WO2022013143A1 - Oligosaccharide production - Google Patents

Oligosaccharide production Download PDF

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Publication number
WO2022013143A1
WO2022013143A1 PCT/EP2021/069310 EP2021069310W WO2022013143A1 WO 2022013143 A1 WO2022013143 A1 WO 2022013143A1 EP 2021069310 W EP2021069310 W EP 2021069310W WO 2022013143 A1 WO2022013143 A1 WO 2022013143A1
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WIPO (PCT)
Prior art keywords
sucrose
hmo
lactose
cell
transferase
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PCT/EP2021/069310
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French (fr)
Inventor
Margit Pedersen
Manos PAPADAKIS
Peter Becker
Eric Samain
Pauline Peltier-Pain
Katrine Bych KAMPMANN
Ted JOHANSON
Elise Champion
Gyula Dekany
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Glycom A/S
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Priority claimed from US16/927,700 external-priority patent/US11293042B2/en
Application filed by Glycom A/S filed Critical Glycom A/S
Priority to CN202180049624.0A priority Critical patent/CN115803443A/en
Priority to EP21748487.2A priority patent/EP4179102A1/en
Publication of WO2022013143A1 publication Critical patent/WO2022013143A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/26—Klebsiella (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to the field of biotechnology, notably to a microbial production of oligosaccharides, particularly of human milk oligosaccharides (HMOs), using a genetically modified microorganism, particularly E. coli , using sucrose as its exclusive carbon source.
  • HMOs human milk oligosaccharides
  • oligosaccharides of interest would be synthesized by enzymatic glycosylation of sugar acceptors mediated by one or more heterologous glycosyl transferases of the microorganisms, and the one or more activated sugar nucleotides necessary for glycosylation would be produced by the same microorganism through overexpressing one or more genes encoding endogenous activated sugar nucleotide producing enzymes.
  • the metabolic pathways of such syntheses require a carbon source which is mainly a simple carbon building block, typically glycerol or glucose (see e.g.
  • lactose can be the carbon source if it also serves as an acceptor (Lee et al. Microb. Cell Fact. 11:48 (2012)).
  • antibiotic- resistance selection marker genes have been utilized to separate the transformed microorganisms from the non-transformed ones in the inoculum and the fermentation broth.
  • the use of antibiotics has been avoided by integrating the genes coding for enzymes involved in the de novo biosynthesis of the donor sugar in the chromosome of the microorganisms (Baumgartner et al. Microb. Cell Fact. 12:40 (2013)).
  • sucrose As a carbon and energy source, but most of them are pathogenic.
  • the E. coli strains used mainly in industry to synthesize chemical materials cannot live and grow on sucrose (Bruschi et al. Biotechnol. Adv. 30, 1001 (2012)).
  • sucrose can be a cheaper carbon and energy source.
  • attempts have been made to create suc + strains of E. coli that can live and grow on sucrose e.g. GB 2155935 A, Sabri et al. Appl. Environ. Microbiol 79, 478 (2013)
  • produce industrially profitable products by them such as amino acids, biofuel, carotenoids etc. (e.g.
  • WO 2012/007481 describes E. coli transformants that express either a sucrose phosphorylase or a sucrose invertase in combination with a fructokinase. Thereby, the microorganism is able to produce 2’-fucosyllactose, utilizing sucrose as its main carbon source.
  • WO 2014/067696, WO 2015/150328, WO 2019/043029 and WO 2019/076941 describe E.coii transformants comprising a csc-gene cluster that enables them to grow on sucrose and produces fucose, 2’-FL, sialyllactose and sialic acid, respectively.
  • a first aspect of the invention relates to a process for making an oligosaccharide, preferably of 3-8 monosaccharide units, more preferably of 3-5 monosaccharide units, particularly a HMO, by glycosylating a carbohydrate acceptor which is not sucrose, preferably lactose, comprising the steps of: a) providing a cell, preferably an E.coii cell, that can internalize said acceptor into said cell and comprises
  • a recombinant gene encoding a glycosyl transferase which is able to transfer a glycosyl residue of an activated sugar nucleotide to said acceptor, internalized in said cell, or to an intermediate in the biosynthetic pathway to said oligosaccharide from said acceptor and
  • said process being characterized in that said cell also comprises one or more genes encoding a sucrose utilization system, preferably encoding a heterologous sucrose utilization system, more preferably encoding a heterologous PTS-dependent sucrose utilization transport system, still more preferably scr genes, so that said cell can use sucrose as a carbon source, preferably the main carbon source, more preferably the sole carbon source, for making said activated sugar nucleotide and as an energy source, preferably the main energy source, more preferably the sole energy source, for making said oligosaccharide.
  • a sucrose utilization system preferably encoding a heterologous sucrose utilization system, more preferably encoding a heterologous PTS-dependent sucrose utilization transport system, still more preferably scr genes
  • a second aspect of the invention relates to a cell, preferably an E.coii cell, that can internalize a carbohydrate acceptor, which is not sucrose, preferably lactose, into said cell and that comprises: - a recombinant gene encoding a glycosyl transferase which is able to transfer a glycosyl residue of an activated sugar nucleotide to said acceptor, internalized in said cell, or to an intermediate in the biosynthetic pathway to said oligosaccharide from said acceptor
  • sucrose utilization system preferably a heterologous sucrose utilization system, more preferably scr genes, so that said cell can use sucrose as a carbon source, preferably the main carbon source, more preferably the sole carbon source.
  • FIG. 1 is intended to illustrate the invention further. It is not intended to limit the subject matter of the invention thereto.
  • the engineered microorganism is a fully metabolically active cell in which the growth and the oligosaccharide synthesis may proceed simultaneously.
  • the cell comprises a heterologous phosphotransferase (PTS)-dependent sucrose utilization transport system containing a sucrose specific porin (facilitates the sucrose diffusion through the outer membrane), a sucrose transport protein (provides intracellular sucrose-6-phosphate from extracellular sucrose) and a sucrose-e- phosphate hydrolase (provides glucose-6-phosphate and fructose).
  • PTS heterologous phosphotransferase
  • the oxidation of glucose-e- phosphate and fructose provides biological energy source by the organism’s own metabolic system.
  • glucose-6-phosphate and fructose serve as carbon source for producing sugar nucleotides in the cell’s natural biosynthetic pathway or in any heterologous pathway expressed in the cell.
  • the so-produced sugar nucleotides are donors for glycosylating carbohydrate acceptors (e.g. lactose), internalized through a specific permease by the cell, and thereby manufacturing oligosaccharides of interest.
  • the glycosylation is mediated by one or more glycosyl transferases which are directly produced by expressing heterologous genes from plasmid or the chromosome.
  • the organism lacks any enzyme degrading either the acceptor or the oligosaccharide product in the cell.
  • Figure 2 Illustrate the ability to grow on sucrose with a heterologous PTS-dependent sucrose utilization transport system in the cell.
  • the MDO strain does not contain the PTS-dependent sucrose utilization transport system, whereas MPLNnT does grow on sucrose.
  • an exogenous mono- or disaccharide acceptor preferably lactose
  • a suitable genetically transformed microorganism particularly E.coli
  • this carbohydrate acceptor can be glycosylated in the microorganism using sucrose as its carbon and energy source, and that an exogenous oligosaccharide can be produced and separated in good yield.
  • oligosaccharide-producing microorganism that can live on sucrose, utilize sucrose for the metabolic syntheses of the necessary nucleotide sugar donors for glycosylation, can internalize simple carbohydrate acceptors and perform glycosylation reactions on them for synthesizing more complex oligosaccharides.
  • the invention therefore, in a first aspect, involves a process of making an oligosaccharide by: a) providing a cell of a microorganism, preferably an E.coli cell, that can internalize sucrose and a carbohydrate acceptor, preferably lactose, into said cell and that comprises: a recombinant gene encoding a glycosyl transferase which can transfer a glycosyl residue of an activated sugar nucleotide to the acceptor within the cell or to an intermediate in the biosynthetic pathway to said oligosaccharide from said acceptor, and a biosynthetic pathway for making the activated sugar nucleotide from sucrose, b) culturing the cell in an aqueous culture medium in the presence of the acceptor and sucrose, and c) separating the oligosaccharide product from the cell from the culture medium or from both.
  • a microorganism preferably an E.coli cell
  • a carbohydrate acceptor
  • the process features the cell being transformed with one or more foreign genes encoding a sucrose utilization system that allows the cell to use sucrose as a carbon source, preferably the main carbon source, more preferably the sole carbon source, for the biosynthesis of the activated sugar nucleotide by the cell.
  • the sucrose utilization system with which the cell is transformed, also preferably allows the cell to use sucrose as an energy source, preferably the main energy source, more preferably the sole energy source, for the biosynthesis of the oligosaccharide.
  • the term “carbohydrate acceptor” or “acceptor” preferably means a mono- or disaccharide other than sucrose and its glycosides.
  • a monosaccharide acceptor or a monosaccharide part of a disaccharide acceptor can comprise any 5-6 carbon atom sugar moiety that is an aldose (e.g. D-glucose, D-galactose, D-mannose, D-ribose, D- arabinose, L-arabinose, D-xylose, etc.), ketose (e.g. D-fructose, D-sorbose, D-tagatose, etc.), deoxysugar (e.g.
  • L-rhamnose, L-fucose, etc. or deoxy-aminosugar (e.g. N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.).
  • deoxy-aminosugar e.g. N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.
  • the C-1 (in case of aldoses) or C-2 (in the case of ketoses) anomeric carbon atom at the reducing end of the mono- or disaccharide residue is linked to the non-sugar moiety by a covalent bond or a linker forming a 0-, N-, S- or C-glycoside.
  • the aglycon of these glycosidic derivatives, with or without a linker is one of the following groups: a) -ORA, wherein RA is a linear or branched hydrocarbon chain having, when saturated, 1- 24, preferably 1-6 carbon atoms (such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s- butyl, t-butyl, n-hexyl, etc.) or, when unsaturated, 2-24, preferably 2-6 carbon atoms (such as vinyl, allyl, propargyl, etc.), or R A means an aryl moiety (a homoaromatic group such as phenyl or naphthyl), or R A means a group removable by hydrogenolysis, that is a group whose bond attached to the oxygen can be cleaved by addition of hydrogen in the presence of catalytic amounts of palladium, Raney nickel or
  • Suitable groups include benzyl, diphenylmethyl (benzhydryl), 1-naphthylmethyl, 2-naphthylmethyl or triphenylmethyl (trityl) groups.
  • Any of the above mentioned R A groups can be optionally substituted by one or more groups selected from: alkyl (only for aryl and group removable by hydrogenolysis), hydroxy, alkoxy, carboxy, oxo, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylamino, arylcarbonyl, amino, mono and dialkylamino, carbamoyl, mono- and dialkyl-amino-carbonyl, alkyl- carbonylamino, cyano, alkanoyloxy, nitro, alkylthio and halogen; in case of a group removable by hydrogenolysis, such substitution, if present, is preferably on the aromatic ring(s);
  • the amino acid is selected from the group consisting of: a-amino acids and b-amino acids such as Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, hydroxyproline, a-methylserine, b-alanine, etc.
  • a-amino acids and b-amino acids such as Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, hydroxyproline, a-methylserine, b-alanine, etc.
  • These amino acids can be either directly or via a linker as defined above (e.g.
  • O-Glycosides can be formed involving OH-containing amino acids such as serine, threonine, hydroxyproline, etc.
  • N- glycosides can be made using the a-, b-, etc. amino group of any amino acid or the additional amino group of the side chain of e.g.
  • lysine, asparagine or glutamine S-glycosides can be made using cysteine, while C-glycosides (C-glycans) contain a C-C bond coupling a C- atom of the amino acid to the anomeric carbon atom of the non-reducing end of the oligosaccharide part; g) a polyethylene glycol residue.
  • Polyethylene glycol (PEG) is a water soluble polyether of molecular formula C 2n H 4n+2 0 n+i , having oxyethylene (-CH 2 -0-CH 2 - or CH 2 -CH 2 -0-) repeating units and wherein n is 2 to 100, preferably 2 to 50, particularly 2 to 25, more particularly 2 to 10.
  • Lower molecular weight PEGs are available in a purified form and are referred to as a “monodisperse PEG", and are also available as mixtures of PEGs and are referred to as a “polydisperse PEG”. With regard to their geometry, PEGs can be in a linear, branched, star or comb configuration. Linear PEGs are preferably lower molecular weight PEGs (i.e. , n is 2 to 10, preferably 3 to 6). Branched PEGs preferably have 3 to 10 linear, preferably lower molecular weight, PEG chains emanating from a central core group. Star PEGs preferably have 10 to 100 linear or branched, preferably lower molecular weight, PEG chains emanating from a central core group.
  • Comb PEGs have multiple linear, branched and/or star, preferably lower molecular weight, PEG chains bonded to a polymer backbone.
  • Terminal primary hydroxy group of PEGs can be bonded by an ether bond with an alkyl group, preferably methyl.
  • their terminal hydroxy group can be replaced by amino, alkyl amino, dialkyl amino, acylamino, thiol or alkyl thio groups or their terminal hydroxymethyl group can be oxidized to a carboxyl, which can be esterified or be present in amide form with ammonia or a primary or secondary amine.
  • the attachment is a glycosidic-like bond; h) a polyvinyl alcohol residue.
  • Polyvinyl alcohol is a water-soluble polymer of molecular formula (C 2 H 4 0) X having -CH 2 -CH(OH)- monomer units, wherein x is any desired integer.
  • any of the OH-groups can be glycosylated; i) an a,b-unsaturated amido group of formula A wherein Qi , Q 2 , Q 3 and Q 4 are, independently, H and Ci-C 6 -alkyl, which alkyl optionally can be substituted with halogen, OH, nitro or phenyl groups.
  • the residue of formula A via its N atom, is linked to the sugar by a covalent bond, preferably to the anomeric carbon atom of the carbohydrate in the form of an N-glycoside; or j) an a,b-unsaturated carbonyl group of formula B
  • the carbohydrate acceptor is a galactosyl disaccharide, particularly lactose.
  • oligosaccharide product or “oligosaccharide” preferably means a glycosylated derivative of a carbohydrate acceptor disclosed above wherein a glycosyl residue is attached to the carbohydrate moiety of the carbohydrate acceptor by interglycosidic linkage.
  • an oligosaccharide product is of 3- 8 monosaccharide units, particularly of 3-5 monosaccharide units, more particularly of 4 monosaccharide units or of 3 monosaccharide units.
  • the oligosaccharide product of this invention is a recombinant product, i.e.
  • an intermediate in the biosynthetic pathway in relation to an oligosaccharide or more specifically an HMO, is to be understood as an intermediate oligosaccharide or HMO in the reaction steps needed to produce the desired oligosaccharide or HMO.
  • LNT-II is an intermediate in the biosynthetic pathway to LNT or LNnT.
  • the genetically modified microorganism or cell used in the process of some embodiments of this invention can be selected from the group consisting of bacteria and yeasts, preferably a bacterium.
  • Bacteria are preferably selected from the group of: Escherichia coii, Bacillus spp. (e.g.
  • Bacillus subtil is), Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Neisseria gonorrhoeae, Neisseria meningitis, Lactobacillus spp., Lactococcus spp., Enterococcus spp., Bifidobacterium spp., Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., Pseudomonas, among which E. coii is preferred.
  • the microorganism or cell preferably an E.coii cell
  • the alteration can result in a change in the original characteristics of the wild type cell, e.g. the modified cell is able to perform additional chemical transformation due to the introduced new genetic material that encodes the expression of an enzymes not being in the wild type cell, or is not able to carry out transformation like degradation due to removal or deactivation of gene/genes.
  • a genetically modified cell contains at least one artificial alteration in its genome compared to its naturally occurring (wild type) variant.
  • a nucleic acid construct is added to the cell by way of integration into the genome or by addition via plasmid, or a nucleic acid sequence is deleted from or changed in the genome of the cell.
  • the so-transformed cell has a genotype that is different from that before the alteration and, therefore, the modified cell shows modified feature(s).
  • the genetically modified cell can perform at least one additional or altered biochemical reaction, when cultured or fermented, due to the introduction of a heterologous nucleic acid sequence or the modification of a native nucleic acid sequence that encodes an enzyme that is not expressed in the wild type cell, or the genetically modified cell cannot perform a biochemical reaction due to the deletion, addition or modification of a nucleic acid sequence that encodes an enzyme found in the wild type cell.
  • the genetically modified cell can be constructed by well-known, conventional genetic engineering techniques (e.g. Green and Sambrook: Molecular Cloning: A laboratory Manual, 4 th ed., Cold Spring Harbor Laboratory Press (2012); Current protocols in molecular biology (Ausubel et al. eds.), John Wiley and Sons (2010)).
  • nucleic acid sequence encoding a polypeptide having a glycosyl transferase activity or “nucleic acid sequence encoding glycosyl transferase” means a gene, a functional fragment thereof or a codon-optimized version thereof that express a polypeptide having a glycosyl transferase activity.
  • gene in the present context relates to a coding nucleic acid sequence and includes genomic DNA and cDNA.
  • promoter means a nucleic acid sequence involved in the binding of RNA polymerase to initiate transcription of an operably linked gene, wherein the gene includes a coding DNA sequence and other (non-coding) sequences, e.g. the 5’-untranslated region (5’- UTR) located upstream of the coding sequence, which comprises a ribosomal binding site.
  • a promoter in some embodiments of this invention is an isolated DNA sequence, i.e. not an integrated DNA fragment of the genomic DNA.
  • the nucleotide sequence of a promoter corresponds to, or have at least 80 % identity, preferably 90%to 99.9 % identity, such as 95%, 98%, and even more preferably 100% identity with the nucleotide sequence of a fragment of bacterial genomic DNA that is regarded as promoter region of a gene, e.g. a promoter region of a glp operon or lac operon of E.coli.
  • a preferred promoter is the promoter of SEQ ID NO: 5.
  • glp operon means a functioning unit of genomic DNA or cDNA containing a cluster of genes under the control of a single promoter.
  • glp operon is meant a cluster of genes involved in the respiratory metabolism of glycerol of bacteria.
  • a glp operon promoter sequence comprised in a DNA construct corresponds to or has at least 80 % identity, preferably 90-99.9 % identity, such as 95%, 98%, and even more preferably 100% identity with the nucleotide sequence of a fragment of the genomic DNA regarded as a promoter region of the corresponding glp operon of E. coir, in particular, the isolated sequence of a promoter of a glp operon of some embodiments of the invention corresponds to, or has said percent of identity with a fragment of the genomic sequence upstream the sequence having GenBank ID:
  • E. coli genome is referred herein to the complete genomic DNA sequence of E coli K-12 MG1655 (GenBank ID:U00096.3).
  • a promoter of the glp operon is operably linked to the heterologous glycosyl transferase genes and/or the heterologous scr genes.
  • those genes are expressed under a glpF promoter (WO 2019/123324, the content of which is incorporated herein by reference), in particular the glpF promoter of SEQ ID NO: 5.
  • the process of some embodiments of this invention also involves transporting the exogenous carbohydrate acceptor, preferably lactose, into the genetically modified microorganism for glycosylation to produce a foreign oligosaccharide of interest, preferably without adversely affecting the basic functions of the cell or destroying its integrity.
  • the transport takes place via a passive mechanism, during which the exogenous acceptor diffuses passively across the plasma membrane of the cell. Diffusion of the acceptor into the microorganism is a function of the concentration differences between the fermentation broth and the extra- and intracellular space of the cell with respect to the acceptor, whereby the acceptor passes from the place of higher concentration to the place of lower concentration.
  • the acceptor is internalized with the aid of an active transport.
  • the genetically modified microorganism contains transporter proteins, called permeases, which act as enzymes and with which the microorganism is able to admit exogenous substances and to concentrate them in the cytoplasm.
  • lactose permease acts specifically on galactose, simple galactosyl disaccharides such as lactose and their glycosides.
  • the specificity towards the sugar moiety of the exogenous carbohydrate acceptor to be internalized can be altered by mutation of the microorganism by means of conventional recombinant DNA manipulation techniques.
  • the internalization of exogenous lactose or its derivative takes place via an active transport mechanism mediated by a lactose permease.
  • the genetically modified microorganism preferably lacks any enzyme activity (such as LacZ) that would degrade the acceptor.
  • the microorganism is not able to hydrolyze or degrade the oligosaccharide product or any oligosaccharide intermediate in the biosynthetic pathway to the oligosaccharide product.
  • the genetically modified cell used in the process of the invention comprises one or more endogenous or recombinant genes encoding one or more glycosyl transferase enzymes that are able to transfer the glycosyl residue of an activated sugar nucleotide to the internalized acceptor.
  • the gene or an equivalent DNA sequence thereof, if it is recombinant, can be introduced into the cell by conventional techniques, e.g. using an expression vector or by chromosomal integration.
  • the origin of the heterologous nucleic acid sequence can be any animal (including human) or plant, eukaryotic cells such as those from Saccharomyces cerevisiae, Saccharomyces pombe, Candida albicans or from algae, prokaryotic cells such as those originated from E.
  • coli Bacteroides fragilis, Photo bacterium sp., Bacillus subtilis, Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Rhizobium meliloti, Neisseria gonorrhoeae and Neisseria meningitidis, or virus.
