleotide sequence. A recombinant microbial cell may contain a gene not found in its native (non-recombinant) precursor. A recombinant cell may also contain a variant of a gene found in the recombinant cell's native precursor that has been modified and reintroduced into the cell by technical means. The term "recombinant" also encompasses microbial cells that contain a nucleotide sequence endogenous to the bacterial cell and that have been modified without removing the nucleic acid molecule containing the nucleotide sequence from the bacterial cell. Such modifications include those obtained by gene replacement, site-directed mutagenesis, and related techniques. Thus, a "recombinant polypeptide" is one produced by a recombinant cell.

[0041] As used herein, a "heterologous nucleotide sequence" or "heterologous nucleic acid" refers to a sequence derived from a source foreign to a particular host cell (e.g., derived from a different species) or, if derived from the same source, modified from its original form. Thus, a heterologous nucleotide sequence can be a nucleotide sequence in which a heterologous protein-encoding nucleotide sequence is operably linked to a promoter, where the protein-encoding nucleotide sequence and the promoter nucleotide sequence are obtained from different source organisms, or, if derived from the same source organism, either the protein-encoding nucleotide sequence or the promoter has been modified from its original form. A heterologous nucleotide sequence can be stably introduced into the genome of a bacterial cell by, for example, transposition, transfection, transformation, conjugation, or transduction. The applicable technique depends 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). Thus, a "genetically engineered microbial cell" is understood as a microbial cell that has been transformed or transfected, or is capable of being transformed or transfected with an exogenous polynucleotide sequence. Thus, the nucleotide sequence used in the present invention may be, for example, contained in a vector that is stably transformed / transfected or otherwise introduced into the host microbial cell. A wide variety of vectors can be used to obtain polypeptides synthesized in microbial cells. Such vectors include, inter alia, chromosomal, episomal, and virally derived vectors, such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, vectors derived from viruses, and vectors derived from combinations thereof, such as vectors derived from genetic elements of plasmids and bacteriophages, e.g., cosmids and phagemids. Expression system constructs may contain control regions that regulate and generate expression.Generally, any system or vector suitable for maintaining, propagating, or expressing polynucleotides and synthesizing polypeptides in bacterial host cells can be used for expression in this regard. The appropriate nucleotide sequence can be inserted into the expression system by any of a variety of well-known, routine techniques, such as those described in Sambrook et al., supra. 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.

[0042] The genetically engineered microbial cells contain glycerol kinase, an enzyme that catalyzes the phosphorylation of glycerol using adenosine triphosphate (ATP) to produce sn-glycerol 3-phosphate. More specifically, the microbial cells possess a glycerol kinase that is a functional variant of the Escherichia coli (E. coli) K-12 glycerol kinase GlpK.

[0043] Glycerol kinase from Escherichia coli (K12 strain) 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 set forth in SEQ ID NO:2 in the attached sequence listing. The deduced amino acid sequence of E. coli K-12 GlpK is disclosed in the UniProt Knowledge Base (www.uniprot.org), release 2022_01 as of February 23, 2022: entry number P0A6F3. Additionally or alternatively, the amino acid sequence of native E. coli K12 glycerol kinase GlpK is also set forth in SEQ ID NO:1 in the sequence listing. FIG. 2 shows the nucleotide sequence of the protein coding region of Escherichia coli (E. coli) (K12) glpK and the amino acid sequence of Escherichia coli (E. coli) (K12) GlpK deduced from the nucleotide sequence.

[0044] In certain embodiments, a functional variant of the E. coli (K12) glycerol kinase GlpK comprises an amino acid residue comprising a non-ionizable but polar-acting side chain within its amino acid sequence at a position corresponding to amino acid position 55 in the amino acid sequence of E. coli (K12) GlpK set forth in SEQ ID NO:1, and / or the variant comprises an amino acid residue comprising an anionic side chain at a position corresponding to amino acid position 231 in the amino acid sequence of E. coli (K12) GlpK set forth in SEQ ID NO:1.

[0045] Thus, the microbial cell comprises a glycerol kinase that is a functional variant of the Escherichia coli (E. coli) glycerol kinase GlpK, wherein the functional variant possesses an amino acid residue that contains a non-ionized (also called uncharged) but polar-acting side chain at a position corresponding to amino acid position 55 in the amino acid sequence of Escherichia coli (E. coli) GlpK set forth in SEQ ID NO:1.

[0046] Amino acids with non-ionizable but polar side chains belong to the group of amino acids with side chains bearing a functional group containing at least one atom with an electron pair available for hydrogen bonding to water.

[0047] In some embodiments, amino acid residues that contain non-ionizable but polar-acting side chains are hydrophilic amino acids.

[0048] Additionally and / or alternatively, the amino acid containing a non-ionizable but polar-acting side chain is an amide amino acid, preferably selected from the group consisting of L-asparagine and L-glutamine.

[0049] Additionally and / or alternatively, the amino acid containing a non-ionizable but polar side chain is a sulfur-containing amino acid, preferably selected from the group consisting of L-serine, L-threonine, L-cysteine, and L-methionine.

[0050] Additionally and / or alternatively, the amino acid residue containing a non-ionizable but polar-acting side chain is an amino acid selected from the group of amino acids consisting of L-serine, L-threonine, L-cysteine, L-methionine, L-asparagine and L-glutamine.

[0051] Additionally and / or alternatively, the microbial cell comprises a glycerol kinase that is a functional variant of the Escherichia coli (E. coli) (K12) glycerol kinase GlpK, wherein the functional variant possesses an amino acid residue comprising an anionic side chain at a position corresponding to amino acid 231 in the amino acid sequence of E. coli GlpK set forth in SEQ ID NO: 1. The amino acid residue comprising an anionic side chain is preferably selected from the group consisting of the amino acids L-aspartic acid and L-glutamic acid.