  • glycosyl transferase enzyme/enzymes encoded and expressed by the gene(s) or equivalent DNA sequence(s) are preferably glucosyl transferases, galactosyl transferases, N-acetylglucosaminyl transferases, N- acetylgalactosaminyl transferases, glucuronosyl transferases, xylosyl transferases, mannosyl transferases, fucosyl transferases, sialyl transferases and the like.
  • the glycosyl transferases are selected from the group consisting of b-1,3-N-acetylglucosaminyl- transferase, b-1,6-N-acetylglucosaminyl-transferase, b-1, 3-galactosyl-transferase, b-1 ,4- galactosyl-transferase, b-1 ,3-N-acetylgalactosaminyl-transferase, b-1 ,3-glucuronosyl- transferase, a-2, 3-sialyl-transferase, a-2, 6-sialyl-transferase, a-2, 8-sialyl-transferase, a-1,2- fucosyl-transferase, a-1,3-fucosyl-transferase and a-1,4-fucosyl-transfer
  • the glycosyl transferases are selected from b-1,3-N-acetylglucosaminyl transferase, b-1,6-N- acetylglucosaminyl transferase, b-1,3 ⁇ 3 ⁇ 80 ⁇ I transferase, b-1,4-galactosyl transferase, a- 2,3-sialyl transferase, a-2,6-sialyl transferase, a-1,2-fucosyl transferase, a-1,3-fucosyl transferase and a-1,4 fucosyl transferase, that is from those involved in the construction of HMO core structures as well as fucosylated and/or sialylated HMOs and its glycosidic derivatives, wherein the aglycon is a moiety defined above at the group of carbohydrate acceptors.
  • the genes encoding the above-mentioned transferases have been described
  • the genetically modified microorganism or cell used in the process of the invention is capable of producing an oligosaccharide from the internalized precursor/acceptor which is preferably lactose.
  • Said cell possesses enzymatic activity that is necessary for synthesizing an oligosaccharide, from an internalized mono- or disaccharide via consecutive glycosylation steps, wherein the internalized acceptor, e.g. lactose, is glycosylated, in a first glycosylation step, to a trisaccharide, then that trisaccharide is glycosylated, in a second glycosylating step, to a tetrasaccharide, and so forth.
  • the internalized acceptor e.g. lactose
  • glycosylation steps are mediated by respective glycosyl transferases.
  • the phrase “a glycosyl transferase which is able to transfer a glycosyl residue of an activated sugar nucleotide to an intermediate in the biosynthetic pathway to said oligosaccharide from said acceptor” or “a glycosyl transferase which can transfer a glycosyl residue of an activated sugar nucleotide to an intermediate in the biosynthetic pathway to said oligosaccharide from said acceptor” refers to the second, third etc. glycosylation step.
  • lacto-N-tetraose is made from lactose
  • a first glycosyl transferase (a b-1 ,3-N- acetyl-glucosaminyl transferase) transfers GlcNAc of UDP-GIcNAc to the internalized lactose to form lacto-N-triose II
  • a second glycosyl transferase (a b-1,3-galactosyl transferase) transfers Gal of UDP-Gal to the previously formed lacto-N-triose II (LNT-II) to form lacto-N- tetraose.
  • lacto-N-triose II is considered to be an intermediate in the biosynthetic pathway to LNT from lactose.
  • activated sugar nucleotides serve as donors, and the glycosyl transferase in question transfers a monosaccharide of an appropriate donor molecule to the acceptor molecule.
  • Each activated sugar nucleotide generally comprises a phosphorylated glycosyl residue attached to a nucleoside, and the specific glycosyl transferase enzyme accepts only the specific sugar nucleotide.
  • activated sugar nucleotides are involved in the glycosyl transfer: UDP-GIc, UDP-Gal, UDP-GIcNAc, UDP-GalNAc, UDP-glucuronic acid, UDP- Xyl, GDP-Man, GDP-Fuc and CMP-sialic acid, particularly those selected from the group consisting of UDP-Gal, UDP-GIcNAc, GDP-Fuc and CMP-sialic acid.
  • the genetically modified microorganism used in the process of this invention possesses a biosynthetic pathway to the above mentioned activated sugar nucleotides, that is, it has one or more sets of genes encoding one or more enzymes responsible for the synthesis of one or more activated glycosyl nucleotides, ready for glycosylation in glycosyl transferase mediated reaction in the cell, when cultured.
  • the sets of genes are either naturally present in the cell or introduced into the cell by means of recombinant DNA manipulation techniques.
  • the production of the activated glycosyl nucleotides by the cell takes place under the action of enzymes involved in the biosynthetic pathway of that respective sugar nucleotide stepwise reaction sequence starting from a carbon source (for a review for monosaccharide metabolism see e.g. H. H. Freeze and A. D. Elbein: Chapter 4: Glycosylation precursors, in: Essentials of Glycobiology, 2 nd edition (Eds. A. Varki et al.), Cold Spring Harbour Laboratory Press (2009)).
  • the production of the activated sugar nucleotides by the genetically modified microorganism via its own biosynthetic pathway is advantageous compared to in vitro versions of transfer glycosylation, as it avoids using the very expensive sugar nucleotide type donors added exogenously, hence the donors are formed by the cell in situ and the phosphatidyl nucleoside leaving groups are recycled in the cell.
  • the microorganism used in the process of the invention comprises genes encoding a sucrose utilization system, that is the cell has a capability to catabolically utilize sucrose as a carbon source, as well as an energy source.
  • the system that enables the cell to utilize sucrose can be one normally found in the gene pool of that cell, but preferably is a heterologous system (i.e. derived from a different organism and transferred to the host cell by conventional recombinant DNA manipulation techniques).
  • sucrose catabolism i) The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (“PTS”), where sucrose is transported into the microorganism and concomitantly phosphorylated to generate intracellular sucrose-6-phosphate which is hydrolysed to glucose-e- phosphate and fructose that are then involved in the central carbon metabolism of the cell.
  • PEP phosphoenolpyruvate
  • PTS phosphoenolpyruvate
  • sucrose is transported into the microorganism and concomitantly phosphorylated to generate intracellular sucrose-6-phosphate which is hydrolysed to glucose-e- phosphate and fructose that are then involved in the central carbon metabolism of the cell.
  • PTS can be encoded by scr or sac genes; ii) The non-phosphotransferase-dependent system (“non- PTS”), where extracellular sucrose enters the cell with the aid of a proton symport transport system (sucrose permease) and, after transport, is hydrolysed by an invertase enzyme to glucose and fructose followed by phosphorylation.
  • non- PTS The non-phosphotransferase-dependent system
  • sucrose permease proton symport transport system
  • the esc regulon consists of genes encoding the enzymes that are responsible for the non-PTS sucrose utilization.
  • the oligosaccharide-producing microorganism is fed with sucrose that provides energy via glycolysis for growing, reproducing and maintaining its structure.
  • sucrose taken up by the cell provides, via sucrose catabolism, precursors for the synthesis of the activated sugar nucleotide(s) necessary for the glycosylation process that takes place in the cell.
  • the internalized carbohydrate acceptor participates in the glycosyl transferase induced glycosylation reaction, in which a glycosyl residue of an activated nucleotide donor produced by the cell is transferred so that the acceptor is glycosylated.
  • the cell when more than one glycosyl transferase is expressed by the cell, additional glycosylation reactions can occur resulting in the formation of the target oligosaccharide.
  • the cell preferably lacks any enzyme activity which would degrade the oligosaccharide derivatives produced in the cell.
  • the sucrose utilization system is heterologous.
  • the microorganism preferably a bacterium, more preferably an E. coli
  • a sucrose uptake cassette should be introduced, using an appropriate expression plasmid or via chromosome integration, in the sucrose minus cell to make it be sucrose plus.
  • the sucrose pathway genes comprise a PTS- dependent sucrose utilization system, and especially the source regulon is scr.
  • Microorganisms having scr genes are for example Salmonella ssp., Klebsiella pneumoniae, Bacteroides fragilis, Vibrio alginolyticus.
  • the scr genes comprise the following: scrY, scrA, scrB, scrR and scrK.
  • the gene scrA codes for the sucrose transport protein Enzyme ll Scr that provides intracellular sucrose-6-phosphate from extracellular sucrose via an active transport through the cell membrane and the concomitant phosphorylation.
  • the sucrose specific ScrY porin (encoded by scrY) facilitate the sucrose diffusion through the outer membrane.
  • the ScrB invertase enzyme (encoded by scrB) splits the accumulated sucrose-6-phosphate by hydrolysis to glucose-6-phosphate and fructose.
  • a fructokinase ScrK (encoded by scrK) phosphorylates fructose to fructose-e- phosphate, however the presence of this enzyme is not crucial because the fructose can be phosphorylated by other mechanisms owned by the cell.
  • the repressor protein ScrR (encoded by scrR) negatively controls the expression of the scrYAB genes and is induced by sucrose or fructose.
  • the heterologous scr genes are introduced into the microorganism using plasmids, for example by a two-plasmid system where one contains the scrA gene and the other does the scrB gene.
  • the scrY, scrR and optionally the scrK gene can be carried by either plasmids.
  • the heterologous scr genes are introduced into the genome of the microorganism, that is they are inserted in a certain site of the genome (genetic locus) of said microorganism or cell, preferably operably linked to one or more control sequence(s) that is recognized by the host cell.
  • the genes coding for the glycosyl transferases are also inserted in the genome of the host cell.
  • the preferred oligosaccharide producing cells of the invention are plasmid-free.
  • the advantage of having a plasmid free cell is that the production of the oligosaccharide, such as an HMO is increased and carbohydrate by-products are decreased.
  • antibiotics are not added to the fermentation broth and neither during cell bank preparation and inoculum propagation in the process of some embodiments of this invention.
  • the carbohydrate acceptor to be glycosylated by the microorganism in the process of the invention can be a mono- or disaccharide selected from galactose, N-acetyl-glucosamine, a galactosylated monosaccharide, an N-acetyl-glucosaminylated monosaccharide, and glycosidic derivatives thereof defined above. All these carbohydrate derivatives can be easily taken up by a cell having a LacY permease by means of an active transport and accumulate in the cell before being glycosylated (WO 01/04341, Fort et al. J. Chem. Soc., Chem. Comm.
  • the cell is able to transport these carbohydrate acceptors into the cell using its LacY permease, and the cell lacks any enzymes that could degrade these acceptors, especially LacZ.
  • the cell has a deleted or deficient lacA gene on the lac operon.
  • the lad gene for the lac repressor is also deleted or deactivated in the microorganism. In the absence of the functioning repressor, no inducer is needed for any gene under the control of the promoter Plac.
  • the hlyE gene coding for the toxin cytolysin A, is deleted or deactivated in order to eliminate the risk of unintended expression of the toxin and thus to obtain a safer manufacturing strain.
  • the regulatory repressor gene glpR may be deleted or deactivated (see WO 2019/123324), which may result in a higher overall production of the product oligosaccharides, particularly HMOs.
  • the genetically modified cell used in the process of the invention is cultured in an aqueous culture medium in the following phases:
  • sucrose a second (feeding) phase with sucrose which is added continuously ensuring a limited cell growth that lasts until the sucrose and preferably most (e.g. at least 60 %) of the lactose have been consumed which is preferably at least 35 hours, such as at least 45 hours, 50 to 70 hours, or up to about 130 hours.
  • the exogenous carbohydrate acceptor preferably lactose, to be internalized by and glycosylated in the cell
  • the acceptor can be added in this second phase as a pure solid/liquid or in a form of a concentrated aqueous solution or suspension.
  • the oligosaccharide production takes place in this second phase and can take up to 6-7 days.
  • this feeding phase is performed under conditions allowing the production of a culture with a high cell density.
  • a feature of the process of some embodiments of this invention is that there is no need to change the carbon source and/or the energy source between the growth phase and the production phase of the microorganism.
  • the process further comprises the addition of an inducer to the culture medium to induce the expression in the cell of enzyme(s) and/or of protein(s) involved in the transport of the acceptor and/or in the glycosylation of the internalized acceptor and/or in the biosynthesis of the activated sugar nucleotide donors.
  • the inducer is preferably isopropyl b-D-thiogalactoside (IPTG) and is added to the culture medium in the beginning of the feeding phase.
  • IPTG isopropyl b-D-thiogalactoside
  • the use of inducer is not necessary if the cell is of Lad genotype.
  • microorganisms used in the process of invention and described above are highly stable under the process conditions of this invention described above.
  • these microorganisms can be used to produce oligosaccharides using sucrose as their carbon and energy sources at least at the same production rate as, and in a more reliable and reproducible manner than, microorganisms using glycerol and/or glucose as their carbon and/or energy sources.
  • some embodiments of the genetically modified cells used in the process of the invention produce less carbohydrate by-products (e.g.
  • the amount of other HMO components compared to the desired HMO are decreased by at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, when the genetically modified organism of the invention is grown on sucrose as compared to a similar microorganism which does not contain the PTS-dependant sucrose system and therefore is grown on glucose.
  • the DFL/2’-FL ratio is decreased by at least 10%, by at least 15%, such as at least 20%, such as at least 25% when the genetically modified microorganism of the present invention is grown on sucrose, compared to a similar microorganism which does not contain the PTS-dependant sucrose system and therefore is grown on glucose.
  • the use of the sucrose strains is therefore beneficial as it facilitates the purification process.
  • the HMOs made by the sucrose strains are extremely low in the sum of unspecified impurities. The reduced by-product formation provides a bigger upstream process optimisation potential, since the sucrose strain does not waste as much energy in the production.
  • the process of making the oligosaccharide, such as an HMO, using a modified microorganism according to the invention, and growing it on sucrose as carbon source results in an improved carbon/energy utilization compared to when glucose is used as carbon source.
  • the sucrose is more resistant to heat sterilization than e.g. monosaccharide carbon sources like glucose, and thus the HMOs produced by the sucrose strain do not contain such degradation by-products, in particular the HMO product produced by the process of the present invention is essentially free of isomaltose, kojibiose, gentiobiose and nigerose.
  • the oligosaccharide product has accumulated both in the intra- and the extracellular matrix of the microorganism.
  • the product has preferably been transported out of the cell to the supernatant in a passive way, i.e. it can diffuse outside across the cell membrane. This transport can be facilitated by one or more sugar efflux transporters in the cell, i.e. proteins that promote the effluence of sugar derivatives from the cell to the supernatant.
  • the sugar efflux transporter(s) can be homologous or heterologous and can be overexpressed under the conditions of the fermentation to enhance the export of the oligosaccharide derivative produced.
  • the specificity towards the sugar moiety of the product to be secreted can be altered by mutation of the cell by means of conventional recombinant DNA manipulation techniques and protein engineering.
  • the oligosaccharide accumulates in the extracellular matrix.
  • the oligosaccharide can be transported out of the cell to the supernatant by permealizing the cell or disrupting the cell walls in a conventional manner.
  • the oligosaccharide product can then be separated in a conventional manner from the aqueous culture medium, in which it was made by the cell.
  • a first step of separating the oligosaccharide from the culture medium preferably involves separating the oligosaccharide from the microorganism which produced it. This preferably involves clarifying the culture medium to remove suspended particulates and contaminants, particularly cells, cell components, insoluble metabolites and debris produced by culturing the genetically modified microorganism.
  • the aqueous culture medium which contains the oligosaccharide product, can be clarified in a conventional manner.
  • the culture medium is clarified by centrifugation and/or microfiltration and/or ultrafiltration.
  • a second step of separating the oligosaccharide from the culture medium preferably involves removing substantially all the proteins, as well as peptides, amino acids, RNA and DNA and any endotoxins and glycolipids that could interfere with the subsequent separation step, from the aqueous culture medium, preferably after it has been clarified.
  • proteins and related impurities can be removed from the culture medium in a conventional manner.
  • proteins and related impurities are removed from the culture medium by ultrafiltration, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration (i.e., size exclusion chromatography), particularly by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography.
  • size exclusion chromatography proteins and related impurities are retained by a chromatography medium or a selected membrane, while the oligosaccharide product remains in the aqueous culture medium.
  • the oligosaccharide product in the aqueous culture medium can then be separated from sugar-like by-product(s) and from the culture medium, after proteins and related impurities have been removed from the culture medium.
  • This can be suitably done by subjecting the culture medium to chromatographic separation.
  • This separation can be carried out, in case of a neutral oligosaccharide product, in a chromatographic separation column(s), filled with a conventional cationic and/or anionic ion exchange resin.
  • the acidic cationic ion exchange resin can be in monovalent or divalent cationic form and is preferably in H + , K + , Na + , Mg 2+ or Ca 2+ form, particularly Ca 2+ .
  • the chromatographic separation can be carried out in a conventional manner at a pH of the solution of 2 to 9.
  • the eluent used in the chromatographic separation is preferably water, especially demineralized water, but aqueous salt solutions can also be used. Alcohols, such as ethanol, and aqueous alcohol mixtures can also be used. Treatment with ion exchange resins removes charged particles and salts, too.
  • the aqueous solution containing said oligosaccharide can be treated with active charcoal, preferably to remove coloured bodies from the solution.
  • the process of this invention for producing an oligosaccharide preferably having a lactose unit at the reducing end or a glycoside thereof comprises the steps of:
  • the cell also comprises a heterologous sucrose utilization system, preferably a PTS-dependent sucrose utilization system, especially where the source regulon is scr, to provide sucrose as a carbon source for biosynthesis of said activated sugar nucleotide by said cell.
  • a heterologous sucrose utilization system preferably a PTS-dependent sucrose utilization system, especially where the source regulon is scr, to provide sucrose as a carbon source for biosynthesis of said activated sugar nucleotide by said cell.
  • the genetically modified cell used in this preferred process, can have more than one recombinant gene, encoding more than one glycosyl transferase enzyme which is able to transfer the glycosyl residue of an activated sugar nucleotide to an internalized acceptor molecule or the previously glycosylated acceptor made by the same cell, so the oligosaccharide product is formed from the internalized acceptor by multiple glycosyl transfer mediated by multiple glycosyl transferases expressed by the cell. Accordingly, the resulting oligosaccharide product can be a glycosylated lactose or a glycoside thereof.
  • the glycosylated lactose is preferably an N-acetyl-glucosaminylated, galactosylated, fucosylated and/or sialylated lactose.
  • the cell comprises one or more recombinant genes encoding an N-acetyl-glucosaminyl transferase, a galactosyl transferase, a sialyl transferase and/or a fucosyl transferase, and also comprise a biosynthetic pathway to generate the corresponding activated sugar type nucleotides, that is UDP-GIcNAc, UDP-Gal, GDP-Fuc and/or CMP-sialic acid, used as sugar doners for the transferases.
  • the oligosaccharide product made by this process is characterized by formula 1
  • Y is OH or a non-sugar aglycon defined above, preferably OH,
  • Ri is fucosyl or H
  • R 2 is fucosyl or H
  • R 3 is selected from H, sialyl, N-acetyl-lactosaminyl and lacto-N-biosyl groups, wherein the N-acetyl-lactosaminyl group can carry a glycosyl residue comprising one or more N- acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; each of the N-acetyl- lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,
  • R 4 is selected from H, sialyl and N-acetyl-lactosaminyl groups optionally substituted with a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto- N-biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue, provided that at least one of the Ri, R 2 , R 3 and R 4 groups is different from H.
  • the compound of formula 1 made by this process can be characterized by formula 1a, 1b or 1c wherein Y, Ri and R 2 are as defined above, preferably OH,
  • R 3a is an N-acetyl-lactosaminyl group optionally substituted with a glycosyl residue comprising one N-acetyl-lactosaminyl and/or one lacto-N-biosyl group; each of the N- acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,
  • R 4a is H or an N-acetyl-lactosaminyl group optionally substituted with a lacto-N-biosyl group; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,
  • R 3b is a lacto-N-biosyl group optionally substituted with one or more sialyl and/or fucosyl residue(s),
  • R 4b is H or an N-acetyl-lactosaminyl group optionally substituted with one or two N-acetyl- lactosaminyl and/or one lacto-N-biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residues,
  • R 5 is, independently, H or sialyl, and wherein at least one of Ri, R 2 or R 5 is not H.
  • N-acetyl-lactosaminyl group in the glycosyl residue of R 3a is attached to another N- acetyl-lactosaminyl group with a 1-3 interglycosidic linkage
  • lacto-N-biosyl group in the glycosyl residue of R3 a is attached to the N-acetyl- lactosaminyl group with a 1-3 interglycosidic linkage
  • the lacto-N-biosyl group in the glycosyl residue of F a is attached to the N-acetyl- lactosaminyl group with a 1-3 interglycosidic linkage
  • N-acetyl-lactosaminyl group in the glycosyl residue is attached to another N- acetyl-lactosaminyl group with a 1-3 or a 1-6 interglycosidic linkage
  • the lacto-N-biosyl group in the glycosyl residue of is attached to the N-acetyl- lactosaminyl group with a 1-3 interglycosidic linkage.
  • the compounds according to formulae 1a, 1b and 1c made by the process are human milk oligosaccharides (when Y is OH) or glycosides thereof (when Y is non sugar aglycon).
  • the preferred compounds of formula 1a made by the process are selected from lacto-N- neotetraose (LNnT), para-lacto-N-hexaose (pLNH), para-lacto-N-neohexaose (pLNnH), lacto-N- neohexaose (LNnH), para-lacto-N-octaose (pLNO), lacto-N-neooctaose (pLNnO) and glycosides thereof, all of which can optionally be substituted with one or more sialyl and/or fucosyl residue.
  • the preferred compounds of formula 1b made by the process is selected from lacto-N-tetraose (LNT), lacto-N-hexaose (LNH), lacto-N-octaose (LNO), iso-lacto-N-octaose (iLNO), lacto-N-decaose (LND), lacto-N-neodecaose (LNnD) and glycosides thereof, all of them can optionally be substituted with one or more sialyl and/or fucosyl residue.
  • the compounds of subformulae 1a, 1b or 1c are selected from the group of: 2’-fucosyllactose (2’-FL), 3-fucosy I lactose (3-FL), 2’, 3- difucosyllactose (2’,3-DFL), 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), 3’-sialyl-3- fucosyllactose (3’S3FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N- fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto
  • HMO human milk oligosaccharide
  • the HMOs consist of a lactose unit at the reducing end and one or more from the following monosaccharide units: N-acetyl-glucosamine, galactose, fucose and sialic acid (see Urashima et al.: Milk Oligosaccharides, Nova Biomedical Books, New York, 2011, ISBN: 978-1-61122- 831-1; Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)).
  • the cell then comprises one or more recombinant genes encoding b-1,3-N-acetyl-glucosaminyl transferase, b-1,6-N-acetyl-glucosaminyl transferase, b-1,3 ⁇ 3 ⁇ 80 ⁇ I transferase, b-1,4- galactosyl transferase, a-2,3-sialyl transferase, a-2,6-sialyl transferase, a-1 ,2-fucosyl transferase, a-1,3-fucosyl transferase and/or a-1, 4 fucosyl transferase, and also comprise a biosynthetic pathway to produce the corresponding activated sugar type nucleotides, that is UDP-GIcNAc, UDP-Gal, GDP-Fuc and/or CMP-sialic acid.
  • the process of the invention relates to the production of neutral (non-acidic) HMOs.
  • the neutral HMOs do not contain sialic acid residue in their structure and therefore can be non-fucosylated neutral HMOs (referred to as N-acetylated HMOs or core HMOs) and fucosylated HMOs.
  • the N-acetylated HMOs has only three types of monosaccharide in their structure: glucose, galactose and N-acetyl-glucosamine, and the N- acetylated HMOs are preferably selected from the group consisting of lacto-N-neotetraose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-neohexaose, para-lacto-N-octaose, lacto-N-neooctaose, lacto-N-tetraose, lacto-N-hexaose, lacto-N-octaose, iso-lacto-N-octaose, lacto-N-decaose and lacto-N-neodecaose, more preferably lacto-N-neotetraos
  • the group of fucosylated neutral HMOs encompasses N-acetylated HMOs disclosed above that are fucosylated and difucosylated lactoses (2’-FL, 3-FL, DFL). According, the process, in a preferred embodiment, relates to the production of neutral HMOs comprising:
  • a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to the internalized lactose or to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc,
  • the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc and/or GDP-Fuc, and optionally UDP-Gal by the cell, and as energy source for making said neutral HMO.
  • a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc and/or GDP-Fuc, and optionally UDP-Gal by the cell, and as energy source for making said neutral HMO.
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, and particularly does not contain scrK.
  • the process of this invention for producing an N- acetylated HMO preferably of 3-6 monosaccharide units, comprises the steps of:
  • a recombinant gene encoding a N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to internalized lactose or to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose
  • a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to the N-acetyl-glucosaminylated lactose to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose
  • a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to the N-acetyl-glucosaminylated lactose to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose
  • the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc and optionally UDP-Gal by the cell, and as energy source for making said N-acetylated HMO.
  • a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc and optionally UDP-Gal by the cell, and as energy source for making said N-acetylated HMO.
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, and particularly does not contain scrK.
  • the product is preferably lacto-N-triose II, and if a b-1,3- or a b-I, ⁇ qIqo ⁇ I transferase is also present in the cell, the product is preferably LNT or LNnT, respectively.
  • the process of this invention for producing a fucosylated N-acetylated HMO comprises the steps of:
  • a recombinant gene encoding a N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-GIcNAc;
  • a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc, and
  • a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-Gal,
  • the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc, GDP-Fuc and optionally UDP-Gal by the cell, and as energy source for making said fucosylated N-acetylated HMO.
  • a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc, GDP-Fuc and optionally UDP-Gal by the cell, and as energy source for making said fucosylated N-acetylated HMO.
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, and particularly does not contain scrK.
  • the process of this invention for producing a fucosylated lactose comprises the steps of:
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence and particularly does not contain scrK.
  • the product is preferably 2’- fucosyllactose (2’-FL)
  • the fucosyl transferase is an a-1,3-fucosyl transferase
  • the product is preferably 3-fucosyllactose (3-FL)
  • DFL difucosyllactose
  • the process of this invention for producing a sialylated HMO preferably of 3-5 monosaccharide units, comprises the steps of:
  • a recombinant gene encoding a sialyl transferase enzyme which is able to transfer the sialyl residue of CMP-sialic acid to internalized lactose or to an intermediate in the biosynthetic pathway to said sialylated HMO from lactose, and
  • the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of CMP-sialic acid by the cell, and as energy source for making said sialylated HMO.
  • a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of CMP-sialic acid by the cell, and as energy source for making said sialylated HMO.
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence, and particularly does not contain scrK.
  • the product is preferably 3’-sialyllactose
  • the sialyl transferase is an a-2,6-sialyl transferase
  • the product is preferably 6’- sialyllactose
  • the process of this invention for producing a fucosylated and sialylated HMO, preferably of 3-5 monosaccharide units comprises the steps of:
  • - a recombinant gene encoding a sialyl transferase enzyme which is able to transfer the sialyl residue of CMP-sialic acid to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated and sialylated HMO from lactose
  • - a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated and sialylated HMO from lactose
  • a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated and sialylated HMO from lactose
  • the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of CMP-sialic acid and GDP-Fuc by the cell, and as energy source for making said fucosylated and sialylated HMO.
  • a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of CMP-sialic acid and GDP-Fuc by the cell, and as energy source for making said fucosylated and sialylated HMO.
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence, and particularly does not contain scrK.
  • the product is preferably 3’-sialyl-3-fucosyllactose.
  • the process of this invention for producing a fucosylated LNT or LNnT comprises the steps of:
  • the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc, UDP-Gal and GDP-Fuc by the cell, and as energy source for making said fucosylated LNT or LNnT.
  • a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc, UDP-Gal and GDP-Fuc by the cell, and as energy source for making said fucosylated LNT or LNnT.
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectably or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence, and particularly does not contain scrK.
  • a second aspect of the invention relates to providing a genetically modified microorganism that can internalize sucrose and a carbohydrate acceptor, which is not sucrose, preferably lactose, or a glycoside thereof, into said microorganism and that comprises: a recombinant gene encoding a glycosyl transferase which can transfer a glycosyl residue of an activated sugar nucleotide to the acceptor within the microorganism, a biosynthetic pathway for making the activated sugar nucleotide from sucrose, and one or more genes encoding a heterologous PTS-dependent sucrose utilization system, preferably scr genes, so that said cell can use sucrose as a carbon source, preferably the main carbon source, more preferably the sole carbon source, for making said activated sugar nucleotide and as an energy source, preferably the main energy source, more preferably the sole energy source, thereby making an oligosaccharide or a glycoside thereof comprising the internalized carbohydrate acceptor
  • the aglycon moieties of the glycosides of the internalized carbohydrate acceptors or the oligosaccharide products are disclosed in the first aspect.
  • the genetically modified microorganism or cell of this invention can be selected from the group consisting of bacteria and yeasts, preferably a bacterium.
  • Bacteria are preferably selected from the group of: Escherichia coii, Bacillus spp. (e.g.
  • Bacillus subtilis Bacillus subtilis), Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Neisseria gonorrhoeae, Neisseria meningitis, Lactobacillus spp., Lactococcus spp., Enterococcus spp., Bifidobacterium spp., Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., Pseudomonas, among which E.
  • the microorganism or cell preferably an E.coli cell
  • the microorganism or cell is genetically modified so that there is at least one alteration in its DNA sequence.
  • the alteration can result in a change in the original characteristics of the wild type cell, e.g. the modified cell is able to perform additional chemical transformation due to the introduced new genetic material that encodes the expression of an enzymes not being in the wild type cell, or is not able to carry out transformation like degradation due to removal or deactivation of gene/genes.
  • a genetically modified cell contains at least one artificial alteration in its genome compared to its naturally occurring (wild type) variant.
  • a nucleic acid construct is added to the cell by way of integration into the genome or by addition via plasmid, or a nucleic acid sequence is deleted from or changed in the genome of the cell.
  • the so-transformed cell has a genotype that is different from that before the alteration and, therefore, the modified cell shows modified feature(s).
  • the genetically modified cell can perform at least one additional or altered biochemical reaction, when cultured or fermented, due to the introduction of a heterologous nucleic acid sequence or the modification of a native nucleic acid sequence that encodes an enzyme that is not expressed in the wild type cell, or the genetically modified cell cannot perform a biochemical reaction due to the deletion, addition or modification of a nucleic acid sequence that encodes an enzyme found in the wild type cell.
  • the genetically modified cell can be constructed by well-known, conventional genetic engineering techniques (e.g. Green and Sambrook: Molecular Cloning: A laboratory Manual, 4 th ed., Cold Spring Harbor Laboratory Press (2012); Current protocols in molecular biology (Ausubel et al. eds.), John Wiley and Sons (2010)).
  • the terms “host cell”, “recombinant microorganism or cell” or “genetically modified microorganism or cell” are used interchangeably to designate such constructs.
  • promoter means a nucleic acid sequence involved in the binding of RNA polymerase to initiate transcription of an operably linked gene, wherein the gene includes a coding DNA sequence and other (non-coding) sequences, e.g. the 5’-untranslated region (5’- UTR) located upstream of the coding sequence, which comprises a ribosomal binding site.
  • a promoter in some embodiments of this invention is an isolated DNA sequence, i.e. not an integrated DNA fragment of the genomic DNA.
  • the nucleotide sequence of a promoter corresponds to, or have at least 80 % identity, preferably 90-99.9 % identity with the nucleotide sequence of a fragment of bacterial genomic DNA that is regarded as promoter region of a gene, e.g. a promoter region of a glp operon or lac operon of E.coli.
  • promoter region of a gene e.g. a promoter region of a glp operon or lac operon of E.coli.
  • glp operon is meant a cluster of genes involved in the respiratory metabolism of glycerol of bacteria.
  • a glp operon promoter sequence comprised in a DNA construct corresponds to or has at least 80 % identity, preferably 90-99.9 % identity with the nucleotide sequence of a fragment of the genomic DNA regarded as a promoter region of the corresponding glp operon of E. coir, in particular, the isolated sequence of a promoter of a glp operon of some embodiments of the invention corresponds to, or has said percent of identity with a fragment of the genomic sequence upstream the sequence having GenBank ID:
  • E. coli genome is referred herein to the complete genomic DNA sequence of E coli K-12 MG1655 (GenBank ID:U00096.3).
  • a promoter of the glp operon is operably linked to the heterologous glycosyl transferase genes and/or the heterologous scr genes.
  • those genes are expressed under a glpF promoter described in WO 2019/123324, the content of which is incorporated herein by reference, even more preferably by the glpF promoter of SEQ ID NO: 5.
  • the genetically modified microorganism or cell of this invention is able to transport the exogenous carbohydrate acceptor, preferably lactose, into the cell for glycosylation to produce a foreign oligosaccharide of interest, preferably without adversely affecting the basic functions of the cell or destroying its integrity.
  • the transport takes place via a passive mechanism, during which the exogenous acceptor diffuses passively across the plasma membrane of the cell. Diffusion of the acceptor into the microorganism is a function of the concentration differences between the fermentation broth and the extra- and intracellular space of the cell with respect to the acceptor, whereby the acceptor passes from the place of higher concentration to the place of lower concentration.
  • the acceptor is internalized with the aid of an active transport.
  • the genetically modified microorganism contains transporter proteins, called permeases, which act as enzymes and with which the microorganism is able to admit exogenous substances and to concentrate them in the cytoplasm.
  • permeases transporter proteins
  • lactose permease (LacY) acts specifically on galactose, simple galactosyl disaccharides such as lactose and their glycosides.
  • the specificity towards the sugar moiety of the exogenous carbohydrate acceptor to be internalized can be altered by mutation of the microorganism by means of conventional recombinant DNA manipulation techniques.
  • the internalization of exogenous lactose or its derivative takes place via an active transport mechanism mediated by a lactose permease.
  • the genetically modified microorganism preferably lacks any enzyme activity (such as LacZ) that would degrade the acceptor. Likewise, the microorganism is not able to hydrolyze or degrade the oligosaccharide product or any oligosaccharide intermediate in the biosynthetic pathway to the oligosaccharide product.
  • any enzyme activity such as LacZ
  • the genetically modified cell of the invention comprises one or more endogenous or recombinant genes encoding one or more glycosyl transferase enzymes that are able to transfer the glycosyl residue of an activated sugar nucleotide to the internalized acceptor.
  • the gene or an equivalent DNA sequence thereof if it is recombinant, can be introduced into the cell by conventional techniques, e.g. using an expression vector or by chromosomal integration.
  • the origin of the heterologous nucleic acid sequence can be any animal (including human) or plant, eukaryotic cells such as those from Saccharomyces cerevisae, Saccharomyces pombe,
  • Candida albicans or from algae prokaryotic cells such as those originated from E. coli, Bacteroides fragilis, Photo bacterium sp. , Bacillus subtilis, Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Rhizobium meliloti, Neisseria gonorrhoeae and Neisseria meningitidis, or virus.
  • glycosyl transferase enzyme/enzymes encoded and expressed by the gene(s) or equivalent DNA sequence(s) are preferably glucosyl transferases, galactosyl transferases, N-acetylglucosaminyl transferases, N- acetylgalactosaminyl transferases, glucuronosyl transferases, xylosyl transferases, mannosyl transferases, fucosyl transferases, sialyl transferases and the like.
  • the glycosyl transferases are selected from the group consisting of b-1,3-N-acetylglucosaminyl- transferase, b-1,6-N-acetylglucosaminyl-transferase, b-1, 3-galactosyl-transferase, b-1 ,4- galactosyl-transferase, b-1 ,3-N-acetylgalactosaminyl-transferase, b-1 ,3-glucuronosyl- transferase, a-2, 3-sialyl-transferase, a-2, 6-sialyl-transferase, a-2, 8-sialyl-transferase, a-1,2- fucosyl-transferase, a-1,3-fucosyl-transferase and a-1,4-fucosyl-transfer
  • the glycosyl transferases are selected from b-1,3-N-acetylglucosaminyl transferase, b-1,6-N- acetylglucosaminyl transferase, b-1,3 ⁇ 3 ⁇ 80 ⁇ I transferase, b-I, ' 4-galactosyl transferase, a- 2,3-sialyl transferase, a-2,6-sialyl transferase, a-1,2-fucosyl transferase, a-1,3-fucosyl transferase and a-1,4 fucosyl transferase, that is from those involved in the construction of HMO core structures as well as fucosylated and/or sialylated HMOs and its glycosidic derivatives, wherein the aglycon is a moiety defined above at the group of carbohydrate acceptors.
  • the genes encoding the above-mentioned transferases have
  • the genetically modified microorganism or cell of the invention is capable of producing an oligosaccharide from an internalized precursor/acceptor which is preferably lactose and not sucrose.
  • Said cell possesses enzymatic activity that is necessary for synthesizing an oligosaccharide, from an internalized mono- or disaccharide, or a glycoside thereof, via consecutive glycosylation steps, wherein the internalized acceptor, e.g. lactose, is glycosylated, in a first glycosylation step, to a trisaccharide, then that trisaccharide is glycosylated, in a second glycosylating step, to a tetrasaccharide, and so forth.
  • the glycosylation steps are mediated by respective glycosyl transferases.
  • activated sugar nucleotides serve as donors, and the glycosyl transferase in question transfers a monosaccharide of an appropriate donor molecule to the acceptor molecule.
  • Each activated sugar nucleotide generally comprises a phosphorylated glycosyl residue attached to a nucleoside, and the specific glycosyl transferase enzyme accepts only the specific sugar nucleotide.
  • activated sugar nucleotides are involved in the glycosyl transfer: UDP-GIc, UDP-Gal, UDP- GlcNAc, UDP-GalNAc, UDP-glucuronic acid, UDP-Xyl, GDP-Man, GDP-Fuc and CMP-sialic acid, particularly those selected from the group consisting of UDP-Gal, UDP-GIcNAc, GDP-Fuc and CMP-sialic acid.
  • the genetically modified microorganism of this invention possesses a biosynthetic pathway to the above mentioned activated sugar nucleotides, that is, it has one or more sets of genes encoding one or more enzymes capable of the synthesis of one or more activated glycosyl nucleotides, ready for glycosylation in glycosyl transferase mediated reaction in the cell, when cultured.
  • the sets of genes are either naturally present in the cell or introduced into the cell by means of recombinant DNA manipulation techniques.
  • the production of the activated glycosyl nucleotides by the cell takes place under the action of enzymes involved in the biosynthetic pathway of that respective sugar nucleotide stepwise reaction sequence starting from a carbon source (for a review for monosaccharide metabolism see e.g. H. H. Freeze and A. D. Elbein: Chapter 4: Glycosylation precursors, in: Essentials of Glycobiology, 2 nd edition (Eds. A. Varki et al.), Cold Spring Harbour Laboratory Press (2009)).
  • the production of the activated sugar nucleotides by the genetically modified microorganism via its own biosynthetic pathway is advantageous compared to in vitro versions of transfer glycosylation, as it avoids using the very expensive sugar nucleotide type donors added exogenously, hence the donors are formed by the cell in situ and the phosphatidyl nucleoside leaving groups are recycled in the cell.
  • the microorganism of the invention comprises genes encoding a sucrose utilization system, that is the cell has a capability to catabolically utilize sucrose as a carbon source, as well as an energy source.
  • the system that enables the cell to utilize sucrose can be one normally found in the gene pool of that cell but preferably is a heterologous system (i.e. derived from a different organism and transferred to the host cell by conventional recombinant DNA manipulation techniques).
  • a heterologous system i.e. derived from a different organism and transferred to the host cell by conventional recombinant DNA manipulation techniques.
  • two kinds of sucrose catabolism can be used.
  • sucrose is transported into the microorganism and concomitantly phosphorylated to generate intracellular sucrose-6-phosphate which is hydrolysed to glucose-6-phosphate and fructose that are then involved in the central carbon metabolism of the cell.
  • PTS can be encoded by scr or sac genes.
  • non-PTS extracellular sucrose enters the cell with the aid of a proton symport transport system (sucrose permease) and, after transport, is hydrolysed by an invertase enzyme to glucose and fructose followed by phosphorylation.
  • the esc regulon consists of genes encoding the enzymes that are responsible for the non-PTS sucrose utilization.
  • the sucrose utilization system is heterologous.
  • the microorganism preferably a bacterium, more preferably an E. coli, such as K-12 or BL21
  • a sucrose uptake cassette should be introduced, using an appropriate expression plasmid or via chromosome integration, in the sucrose minus cell to make it be sucrose plus.
  • the sucrose pathway genes comprise a PTS-dependent sucrose utilization system, and especially the source regulon is scr.
  • Microorganisms having scr genes are for example Salmonella ssp., Klebsiella pneumoniae, Bacteroides fragilis, Vibrio alginolyticus.
  • the scr genes comprise the following: scrY, scrA, scrB, scrR and scrK.
  • the gene scrA codes for the sucrose transport protein Enzyme ll Scr that provides intracellular sucrose-6-phosphate from extracellular sucrose via an active transport through the cell membrane and the concomitant phosphorylation.
  • the sucrose specific ScrY porin (encoded by scrY) facilitate the sucrose diffusion through the outer membrane.
  • the ScrB invertase enzyme (encoded by scrB) splits the accumulated sucrose-6-phosphate by hydrolysis to glucose-6-phosphate and fructose.
  • a fructokinase ScrK (encoded by scrK) phosphorylates fructose to fructose-e- phosphate, however the presence of this enzyme is not crucial because the fructose can be phosphorylated by other mechanisms owned by the cell.
  • the repressor protein ScrR (encoded by scrR) negatively controls the expression of the scrYAB genes and is induced by sucrose or fructose.
  • the heterologous scr genes are introduced into the microorganism using plasmids, for example by a two-plasmid system where one contains the scrA gene and the other plasmid contains the scrB gene.
  • the scrY, scrR and optionally the scrK gene can be carried by either plasmids.
  • the scr genes are selected from the sequences listed in table 1.
  • the scrY and scrA is on one plasmid and encodes the polypeptides of WP_004147437.1 (SEQ ID NO: 1) and AHM80898.1 (SEQ ID NO: 2), respectively, and the scrB and scrR is on the second plasmid encodes the polypeptides of CAA47974.1 (SE ID NO:
  • the genetically modified microorganism comprises 4 genes which encode PTS-dependent sucrose utilization system, where the genes encode i) sucrose porin scrY of SEQ ID NO: 1 or a homologue thereof which is at least 85% identical to SEQ ID NO: 1, such as 90%, 95% or 98% identical to SEQ ID NO: 1; and ii) PTS permease scrA of SEQ ID NO: 2 or a homologue thereof which is at least 85% identical to SEQ ID NO: 2, such as 90%, 95% or 98% identical to SEQ ID NO: 2; and iii) sucrose-6-phosphate hydrolase scrB of SEQ ID NO: 3 or a homologue thereof which is at least 85% identical to SEQ ID NO: 3, such as 90%, 95% or 98% identical to SEQ ID NO: 3; and iv) repressor protein scrR of SEQ ID NO: 4 or a homologue thereof which is at least 85% identical to SEQ ID NO: 4 such as 90%, 95% or 98% identical to SEQ ID NO: 1
  • the heterologous scr genes are introduced into the genome of the microorganism, that is they are chromosomally integrated by insertion in a certain site of the genome (genetic locus) of said microorganism or cell, preferably operably linked to one or more control sequence(s) that is recognized by the host cell.