[0052] To harbor any one of the functional variants of Escherichia coli (E. coli) (K12) glycerol kinase GlpK, a 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. Thus, the genetically engineered microbial cell contains an exogenous functional gene that encodes and expresses a functional variant of Escherichia coli (E. coli) (K12) glycerol kinase GlpK.

[0053] In some embodiments, the exogenous gene encoding the functional variant of Escherichia coli (E. coli) (K12) GlpK is integrated into a 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 (E. coli) (K12) glycerol kinase GlpK is contained in an episomal nucleic acid molecule present in the genetically engineered microbial cell.

[0054] In some embodiments, the microbial cell is 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 Escherichia coli (E. coli) (K12) GlpK.

[0055] The genetically engineered microbial cell possesses a glycerol kinase that is one of the functional variants of Escherichia coli (E. coli) K-12 glycerol kinase GlpK described herein. The microbial cell does not possess a native or endogenous glycerol kinase that is not one of the functional variants of Escherichia coli (E. coli) (K12) glycerol kinase GlpK and that possesses higher enzymatic activity than any one of the functional variants of Escherichia coli (E. coli) (K12) glycerol kinase GlpK described herein. Thus, in some embodiments, the genetically engineered microbial cell comprises a deletion or functional inactivation of its endogenous gene(s) encoding glycerol kinase.

[0056] Functional inactivation can be the deletion and / or inactivation of one or more expression control sequences of the native glycerol kinase gene(s) of the microbial cell, such that expression of the native glycerol kinase gene(s) of the microbial cell is abolished. Additionally or alternatively, the functional inactivation can be the deletion or alteration of the protein coding region of the native glycerol kinase gene(s) of the microbial cell, for example, by introducing a frameshift mutation, a mutation that impairs translation initiation, a mutation that introduces a stop codon, or by altering the amino acid sequence of a polypeptide encoded by an altered nucleotide sequence such that the resulting polypeptide does not possess glycerol kinase activity.

[0057] In some embodiments, the genetically engineered microbial cell comprises a functional exogenous gene encoding a functional variant of the Escherichia coli (E. coli) (K12) glycerol kinase GlpK disclosed herein in addition to a deletion or functional inactivation of the microbial cell's endogenous glycerol kinase gene(s).

[0058] In some embodiments, the genetically engineered microbial cell is genetically engineered such that the protein coding region of its native glycerol kinase gene is altered to encode a functional variant of Escherichia coli (E. coli) (K12) GlpK. When the genetically engineered microbial cell is an Escherichia coli (E. coli) cell, alteration of the protein coding region of the native or endogenous glycerol kinase gene of the microbial cell is one option.

[0059] In some embodiments, the microbial cell is a genetically engineered microbial cell that has been genetically engineered in that an exogenous gene encoding a functional variant of Escherichia coli (E. coli) (K12) glycerol kinase has replaced one or more native / endogenous glycerol kinase genes of the microbial cell. Replacing the protein coding region of the native or endogenous glycerol kinase gene of the microbial cell is an alternative to modifying the protein coding region of the native or endogenous glycerol kinase-encoding gene of the microbial cell when the genetically engineered microbial cell is an Escherichia coli (E. coli) cell.

[0060] The genetically engineered microbial cell further comprises a metabolic pathway for the intracellular biosynthesis of the desired oligosaccharide, and thus further comprises a metabolic pathway for the intracellular biosynthesis of a nucleotide-activated monosaccharide as a donor substrate for its monosaccharide moiety for transfer to an acceptor molecule, all the enzymes and transporters necessary to provide the acceptor molecule for obtaining the monosaccharide moiety, and a glycosyltransferase for the transfer of the monosaccharide moiety from the nucleotide-activated monosaccharide to the acceptor molecule.

[0061] The genetically engineered microbial cells contain metabolic pathways for the intracellular biosynthesis of nucleotide-activated monosaccharides, which serve as donor substrates for their monosaccharide moieties, and glycosyltransferases that transfer the monosaccharide moiety from the donor substrate to an acceptor molecule.

[0062] In some embodiments, the nucleotide-activated monosaccharide is selected from the group consisting of 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).

[0063] Nucleotide-activated monosaccharides can be synthesized by the genetically engineered microbial cells via a de novo pathway. Additionally and / or alternatively, the microbial cells can use a salvage pathway to provide the nucleotide-activated monosaccharides.

[0064] In embodiments in which the nucleotide-activated monosaccharide is synthesized by a de novo pathway, the microbial cell possesses an enzyme required for the de novo biosynthetic pathway of the nucleotide-activated monosaccharide. In some embodiments, the microbial cell has been genetically engineered to possess at least one exogenous gene encoding an enzyme required for the de novo biosynthetic pathway of the nucleotide-activated monosaccharide.

[0065] At least one gene encoding an enzyme required for the de novo biosynthetic pathway of a nucleotide-activated monosaccharide may be endogenous to the microbial cell or may be introduced into the microbial cell from an exogenous source for expression. Expression, i.e., functional transcription and translation, of the protein encoded by the endogenous gene can be modified by genetically engineering the microbial cell. For example, altering the expression of an endogenous gene can be achieved by modifying the gene's transcription promoter, by modifying the gene's ribosome binding site, and / or by modifying the codon usage of the gene's protein-coding nucleotide sequence. Furthermore, the gene's protein-coding nucleotide sequence can be modified so that the activity and / or specificity of the enzyme encoded by the gene is favorable for the desired biosynthetic pathway. Those skilled in the art will know which gene(s) need to be expressed in a genetically modified organism for the de novo synthesis of a nucleotide-activated sugar donor molecule.