  • scrY and scrA are operably linked to one promoter element (an operon) and scrB and scrR is operably linked to another promoter element.
  • the sequence of the two promoter elements may be identical for the two operons.
  • the promoter element is pglpF, in particular the pglpF promoter of SEQ ID NO: 5.
  • Other pglpF promoters are described in WO2019/123324, a preferred pglpF promoter is the one of SEQ ID NO:5.
  • the scr genes are introduced into the mixed acid fermentation pathway.
  • Mixed acid fermentation results in a mixture of end products including lactate, acetate, succinate, formate and ethanol.
  • the formation of these end products is driven by known pathways into which the scr genes can be introduced.
  • the genes coding for the glycosyl transferases are also inserted in the genome of the host cell (that is, chromosomally integrated).
  • oligosaccharide preferably the HMO
  • the genetically modified cell of the invention it is (they are) also inserted in the genome of the host cell.
  • the most preferred oligosaccharide producing cells of the invention are plasmid-free.
  • control sequences or promoters operably linked to heterologous DNA sequences encoding the glycosyl transferases necessary for making the oligosaccharide product or the proteins necessary for the sucrose uptake can be any suitable promoter.
  • a promoter of the glp operon is used, preferably glpF or its variant (WO 2019/123324, the content of which is incorporated herein by reference).
  • the carbohydrate acceptor to be glycosylated by the microorganism of the invention can be a mono- or disaccharide selected from galactose, N-acetyl-glucosamine, a galactosylated monosaccharide, an N-acetyl-glucosaminylated monosaccharide, a lactose, and glycosidic derivatives thereof defined above. All these carbohydrate derivatives can be easily taken up by a cell having a LacY permease by means of an active transport and accumulate in the cell before being glycosylated (WO 01/04341, Fort et al. J. Chem. Soc., Chem. Comm.
  • the cell is able to transport these carbohydrate acceptors into the cell using its LacY permease, and the cell lacks any enzymes that could degrade these acceptors, especially LacZ, this can be achieved by deleting or mutating the lacz gene to render it incapable of expressing functional LacZ.
  • the cell has a deleted or deficient lacA gene on the lac operon.
  • the lad gene for the lac repressor is also deleted or deactivated in the microorganism. In the absence of the functioning repressor, no inducer is needed for expressing any gene under the control of the promoter Plac.
  • the hlyE gene coding for the toxin cytolysin A, is deleted or deactivated in order to eliminate the risk of unintended expression of the toxin and thus to obtain a safer manufacturing strain.
  • the regulatory repressor gene glpR may be deleted or deactivated (WO 2019/123324), which may result in a higher overall production of the product oligosaccharides, particularly HMOs.
  • microorganisms of invention described above are highly stable under the process conditions of this invention described above. As a result, it is believed that these microorganisms can be used to produce oligosaccharides using sucrose as their carbon and energy sources at least at the same production rate as, and in a more reliable and reproducible manner than microorganisms using glycerol and/or glucose as their carbon and/or energy sources.
  • the genetically modified cells used in the process of the invention produce less carbohydrate by-products while the HMO titre and yield can be at least retained or even slightly increased, compared to the glucose or glycerol utilizing strains.
  • the use of the sucrose strains is therefore beneficial as it facilitates the purification process.
  • the HMOs made by the sucrose strains are extremely low in the sum of unspecified impurities.
  • the reduced by-product formation provides a bigger upstream process optimisation potential, since the sucrose strain does not waste as much energy in the production.
  • the sucrose is more resistant to heat sterilization than e.g. monosaccharide carbon sources like glucose, and thus the HMOs produced by the sucrose strain do not contain such degradation by-products.
  • the HMO product is free of isomaltose, kojibiose, gentiobiose and nigerose.
  • the genetically modified cell of the invention can have more than one recombinant gene, encoding more than one glycosyl transferase enzyme which is able to transfer the glycosyl residue of an activated sugar nucleotide to an internalized acceptor molecule or the previously glycosylated acceptor made by the same cell, so the oligosaccharide product is formed from the internalized acceptor by multiple glycosyl transfer mediated by multiple glycosyl transferases expressed by the cell. Accordingly, the resulting oligosaccharide product can be a glycosylated lactose or a glycoside thereof.
  • the glycosylated lactose is preferably an N-acetyl- glucosaminylated, galactosylated, fucosylated and/or sialylated lactose.
  • the cell comprises one or more recombinant genes encoding an N-acetyl- glucosaminyl transferase, a galactosyl transferase, a sialyl transferase and/or a fucosyl transferase, and also comprise a biosynthetic pathway to the corresponding activated sugar type nucleotides, that is UDP-GIcNAc, UDP-Gal, GDP-Fuc and/or CMP-sialic acid.
  • the genetically modified E. coli cell is suitable for producing a neutral HMO, preferably of 3-6 monosaccharide units, and comprises
  • a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to internalized lactose or to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc,
  • a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc and/or GDP-Fuc, and optionally UDP-Gal by the cell, and as energy source for making said neutral HMO.
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence and particularly does not contain scrK.
  • the genetically modified E. coli cell suitable for producing an N-acetylated HMO comprises: - a recombinant gene encoding a N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to internalized lactose or to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose,
  • a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to the N-acetyl-glucosaminylated lactose or to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose, and
  • the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc and optionally UDP-Gal by the cell, and as energy source for making said N- acetylated HMO.
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence, and particularly does not contain scrK.
  • the product is preferably lacto-N-triose II, and if a b-1,3- or a b-1,4 ⁇ 3 ⁇ 80 ⁇ I transferase is also present in the cell, the product is preferably LNT or LNnT, respectively.
  • the genetically modified E. coli cell is suitable for producing a fucosylated N-acetylated HMO, preferably LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II or LNDFH-III, and comprises:
  • a recombinant gene encoding an N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-GIcNAc;
  • a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc, and
  • a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-Gal, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc, GDP-Fuc and UDP-Gal by the cell, and as energy source for making said fucosylated N-acetylated HMO.
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence and particularly does not contain scrK.
  • the genetically modified E. coli cell is suitable for producing a fucosylated HMO, preferably 2’-FL, 3-FL or DFL, and comprises:
  • the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of GDP-Fuc by the cell, and as energy source for making said fucosylated HMO.
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence and particularly does not contain scrK.
  • the product is preferably 2’- fucosyllactose
  • the fucosyl transferase is an a-1,3-fucosyl transferase
  • the product is preferably 3-fucosyllactose
  • both a-1,2- and a-1,3-fucosyl transferases are expressed in the cell, the product is preferably difucosyllactose.
  • the genetically modified E. coli cell suitable for producing a sialylated HMO preferably of 3-5 monosaccharide units, more preferably a sialylated lactose, comprises:
  • a recombinant gene encoding a sialyl transferase enzyme which is able to transfer the sialyl residue of CMP-sialic acid to internalized lactose or to an intermediate in the biosynthetic pathway to said sialylated HMO from lactose, and
  • the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of CMP-sialic acid by the cell, and as energy source for making said sialylated HMO.
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence, and particularly does not contain scrK.
  • the product is preferably 3’-sialyllactose
  • the sialyl transferase is an a-2,6-sialyl transferase
  • the product is preferably 6’- sialyllactose
  • the genetically modified E. coli cell suitable for producing a fucosylated and sialylated HMO, preferably of 3-5 monosaccharide units, more preferably a sialyl-fucosyl-lactose comprises:
  • a recombinant gene encoding a sialyl transferase enzyme which is able to transfer the sialyl residue of CMP-sialic acid to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated and sialylated HMO from lactose,
  • the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of CMP-sialic acid and GDP-Fuc by the cell, and as energy source for making said fucosylated and sialylated HMO.
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, and particularly does not contain scrK.
  • the product is preferably 3’-sialyl-3-fucosyllactose.
  • the genetically modified E. coli cell is suitable for producing a fucosylated LNT or LNnT and comprises:
  • the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc, UDP-Gal and GDP-Fuc by the cell, and as energy source for making said fucosylated LNT or LNnT.
  • the heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, and particularly does not contain scrK.
  • a genetically modified microorganism for making an oligosaccharide or a glycoside of said oligosaccharide comprising: a. a recombinant gene encoding a glycosyl transferase which can transfer a glycosyl residue of an activated sugar nucleotide to a carbohydrate acceptor or a glycoside of said acceptor, b. a biosynthetic pathway for making said activated sugar nucleotide from sucrose, and c.
  • nucleic acid sequences which encode PTS-dependent sucrose utilization system, where the nucleic acid sequences encode: i) sucrose porin scrY of SEQ ID NO: 1 or a homologue thereof which is at least 85% identical to SEQ ID NO: 1, such as 90%, 95% or 98% identical to SEQ ID NO: 1; and ii) PTS permease scrA of SEQ ID NO: 2 or a homologue thereof which is at least 85% identical to SEQ ID NO: 2, such as 90%, 95% or 98% identical to SEQ ID NO: 2; and iii) sucrose-6-phosphate hydrolase scrB of SEQ ID NO: 3 or a homologue thereof which is at least 85% identical to SEQ ID NO: 3, such as 90%, 95% or 98% identical to SEQ ID NO: 3; and iv) repressor protein scrR of SEQ ID NO: 4 or a homologue thereof which is at least 85% identical to SEQ ID NO: 4 such as 90%, 95% or 98% identical to SEQ ID NO:
  • a genetically modified microorganism for making an oligosaccharide or a glycoside of said oligosaccharide comprising:
  • a recombinant gene encoding a glycosyl transferase which can transfer a glycosyl residue of an activated sugar nucleotide to a carbohydrate acceptor or a glycoside of said acceptor,
  • heterologous PTS-dependent sucrose utilization system comprises proteins encoded by scr genes scrY for a sucrose porin, scrA for a PTS permease, scrB for a sucrose-6-phosphate hydrolase and scrR for a repressor protein.
  • glycosyl transferase can transfer a glycosyl residue of an activated sugar nucleotide to an intermediate in the biosynthetic pathway to said oligosaccharide from said acceptor.
  • R A IS a linear or branched hydrocarbon chain having, when saturated, 1-24, preferably 1-6 carbon atoms or, when unsaturated, 2-24, preferably 2-6 carbon atoms, or RA means an aryl moiety, or RA means a group removable by hydrogenolysis,
  • X is N or S
  • RB means linear or branched hydrocarbon chain having, when saturated, 1-24, preferably 1-6 carbon atoms or, when unsaturated, 2-24, preferably 2-6 carbon atoms, or R B means an aryl moiety, or R B means a benzyl group,
  • each R' independently is an electron withdrawing group selected from -CN, -COOH, -COO-alkyl, -CO-alkyl, -CONH2, -CONH-alkyl and - CON(alkyl)2, or wherein the two R'-groups are linked together and form -CO-(CH2)2-4-CO- and thus form with the carbon atom, to which they are attached, a 5-7 membered cycloalkan-1,3-dione, in which dione any of its methylene groups is optionally substituted with 1 or 2 alkyl groups, and wherein R" is H or alkyl,
  • lacZ is deleted or deactivated, and optionally one or more of the following genes are deleted or deactivated: glpR, lacl, lacA and hlyE.
  • the scr genes are operably linked to a glp, such as a glpF promoter, such as the glpF promoter of SEQ ID NO: 5.
  • R A IS a linear or branched hydrocarbon chain having, when saturated, 1- 24, preferably 1-6 carbon atoms or, when unsaturated, 2-24, preferably 2-6 carbon atoms, or R A means an aryl moiety, or R A means a group removable by hydrogenolysis,
  • X is N or S
  • RB means linear or branched hydrocarbon chain having, when saturated, 1-24, preferably 1-6 carbon atoms or, when unsaturated, 2-24, preferably 2-6 carbon atoms, or R B means an aryl moiety, or R B means a benzyl group,
  • each R' independently is an electron withdrawing group selected from -CN, -COOH, -COO-alkyl, -CO-alkyl, -CONH2, -CONH-alkyl and - CON(alkyl)2, or wherein the two R'-groups are linked together and form -CO-(CH2)2-4- CO- and thus form with the carbon atom, to which they are attached, a 5-7 membered cycloalkan-1,3-dione, in which dione any of its methylene groups is optionally substituted with 1 or 2 alkyl groups, and wherein R" is H or alkyl,
  • Q2, Q3 and Q4 are, independently, H and Ci-C6-alkyl, which alkyl is optionally substituted with halogen, OH, nitro or phenyl groups,
  • Ri is fucosyl or H
  • R2 is fucosyl or H
  • R3IS selected from H, sialyl, N-acetyl-lactosaminyl and lacto-N-biosyl groups, wherein the N- acetyl-lactosaminyl group optionally carries a glycosyl residue comprising one or more N- acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; each of the N-acetyl- lactosaminyl and lacto-N-biosyl groups is optionally substituted with one or more sialyl and/or fucosyl residue,
  • R4 is selected from H, sialyl and N-acetyl-lactosaminyl groups optionally substituted with a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N- biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups is optionally substituted with one or more sialyl and/or fucosyl residue, provided that at least one of the Ri, R2, Rsand R4 groups is different from H.
  • oligosaccharide of formula 1 represents a human milk oligosaccharide (HMO).
  • the E. coli comprises: (i) - a recombinant gene encoding an N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to an internalized lactose or to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-GIcNAc; and/or - a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to an internalized lactose or to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc,
  • a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer a galactosyl residue of UDP-Gal to N-acetyl-glucosaminylated lactose or to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose, and
  • the N-acetyl-glucosaminyl transferase is a b-1,3-N-acetyl-glucosaminyl transferase, no recombinant gene encoding a galactosyl transferase is present in the cell, and the N- acetylated HMO is lacto-N-triose II,
  • the N-acetyl-glucosaminyl transferase is a b-1,3-N-acetyl-glucosaminyl transferase
  • the galactosyl transferase is a b-1,3 ⁇ 3 ⁇ 80 ⁇ I transferase
  • the N-acetylated HMO is lacto- N-tetraose
  • the N-acetyl-glucosaminyl transferase is a b-1,3-N-acetyl-glucosaminyl transferase
  • the galactosyl transferase is a b-1,4 ⁇ 3 ⁇ 80 ⁇ I transferase
  • the N-acetylated HMO is lacto- N-neotetraose.
  • microorganism is E. coli and the HMO is an N-acetylated HMO, and the E. coli comprises:
  • a recombinant gene encoding an N-acetyl-glucosaminyl transferase enzyme which is able to transfer a GlcNAc of UDP-GIcNAc to an internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-GIcNAc;
  • a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer a fucosyl residue of GDP-Fuc to an internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc, and
  • a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer a galactosyl residue of UDP-Gal to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-Gal.
  • microorganism is E. coli and the HMO is 2'-FL, 3-FL or DFL, and the E. coli comprises:
  • a recombinant gene encoding a sialyl transferase enzyme which is able to transfer a sialyl residue of CMP-sialic acid to an internalized lactose or to an intermediate in the biosynthetic pathway to said sialylated HMO from lactose, and
  • a recombinant gene encoding a sialyl transferase enzyme which is able to transfer a sialyl residue of CMP-sialic acid to an internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated and sialylated HMO from lactose, and
  • a process for making an oligosaccharide or a glycoside of said oligosaccharide by glycosylating a carbohydrate acceptor, or a glycoside of said acceptor, wherein the carbohydrate acceptor is not sucrose comprising the steps of: a) providing of a genetically modified microorganism of any one of embodiments 1 to 30, b) culturing said cell in the presence of said acceptor and sucrose, and c) separating said oligosaccharide from said cell, from the culture medium or from both.
  • oligosaccharide is a human milk oligosaccharide (HMO).
  • neutral HMO is an N-acetylated HMO selected from the group consisting of lacto-N-neotetraose, para-lacto-N-hexaose, para-lacto-N- neohexaose, lacto-N-neohexaose, para-lacto-N-octaose, lacto-N-neooctaose, lacto-N-tetraose, lacto-N-hexaose, lacto-N-octaose, iso-lacto-N-octaose, lacto-N-decaose and lacto-N- neodecaose, more preferably lacto-N-neotetraose, lacto-N-tetraose, lacto-N-hexaose, para- lacto-N-hexaose, para- lacto-
  • Example 1 Comparative test for making LNnT by a glycerol or sucrose utilizing E. coli
  • strains were constructed from Escherichia coli K12 strain DH1 which was obtained from the Deutsche Sammlung von Mikroorganismen (reference DSM 5346) by deleting the genes: lacZ nanKETA lacA melA wcaJ mdoH, by inserting a Plac promoter, and maintaining genes involved in the UDP-GIcNAc and UDP-Gal biosynthesis.
  • the glycerol utilizing strain (strain I) contains a pBBR3 -IgtA-tet plasmid carrying N.
  • sucrose utilizing strain contains the two following plasmids:
  • pBS-scrBR-galT-amp which is a pUC derivative carrying the galT gene encoding an b-1,4- galactosyl transferase, the s crR gene encoding a sucrose repressor, the scrB gene encoding a sucrose-6-phosphate hydrolase and the ampicillin resistance gene;
  • pBBR3-scrYA-lgtA-tet which is a pBBR1-MCS3 derivative carrying the IgtA gene encoding a b-I ⁇ -N-qob ⁇ g ⁇ IuooeqGh ⁇ hgI transferase, the scrA gene encoding a PTS system sucrose- specific EIIBC component, the scrY gene encoding a sucrose porin and the tetracycline resistance gene.
  • Glucose, glycerol, sucrose and lactose were each sterilized at 120 °C.
  • Isopropyl thio ⁇ -D- galactopyranoside (IPTG) was filter sterilized.
  • the culture was carried out in a 3 I fermenter containing «0.9 I of mineral culture medium (Samain et al. J. Biotechnol. 72, 33 (1999); does not contain antibiotics for the sucrose system).
  • the temperature was kept at 33 °C and the pH regulated at 6.8 with 28% NH 4 OH.
  • the inoculum of the producing strain consisted in a LB medium (20 ml) supplemented with ampicillin and tetracycline for strain I or M9 medium (20 ml) supplemented with sucrose for strain II was added to the fermenter.
  • the exponential growth phase started with the inoculation and stopped until exhaustion of the carbon source (glucose for strain I or sucrose for strain II, «17.5 g/l) initially added to the medium.
  • a lactose solution 70 g of lactose/500 ml of water
  • the carbon source 500 g/l solution, 4.5 g/h of glycerol for strain I and 3 g/h of sucrose for strain II.
  • the inducer isopropyl thio ⁇ -D-galactopyranoside, IPTG, 1-2 ml of a 50 mg/ml solution
  • the glycerol-fed fermentation (strain I) lasted for 90 hours after which the cells died (LNnT titre: 45 g/l).
  • the sucrose-fed fermentation (strain II) produced an LNnT concentration of 56 g/l after 116 hours.
  • the strain was constructed from Escherichia coli K12 strain DH1 which was obtained from the Deutsche Sammlung von Mikroorganismen (reference DSM 5346) by deleting the genes: lacZ nanKETA lacA melA wcaJ mdoH and by inserting a Plac promoter to upstream the gmd gene.
  • the strain contains the following plasmids:
  • pBS-futC-scrBR-amp which is a pUC derivative carrying the futC gene encoding an a-1,2- fucosyl transferase, the s crR gene encoding a sucrose repressor, the scrB gene encoding a sucrose-6-phosphate hydrolase and the ampicillin resistance gene;
  • pBBR3-GMAB-scrYA-tet which is a pBBR1-MCS3 derivative carrying the manB, manC, gmd and wcaG genes involved in the GDP-Fuc biosynthesis, the scrA gene encoding a PTS system sucrose-specific EIIBC component, the scrY gene encoding a sucrose porin and the tetracycline resistance gene.
  • IPTG Isopropyl thio ⁇ -D-galactopyranoside
  • the culture was carried out in a 2 I fermenter containing «0.9 I of mineral culture medium (Samain et al. J. Biotechnol. 72, 33 (1999); does not contain antibiotics).
  • the temperature was kept at 33 °C and the pH regulated at 6.8 with 28% NH 4 OH.
  • the inoculum of the producing strain consisted in a M9 medium supplemented with sucrose (20 ml) was added to the fermenter.
  • the exponential growth phase started with the inoculation and stopped until exhaustion of sucrose ( «22 g/l) initially added to the medium.
  • the inducer isopropyl thio ⁇ -D- galactopyranoside, IPTG, 1-2 ml of a 50 mg/ml solution
  • a feeding with a lactose + sucrose solution (160 g of lactose + 500 g of sucrose/I in water) started with a rate of 9 ml/h for 6 hours, then was continued for 115 hours at a rate of 6 ml/h.
  • the concentration of 21- FL was around 100 g/l in the supernatant at the end of the fermentation.
  • Example 3 Comparative test for making 2’-FL by a glucose or sucrose utilizing E. coli
  • Gene targeting in the chromosomal DNA was done using standard DNA manipulation techniques, e.g. as disclosed in Warming et al. Nucleic Acids Res. 33, e36 (2005). Insertion of genetic cassettes in the chromosomal DNA was done by gene Gorging as described by Herring et al. Gene 311, 153 (2003).