[0066] In embodiments in which the salvage pathway is used for the biosynthesis of a nucleotide-activated monosaccharide as a donor substrate, the microbial cell contains an enzyme that catalyzes the coupling of a nucleotide with a monosaccharide. An example of such an enzyme is the bifunctional fucokinase / L-fucose-1-phosphate-guanylyltransferase 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 coupling of a nucleotide with a monosaccharide is the N-acetylneuraminic acid cytidylyltransferase NeuA from Neisseria meningitidis, which forms CMP-neuraminic acid.

[0067] In some embodiments, the microbial cells harboring a salvage pathway for providing a nucleotide-activated monosaccharide as a donor substrate contain exogenous genes that encode and express enzymes that catalyze the coupling of a nucleotide with a monosaccharide.

[0068] Monosaccharides used as substrates for the formation of nucleotide-activated monosaccharides in salvage pathways can be internalized by microbial cells using 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 (E. coli). Monosaccharide import proteins can be encoded by and expressed from endogenous genes or encoded by and expressed from exogenous genes.

[0069] For the biosynthesis of a desired oligosaccharide, the acceptor molecule is a sugar, i.e., an acceptor sugar. The acceptor molecule can be selected from the group consisting of a monosaccharide, a disaccharide, and an oligosaccharide. In some embodiments, the acceptor sugar is a disaccharide, which is converted to a trisaccharide. In some embodiments, the acceptor sugar is a trisaccharide, which is converted to a tetrasaccharide. In some embodiments, the acceptor sugar is a tetrasaccharide, which is converted to a pentasaccharide. In some embodiments, the acceptor sugar is a pentasaccharide, which is converted to a hexasaccharide.

[0070] In some embodiments, the acceptor sugar or a precursor of the acceptor sugar is internalized by the microbial cells, preferably by utilizing a specific transporter present in the microbial cell's plasma membrane. In a process for producing an oligosaccharide of interest using engineered microbial cells, the engineered microbial cells are cultured in the presence of the acceptor sugar or a precursor of the acceptor sugar. The acceptor sugar or a precursor thereof present in the culture medium is internalized by the engineered microbial cells and utilized in the biosynthesis of the oligosaccharide of interest.

[0071] Additionally and / or alternatively, the acceptor sugar is synthesized intracellularly by the genetically engineered microbial cell.

[0072] In some embodiments, an acceptor sugar is synthesized intracellularly by a microbial cell from a monosaccharide that is internalized by the microbial cell and extended with a monosaccharide moiety to yield a disaccharide, which can be further extended by the addition of additional monosaccharide moieties to synthesize an oligosaccharide consisting of three, four, five, six, or more monosaccharide moieties intracellularly, which then constitutes an acceptor sugar for biosynthesis of a desired oligosaccharide. For example, a microbial cell may internalize glucose and convert it to lactose by the addition of a galactose moiety. The lactose can then be converted to an oligosaccharide, such as, for example, 2'-FL, 3-FL, LNT-II, LNT, LNnT, 3'-SL, or 6'-SL. Each of the oligosaccharides can be an oligosaccharide of interest or can constitute an acceptor sugar.

[0073] In some embodiments, the microbial cells are genetically engineered to harbor an enzyme that catalyzes the conversion of an acceptor sugar, i.e., a disaccharide or oligosaccharide, to a desired oligosaccharide by transferring a monosaccharide moiety from a donor substrate to the acceptor sugar. Thus, the microbial cells contain and express at least one exogenous, homologous, or heterologous gene encoding an enzyme that catalyzes the conversion of the acceptor sugar to a desired oligosaccharide.

[0074] A gene or equivalent functional nucleotide sequence encoding the enzymatic ability of a microbial cell to catalyze the conversion of an acceptor sugar to a desired oligosaccharide, i.e., add a monosaccharide moiety from a donor substrate to the acceptor sugar, can be a homologous nucleotide sequence or a heterologous nucleotide sequence. As used herein with respect to a nucleotide sequence, the term "homologous" refers to a nucleotide sequence that is native to the species to which the microbial cell for producing the desired oligosaccharide belongs. As used herein with respect to a nucleotide sequence, the term "heterologous" refers to an artificially produced nucleotide sequence or a nucleotide sequence that is derived from a species other than that to which the microbial cell for producing the desired oligosaccharide belongs. A heterologous nucleotide sequence derived from a species other than that of the microbial cell for producing the desired oligosaccharide can originate from a plant, an animal, including a human, a bacterium, an archaea, a fungus, or a virus.

[0075] Enzymes that catalyze the transfer of a monosaccharide moiety to an acceptor molecule are glycosyltransferases or transglycosidases. While transglycosidases catalyze the transfer of a monosaccharide moiety from one sugar to another, glycosyltransferases catalyze the transfer of a monosaccharide moiety from a nucleotide-activated sugar as a donor substrate to an acceptor sugar. The glycosyltransferase may be selected from the group consisting of galactosyltransferase, glucosyltransferase, fucosyltransferase, sialyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, glucuronosyltransferase, 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, α-1,4-fucosyltransferase, α-2,3-sialyltransferase, α-2,6-sialyltransferase. erase), α-2,8-sialyltransferase, β-1,3-N-acetylglucosaminyltransferase, β-1,4-N-acetylglucosaminyltransferase, α-1,3-N-acetyl-galactosaminyltransferase, β-1,3-N-acetyl-galactosaminyltransferase, β-1,4-N-acetyl-galactosaminyltransferase, α-1,2-mannosyltransferase, and β-1,4-xylosyltransferase.

[0076] It is understood that a specific glycosyltransferase catalyzes the transfer of the respective monosaccharide moiety from a nucleotide-activated monosaccharide to an acceptor molecule.

[0077] Thus, some genetically engineered microbial cells possess metabolic pathways to provide GDP-Fuc, as well as fucosylated oligosaccharides of interest, such as 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), and difucosyl-lacto-N-neohexaose (DF-LNnH). , lacto-N-difucosylhexaose I, lacto-N-difucosylhexaose II, para-lacto-N-fucosylhexaose (paraLNH), fucosyl-lacto-N-sialylpentaose a (F-LST-a), fucosyl-lacto-N-sialylpentaose b (F-LST-b), fucosyl-lacto-N-sialylpentaose c (F-LST-c), fucosyl-lacto-N-sialylpentaose c, disialyl-lacto-N-fucopentaose, 3-fucosyl-3'-sialyllactose (3F-3'-SL), 3-fucosyl-6'-sialyllactose (3F-6'-SL), lacto-N-neodifucohexaose I, disialyl-lacto-N-fucopentaose (DS-LNFP) It further possesses fucosyltransferases for the biosynthesis of N-neodifucohexaose I (LNnDFH I) or lacto-N-neodifucohexaose I (LNnDFH I).

[0078] Some genetically engineered microbial cells possess metabolic pathways to provide CMP-NeuNAc, as well as sialylated oligosaccharides of interest, such as 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), sialyllactose-N-tetraose a (LST-a), sialyllactose-N-tetraose b (LST-b), sialyllactose-N-tetraose c (LST-c), disialyllacto-N-tetraose (DS-LNT), fucosyl-lacto-N-sialylpentaenoate (FNT), and sialylated saccharides of interest. The fucosyl lacto-N-sialylpentaose further possesses sialyltransferases for the biosynthesis of fucosyl-lacto-N-sialylpentaose a (F-LST-a), fucosyl-lacto-N-sialylpentaose b (F-LST-b), fucosyl-lacto-N-sialylpentaose c (F-LST-c), fucosyl-lacto-N-sialylpentaose c, disialyl-lacto-N-fucopentaose, 3-fucosyl-3'-sialyllactose (3F-3'-SL), 3-fucosyl-6'-sialyllactose (3F-6'-SL), lacto-N-neodifucohexaose I, disialyl-lacto-N-fuco-pentaose (DS-LNFP V), or lacto-N-neodifucohexaose(hxaose) I (LNnDFH I).

[0079] Some genetically engineered microbial cells possess metabolic pathways to provide UDP-GlcNAc, as well as N-acetylglucosamine-containing oligosaccharides, such as 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), lacto-N-triose II (LNFP-II ... -N-fucopentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP-V), lacto-N-neofucopentaose V (LNnFP-V), lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para-lacto-N-hexaose (paraLNH), para-lacto-N-neohexaose (paraLNnH), difucosyl-lacto-N-neohexaose (DF-LNnH), lacto -N-Difucosylhexaose I, lacto-N-difucosylhexaose II, para-lacto-N-fucosylhexaose (paraLNH), fucosyl-lacto-N-sialylpentaose a (F-LST-a), fucosyl-lacto-N-sialylpentaose b (F-LST-b), fucosyl-lacto-N-sialylpentaose c (F-LST-c), fucosyl-lacto-N-sialylpentaose c, disialyl-lacto-N-fucopentaose , 3-fucosyl-3'-sialyllactose (3F-3'-SL), 3-fucosyl-6'-sialyllactose (3F-6'-SL), lacto-N-neodifucohexaose I, sialyllactose-N-tetraose a (LST-a), sialyllactose-N-tetraose b (LST-b), lacto-N-tetraose c (LST-c), disialyl-lacto-N-tetraose (DS-LNT), disialyl-lacto-N-fucopentaose (DS-LNFP V), or lacto-N-neodifucohexaose(hxaose) I (LNnDFH I).

[0080] Some genetically engineered microbial cells possess metabolic pathways to provide UDP-Gal as well as galactose moiety-containing oligosaccharides, such as 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), lacto-N-fucopentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP-V), lact para-N-neofucopentaose V (LNnFP-V), lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para-lacto-N-hexaose (paraLNH), para-lacto-N-neohexaose (paraLNnH), difucosyl-lacto-N-neohexaose (DF-LNnH), lacto-N-difucosylhexaose I, lacto-N-difucosylhexaose II, para-lacto-N-fuco Fucosylhexaose (paraLNH), fucosyl-lacto-N-sialylpentaose a (F-LST-a), fucosyl-lacto-N-sialylpentaose b (F-LST-b), fucosyl-lacto-N-sialylpentaose c (F-LST-c), fucosyl-lacto-N-sialylpentaose c, disialyl-lacto-N-fucopentaose, 3-fucosyl-3'-sialyllactose (3F-3'-SL), 3-fucosyl-6'-sialyllactose It also possesses galactosyltransferases for the biosynthesis of lactose (3F-6'-SL), lacto-N-neodifuco-hexaose I, 3'-sialyllactose (3-SL), 6'-sialyllactose (6-SL), sialyllactose-N-tetraose a (LST-a), sialyllactose-N-tetraose b (LST-b), sialyllactose-N-tetraose c (LST-c), disialyl-lacto-N-tetraose (DS-LNT), disialyl-lacto-N-fucopentaose (DS-LNFP V), lacto-N-neodifucohexaose (hxaose) I (LNnDFH I), 3'-galactosyllactose (3'-GL), 6'-galactosyllactose (6'-GL), etc.

[0081] In some embodiments, the engineered microbial cell possesses a sugar transporter in its cell membrane that translocates an oligosaccharide of interest from the cytoplasm of the microbial cell across the cell membrane into the medium or, in the case of Gram-negative bacteria, into the periplasmic space.

[0082] Sugar transporters that mediate the transfer of carbohydrates, e.g., oligosaccharides of interest, across cell membranes may consist of a single polypeptide or may consist of multiple polypeptides (each considered a subunit) that form homomeric or heteromeric complexes that transfer the oligosaccharide of interest across the cell membrane.