  • the strains were derived from an E. coli platform strain that was constructed from E. coli K12 DH1 (genotype: F ⁇ , A , gyrA96, recA1, relA1, endA1, thi-1, hsdRU, supE44, obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), www.dsmz.de, reference DSM 4235) by disrupting (deletions of) the genes lacZ, nanKETA , lacA, melA, wcaJ, mdoH and by inserting a P lac promoter upstream the gmd gene.
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
  • strain 1 was further modified by inserting a) scrYA from Klebsiella pneumoniae (GeneBank accession ID: X57401.1 or SEQ ID NO: 6, herein) in another locus involved in sugar metabolism and expressed under the control of a glpF promoter with a modified SD sequence (PglpF_SD1 , see WO 2019/123324 and SEQ ID NO: 5 herein), b) scrBR from Salmonella thyphimurium (GeneBank accession ID: X67750.1, or SEQ ID NO: 7 herein) in one of the loci that enables sugar metabolism under the control of the Pscr promoter (strain 2).
  • the strains are plasmid-free.
  • the fermentations were carried out in 250 ml fermenters (DASBOX Mini Bioreactor system, Eppendorf) starting with 100 ml of defined mineral culture medium, consisting of 25 g/kg carbon source (glucose or sucrose), (NH ⁇ HPO ⁇ KH2PO4, MgS0 4 x 7H2O, KOH, NaOH, trace element solution, citric acid, antifoam and thiamine.
  • the trace element solution contained Mn, Cu, Fe, Zn as sulfate salts and citric acid. Lactose was added pre-inoculation at 80 g/kg.
  • the pH throughout fermentation was controlled at 6.8 by titration with 14 % NH4OH solution.
  • Aeration was at 1 vvm using air and dissolved oxygen was controlled above 20 % of air saturation. Fermentations were started by inoculation with 2 % (v/v) of pre-cultures grown in a similar medium. After depletion of the carbon source contained in the batch medium, a sterile feed solution containing glucose or sucrose, MgSCL x 7H2O, trace metal solution and lactose was fed continuously at a constant feed rate in a carbon-limited manner. The lactose concentration in the broth was maintained above 15 g/kg throughout and until the end of the fermentations. End- of-fermentation was at approximately 120 hours.
  • Example 4 Comparative test for making 2’-FL by a glucose or sucrose utilizing E. coli
  • strain 2 This test comprised strain 1, disclosed in Example 3 above, and a modified strain 2, referred to as strain 2’, which differs from strain 2 disclosed in Example 3 above in that the regulatory gene glpR was not deleted and thus remained functional.
  • the strains were fermented separately, under equivalent industrialized conditions based on the conditions disclosed in Example 3, with glucose for strain 1 and sucrose for strain 21.
  • the product 2’-FL was purified and isolated from the broths using identical unit step operations, without crystallization. Analysis of the purified samples showed substantial difference in the presence of specific carbohydrate impurities.
  • Product of strain 1 (fermented with glucose) had more than 2 w/w% of glucose degradation products, containing (amongst other products) isomaltose, kojibiose, gentiobiose and nigerose, while the product of strain 2’ (fermented with sucrose) was totally free of these impurities.
  • sucrose-manufactured product is extremely low in the sum of unspecified impurities compared to the glucose-manufactured product, i.e. 0.05 w/w% vs 0.26 w/w%.
  • the overall advantage of the sucrose technology is thereby two-fold, firstly to obtain a product with higher purity (thereby alleviating the need for costly crystallisation) and secondly, a 2 % higher carbon efficiency, since the carbon source upon heat sterilisation was not converted into non-metabolisable carbohydrate impurities.
  • a strain was derived from an E. coli platform strain that was constructed from E. coli K12 DH1 (genotype: F ⁇ , A , gyrA96, recA1, relA1, endA1, thi-1, hsdRU, supE44, obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), www.dsmz.de, reference DSM 4235) by disrupting (deletions of) the genes lacZ, nanKETA , lacA, melA, wcaJ, mdoH and by inserting a P lac promoter upstream the gmd gene.
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
  • codon optimized IgtA coding sequence for b1,3-N-acetyl glucosaminyl transferase were integrated into the genome (chromosome) of the E. coli platform strain in loci related to sugar metabolism and expressed under the control of the glpF promoter; two copies of codon optimized galTK encoding b1,3-galactosyl transferase (WO 2020/115671) was integrated into the genome of the E. coli platform strain in another loci involved in sugar metabolism and expressed under the control of the glpF promoter.
  • sucrose operon scrYABR (while split into scrYA and scrBR) was done at two sites in the mixed-acid fermentation pathway, thereby deleting genes that cause formation of undesired fermentative metabolites under anaerobic conditions, but not under normal aerobic fermentation conditions.
  • an expression cassette containing the glpF promoter linked to a codon optimized DNA encoding a putative MFS transporter protein from Rosenbergiella nectarea (GenBank accession ID: WP_092672081.1), which facilitates the export of LNT from the cell, was inserted in a locus involved in the utilization of alternative carbon sources.
  • lad and hlyE were deleted.
  • the so-constructed strain is plasmid-free and able to grow on glucose and sucrose.
  • the fermentations were carried out in 250 ml fermenters (DASBOX Mini Bioreactor system, Eppendorf) starting with 100 ml of defined mineral culture medium, consisting of 30 g/kg carbon source (glucose or sucrose), (NFU ⁇ HPCL, KH 2 PO 4 , MgSCL x TFhO, KOH, NaOH, trace element solution, citric acid, antifoam and thiamine.
  • the trace element solution contained Mn, Cu, Fe, Zn as sulfate salts and citric acid.
  • Lactose was provided by bolus addition of 80 g/kg approximately 5 hours after the end of the batch phase, i.e. at approximately 18-19 h elapsed fermentation time (ELT).
  • sucrose-utilising strain in fermentations with sucrose versus glucose as carbon source.
  • the processes were identical except for the type of carbon source used.
  • the data demonstrate clearly superiority of sucrose in terms of LNT productivity and carbon yield.
  • the table 3 below shows that the average carbon yield was increased by 17% when using sucrose instead of glucose. This is also reflected by the 19 % higher final LNT titre at end-of-fermentation and the increased lactose consumption rate (lactose was only added by bolus addition and not in the feed solution).
  • the use of the sucrose technology is therefore beneficial as it not only facilitates the purification process due to the higher final titre, but also leads to a more efficient carbon source conversion to product.
  • Table 3 LNT production from strains with and without heterologous PTS-dependent sucrose utilization system.
  • the background strain used for constructing the above strains are not capable of growing on sucrose.
  • the background strain, MDO was constructed from Escherichia coli K12 DH1.
  • the E. coli K12 DH1 genotype is: F , k, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44.
  • MDO has the following modifications: lacZ deletion of 1,5kbp, iacA ⁇ deletion of 0,5kbp, nanKETA : deletion of 3,3kbp, melA ⁇ deletion of 0,9kbp, wcaJ ⁇ deletion of 0,5kbp, mdoH. deletion of 0,5kbp, and insertion of Plac promoter upstream of the gmd gene.
  • the strain MPLNnT can produce the tetrasaccharide HMO, LNnT, while the MDO strain does not form a product of biotechnological interest.
  • the strain MPLNnT bears the genes that are responsible for the biosynthesis of LNnT, namely IgtA (encoding a beta-1, 3-N- acetyloglucosamine transferase) from N. meningitidis and galT (encoding a beta-1, 4- galactosyltransferase) from H. pylori, as well as four additional genes conferring the ability to grow on sucrose as the sole carbon and energy source.
  • the strains were grown in 96-well microtiter plates using a 4-day protocol. During the first 24 hours, cells were grown to high densities while in the next 72 hours cells were transferred to a medium containing sucrose as the sole carbon and energy source. Specifically, during day 1, fresh inoculums were prepared using a Luria-Bertani broth containing 20% glucose. After 24 hours of incubation of the prepared cultures at 34 °C, cells were transferred to a basal minimal medium (200 uL) supplemented with magnesium sulphate and thiamine to which an initial bolus of 20 % glucose solution and 15 g/L sucrose solution as carbon source was provided to the cells.
  • a basal minimal medium 200 uL
  • the MPLNnT stain has also been subjected to fermentation and has shown to be capable of producing between 83 and 85% LNnT relative to the total amount of HMO produced when grown on sucrose.
  • the plasmid containing strain of example 2 and the chromosomal strain 2’ of example 4 were compared in a head-to-head analysis.
  • the chromosomal strain 2’ of example 4 has two copies of futC, which is necessary to achieve levels of futC which is similar to those obtained from the high expression plasmid pBS in example 2.
  • the expression plasmids in the strain of example 2 have antibiotic markers (amp and tet) to secure that the plasmids are maintained within the cell when propagated, such markers are not needed in chromosomal strains, which is an advantage in production since antibiotics can be avoided in cell banking and during propagation.
  • the strains were fermented separately, under equivalent industrialized conditions based on the conditions disclosed in Example 3, with sucrose for both strains.
  • the product 2’-FL was purified and isolated from the broths using identical unit step operations, without crystallization.
  • the 2’FL yield is slightly higher (but statistically significant) for the chromosomal strain.
  • the amount of DFL by-product is reduced for the chromosomal strain in that the DFL/2’FL ratio is lower, this is a benefit in the down-stream processing since less by-product needs to be removed to obtain pure 2’FL.

Abstract

The invention relates to a genetically modified microorganism for making an oligosaccharide, preferably of 3-8 monosaccharide units, more preferably of 3-5 monosaccharide units, particularly a HMO, which comprises one or more genes encoding a sucrose utilization system, so the microorganism can use sucrose as a carbon and energy source.

Description

OLIGOSACCHARIDE PRODUCTION
FIELD OF THE INVENTION
The present invention relates to the field of biotechnology, notably to a microbial production of oligosaccharides, particularly of human milk oligosaccharides (HMOs), using a genetically modified microorganism, particularly E. coli , using sucrose as its exclusive carbon source.
BACKGROUND OF THE INVENTION
The fermentative syntheses of foreign or exogenous oligosaccharides using recombinant microorganisms have recently become of great commercial and industrial interest. In such syntheses, oligosaccharides of interest would be synthesized by enzymatic glycosylation of sugar acceptors mediated by one or more heterologous glycosyl transferases of the microorganisms, and the one or more activated sugar nucleotides necessary for glycosylation would be produced by the same microorganism through overexpressing one or more genes encoding endogenous activated sugar nucleotide producing enzymes. The metabolic pathways of such syntheses require a carbon source which is mainly a simple carbon building block, typically glycerol or glucose (see e.g. WO 01/04341 , Priem et al. Glycobiology 12, 235 (2002), Fort et al. Chem. Comm. 2558 (2005), Drouillard et al. Angew. Chem. Int. Ed. 45, 1778 (2006), WO 2010/070104, WO 2012/112777, WO 2013/182206, WO 2014/048439). In some syntheses, lactose can be the carbon source if it also serves as an acceptor (Lee et al. Microb. Cell Fact. 11:48 (2012)). As the microorganisms have been genetically manipulated, antibiotic- resistance selection marker genes have been utilized to separate the transformed microorganisms from the non-transformed ones in the inoculum and the fermentation broth. However, the use of antibiotics has been avoided by integrating the genes coding for enzymes involved in the de novo biosynthesis of the donor sugar in the chromosome of the microorganisms (Baumgartner et al. Microb. Cell Fact. 12:40 (2013)).
Around 50 % of wild-type E. coli are able to utilize sucrose as a carbon and energy source, but most of them are pathogenic. The E. coli strains used mainly in industry to synthesize chemical materials cannot live and grow on sucrose (Bruschi et al. Biotechnol. Adv. 30, 1001 (2012)). However, in some cases, sucrose can be a cheaper carbon and energy source. For this reason, attempts have been made to create suc+ strains of E. coli that can live and grow on sucrose (e.g. GB 2155935 A, Sabri et al. Appl. Environ. Microbiol 79, 478 (2013)) and produce industrially profitable products by them such as amino acids, biofuel, carotenoids etc. (e.g. EP- A-1149911, EP-A-2239336, EP-A-2371952, EP-A-2405006, WO 2010/051849, WO 2012/078311, Kim et al. Biores. Technol. 130, 288 (2013)). However, these suc+ transformants have generally been less productive than sue strains (Khamduang et al. J. Ind. Microbiol. Biotechnol. 36, 1267 (2009)).
WO 2012/007481 describes E. coli transformants that express either a sucrose phosphorylase or a sucrose invertase in combination with a fructokinase. Thereby, the microorganism is able to produce 2’-fucosyllactose, utilizing sucrose as its main carbon source. Furthermore, WO 2014/067696, WO 2015/150328, WO 2019/043029 and WO 2019/076941 describe E.coii transformants comprising a csc-gene cluster that enables them to grow on sucrose and produces fucose, 2’-FL, sialyllactose and sialic acid, respectively.
There has been, however, a continuing need for alternative processes for making oligosaccharides, particularly HMOs, using transformed microorganisms that are able to utilize more effectively sucrose as a carbon and energy source.
SUMMARY OF THE INVENTION
A first aspect of the invention relates to a process for making an oligosaccharide, preferably of 3-8 monosaccharide units, more preferably of 3-5 monosaccharide units, particularly a HMO, by glycosylating a carbohydrate acceptor which is not sucrose, preferably lactose, comprising the steps of: a) providing a cell, preferably an E.coii cell, that can internalize said acceptor into said cell and comprises
- a recombinant gene encoding a glycosyl transferase which is able to transfer a glycosyl residue of an activated sugar nucleotide to said acceptor, internalized in said cell, or to an intermediate in the biosynthetic pathway to said oligosaccharide from said acceptor and
- a biosynthetic pathway to make said activated sugar nucleotide in said cell, b) culturing said cell in the presence of said acceptor and sucrose, and c) separating said oligosaccharide from said cell, from the culture medium or from both, said process being characterized in that said cell also comprises one or more genes encoding a sucrose utilization system, preferably encoding a heterologous sucrose utilization system, more preferably encoding a heterologous PTS-dependent sucrose utilization transport system, still more preferably scr genes, so that said cell can use sucrose as a carbon source, preferably the main carbon source, more preferably the sole carbon source, for making said activated sugar nucleotide and as an energy source, preferably the main energy source, more preferably the sole energy source, for making said oligosaccharide.
A second aspect of the invention relates to a cell, preferably an E.coii cell, that can internalize a carbohydrate acceptor, which is not sucrose, preferably lactose, into said cell and that comprises: - a recombinant gene encoding a glycosyl transferase which is able to transfer a glycosyl residue of an activated sugar nucleotide to said acceptor, internalized in said cell, or to an intermediate in the biosynthetic pathway to said oligosaccharide from said acceptor
- a biosynthetic pathway to make said activated sugar nucleotide in said cell, and
- one or more genes encoding a sucrose utilization system, preferably a heterologous sucrose utilization system, more preferably scr genes, so that said cell can use sucrose as a carbon source, preferably the main carbon source, more preferably the sole carbon source.
BRIEF DESCRIPTION OF THE FIGURE
Figure 1 is intended to illustrate the invention further. It is not intended to limit the subject matter of the invention thereto.
The engineered microorganism is a fully metabolically active cell in which the growth and the oligosaccharide synthesis may proceed simultaneously. The cell comprises a heterologous phosphotransferase (PTS)-dependent sucrose utilization transport system containing a sucrose specific porin (facilitates the sucrose diffusion through the outer membrane), a sucrose transport protein (provides intracellular sucrose-6-phosphate from extracellular sucrose) and a sucrose-e- phosphate hydrolase (provides glucose-6-phosphate and fructose). The oxidation of glucose-e- phosphate and fructose provides biological energy source by the organism’s own metabolic system. Also, glucose-6-phosphate and fructose serve as carbon source for producing sugar nucleotides in the cell’s natural biosynthetic pathway or in any heterologous pathway expressed in the cell. The so-produced sugar nucleotides are donors for glycosylating carbohydrate acceptors (e.g. lactose), internalized through a specific permease by the cell, and thereby manufacturing oligosaccharides of interest. The glycosylation is mediated by one or more glycosyl transferases which are directly produced by expressing heterologous genes from plasmid or the chromosome. The organism lacks any enzyme degrading either the acceptor or the oligosaccharide product in the cell.
Figure 2 Illustrate the ability to grow on sucrose with a heterologous PTS-dependent sucrose utilization transport system in the cell. The MDO strain does not contain the PTS-dependent sucrose utilization transport system, whereas MPLNnT does grow on sucrose.
DETAILED DESCRIPTION OF THE INVENTION
It has been surprisingly discovered that an exogenous mono- or disaccharide acceptor, preferably lactose, can be internalized in a suitable genetically transformed microorganism, particularly E.coli, by a transport mechanism of the microorganism, so that this carbohydrate acceptor can be glycosylated in the microorganism using sucrose as its carbon and energy source, and that an exogenous oligosaccharide can be produced and separated in good yield. Thereby, an efficient, cheap and easily up-scalable process for producing oligosaccharides can be obtained. In order to make the process successful, a special oligosaccharide-producing microorganism is needed that can live on sucrose, utilize sucrose for the metabolic syntheses of the necessary nucleotide sugar donors for glycosylation, can internalize simple carbohydrate acceptors and perform glycosylation reactions on them for synthesizing more complex oligosaccharides.
The invention therefore, in a first aspect, involves a process of making an oligosaccharide by: a) providing a cell of a microorganism, preferably an E.coli cell, that can internalize sucrose and a carbohydrate acceptor, preferably lactose, into said cell and that comprises: a recombinant gene encoding a glycosyl transferase which can transfer a glycosyl residue of an activated sugar nucleotide to the acceptor within the cell or to an intermediate in the biosynthetic pathway to said oligosaccharide from said acceptor, and a biosynthetic pathway for making the activated sugar nucleotide from sucrose, b) culturing the cell in an aqueous culture medium in the presence of the acceptor and sucrose, and c) separating the oligosaccharide product from the cell from the culture medium or from both.
The process features the cell being transformed with one or more foreign genes encoding a sucrose utilization system that allows the cell to use sucrose as a carbon source, preferably the main carbon source, more preferably the sole carbon source, for the biosynthesis of the activated sugar nucleotide by the cell. The sucrose utilization system, with which the cell is transformed, also preferably allows the cell to use sucrose as an energy source, preferably the main energy source, more preferably the sole energy source, for the biosynthesis of the oligosaccharide.