[0083] In some embodiments, the sugar transporter is a member selected from the group consisting of 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.

[0084] In certain embodiments, the sugar transporter is encoded by and expressed from 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.

[0085] The exogenous gene(s) encoding the transporter or transporter complex that facilitates transport of the oligosaccharide of interest across the cell membrane can be either endogenous or exogenous to the genetically engineered microbial cell.

[0086] In some embodiments, the sugar transporter gene is endogenous to the genetically engineered bacterial cell. Functional elements directing the synthesis of the protein encoded by the open reading frame of the sugar transporter gene(s) can be modified so that the level of transcription of the open reading frame or translation of the mRNA leading to the synthesis of the transporter protein differs from that naturally found in the preceding cell. Modification of the functional element can increase or decrease the transcription and / or translation level compared to the unmodified state. Modification can be a single nucleotide modification or a complete replacement of the functional element. In either case, the non-native level of transcription and / or translation is optimized for transport of the intracellular oligosaccharide of interest across the inner membrane.

[0087] In another embodiment, the gene or genes encoding the sugar transporter or transporter complex are exogenous to the genetically engineered microbial cell. In such embodiments, the protein coding region is operably linked to naturally occurring or artificial functional elements, such as a promoter or ribosome binding site, in such a way that the transcription and translation levels of the transporter are optimized for transporting the oligosaccharide of interest across the inner membrane.

[0088] Transcription of the gene(s) encoding the sugar transporter or a subunit of the sugar transporter can be either constitutive or regulated. Those skilled in the art will know how to select and use promoters and / or combinations of promoters with additional transcriptional regulators to achieve optimal expression of the gene(s) encoding the sugar transporter.

[0089] In embodiments in which the gene(s) encoding the sugar transporter are heterologous to the microbial cell for production of the oligosaccharide of interest, the nucleotide sequence of the gene(s) may be altered compared to the naturally occurring nucleotide sequence, but may encode a polypeptide possessing an unaltered amino acid sequence.

[0090] A sugar transporter for translocating an oligosaccharide of interest across a cell membrane can have its native amino acid sequence, i.e., the amino acid sequence found in nature. Alternatively, the amino acid sequence of the sugar transporter can be modified compared to its native amino acid sequence so that the resulting variant has enhanced activity with respect to translocating the oligosaccharide of interest and / or other beneficial characteristics, such as enhanced protein stability or more stable integration into the inner membrane.

[0091] In certain embodiments, the nucleotide sequence encoding the sugar transport protein(s) is artificial, and therefore the amino acid sequence of the resulting protein(s) that acts as a transporter for exporting the oligosaccharide of interest from the cytoplasm to the periplasm is not found in nature.

[0092] 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 to methylglyoxal and phosphate. Surprisingly, it has been found that deletion or functional inactivation of an endogenous gene of a microbial cell encoding a methylglyoxal synthase further improves the fermentative production of a desired oligosaccharide by a microbial cell harboring a functional variant of a naturally occurring E. coli glycerol kinase GlpK, the amino acid sequence of which is set forth in SEQ ID NO: 1, wherein the functional variant possesses an amino acid residue comprising a non-ionized but polar-acting side chain at a position in its amino acid sequence corresponding to amino acid position 55 in the amino acid sequence of naturally occurring E. coli GlpK and / or wherein the functional variant possesses an amino acid comprising an anionic side chain at a position in its amino acid sequence corresponding to amino acid position 231 in the amino acid sequence of naturally occurring E. coli GlpK.

[0093] The genetically engineered microbial cells described hereinabove can be used for the fermentative production of oligosaccharides of interest.

[0094] Accordingly, further disclosed is a method for the fermentative production of an oligosaccharide of interest, comprising: A genetically engineered microbial cell according to a first aspect for producing a desired oligosaccharide, i.e., a genetically engineered microbial cell capable of intracellularly synthesizing a desired oligosaccharide when cultured in the presence of glycerol as a sole carbon source, comprising a glycerol permease, a functional variant of native Escherichia coli (E. coli) glycerol kinase GlpK, the amino acid sequence of which is set forth in SEQ ID NO: 1 (this functional variant has an amino acid sequence corresponding to amino acid position 55 in the amino acid sequence of native Escherichia coli (E. coli) GlpK). a genetically engineered microbial cell that possesses an amino acid residue containing a non-ionizable but polar-active side chain at a position in its amino acid sequence corresponding to amino acid 231 in the amino acid sequence of native Escherichia coli (E. coli) GlpK, and / or the functional variant possesses an amino acid containing an anionic side chain at a position in its amino acid sequence corresponding to amino acid 231 in the amino acid sequence of native Escherichia coli (E. coli) GlpK), a metabolic pathway for the intracellular biosynthesis of a nucleotide-activated monosaccharide as a donor substrate for the monosaccharide moiety, and a glycosyltransferase for the transfer of the monosaccharide moiety from the nucleotide-activated monosaccharide to an acceptor molecule; Culturing the genetically engineered microbial cells in a culture medium (containing glycerol as a carbon source for the genetically engineered microbial cells) and under conditions that allow the genetically engineered microbial cells to synthesize the desired oligosaccharides intracellularly; and Optionally, recovering the oligosaccharides of interest from the microbial cells and / or culture medium.