In accordance with this invention, the term “carbohydrate acceptor” or “acceptor” preferably means a mono- or disaccharide other than sucrose and its glycosides. A monosaccharide acceptor or a monosaccharide part of a disaccharide acceptor can comprise any 5-6 carbon atom sugar moiety that is an aldose (e.g. D-glucose, D-galactose, D-mannose, D-ribose, D- arabinose, L-arabinose, D-xylose, etc.), ketose (e.g. D-fructose, D-sorbose, D-tagatose, etc.), deoxysugar (e.g. L-rhamnose, L-fucose, etc.) or deoxy-aminosugar (e.g. N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.). In a glycoside-type carbohydrate acceptor the sugar moiety is attached to a non-sugar residue (aglycon) by either a covalent bond, which is a direct linkage between the glycosidic carbon atom of the sugar residue and any atom of the non-sugar moiety, or by a linker, which consists of one, two, three or four atoms such as -0-, - C-, -NH-, -N(OH)-, -S-, -C(=0)-, -C(=S)-, -C(=NH)-, -C(=N-OH)-, -C(=0)-0-, -0-C(=0)-, -C(=0)- S-, -S-C(=0)-, -C(=S)-0-, -0-C(=S)-, -C(=S)-S-, -S-C(=S )-, -C(=0)-NH-, -NH-C(=0)-, -C(=NH)- 0-, -0-C(=NH)-, -C(=S)-NH-, -NH-C(=S)-, -C(=NH)-S- and -S-C(=NH). Thus, the C-1 (in case of aldoses) or C-2 (in the case of ketoses) anomeric carbon atom at the reducing end of the mono- or disaccharide residue is linked to the non-sugar moiety by a covalent bond or a linker forming a 0-, N-, S- or C-glycoside. Preferably, the aglycon of these glycosidic derivatives, with or without a linker, is one of the following groups: a) -ORA, wherein RA is a linear or branched hydrocarbon chain having, when saturated, 1- 24, preferably 1-6 carbon atoms (such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s- butyl, t-butyl, n-hexyl, etc.) or, when unsaturated, 2-24, preferably 2-6 carbon atoms (such as vinyl, allyl, propargyl, etc.), or RA means an aryl moiety (a homoaromatic group such as phenyl or naphthyl), or RA means a group removable by hydrogenolysis, that is a group whose bond attached to the oxygen can be cleaved by addition of hydrogen in the presence of catalytic amounts of palladium, Raney nickel or another appropriate metal catalyst known for use in hydrogenolysis, resulting in the regeneration of an OH group; such protecting groups are well known to the skilled man and are discussed in Protective Groups in Organic Synthesis, PGM Wuts and TW Greene, John Wiley & Sons 2007. Suitable groups include benzyl, diphenylmethyl (benzhydryl), 1-naphthylmethyl, 2-naphthylmethyl or triphenylmethyl (trityl) groups. Any of the above mentioned RA groups can be optionally substituted by one or more groups selected from: alkyl (only for aryl and group removable by hydrogenolysis), hydroxy, alkoxy, carboxy, oxo, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylamino, arylcarbonyl, amino, mono and dialkylamino, carbamoyl, mono- and dialkyl-amino-carbonyl, alkyl- carbonylamino, cyano, alkanoyloxy, nitro, alkylthio and halogen; in case of a group removable by hydrogenolysis, such substitution, if present, is preferably on the aromatic ring(s); b) -X-RB, wherein X is N or S, and RB means linear or branched hydrocarbon chain having, when saturated, 1-24, preferably 1-6 carbon atoms (such as methyl, ethyl, n-propyl, i-propyl, n- butyl, i-butyl, s-butyl, t-butyl, n-hexyl, etc.) or, when unsaturated, 2-24, preferably 2-6 carbon atoms (such as vinyl, allyl, propargyl, etc.), or RB means an aryl moiety (a homoaromatic group such as phenyl or naphthyl), or RB means a benzyl group. Any of the above mentioned RB groups can be optionally substituted by one or more groups selected from: alkyl (only for aryl and benzyl), hydroxy, alkoxy, carboxy, oxo, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, aryloxy- carbonyl, aryloxy, arylamino, arylcarbonyl, amino, mono and dialkylamino, carbamoyl, mono- and dialkyl-amino-carbonyl, alkyl-carbonylamino, cyano, alkanoyloxy, nitro, alkylthio and halogen; c) a group that links the anomeric carbon atom and the adjacent carbon atom to each other by a -NH-C(=0)-0-bridge, thus forming a fused 5-membered ring as depicted below in case of an aldose:
Figure imgf000008_0001
d) azide; e) -NH-C(R”)=C(R’)2, wherein each R’ independently is an electron withdrawing group selected from -CN, -COOH, -COO-alkyl, -CO-alkyl, -CONH2, -CONH-alkyl and -CON(alkyl)2, or wherein the two R’-groups are linked together and form -CO-(CH2)2-4-CO- and thus form with the carbon atom, to which they are attached, a 5-7 membered cycloalkan-1,3-dione, in which dione any of the methylene groups is optionally substituted with 1 or 2 alkyl groups, and wherein R” is H or alkyl; f) a residue of an amino acid, which can be any natural or non-natural amino acid, that is an alkanoic acid derivative having at least one amino group as a substituent. Preferably, the amino acid is selected from the group consisting of: a-amino acids and b-amino acids such as Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, hydroxyproline, a-methylserine, b-alanine, etc. These amino acids can be either directly or via a linker as defined above (e.g. for urea-linked glycopeptides see WO 2009/040363) bound to the carbohydrate at its C-1 (in case of aldoses) or C-2 (in the case of ketoses) anomeric carbon atom thus forming 0-, N-, S- or C-glycosides. O-Glycosides (O-glycans) can be formed involving OH-containing amino acids such as serine, threonine, hydroxyproline, etc., N- glycosides (N-glycans) can be made using the a-, b-, etc. amino group of any amino acid or the additional amino group of the side chain of e.g. lysine, asparagine or glutamine, S-glycosides can be made using cysteine, while C-glycosides (C-glycans) contain a C-C bond coupling a C- atom of the amino acid to the anomeric carbon atom of the non-reducing end of the oligosaccharide part; g) a polyethylene glycol residue. Polyethylene glycol (PEG) is a water soluble polyether of molecular formula C2nH4n+20n+i, having oxyethylene (-CH2-0-CH2- or CH2-CH2-0-) repeating units and wherein n is 2 to 100, preferably 2 to 50, particularly 2 to 25, more particularly 2 to 10. Lower molecular weight PEGs are available in a purified form and are referred to as a “monodisperse PEG", and are also available as mixtures of PEGs and are referred to as a “polydisperse PEG". With regard to their geometry, PEGs can be in a linear, branched, star or comb configuration. Linear PEGs are preferably lower molecular weight PEGs (i.e. , n is 2 to 10, preferably 3 to 6). Branched PEGs preferably have 3 to 10 linear, preferably lower molecular weight, PEG chains emanating from a central core group. Star PEGs preferably have 10 to 100 linear or branched, preferably lower molecular weight, PEG chains emanating from a central core group. Comb PEGs have multiple linear, branched and/or star, preferably lower molecular weight, PEG chains bonded to a polymer backbone. Terminal primary hydroxy group of PEGs can be bonded by an ether bond with an alkyl group, preferably methyl. In addition, their terminal hydroxy group can be replaced by amino, alkyl amino, dialkyl amino, acylamino, thiol or alkyl thio groups or their terminal hydroxymethyl group can be oxidized to a carboxyl, which can be esterified or be present in amide form with ammonia or a primary or secondary amine. The attachment is a glycosidic-like bond; h) a polyvinyl alcohol residue. Polyvinyl alcohol (PVA) is a water-soluble polymer of molecular formula (C2H40)X having -CH2-CH(OH)- monomer units, wherein x is any desired integer. When attached to carbohydrate, any of the OH-groups can be glycosylated; i) an a,b-unsaturated amido group of formula A
Figure imgf000009_0001
wherein Qi, Q2, Q3 and Q4 are, independently, H and Ci-C6-alkyl, which alkyl optionally can be substituted with halogen, OH, nitro or phenyl groups. The residue of formula A, via its N atom, is linked to the sugar by a covalent bond, preferably to the anomeric carbon atom of the carbohydrate in the form of an N-glycoside; or j) an a,b-unsaturated carbonyl group of formula B
Figure imgf000009_0002
B wherein Qi, Q2 and Q3 are as defined at residue of formula A.
Preferably, the carbohydrate acceptor is a galactosyl disaccharide, particularly lactose.
Also in accordance with this invention, the term “oligosaccharide product" or “oligosaccharide” preferably means a glycosylated derivative of a carbohydrate acceptor disclosed above wherein a glycosyl residue is attached to the carbohydrate moiety of the carbohydrate acceptor by interglycosidic linkage. Preferably, an oligosaccharide product is of 3- 8 monosaccharide units, particularly of 3-5 monosaccharide units, more particularly of 4 monosaccharide units or of 3 monosaccharide units. The oligosaccharide product of this invention is a recombinant product, i.e. it is made by a genetically transformed microorganism and is foreign or heterologous to the microorganism. The term “an intermediate in the biosynthetic pathway" in relation to an oligosaccharide or more specifically an HMO, is to be understood as an intermediate oligosaccharide or HMO in the reaction steps needed to produce the desired oligosaccharide or HMO. For example, LNT-II is an intermediate in the biosynthetic pathway to LNT or LNnT.
The genetically modified microorganism or cell used in the process of some embodiments of this invention can be selected from the group consisting of bacteria and yeasts, preferably a bacterium. Bacteria are preferably selected from the group of: Escherichia coii, Bacillus spp. (e.g. Bacillus subtil is), Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Neisseria gonorrhoeae, Neisseria meningitis, Lactobacillus spp., Lactococcus spp., Enterococcus spp., Bifidobacterium spp., Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., Pseudomonas, among which E. coii is preferred.
Further in accordance with some embodiments of this invention, the microorganism or cell, preferably an E.coii cell, is genetically modified so that there is at least one alteration in its DNA sequence. The alteration can result in a change in the original characteristics of the wild type cell, e.g. the modified cell is able to perform additional chemical transformation due to the introduced new genetic material that encodes the expression of an enzymes not being in the wild type cell, or is not able to carry out transformation like degradation due to removal or deactivation of gene/genes. In this regard, a genetically modified cell contains at least one artificial alteration in its genome compared to its naturally occurring (wild type) variant. By the alteration, either a nucleic acid construct is added to the cell by way of integration into the genome or by addition via plasmid, or a nucleic acid sequence is deleted from or changed in the genome of the cell. Whatever is the case, the so-transformed cell has a genotype that is different from that before the alteration and, therefore, the modified cell shows modified feature(s). Preferably, the genetically modified cell can perform at least one additional or altered biochemical reaction, when cultured or fermented, due to the introduction of a heterologous nucleic acid sequence or the modification of a native nucleic acid sequence that encodes an enzyme that is not expressed in the wild type cell, or the genetically modified cell cannot perform a biochemical reaction due to the deletion, addition or modification of a nucleic acid sequence that encodes an enzyme found in the wild type cell. The genetically modified cell can be constructed by well-known, conventional genetic engineering techniques (e.g. Green and Sambrook: Molecular Cloning: A laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press (2012); Current protocols in molecular biology (Ausubel et al. eds.), John Wiley and Sons (2010)).
The terms “host cell”, “recombinant microorganism or cell” or “genetically modified microorganism or cell” are used interchangeably to designate such constructs. The term “nucleic acid sequence encoding a polypeptide having a glycosyl transferase activity” or “nucleic acid sequence encoding glycosyl transferase” means a gene, a functional fragment thereof or a codon-optimized version thereof that express a polypeptide having a glycosyl transferase activity.
The term “gene” in the present context relates to a coding nucleic acid sequence and includes genomic DNA and cDNA.
The term “promoter" means a nucleic acid sequence involved in the binding of RNA polymerase to initiate transcription of an operably linked gene, wherein the gene includes a coding DNA sequence and other (non-coding) sequences, e.g. the 5’-untranslated region (5’- UTR) located upstream of the coding sequence, which comprises a ribosomal binding site. A promoter in some embodiments of this invention is an isolated DNA sequence, i.e. not an integrated DNA fragment of the genomic DNA. The nucleotide sequence of a promoter corresponds to, or have at least 80 % identity, preferably 90%to 99.9 % identity, such as 95%, 98%, and even more preferably 100% identity with the nucleotide sequence of a fragment of bacterial genomic DNA that is regarded as promoter region of a gene, e.g. a promoter region of a glp operon or lac operon of E.coli. A preferred promoter is the promoter of SEQ ID NO: 5.
The term “operon” means a functioning unit of genomic DNA or cDNA containing a cluster of genes under the control of a single promoter. By “glp operon” is meant a cluster of genes involved in the respiratory metabolism of glycerol of bacteria. Preferably, a glp operon promoter sequence comprised in a DNA construct corresponds to or has at least 80 % identity, preferably 90-99.9 % identity, such as 95%, 98%, and even more preferably 100% identity with the nucleotide sequence of a fragment of the genomic DNA regarded as a promoter region of the corresponding glp operon of E. coir, in particular, the isolated sequence of a promoter of a glp operon of some embodiments of the invention corresponds to, or has said percent of identity with a fragment of the genomic sequence upstream the sequence having GenBank ID:
EG 10396 ( glpFKX ), EG10391 ( glpABC ), EG10394 ( glpD ), EG10401 ( glpTQ ). The E. coli genome is referred herein to the complete genomic DNA sequence of E coli K-12 MG1655 (GenBank ID:U00096.3).
In some embodiments, a promoter of the glp operon is operably linked to the heterologous glycosyl transferase genes and/or the heterologous scr genes. Preferably, those genes are expressed under a glpF promoter (WO 2019/123324, the content of which is incorporated herein by reference), in particular the glpF promoter of SEQ ID NO: 5. The process of some embodiments of this invention also involves transporting the exogenous carbohydrate acceptor, preferably lactose, into the genetically modified microorganism for glycosylation to produce a foreign oligosaccharide of interest, preferably without adversely affecting the basic functions of the cell or destroying its integrity. In one embodiment, the transport takes place via a passive mechanism, during which the exogenous acceptor diffuses passively across the plasma membrane of the cell. Diffusion of the acceptor into the microorganism is a function of the concentration differences between the fermentation broth and the extra- and intracellular space of the cell with respect to the acceptor, whereby the acceptor passes from the place of higher concentration to the place of lower concentration. In another embodiment, the acceptor is internalized with the aid of an active transport. In such a case, the genetically modified microorganism contains transporter proteins, called permeases, which act as enzymes and with which the microorganism is able to admit exogenous substances and to concentrate them in the cytoplasm. Specifically, lactose permease (LacY) acts specifically on galactose, simple galactosyl disaccharides such as lactose and their glycosides. The specificity towards the sugar moiety of the exogenous carbohydrate acceptor to be internalized can be altered by mutation of the microorganism by means of conventional recombinant DNA manipulation techniques. In a preferred embodiment, the internalization of exogenous lactose or its derivative takes place via an active transport mechanism mediated by a lactose permease. The genetically modified microorganism preferably lacks any enzyme activity (such as LacZ) that would degrade the acceptor. Likewise, the microorganism is not able to hydrolyze or degrade the oligosaccharide product or any oligosaccharide intermediate in the biosynthetic pathway to the oligosaccharide product.
Moreover, the genetically modified cell used in the process of the invention comprises one or more endogenous or recombinant genes encoding one or more glycosyl transferase enzymes that are able to transfer the glycosyl residue of an activated sugar nucleotide to the internalized acceptor. The gene or an equivalent DNA sequence thereof, if it is recombinant, can be introduced into the cell by conventional techniques, e.g. using an expression vector or by chromosomal integration. The origin of the heterologous nucleic acid sequence can be any animal (including human) or plant, eukaryotic cells such as those from Saccharomyces cerevisiae, Saccharomyces pombe, Candida albicans or from algae, prokaryotic cells such as those originated from E. coli, Bacteroides fragilis, Photo bacterium sp., Bacillus subtilis, Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Rhizobium meliloti, Neisseria gonorrhoeae and Neisseria meningitidis, or virus. The glycosyl transferase enzyme/enzymes encoded and expressed by the gene(s) or equivalent DNA sequence(s) are preferably glucosyl transferases, galactosyl transferases, N-acetylglucosaminyl transferases, N- acetylgalactosaminyl transferases, glucuronosyl transferases, xylosyl transferases, mannosyl transferases, fucosyl transferases, sialyl transferases and the like. In a preferred embodiment, the glycosyl transferases are selected from the group consisting of b-1,3-N-acetylglucosaminyl- transferase, b-1,6-N-acetylglucosaminyl-transferase, b-1, 3-galactosyl-transferase, b-1 ,4- galactosyl-transferase, b-1 ,3-N-acetylgalactosaminyl-transferase, b-1 ,3-glucuronosyl- transferase, a-2, 3-sialyl-transferase, a-2, 6-sialyl-transferase, a-2, 8-sialyl-transferase, a-1,2- fucosyl-transferase, a-1,3-fucosyl-transferase and a-1,4-fucosyl-transferase. More preferably, the glycosyl transferases are selected from b-1,3-N-acetylglucosaminyl transferase, b-1,6-N- acetylglucosaminyl transferase, b-1,3^3ΐ80^^I transferase, b-1,4-galactosyl transferase, a- 2,3-sialyl transferase, a-2,6-sialyl transferase, a-1,2-fucosyl transferase, a-1,3-fucosyl transferase and a-1,4 fucosyl transferase, that is from those involved in the construction of HMO core structures as well as fucosylated and/or sialylated HMOs and its glycosidic derivatives, wherein the aglycon is a moiety defined above at the group of carbohydrate acceptors. The genes encoding the above-mentioned transferases have been described in the literature.
The genetically modified microorganism or cell used in the process of the invention is capable of producing an oligosaccharide from the internalized precursor/acceptor which is preferably lactose. Said cell possesses enzymatic activity that is necessary for synthesizing an oligosaccharide, from an internalized mono- or disaccharide via consecutive glycosylation steps, wherein the internalized acceptor, e.g. lactose, is glycosylated, in a first glycosylation step, to a trisaccharide, then that trisaccharide is glycosylated, in a second glycosylating step, to a tetrasaccharide, and so forth. The glycosylation steps are mediated by respective glycosyl transferases. In this regard, the phrase “a glycosyl transferase which is able to transfer a glycosyl residue of an activated sugar nucleotide to an intermediate in the biosynthetic pathway to said oligosaccharide from said acceptor” or “a glycosyl transferase which can transfer a glycosyl residue of an activated sugar nucleotide to an intermediate in the biosynthetic pathway to said oligosaccharide from said acceptor” refers to the second, third etc. glycosylation step.
For example, if lacto-N-tetraose is made from lactose, a first glycosyl transferase (a b-1 ,3-N- acetyl-glucosaminyl transferase) transfers GlcNAc of UDP-GIcNAc to the internalized lactose to form lacto-N-triose II, then a second glycosyl transferase (a b-1,3-galactosyl transferase) transfers Gal of UDP-Gal to the previously formed lacto-N-triose II (LNT-II) to form lacto-N- tetraose. In this regard, lacto-N-triose II is considered to be an intermediate in the biosynthetic pathway to LNT from lactose.
In the glycosyl transferase mediated glycosylation processes of this invention, activated sugar nucleotides serve as donors, and the glycosyl transferase in question transfers a monosaccharide of an appropriate donor molecule to the acceptor molecule. Each activated sugar nucleotide generally comprises a phosphorylated glycosyl residue attached to a nucleoside, and the specific glycosyl transferase enzyme accepts only the specific sugar nucleotide. Thus, preferably the following activated sugar nucleotides are involved in the glycosyl transfer: UDP-GIc, UDP-Gal, UDP-GIcNAc, UDP-GalNAc, UDP-glucuronic acid, UDP- Xyl, GDP-Man, GDP-Fuc and CMP-sialic acid, particularly those selected from the group consisting of UDP-Gal, UDP-GIcNAc, GDP-Fuc and CMP-sialic acid.
The genetically modified microorganism used in the process of this invention possesses a biosynthetic pathway to the above mentioned activated sugar nucleotides, that is, it has one or more sets of genes encoding one or more enzymes responsible for the synthesis of one or more activated glycosyl nucleotides, ready for glycosylation in glycosyl transferase mediated reaction in the cell, when cultured. The sets of genes are either naturally present in the cell or introduced into the cell by means of recombinant DNA manipulation techniques. The production of the activated glycosyl nucleotides by the cell takes place under the action of enzymes involved in the biosynthetic pathway of that respective sugar nucleotide stepwise reaction sequence starting from a carbon source (for a review for monosaccharide metabolism see e.g. H. H. Freeze and A. D. Elbein: Chapter 4: Glycosylation precursors, in: Essentials of Glycobiology, 2nd edition (Eds. A. Varki et al.), Cold Spring Harbour Laboratory Press (2009)).
It should be emphasized, that the production of the activated sugar nucleotides by the genetically modified microorganism via its own biosynthetic pathway is advantageous compared to in vitro versions of transfer glycosylation, as it avoids using the very expensive sugar nucleotide type donors added exogenously, hence the donors are formed by the cell in situ and the phosphatidyl nucleoside leaving groups are recycled in the cell.
In addition, the microorganism used in the process of the invention comprises genes encoding a sucrose utilization system, that is the cell has a capability to catabolically utilize sucrose as a carbon source, as well as an energy source. The system that enables the cell to utilize sucrose can be one normally found in the gene pool of that cell, but preferably is a heterologous system (i.e. derived from a different organism and transferred to the host cell by conventional recombinant DNA manipulation techniques). Typically, two kinds of sucrose catabolism can be used: i) The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (“PTS”), where sucrose is transported into the microorganism and concomitantly phosphorylated to generate intracellular sucrose-6-phosphate which is hydrolysed to glucose-e- phosphate and fructose that are then involved in the central carbon metabolism of the cell. PTS can be encoded by scr or sac genes; ii) The non-phosphotransferase-dependent system (“non- PTS”), where extracellular sucrose enters the cell with the aid of a proton symport transport system (sucrose permease) and, after transport, is hydrolysed by an invertase enzyme to glucose and fructose followed by phosphorylation. In this regard, the esc regulon consists of genes encoding the enzymes that are responsible for the non-PTS sucrose utilization.
During fermentation in the process of this invention, the oligosaccharide-producing microorganism is fed with sucrose that provides energy via glycolysis for growing, reproducing and maintaining its structure. In addition, the sucrose taken up by the cell provides, via sucrose catabolism, precursors for the synthesis of the activated sugar nucleotide(s) necessary for the glycosylation process that takes place in the cell. The internalized carbohydrate acceptor participates in the glycosyl transferase induced glycosylation reaction, in which a glycosyl residue of an activated nucleotide donor produced by the cell is transferred so that the acceptor is glycosylated. Optionally, when more than one glycosyl transferase is expressed by the cell, additional glycosylation reactions can occur resulting in the formation of the target oligosaccharide. Of course, the cell preferably lacks any enzyme activity which would degrade the oligosaccharide derivatives produced in the cell.
In a preferred realization of the process for making an oligosaccharide product, the sucrose utilization system is heterologous. This is the case when the microorganism, preferably a bacterium, more preferably an E. coli , is a strain that is optimized for an industrially profitable transformation like oligosaccharide production, because such a strain generally has no ability to utilize sucrose. Therefore, a sucrose uptake cassette should be introduced, using an appropriate expression plasmid or via chromosome integration, in the sucrose minus cell to make it be sucrose plus. More preferably, the sucrose pathway genes comprise a PTS- dependent sucrose utilization system, and especially the source regulon is scr. Microorganisms having scr genes are for example Salmonella ssp., Klebsiella pneumoniae, Bacteroides fragilis, Vibrio alginolyticus.
The scr genes comprise the following: scrY, scrA, scrB, scrR and scrK. The gene scrA codes for the sucrose transport protein Enzyme llScr that provides intracellular sucrose-6-phosphate from extracellular sucrose via an active transport through the cell membrane and the concomitant phosphorylation. The sucrose specific ScrY porin (encoded by scrY) facilitate the sucrose diffusion through the outer membrane. The ScrB invertase enzyme (encoded by scrB) splits the accumulated sucrose-6-phosphate by hydrolysis to glucose-6-phosphate and fructose. Optionally, a fructokinase ScrK (encoded by scrK) phosphorylates fructose to fructose-e- phosphate, however the presence of this enzyme is not crucial because the fructose can be phosphorylated by other mechanisms owned by the cell. The repressor protein ScrR (encoded by scrR) negatively controls the expression of the scrYAB genes and is induced by sucrose or fructose.
In one embodiment, the heterologous scr genes are introduced into the microorganism using plasmids, for example by a two-plasmid system where one contains the scrA gene and the other does the scrB gene. The scrY, scrR and optionally the scrK gene can be carried by either plasmids. In a preferred embodiment, the heterologous scr genes are introduced into the genome of the microorganism, that is they are inserted in a certain site of the genome (genetic locus) of said microorganism or cell, preferably operably linked to one or more control sequence(s) that is recognized by the host cell.
Also preferably, the genes coding for the glycosyl transferases are also inserted in the genome of the host cell. In this regard, the preferred oligosaccharide producing cells of the invention are plasmid-free. The advantage of having a plasmid free cell is that the production of the oligosaccharide, such as an HMO is increased and carbohydrate by-products are decreased. Also preferably, antibiotics are not added to the fermentation broth and neither during cell bank preparation and inoculum propagation in the process of some embodiments of this invention.
The carbohydrate acceptor to be glycosylated by the microorganism in the process of the invention can be a mono- or disaccharide selected from galactose, N-acetyl-glucosamine, a galactosylated monosaccharide, an N-acetyl-glucosaminylated monosaccharide, and glycosidic derivatives thereof defined above. All these carbohydrate derivatives can be easily taken up by a cell having a LacY permease by means of an active transport and accumulate in the cell before being glycosylated (WO 01/04341, Fort et al. J. Chem. Soc., Chem. Comm. 2558 (2005), EP-A-1911850, WO 2013/182206, WO 2014/048439). This is because the cell is able to transport these carbohydrate acceptors into the cell using its LacY permease, and the cell lacks any enzymes that could degrade these acceptors, especially LacZ. Preferably the cell has a deleted or deficient lacA gene on the lac operon.
According to another preferred embodiment, the lad gene for the lac repressor is also deleted or deactivated in the microorganism. In the absence of the functioning repressor, no inducer is needed for any gene under the control of the promoter Plac.
According to one embodiment, the hlyE gene, coding for the toxin cytolysin A, is deleted or deactivated in order to eliminate the risk of unintended expression of the toxin and thus to obtain a safer manufacturing strain.
In one embodiment, if a glp promoter is used to express one or more recombinant genes in the microorganism or cell according to the invention (see above), the regulatory repressor gene glpR may be deleted or deactivated (see WO 2019/123324), which may result in a higher overall production of the product oligosaccharides, particularly HMOs.
According to one embodiment, the genetically modified cell used in the process of the invention, particularly a LacZ E. cdi cell, is cultured in an aqueous culture medium in the following phases:
(a) an exponential cell growth phase ensured by sucrose at least 12 hours, such as around 18 hours or 20-25 hours, and then
(b) a second (feeding) phase with sucrose which is added continuously ensuring a limited cell growth that lasts until the sucrose and preferably most (e.g. at least 60 %) of the lactose have been consumed which is preferably at least 35 hours, such as at least 45 hours, 50 to 70 hours, or up to about 130 hours.
During the feeding phase, the exogenous carbohydrate acceptor, preferably lactose, to be internalized by and glycosylated in the cell, can be added to the culture medium at once, sequentially or continuously. The acceptor can be added in this second phase as a pure solid/liquid or in a form of a concentrated aqueous solution or suspension. The oligosaccharide production takes place in this second phase and can take up to 6-7 days. Preferably, this feeding phase is performed under conditions allowing the production of a culture with a high cell density.
A feature of the process of some embodiments of this invention is that there is no need to change the carbon source and/or the energy source between the growth phase and the production phase of the microorganism.
Optionally, in the case that the cells are still in possession of the lactose repressor gene lad, the process further comprises the addition of an inducer to the culture medium to induce the expression in the cell of enzyme(s) and/or of protein(s) involved in the transport of the acceptor and/or in the glycosylation of the internalized acceptor and/or in the biosynthesis of the activated sugar nucleotide donors. The inducer is preferably isopropyl b-D-thiogalactoside (IPTG) and is added to the culture medium in the beginning of the feeding phase. However, the use of inducer is not necessary if the cell is of Lad genotype.
It is believed that some embodiments of the microorganisms used in the process of invention and described above are highly stable under the process conditions of this invention described above. As a result, it is believed that these microorganisms can be used to produce oligosaccharides using sucrose as their carbon and energy sources at least at the same production rate as, and in a more reliable and reproducible manner than, microorganisms using glycerol and/or glucose as their carbon and/or energy sources. Beneficially, some embodiments of the genetically modified cells used in the process of the invention produce less carbohydrate by-products (e.g. 2’-FL, DFL, LNT or LNT-II, depending on what the desired product is) while the HMO titre and yield can be at least retained or even slightly increased, compared to the glucose or glycerol utilizing strains. These effects are increased even further when the heterologous PTS-dependent sucrose utilization system is incorporated into the genome of the host cell. In one embodiment of the invention the amount of other HMO components compared to the desired HMO are decreased by at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, when the genetically modified organism of the invention is grown on sucrose as compared to a similar microorganism which does not contain the PTS-dependant sucrose system and therefore is grown on glucose. In one embodiment of the invention the DFL/2’-FL ratio is decreased by at least 10%, by at least 15%, such as at least 20%, such as at least 25% when the genetically modified microorganism of the present invention is grown on sucrose, compared to a similar microorganism which does not contain the PTS-dependant sucrose system and therefore is grown on glucose. The use of the sucrose strains is therefore beneficial as it facilitates the purification process. Furthermore, the HMOs made by the sucrose strains are extremely low in the sum of unspecified impurities. The reduced by-product formation provides a bigger upstream process optimisation potential, since the sucrose strain does not waste as much energy in the production. This means that the process of making the oligosaccharide, such as an HMO, using a modified microorganism according to the invention, and growing it on sucrose as carbon source results in an improved carbon/energy utilization compared to when glucose is used as carbon source. Additionally, the sucrose is more resistant to heat sterilization than e.g. monosaccharide carbon sources like glucose, and thus the HMOs produced by the sucrose strain do not contain such degradation by-products, in particular the HMO product produced by the process of the present invention is essentially free of isomaltose, kojibiose, gentiobiose and nigerose.
At the end of the fermentation process, the oligosaccharide product has accumulated both in the intra- and the extracellular matrix of the microorganism. The product has preferably been transported out of the cell to the supernatant in a passive way, i.e. it can diffuse outside across the cell membrane. This transport can be facilitated by one or more sugar efflux transporters in the cell, i.e. proteins that promote the effluence of sugar derivatives from the cell to the supernatant. The sugar efflux transporter(s) can be homologous or heterologous and can be overexpressed under the conditions of the fermentation to enhance the export of the oligosaccharide derivative produced. The specificity towards the sugar moiety of the product to be secreted can be altered by mutation of the cell by means of conventional recombinant DNA manipulation techniques and protein engineering. Preferably, the oligosaccharide accumulates in the extracellular matrix. Alternatively, the oligosaccharide can be transported out of the cell to the supernatant by permealizing the cell or disrupting the cell walls in a conventional manner.
The oligosaccharide product can then be separated in a conventional manner from the aqueous culture medium, in which it was made by the cell.
A first step of separating the oligosaccharide from the culture medium preferably involves separating the oligosaccharide from the microorganism which produced it. This preferably involves clarifying the culture medium to remove suspended particulates and contaminants, particularly cells, cell components, insoluble metabolites and debris produced by culturing the genetically modified microorganism. In this step, the aqueous culture medium, which contains the oligosaccharide product, can be clarified in a conventional manner. Preferably, the culture medium is clarified by centrifugation and/or microfiltration and/or ultrafiltration.
A second step of separating the oligosaccharide from the culture medium preferably involves removing substantially all the proteins, as well as peptides, amino acids, RNA and DNA and any endotoxins and glycolipids that could interfere with the subsequent separation step, from the aqueous culture medium, preferably after it has been clarified. In this step, proteins and related impurities can be removed from the culture medium in a conventional manner. For example, proteins and related impurities are removed from the culture medium by ultrafiltration, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration (i.e., size exclusion chromatography), particularly by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography. With the exception of size exclusion chromatography, proteins and related impurities are retained by a chromatography medium or a selected membrane, while the oligosaccharide product remains in the aqueous culture medium.
If desired, the oligosaccharide product in the aqueous culture medium can then be separated from sugar-like by-product(s) and from the culture medium, after proteins and related impurities have been removed from the culture medium. This can be suitably done by subjecting the culture medium to chromatographic separation. This separation can be carried out, in case of a neutral oligosaccharide product, in a chromatographic separation column(s), filled with a conventional cationic and/or anionic ion exchange resin. The acidic cationic ion exchange resin can be in monovalent or divalent cationic form and is preferably in H+, K+, Na+, Mg2+ or Ca2+ form, particularly Ca2+. The chromatographic separation can be carried out in a conventional manner at a pH of the solution of 2 to 9. The eluent used in the chromatographic separation is preferably water, especially demineralized water, but aqueous salt solutions can also be used. Alcohols, such as ethanol, and aqueous alcohol mixtures can also be used. Treatment with ion exchange resins removes charged particles and salts, too.
Optionally, at any phase of the separation of the oligosaccharide product from the culture medium, the aqueous solution containing said oligosaccharide can be treated with active charcoal, preferably to remove coloured bodies from the solution.
According to a preferred embodiment, the process of this invention for producing an oligosaccharide, preferably having a lactose unit at the reducing end or a glycoside thereof comprises the steps of:
(i) providing a genetically modified cell comprising
- a recombinant gene encoding a glycosyl transferase enzyme which is able to transfer the glycosyl residue of an activated sugar nucleotide to the internalized lactose or glycoside thereof,
- a biosynthetic pathway to the activated sugar nucleotide,
(ii) culturing the genetically modified cell in the presence of the exogenous lactose or glycoside thereof and sucrose inducing
- internalization of the exogenous lactose or glycoside thereof via an active transport mechanism by the genetically modified cell, and
- formation of the oligosaccharide having a lactose unit at the reducing end or a glycoside thereof from the internalized lactose or glycoside thereof by a glycosyl transfer mediated by the glycosyl transferase enzyme expressed by the cell, (iii) isolating the oligosaccharide product from the cell, from the culture medium or from both, characterized in that the cell also comprises a heterologous sucrose utilization system, preferably a PTS-dependent sucrose utilization system, especially where the source regulon is scr, to provide sucrose as a carbon source for biosynthesis of said activated sugar nucleotide by said cell.
The genetically modified cell, used in this preferred process, can have more than one recombinant gene, encoding more than one glycosyl transferase enzyme which is able to transfer the glycosyl residue of an activated sugar nucleotide to an internalized acceptor molecule or the previously glycosylated acceptor made by the same cell, so the oligosaccharide product is formed from the internalized acceptor by multiple glycosyl transfer mediated by multiple glycosyl transferases expressed by the cell. Accordingly, the resulting oligosaccharide product can be a glycosylated lactose or a glycoside thereof. The glycosylated lactose is preferably an N-acetyl-glucosaminylated, galactosylated, fucosylated and/or sialylated lactose. In order to produce these derivatives the cell comprises one or more recombinant genes encoding an N-acetyl-glucosaminyl transferase, a galactosyl transferase, a sialyl transferase and/or a fucosyl transferase, and also comprise a biosynthetic pathway to generate the corresponding activated sugar type nucleotides, that is UDP-GIcNAc, UDP-Gal, GDP-Fuc and/or CMP-sialic acid, used as sugar doners for the transferases.
More preferably, the oligosaccharide product made by this process is characterized by formula 1
Figure imgf000020_0001
1 wherein Y is OH or a non-sugar aglycon defined above, preferably OH,
Ri is fucosyl or H,
R2 is fucosyl or H,
R3 is selected from H, sialyl, N-acetyl-lactosaminyl and lacto-N-biosyl groups, wherein the N-acetyl-lactosaminyl group can carry a glycosyl residue comprising one or more N- acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; each of the N-acetyl- lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,
R4 is selected from H, sialyl and N-acetyl-lactosaminyl groups optionally substituted with a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto- N-biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue, provided that at least one of the Ri, R2, R3 and R4 groups is different from H.
Even more preferably, the compound of formula 1 made by this process can be characterized by formula 1a, 1b or 1c
Figure imgf000021_0001
wherein Y, Ri and R2 are as defined above, preferably OH,
R3a is an N-acetyl-lactosaminyl group optionally substituted with a glycosyl residue comprising one N-acetyl-lactosaminyl and/or one lacto-N-biosyl group; each of the N- acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,
R4a is H or an N-acetyl-lactosaminyl group optionally substituted with a lacto-N-biosyl group; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,
R3b is a lacto-N-biosyl group optionally substituted with one or more sialyl and/or fucosyl residue(s),
R4b is H or an N-acetyl-lactosaminyl group optionally substituted with one or two N-acetyl- lactosaminyl and/or one lacto-N-biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residues,
R5 is, independently, H or sialyl, and wherein at least one of Ri, R2 or R5 is not H.
Still more preferably, the compounds according to formulae 1a or 1b made by this process are characterized in that:
- the N-acetyl-lactosaminyl group in the glycosyl residue of R3a is attached to another N- acetyl-lactosaminyl group with a 1-3 interglycosidic linkage, - the lacto-N-biosyl group in the glycosyl residue of R3a is attached to the N-acetyl- lactosaminyl group with a 1-3 interglycosidic linkage,
- the lacto-N-biosyl group in the glycosyl residue of F a is attached to the N-acetyl- lactosaminyl group with a 1-3 interglycosidic linkage,
- the N-acetyl-lactosaminyl group in the glycosyl residue
Figure imgf000022_0001
is attached to another N- acetyl-lactosaminyl group with a 1-3 or a 1-6 interglycosidic linkage,
- the lacto-N-biosyl group in the glycosyl residue of
Figure imgf000022_0002
is attached to the N-acetyl- lactosaminyl group with a 1-3 interglycosidic linkage.
Yet more preferably, the compounds according to formulae 1a, 1b and 1c made by the process are human milk oligosaccharides (when Y is OH) or glycosides thereof (when Y is non sugar aglycon).
The preferred compounds of formula 1a made by the process are selected from lacto-N- neotetraose (LNnT), para-lacto-N-hexaose (pLNH), para-lacto-N-neohexaose (pLNnH), lacto-N- neohexaose (LNnH), para-lacto-N-octaose (pLNO), lacto-N-neooctaose (pLNnO) and glycosides thereof, all of which can optionally be substituted with one or more sialyl and/or fucosyl residue. The preferred compounds of formula 1b made by the process is selected from lacto-N-tetraose (LNT), lacto-N-hexaose (LNH), lacto-N-octaose (LNO), iso-lacto-N-octaose (iLNO), lacto-N-decaose (LND), lacto-N-neodecaose (LNnD) and glycosides thereof, all of them can optionally be substituted with one or more sialyl and/or fucosyl residue.
Particularly preferred compounds of formula 1a or 1b are characterized in that:
- the fucosyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group is linked to
■ the galactose of the lacto-N-biosyl group with 1-2 interglycosidic linkage and/or
■ the N-acetyl-glucosamine of the lacto-N-biosyl group with 1-4 interglycosidic linkage and/or
■ the N-acetyl-glucosamine of the N-acetyl-lactosaminyl group with 1-3 interglycosidic linkage,
- the sialyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group is linked to
■ the galactose of the lacto-N-biosyl group with 2-3 interglycosidic linkage and/or
■ the N-acetyl-glucosamine of the lacto-N-biosyl group with 2-6 interglycosidic linkage and/or
■ the galactose of the N-acetyl-lactosaminyl group with 2-6 interglycosidic linkage. According to the most preferred aspect, the compounds of subformulae 1a, 1b or 1c are selected from the group of: 2’-fucosyllactose (2’-FL), 3-fucosy I lactose (3-FL), 2’, 3- difucosyllactose (2’,3-DFL), 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), 3’-sialyl-3- fucosyllactose (3’S3FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N- fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP-V), sialyllacto-N-tetraose a (LST-a), sialyllacto-N-tetraose b (LST-b), sialyllacto-N-tetraose c (LST-c), Sialylfucosyllacto-N-tetraose (FLST-a), Fucosylsialyllacto-N-tetraose (FLST-b), Fucosylsialyllacto-N-neotetroase (FLST-c), lacto-N- difuco-hexoase I (LNDFH-I), Lacto-N-difucohexaose II (LNDFH-II), Lacto-N-neodifucohexaose II (LNDFH-III), Disialyllacto-N-tetraose (DS-LNT), Fucosyldisialyllacto-N-tetraose (FDS-LNT I), Disialylfucosyllacto-N-tetraose (FDS-LNT II) and their glycosides, or salts thereof. The glycosides can be alpha or beta-anomers, but preferably beta-anomers.
One preferred carbohydrate acceptor, exogenously added to the culture medium, is lactose, and the preferred oligosaccharide product is a human milk oligosaccharide (HMO). The HMOs consist of a lactose unit at the reducing end and one or more from the following monosaccharide units: N-acetyl-glucosamine, galactose, fucose and sialic acid (see Urashima et al.: Milk Oligosaccharides, Nova Biomedical Books, New York, 2011, ISBN: 978-1-61122- 831-1; Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). In order to produce HMOs the cell then comprises one or more recombinant genes encoding b-1,3-N-acetyl-glucosaminyl transferase, b-1,6-N-acetyl-glucosaminyl transferase, b-1,3^3ΐ80^^I transferase, b-1,4- galactosyl transferase, a-2,3-sialyl transferase, a-2,6-sialyl transferase, a-1 ,2-fucosyl transferase, a-1,3-fucosyl transferase and/or a-1, 4 fucosyl transferase, and also comprise a biosynthetic pathway to produce the corresponding activated sugar type nucleotides, that is UDP-GIcNAc, UDP-Gal, GDP-Fuc and/or CMP-sialic acid.
According to a preferred embodiment, the process of the invention relates to the production of neutral (non-acidic) HMOs. The neutral HMOs do not contain sialic acid residue in their structure and therefore can be non-fucosylated neutral HMOs (referred to as N-acetylated HMOs or core HMOs) and fucosylated HMOs. The N-acetylated HMOs has only three types of monosaccharide in their structure: glucose, galactose and N-acetyl-glucosamine, and the N- acetylated HMOs are preferably selected from the group consisting of lacto-N-neotetraose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-neohexaose, para-lacto-N-octaose, lacto-N-neooctaose, lacto-N-tetraose, lacto-N-hexaose, lacto-N-octaose, iso-lacto-N-octaose, lacto-N-decaose and lacto-N-neodecaose, more preferably lacto-N-neotetraose, lacto-N- tetraose, lacto-N-hexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose and lacto-N- neohexaose. The group of fucosylated neutral HMOs encompasses N-acetylated HMOs disclosed above that are fucosylated and difucosylated lactoses (2’-FL, 3-FL, DFL). According, the process, in a preferred embodiment, relates to the production of neutral HMOs comprising:
(i) providing a genetically modified E. coli cell comprising:
(ia) - a recombinant gene encoding a N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to internalized lactose or to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-GIcNAc; and/or
- a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to the internalized lactose or to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc,
(ib) - optionally a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-Gal,
(ii) culturing the genetically modified E. coli cell in the presence of exogenous lactose and sucrose, thereby inducing:
- internalization of the exogenous lactose, and
- formation, within the cell, of neutral HMO, and then
(iii) separating the neutral HMO from the cell, from the culture medium or from both, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc and/or GDP-Fuc, and optionally UDP-Gal by the cell, and as energy source for making said neutral HMO.
The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, and particularly does not contain scrK.
According to another preferred embodiment, the process of this invention for producing an N- acetylated HMO, preferably of 3-6 monosaccharide units, comprises the steps of:
(i) providing a genetically modified E. coli cell comprising:
- a recombinant gene encoding a N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to internalized lactose or to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose, - optionally a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to the N-acetyl-glucosaminylated lactose to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose, and
- one or more genes encoding a biosynthetic pathway to UDP-GIcNAc and optionally to UDP-Gal,
(ii) culturing the genetically modified E. coli cell in the presence of exogenous lactose and sucrose, thereby inducing:
- internalization of the exogenous lactose, and
- formation, within the cell, of an N-acetylated HMO, and then
(iii) separating the N-acetylated HMO from the cell, from the culture medium or from both, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc and optionally UDP-Gal by the cell, and as energy source for making said N-acetylated HMO.
The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, and particularly does not contain scrK.
If the N-acetyl-glucosaminyl transferase is a b-1,3-N-acetyl-glucosaminyl transferase and no recombinant gene encoding a galactosyl transferase is present in the cell, the product is preferably lacto-N-triose II, and if a b-1,3- or a b-I,^qIqo^^I transferase is also present in the cell, the product is preferably LNT or LNnT, respectively.