[0095] Also disclosed are methods for alleviating the toxicity of methylglyoxal to microbial cells when cultured in the presence of glycerol as a carbon source, and methods for reducing the sensitivity of microbial cells to methylglyoxal toxicity during the fermentative production of a desired oligosaccharide. These methods include providing a genetically engineered microbial cell that synthesizes the desired oligosaccharide intracellularly, deleting or functionally inactivating endogenous glycerol kinase gene(s) in the microbial cell, and transforming the microbial cell to contain and express a functional variant of Escherichia coli (E. coli) glycerol kinase GlpK, wherein the functional variant possesses an amino acid residue containing a non-ionized but polar-acting side chain at a position corresponding to amino acid 55 in the amino acid sequence of E. coli GlpK set forth in SEQ ID NO:1 and / or an amino acid residue containing an anionic side chain at a position corresponding to amino acid 231 in the amino acid sequence of E. coli GlpK set forth in SEQ ID NO:1.

[0096] While the present invention will be described with reference to particular embodiments and with reference to the drawings, the present invention is not limited thereto, but rather only by the claims. Moreover, terms such as first, second, etc. in this specification and claims are used to distinguish between similar elements and are not necessarily intended to describe an order in time, space, sequence, or otherwise. Terms so used are interchangeable under appropriate circumstances, and it will be understood that the embodiments of the invention described herein can operate in orders other than those described or illustrated herein.

[0097] It should be noted that the term "comprising" used in the claims should not be interpreted as being limited to the means listed thereafter, nor does it exclude other elements or steps. Thus, the term should be interpreted as specifying the presence of the mentioned and described 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. Thus, the scope of the expression "a device comprising means A and B" should not be limited to a device consisting of only components A and B. This simply means that, in the context of the present invention, the relevant components of the device are A and B.

[0098] Throughout this specification, a reference to "one embodiment" or "an embodiment" indicates that a feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

[0099] Similarly, in describing exemplary embodiments of the invention, it should be understood that various features of the invention may be grouped together in a single embodiment, figure, or description for the purpose of streamlining the disclosure and / or facilitating understanding of 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 are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the Detailed Description are expressly incorporated into this Detailed Description, with each claim standing on its own as a separate embodiment of the invention.

[0100] Furthermore, some embodiments described herein include some features included in other embodiments but not others, meaning that combinations of features from different embodiments form different embodiments within the scope of the present invention and as understood by those skilled in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0101] Numerous specific details are set forth in the description and drawings provided herein. However, it will be understood that embodiments of the present invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

[0102] The present invention will now be described by way of a detailed description of some embodiments of the present invention. Other embodiments of the present invention can be constructed according to the knowledge of those skilled in the art without departing from the true spirit or technical teachings of the present invention, and the present invention is limited only by the terms of the appended claims. [Example]

[0103] [Example 1] Adaptive laboratory evolution of 3-FL, ΔmgsA, GlpK(A55T)-Escherichia coli (E. coli) An Escherichia coli (E. coli) strain capable of producing 3-FL was subjected to adaptive laboratory evolution experiments to examine the effect of methylglyoxal toxicity curing on growth and 3-FL production using glycerol as the sole carbon and energy source.

[0104] An Escherichia coli (E. coli) strain capable of producing 3-FL had its lacZ gene deleted, expressed a heterologous alpha-1,3-fucosyltransferase gene, and its mgsA gene deleted. All molecular biology procedures were performed according to the procedures described in Sambrook, J. and Russell, D., Molecular Cloning: A Laboratory Manual, 3 (2003).rd This was done as described in [Illegible Text], Cold Spring Harbor Laboratory Press, New York, 2001. To delete the mgsA gene from an Escherichia coli (E. coli) strain capable of producing 3-FL, the upstream and downstream regions near the open reading frame of mgsA were amplified by polymerase chain reaction (PCR) to generate two DNA fragments. 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 adjacent to mgsA. These two DNA fragments were used as templates for a third PCR using primers 1 and 4 (Table 1) to generate a DNA construct, which was subsequently used to delete the mgsA gene according to a modified version of the method described by Datsenko, K.A. and Wanner, B.L. (One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc. Natl. Acid. Sci. 97 (2000) 6640-6645). Individual clones were analyzed by colony PCR using primers 1 and 4 (Table 1). The selected strain, designated 3-FL-2, was subjected to chemical mutagenesis by exposure to N-methyl-N'-nitro-N-nitrosoguanidine, followed by two successive fed-batch cultivations in a bioreactor. The cultivation was carried out as described in WO 2018 / 077892.

[0105] Cells 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 with 0.9% (w / v) NaCl, and 100 μL aliquots were inoculated onto plates containing 2YT medium composed of 16 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl, and 18 g / L agar. After 48 h of incubation at 30°C, isolated colonies were screened for improved 3-FL production in deep-well plates using the same batch medium used in the fed-batch culture described earlier in this specification. For the preculture used in this experiment, plates were incubated for 24 h at 30°C, 800 rpm, and 80% humidity (Microtron, Infors AG, Switzerland). For the main deep-well plate culture, the medium was supplemented with 10 mM lactose, and the culture conditions were the same as those used for the preculture. Sample preparation and analysis by HPLC were performed as described in WO 2018 / 077892.

[0106] A selected Escherichia coli (E. coli) isolate (3-FL-3) obtained in the adaptive laboratory evolution experiment was cultured in a bioreactor in fed-batch mode to evaluate its growth characteristics and 3-FL production, compared with those of its progenitor strain, which was not subjected to the laboratory evolution experiment. The results are shown in Figure 3, and all data points are relative to the control strain.

[0107] [Table 1]

[0108] The selected Escherichia coli (E. coli) isolate 3-FL-3 (■) was measured by measuring the optical density (OD) of the culture medium at a wavelength of 600 nm. 600Analysis of 3-FL-3 (Figure 3A) revealed a 44% reduction in the time to complete the batch phase compared to the progenitor strain (▲). Furthermore, the selected Escherichia coli (E. coli) isolate 3-FL-3 (■) showed an approximately 18% increase 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 its progenitor strain (▲) (Figure 3B).

[0109] A selected Escherichia coli (E. coli) isolate, 3-FL-3, exhibiting faster growth and increased 3-FL production compared to its progenitor strain, was found to contain a mutant glpK gene encoding a variant of GlpK in which the alanine residue at position 55 was replaced with a threonine residue. This variant of E. coli GlpK is designated GlpK(A55T).

[0110] [Example 2] Production of 2'-FL or LNT by GlpK mutant Escherichia coli (E. coli) strains To examine the effect of GlpK(A55T) on the production of HMOs other than 3-FL, Escherichia coli (E. coli) strains capable of producing 2'-FL or LNT were genetically engineered so that their endogenous glpK gene was replaced with a mutant encoding E. coli (K12) GlpK(A55T). All molecular biology procedures used to modify the endogenous glpK gene with the mutant glpK gene in both 2'-FL- and LNT-producing E. coli strains were previously described in Sambrook, J. and Russell, D., Molecular Cloning: A Laboratory Manual, 3 (2013). rd Edition, Cold Spring Harbor Laboratory Press, New York, 2001. Primer 5 (Table 1) was used in the recombination event to alter the nucleotide sequence of the native glpK gene.

[0111] To identify clones harboring the mutant glpK gene, colony PCR was performed using a combination of primers 6 and 8 (Table 1) to confirm the presence of the wild-type glpK gene, and a combination of primers 7 and 8 (Table 1) to confirm the presence of the mutant glpK gene. Bacterial clones identified as harboring a mutant glpK gene were subjected to nucleotide sequence analysis using primers 8 and 9 (Table 1) to confirm the presence of the mutant glpK gene encoding GlpK(A55T). The 2'-FL-producing E. coli strain constructed in this manner was designated E. coli 2'-FL-2 (Table 2). The LNT-producing E. coli strain constructed in this manner was based on a metabolically engineered E. coli BL21(DE3) strain similar to E. coli LNT-1 (Table 2) and was named E. coli LNT-2 (Table 2). To compare growth characteristics and HMO production profiles, both E. coli strains 2'-FL2 and LNT-2 were cultured in a fed-batch process. Fed-batch culture and HPLC analysis were performed under the same conditions as described in WO 2018 / 077892.

[0112] Despite the presence of the endogenous mgsA gene in the E. coli 2'-FL-2 and E. coli LNT-2 strains, they exhibited improved growth (Figures 4A and 5A) and HMO production (Figures 4B and 5B) compared to their respective control strains (▲). For the E. coli 2'-FL-2 strain (Table 2) (■), Figure 4A shows a reduction in the time required to complete the batch phase, and Figure 4B shows an increase in the 2'-FL titer. The reduction in time to complete the batch phase was approximately 35%, while the 2'-FL titer increased by approximately 36%. Figure 5A shows a 41% reduction in the time to complete the batch phase by the E. coli LNT-2 strain (Table 2) (■) compared to the control strain (▲). Furthermore, the LNT-2 titer in the culture supernatant of Escherichia coli (E. coli) 2'-FL-2 (■) increased by approximately 14% (see Figure 5B).

[0113] [Table 2]

[0114] [Example 3] Overcoming growth inhibition via mgsA inactivation in 2'-FL and LNT-producing strains harboring GlpK(A55T) To ensure that methylglyoxal toxicity was not an issue during HMO production using E. coli strains harboring GlpK(A55T), the growth rates of different E. coli strains harboring GlpK(A55T) for 2'-FL or LNT production were recorded under different production conditions. The growth rates of E. coli strains harboring the native mgsA gene were compared with those of progeny strains with a deleted mgsA gene.

[0115] Sudden exposure of Escherichia coli (E. coli) strains harboring feedback-resistant mutants of GlpK to glycerol can result in inhibition of cell growth. During the fermentative production of HMOs, a bacterial preculture obtained in a seed fermenter is usually used to inoculate the main fermenter while the bacterial cells are still in the exponential growth phase. However, it can happen that the bacterial cell culture in the seed fermenter reaches the stationary growth phase before the main fermenter can be inoculated. This leads to the bacterial cells being exposed to high glycerol concentrations in the main fermenter when inoculated, which in turn leads to methylglyoxal toxicity.

[0116] To investigate whether such a scenario could cause growth problems and affect HMO production, E. coli strains harboring GlpK(A55T) and capable of synthesizing 2'-FL or LNT were tested using the following approach. As described in Example 2, fed-batch precultures were prepared to obtain the inoculum used for HMO production in fed-batch mode of operation. As seen in Figure 6, both the 2'-FL-2 (▲) (Table 2; Figure 6A) and LNT-2 (▲) (Table 2; Figure 6B) E. coli strains were unable to grow in the main fermentor when their precultures were derived from fed-batch cultures.

[0117] The experiment was then repeated using progeny 2'-FL-producing or LNT-producing E. coli strains in which the mgsA gene had been further deleted. The same single-stranded DNA-based method described in Example 2 was used to inactivate the mgsA gene in E. coli 2'-FL-2 and E. coli LNT-2 strains. Primer 10 (Table 1) was used to functionally inactivate the mgsA gene by inserting a stop codon and frameshift at position 43. The combination of primers 11 and 13 (Table 1) was used to confirm functional inactivation of the mgsA gene. The combination of primers 12 and 13 (Table 1) was used to confirm the presence of the endogenous mgsA gene. Individual bacterial clones were selected after colony PCR to verify functional inactivation of mgsA and further characterized by nucleotide sequence analysis using primers 13 and 14 (Table 1).

[0118] For the production of 2'-FL, the Escherichia coli (E. coli) ΔmgsA mutant (designated 2'-FL-3) of the Escherichia coli (E. coli) 2'-FL-2 strain (Table 2; Figure 6A) and the Escherichia coli (E. coli) ΔmgsA mutant (designated LNT-3) of the Escherichia coli (E. coli) LNT-2 strain (Table 2; Figure 6B) were used. When challenged with glycerol at the time of inoculation from the stationary growth phase, cell growth was observed for both strains (■).

[0119] These results demonstrate the importance of further increasing the robustness of bacterial HMO-producing strains to be able to cope with variability in ΔmgsA genotype and / or plant operation as a preventative measure.

[0120] [Example 4] Comparison of 2'-FL production by Escherichia coli (E. coli) harboring GlpK(A55T) or GlpK(G231D) Based on the results shown in Examples 1 and 2, we decided to investigate whether other GlpK mutants could also improve HMO production. To test this hypothesis, we compared the 2'-FL productivity of E. coli 2'-FL-2 (Table 2) with that of E. coli 2'-FL-4 (Table 2), which contains the glpK gene encoding the previously reported feedback-resistant form of GlpK (G231D) (Honisch, C. et al. (2004) Genome Res. 14:2495-2502). The Escherichia coli (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 progenitor strain used to construct the Escherichia coli (E. coli) strain 2'-FL-2 (Table 2). To improve the efficiency of the genetic engineering procedure used, the glpK gene mutations G692A and C693T were used in combination instead of the G692A mutation alone, as described in the literature. Both the nucleotide mutation G692A and the combined mutations G692A and C693T encode the target GlpK enzyme with the amino acid substitution G231D. Construction of the E. coli 2'-FL-4 strain (Table 2) followed the same methodology as used in Example 2 for the generation of the E. coli 2'-FL-2 strain (Table 2). Primer 15 (Table 1) was used for glpK gene modification. For clonal selection, either primer combination 16 and 18 (Table 1) or pair 17 and 18 (Table 1) was used to identify mutational glpK mutants and the native glpK gene, respectively. To confirm the desired modifications, the glpK gene was sequenced using primers 18 and 19 (Table 1). Similar to the E. coli 2'-FL-2 strain (Table 2), E. coli 2'-FL-4 (Table 2) was also cultivated in a fed-batch manner for 2'-FL production using the same culture conditions and analytical procedures described in Example 2.The G231D mutation in the GlpK enzyme of the Escherichia coli (E. coli) strain 2'-FL-4 (●) (Table 2) had an even greater positive effect on 2'-FL production (Figure 4B). The 2'-FL titer produced by E. coli 2'-FL-4 (●) increased by 53% compared to the 2'-FL titer produced by the control strain (▲). Furthermore, a 34% reduction in batch time was achieved compared to the control strain (▲).

Claims

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

2. 2. The genetically engineered microbial cell of claim 1, wherein the amino acid residue having a non-ionized but polar 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.

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

4. 4. The genetically engineered microbial cell of 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 Escherichia coli (E. coli) (K12) GlpK.

5. 5. The genetically engineered microbial cell of any one of claims 1 to 4, which is genetically engineered to contain and express a recombinant gene encoding a functional variant of E. coli GlpK.

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

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

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

9. Use of a genetically engineered microbial cell according to any one of claims 1 to 8 for the production of an oligosaccharide of interest.

10. 10. The use according to claim 9, wherein the oligosaccharide of interest is an oligosaccharide selected from the group consisting of human milk oligosaccharides.

11. 1. A method for the fermentative production of an oligosaccharide of interest, comprising: providing a genetically engineered microbial cell according to any one of claims 1 to 8 for the intracellular biosynthesis of said oligosaccharide of interest; Cultivating the genetically engineered microbial cells in a culture broth containing glycerol as a carbon and energy source for the genetically engineered microbial cells; and recovering the target oligosaccharides from the microbial cells and / or the culture broth. A method comprising:

12. 12. The method of claim 11, wherein the glycerol is the sole carbon and energy source exogenously added to the culture broth.

13. 13. The method of claim 11 or 12, wherein the oligosaccharide of interest is an oligosaccharide selected from the group consisting of human milk oligosaccharides.

14. 1. A method for mitigating the toxicity of methylglyoxal to microbial cells, comprising: providing genetically engineered microbial cells for the fermentative production of oligosaccharides of interest; deleting or functionally inactivating the endogenous glycerol kinase gene of said microbial cell; Transforming the microbial cell to contain and express a gene encoding a functional variant of the E. coli (K12) glycerol kinase GlpK, wherein the functional variant has an amino acid residue at a position corresponding to amino acid 55 in the amino acid sequence of E. coli GlpK set forth in SEQ ID NO: 1, wherein the amino acid residue comprises a non-ionizable but polar-acting side chain, and / or the functional variant has an amino acid residue at a position corresponding to amino acid 231 in the amino acid sequence of E. coli GlpK set forth in SEQ ID NO: 1, wherein the anionic amino acid residue comprises an anionic side chain. A method comprising:

15. 1. A method for reducing the responsiveness of a microbial cell to methylglyoxal toxicity during the fermentative production of an oligosaccharide of interest, comprising: providing a genetically engineered microbial cell for the fermentative production of said target oligosaccharide; deleting or functionally inactivating the endogenous glycerol kinase gene of said microbial cell; Transforming the microbial cell to contain and express a gene encoding a functional variant of the E. coli glycerol kinase GlpK, wherein the functional variant comprises an amino acid residue at a position corresponding to amino acid 55 in the amino acid sequence of E. coli GlpK set forth in SEQ ID NO: 1, wherein the amino acid residue comprises a non-ionizable but polar-acting side chain, and / or the functional variant has an amino acid residue at a position corresponding to amino acid 231 in the amino acid sequence of E. coli GlpK set forth in SEQ ID NO: 1, wherein the anionic amino acid residue comprises an anionic side chain. A method comprising:

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