According to another preferred embodiment, the process of this invention for producing a fucosylated N-acetylated HMO, preferably LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II or LNDFH-III, comprises the steps of:
(i) providing a genetically modified E. coli cell comprising:
- a recombinant gene encoding a N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-GIcNAc;
- a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc, and
- a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-Gal,
(ii) culturing the genetically modified E. coli cell in the presence of exogenous lactose and sucrose, thereby inducing:
- internalization of the exogenous lactose, and
- formation, within the cell, of the fucosylated N-acetylated HMO, and then
(iii) separating the fucosylated N-acetylated HMO from the cell, from the culture medium or from both, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc, GDP-Fuc and optionally UDP-Gal by the cell, and as energy source for making said fucosylated N-acetylated HMO.
The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, and particularly does not contain scrK.
According to yet another preferred embodiment, the process of this invention for producing a fucosylated lactose, preferably 2’-FL, 3-FL or DFL, comprises the steps of:
(i) providing a genetically modified E. coli cell comprising:
- a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to internalized lactose, and
- one or more genes encoding a biosynthetic pathway to GDP-Fuc,
(ii) culturing the cell in the presence of exogenous lactose and sucrose, thereby inducing:
- internalization of the exogenous lactose, and
- formation of the fucosylated lactose, and then
(iii) separating the fucosylated lactose product from the cell, from the culture medium or from both, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of GDP-Fuc by the cell, and as energy source for making said fucosylated lactose.
The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence and particularly does not contain scrK. If the fucosyl transferase is an a-1,2-fucosyl transferase, the product is preferably 2’- fucosyllactose (2’-FL), if the fucosyl transferase is an a-1,3-fucosyl transferase, the product is preferably 3-fucosyllactose (3-FL), and if both a-1,2- and a-1,3-fucosyl transferases are expressed in the cell, the product is preferably difucosyllactose (DFL).
According to still another preferred embodiment, the process of this invention for producing a sialylated HMO, preferably of 3-5 monosaccharide units, comprises the steps of:
(i) providing a genetically modified E. coli cell comprising:
- a recombinant gene encoding a sialyl transferase enzyme which is able to transfer the sialyl residue of CMP-sialic acid to internalized lactose or to an intermediate in the biosynthetic pathway to said sialylated HMO from lactose, and
- one or more genes encoding a biosynthetic pathway to CMP-sialic acid,
(ii) culturing the genetically modified E. coli cell in the presence of exogenous lactose and sucrose, thereby inducing:
- internalization of the exogenous lactose, and
- formation of a sialylated HMO, and then
(iii) separating the sialylated HMO from the cell, from the culture medium or from both, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of CMP-sialic acid by the cell, and as energy source for making said sialylated HMO.
The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence, and particularly does not contain scrK.
If the sialyl transferase is an a-2,3-sialyl transferase, the product is preferably 3’-sialyllactose, and if the sialyl transferase is an a-2,6-sialyl transferase, the product is preferably 6’- sialyllactose.
According to another preferred embodiment, the process of this invention for producing a fucosylated and sialylated HMO, preferably of 3-5 monosaccharide units, comprises the steps of:
(i) providing a genetically modified E. coli cell comprising:
- a recombinant gene encoding a sialyl transferase enzyme which is able to transfer the sialyl residue of CMP-sialic acid to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated and sialylated HMO from lactose, - a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated and sialylated HMO from lactose, and
- one or more genes encoding a biosynthetic pathway to CMP-sialic acid and GDP-Fuc,
(ii) culturing the genetically modified E. coli cell in the presence of exogenous lactose and sucrose, thereby inducing:
- internalization of the exogenous lactose, and
- formation of a sialyl-fucosyl-lactose, and then
(iii) separating the fucosylated and sialylated HMO from the cell, from the culture medium or from both, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of CMP-sialic acid and GDP-Fuc by the cell, and as energy source for making said fucosylated and sialylated HMO.
The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence, and particularly does not contain scrK.
If the sialyl transferase is an a-2,3-sialyl transferase and the fucosyl transferase is an a-1,3- fucosyl transferase, the product is preferably 3’-sialyl-3-fucosyllactose.
According to another preferred embodiment, the process of this invention for producing a fucosylated LNT or LNnT, comprises the steps of:
(i) providing a genetically modified E. coli cell comprising:
- a recombinant gene encoding a N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to internalized lactose to make lacto-N-triose II,
- a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to the lacto-N-triose II to make LNT or LNnT,
- a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to the LNT or LNnT,
- one or more genes encoding a biosynthetic pathway to GDP-Fuc
- one or more genes encoding a biosynthetic pathway to UDP-GIcNAc and to UDP-Gal,
(ii) culturing the genetically modified E. coli cell in the presence of exogenous lactose and sucrose, thereby inducing: - internalization of the exogenous lactose, and
- formation of a fucosylated LNT or LNnT, and then
(iii) separating the fucosylated LNT or LNnT from the cell, from the culture medium or from both, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc, UDP-Gal and GDP-Fuc by the cell, and as energy source for making said fucosylated LNT or LNnT.
The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectably or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence, and particularly does not contain scrK.
A second aspect of the invention relates to providing a genetically modified microorganism that can internalize sucrose and a carbohydrate acceptor, which is not sucrose, preferably lactose, or a glycoside thereof, into said microorganism and that comprises: a recombinant gene encoding a glycosyl transferase which can transfer a glycosyl residue of an activated sugar nucleotide to the acceptor within the microorganism, a biosynthetic pathway for making the activated sugar nucleotide from sucrose, and one or more genes encoding a heterologous PTS-dependent sucrose utilization system, preferably scr genes, so that said cell can use sucrose as a carbon source, preferably the main carbon source, more preferably the sole carbon source, for making said activated sugar nucleotide and as an energy source, preferably the main energy source, more preferably the sole energy source, thereby making an oligosaccharide or a glycoside thereof comprising the internalized carbohydrate acceptor in its structure.
The aglycon moieties of the glycosides of the internalized carbohydrate acceptors or the oligosaccharide products are disclosed in the first aspect.
The genetically modified microorganism or cell of this invention can be selected from the group consisting of bacteria and yeasts, preferably a bacterium. Bacteria are preferably selected from the group of: Escherichia coii, Bacillus spp. (e.g. Bacillus subtilis), Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Neisseria gonorrhoeae, Neisseria meningitis, Lactobacillus spp., Lactococcus spp., Enterococcus spp., Bifidobacterium spp., Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., Pseudomonas, among which E. coii is preferred, especially E. coii K-12 orE. coii BL-21 . Further in accordance with some embodiments of this invention, the microorganism or cell, preferably an E.coli cell, is genetically modified so that there is at least one alteration in its DNA sequence. The alteration can result in a change in the original characteristics of the wild type cell, e.g. the modified cell is able to perform additional chemical transformation due to the introduced new genetic material that encodes the expression of an enzymes not being in the wild type cell, or is not able to carry out transformation like degradation due to removal or deactivation of gene/genes. In this regard, a genetically modified cell contains at least one artificial alteration in its genome compared to its naturally occurring (wild type) variant. By the alteration, either a nucleic acid construct is added to the cell by way of integration into the genome or by addition via plasmid, or a nucleic acid sequence is deleted from or changed in the genome of the cell. Whatever is the case, the so-transformed cell has a genotype that is different from that before the alteration and, therefore, the modified cell shows modified feature(s). Preferably, the genetically modified cell can perform at least one additional or altered biochemical reaction, when cultured or fermented, due to the introduction of a heterologous nucleic acid sequence or the modification of a native nucleic acid sequence that encodes an enzyme that is not expressed in the wild type cell, or the genetically modified cell cannot perform a biochemical reaction due to the deletion, addition or modification of a nucleic acid sequence that encodes an enzyme found in the wild type cell. The genetically modified cell can be constructed by well-known, conventional genetic engineering techniques (e.g. Green and Sambrook: Molecular Cloning: A laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press (2012); Current protocols in molecular biology (Ausubel et al. eds.), John Wiley and Sons (2010)). The terms “host cell”, “recombinant microorganism or cell” or “genetically modified microorganism or cell” are used interchangeably to designate such constructs.
The term “promoter" means a nucleic acid sequence involved in the binding of RNA polymerase to initiate transcription of an operably linked gene, wherein the gene includes a coding DNA sequence and other (non-coding) sequences, e.g. the 5’-untranslated region (5’- UTR) located upstream of the coding sequence, which comprises a ribosomal binding site. A promoter in some embodiments of this invention is an isolated DNA sequence, i.e. not an integrated DNA fragment of the genomic DNA. The nucleotide sequence of a promoter corresponds to, or have at least 80 % identity, preferably 90-99.9 % identity with the nucleotide sequence of a fragment of bacterial genomic DNA that is regarded as promoter region of a gene, e.g. a promoter region of a glp operon or lac operon of E.coli. By “operon” is meant a functioning unit of genomic DNA containing a cluster of genes under the control of a single promoter. By “glp operon” is meant a cluster of genes involved in the respiratory metabolism of glycerol of bacteria. Preferably, a glp operon promoter sequence comprised in a DNA construct corresponds to or has at least 80 % identity, preferably 90-99.9 % identity with the nucleotide sequence of a fragment of the genomic DNA regarded as a promoter region of the corresponding glp operon of E. coir, in particular, the isolated sequence of a promoter of a glp operon of some embodiments of the invention corresponds to, or has said percent of identity with a fragment of the genomic sequence upstream the sequence having GenBank ID:
EG 10396 ( glpFKX ), EG10391 ( glpABC ), EG10394 ( glpD ), EG10401 (glpTQ). The E. coli genome is referred herein to the complete genomic DNA sequence of E coli K-12 MG1655 (GenBank ID:U00096.3).
In some embodiments, a promoter of the glp operon is operably linked to the heterologous glycosyl transferase genes and/or the heterologous scr genes. Preferably, those genes are expressed under a glpF promoter described in WO 2019/123324, the content of which is incorporated herein by reference, even more preferably by the glpF promoter of SEQ ID NO: 5.
The genetically modified microorganism or cell of this invention is able to transport the exogenous carbohydrate acceptor, preferably lactose, into the cell for glycosylation to produce a foreign oligosaccharide of interest, preferably without adversely affecting the basic functions of the cell or destroying its integrity. In one embodiment, the transport takes place via a passive mechanism, during which the exogenous acceptor diffuses passively across the plasma membrane of the cell. Diffusion of the acceptor into the microorganism is a function of the concentration differences between the fermentation broth and the extra- and intracellular space of the cell with respect to the acceptor, whereby the acceptor passes from the place of higher concentration to the place of lower concentration. In another embodiment, the acceptor is internalized with the aid of an active transport. In such a case, the genetically modified microorganism contains transporter proteins, called permeases, which act as enzymes and with which the microorganism is able to admit exogenous substances and to concentrate them in the cytoplasm. Specifically, lactose permease (LacY) acts specifically on galactose, simple galactosyl disaccharides such as lactose and their glycosides. The specificity towards the sugar moiety of the exogenous carbohydrate acceptor to be internalized can be altered by mutation of the microorganism by means of conventional recombinant DNA manipulation techniques. In a preferred embodiment, the internalization of exogenous lactose or its derivative takes place via an active transport mechanism mediated by a lactose permease. The genetically modified microorganism preferably lacks any enzyme activity (such as LacZ) that would degrade the acceptor. Likewise, the microorganism is not able to hydrolyze or degrade the oligosaccharide product or any oligosaccharide intermediate in the biosynthetic pathway to the oligosaccharide product.
Moreover, the genetically modified cell of the invention comprises one or more endogenous or recombinant genes encoding one or more glycosyl transferase enzymes that are able to transfer the glycosyl residue of an activated sugar nucleotide to the internalized acceptor. The gene or an equivalent DNA sequence thereof, if it is recombinant, can be introduced into the cell by conventional techniques, e.g. using an expression vector or by chromosomal integration. The origin of the heterologous nucleic acid sequence can be any animal (including human) or plant, eukaryotic cells such as those from Saccharomyces cerevisae, Saccharomyces pombe,
Candida albicans or from algae, prokaryotic cells such as those originated from E. coli, Bacteroides fragilis, Photo bacterium sp. , Bacillus subtilis, Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Rhizobium meliloti, Neisseria gonorrhoeae and Neisseria meningitidis, or virus. The glycosyl transferase enzyme/enzymes encoded and expressed by the gene(s) or equivalent DNA sequence(s) are preferably glucosyl transferases, galactosyl transferases, N-acetylglucosaminyl transferases, N- acetylgalactosaminyl transferases, glucuronosyl transferases, xylosyl transferases, mannosyl transferases, fucosyl transferases, sialyl transferases and the like. In a preferred embodiment, the glycosyl transferases are selected from the group consisting of b-1,3-N-acetylglucosaminyl- transferase, b-1,6-N-acetylglucosaminyl-transferase, b-1, 3-galactosyl-transferase, b-1 ,4- galactosyl-transferase, b-1 ,3-N-acetylgalactosaminyl-transferase, b-1 ,3-glucuronosyl- transferase, a-2, 3-sialyl-transferase, a-2, 6-sialyl-transferase, a-2, 8-sialyl-transferase, a-1,2- fucosyl-transferase, a-1,3-fucosyl-transferase and a-1,4-fucosyl-transferase. More preferably, the glycosyl transferases are selected from b-1,3-N-acetylglucosaminyl transferase, b-1,6-N- acetylglucosaminyl transferase, b-1,3^3ΐ80^^I transferase, b-I,'4-galactosyl transferase, a- 2,3-sialyl transferase, a-2,6-sialyl transferase, a-1,2-fucosyl transferase, a-1,3-fucosyl transferase and a-1,4 fucosyl transferase, that is from those involved in the construction of HMO core structures as well as fucosylated and/or sialylated HMOs and its glycosidic derivatives, wherein the aglycon is a moiety defined above at the group of carbohydrate acceptors. The genes encoding the above-mentioned transferases have been described in the literature.
The genetically modified microorganism or cell of the invention is capable of producing an oligosaccharide from an internalized precursor/acceptor which is preferably lactose and not sucrose. Said cell possesses enzymatic activity that is necessary for synthesizing an oligosaccharide, from an internalized mono- or disaccharide, or a glycoside thereof, via consecutive glycosylation steps, wherein the internalized acceptor, e.g. lactose, is glycosylated, in a first glycosylation step, to a trisaccharide, then that trisaccharide is glycosylated, in a second glycosylating step, to a tetrasaccharide, and so forth. The glycosylation steps are mediated by respective glycosyl transferases.
In the glycosyl transferase mediated glycosylation processes, activated sugar nucleotides serve as donors, and the glycosyl transferase in question transfers a monosaccharide of an appropriate donor molecule to the acceptor molecule. Each activated sugar nucleotide generally comprises a phosphorylated glycosyl residue attached to a nucleoside, and the specific glycosyl transferase enzyme accepts only the specific sugar nucleotide. Thus, preferably the following activated sugar nucleotides are involved in the glycosyl transfer: UDP-GIc, UDP-Gal, UDP- GlcNAc, UDP-GalNAc, UDP-glucuronic acid, UDP-Xyl, GDP-Man, GDP-Fuc and CMP-sialic acid, particularly those selected from the group consisting of UDP-Gal, UDP-GIcNAc, GDP-Fuc and CMP-sialic acid.
The genetically modified microorganism of this invention possesses a biosynthetic pathway to the above mentioned activated sugar nucleotides, that is, it has one or more sets of genes encoding one or more enzymes capable of the synthesis of one or more activated glycosyl nucleotides, ready for glycosylation in glycosyl transferase mediated reaction in the cell, when cultured. The sets of genes are either naturally present in the cell or introduced into the cell by means of recombinant DNA manipulation techniques. The production of the activated glycosyl nucleotides by the cell takes place under the action of enzymes involved in the biosynthetic pathway of that respective sugar nucleotide stepwise reaction sequence starting from a carbon source (for a review for monosaccharide metabolism see e.g. H. H. Freeze and A. D. Elbein: Chapter 4: Glycosylation precursors, in: Essentials of Glycobiology, 2nd edition (Eds. A. Varki et al.), Cold Spring Harbour Laboratory Press (2009)).
It is emphasized that the production of the activated sugar nucleotides by the genetically modified microorganism via its own biosynthetic pathway is advantageous compared to in vitro versions of transfer glycosylation, as it avoids using the very expensive sugar nucleotide type donors added exogenously, hence the donors are formed by the cell in situ and the phosphatidyl nucleoside leaving groups are recycled in the cell.
In addition, the microorganism of the invention comprises genes encoding a sucrose utilization system, that is the cell has a capability to catabolically utilize sucrose as a carbon source, as well as an energy source. The system that enables the cell to utilize sucrose can be one normally found in the gene pool of that cell but preferably is a heterologous system (i.e. derived from a different organism and transferred to the host cell by conventional recombinant DNA manipulation techniques). Typically, two kinds of sucrose catabolism can be used. According to the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (“PTS”), sucrose is transported into the microorganism and concomitantly phosphorylated to generate intracellular sucrose-6-phosphate which is hydrolysed to glucose-6-phosphate and fructose that are then involved in the central carbon metabolism of the cell. PTS can be encoded by scr or sac genes. According to non-phosphotransferase-dependent system (“non-PTS”), extracellular sucrose enters the cell with the aid of a proton symport transport system (sucrose permease) and, after transport, is hydrolysed by an invertase enzyme to glucose and fructose followed by phosphorylation. In this regard, the esc regulon consists of genes encoding the enzymes that are responsible for the non-PTS sucrose utilization.
In a preferred embodiment, the sucrose utilization system is heterologous. This is the case when the microorganism, preferably a bacterium, more preferably an E. coli, such as K-12 or BL21, is a strain that is optimized for an industrially profitable transformation like oligosaccharide production, because such a strain generally has no ability to utilize sucrose. Therefore, a sucrose uptake cassette should be introduced, using an appropriate expression plasmid or via chromosome integration, in the sucrose minus cell to make it be sucrose plus. More preferably, the sucrose pathway genes comprise a PTS-dependent sucrose utilization system, and especially the source regulon is scr. Microorganisms having scr genes are for example Salmonella ssp., Klebsiella pneumoniae, Bacteroides fragilis, Vibrio alginolyticus.
The scr genes comprise the following: scrY, scrA, scrB, scrR and scrK. The gene scrA codes for the sucrose transport protein Enzyme llScr that provides intracellular sucrose-6-phosphate from extracellular sucrose via an active transport through the cell membrane and the concomitant phosphorylation. The sucrose specific ScrY porin (encoded by scrY) facilitate the sucrose diffusion through the outer membrane. The ScrB invertase enzyme (encoded by scrB) splits the accumulated sucrose-6-phosphate by hydrolysis to glucose-6-phosphate and fructose. Optionally, a fructokinase ScrK (encoded by scrK) phosphorylates fructose to fructose-e- phosphate, however the presence of this enzyme is not crucial because the fructose can be phosphorylated by other mechanisms owned by the cell. The repressor protein ScrR (encoded by scrR) negatively controls the expression of the scrYAB genes and is induced by sucrose or fructose.
Table 1 : List of Scr proteins encoded by the scr genes
Figure imgf000034_0001
In one embodiment, the heterologous scr genes are introduced into the microorganism using plasmids, for example by a two-plasmid system where one contains the scrA gene and the other plasmid contains the scrB gene. The scrY, scrR and optionally the scrK gene can be carried by either plasmids.
In one embodiment the scr genes are selected from the sequences listed in table 1.
In one embodiment the scrY and scrA is on one plasmid and encodes the polypeptides of WP_004147437.1 (SEQ ID NO: 1) and AHM80898.1 (SEQ ID NO: 2), respectively, and the scrB and scrR is on the second plasmid encodes the polypeptides of CAA47974.1 (SE ID NO:
3) and CAA47975.1 (SEQ ID NO: 4), respectively.
In one embodiment the genetically modified microorganism comprises 4 genes which encode PTS-dependent sucrose utilization system, where the genes encode i) sucrose porin scrY of SEQ ID NO: 1 or a homologue thereof which is at least 85% identical to SEQ ID NO: 1, such as 90%, 95% or 98% identical to SEQ ID NO: 1; and ii) PTS permease scrA of SEQ ID NO: 2 or a homologue thereof which is at least 85% identical to SEQ ID NO: 2, such as 90%, 95% or 98% identical to SEQ ID NO: 2; and iii) sucrose-6-phosphate hydrolase scrB of SEQ ID NO: 3 or a homologue thereof which is at least 85% identical to SEQ ID NO: 3, such as 90%, 95% or 98% identical to SEQ ID NO: 3; and iv) repressor protein scrR of SEQ ID NO: 4 or a homologue thereof which is at least 85% identical to SEQ ID NO: 4 such as 90%, 95% or 98% identical to SEQ ID NO: 4
In a preferred embodiment, the heterologous scr genes are introduced into the genome of the microorganism, that is they are chromosomally integrated by insertion in a certain site of the genome (genetic locus) of said microorganism or cell, preferably operably linked to one or more control sequence(s) that is recognized by the host cell. Preferably, scrY and scrA are operably linked to one promoter element (an operon) and scrB and scrR is operably linked to another promoter element. The sequence of the two promoter elements may be identical for the two operons. In a referred embodiment the promoter element is pglpF, in particular the pglpF promoter of SEQ ID NO: 5. Other pglpF promoters are described in WO2019/123324, a preferred pglpF promoter is the one of SEQ ID NO:5.
In one embodiment the scr genes are introduced into the mixed acid fermentation pathway. Mixed acid fermentation results in a mixture of end products including lactate, acetate, succinate, formate and ethanol. The formation of these end products is driven by known pathways into which the scr genes can be introduced.
Also preferably, the genes coding for the glycosyl transferases are also inserted in the genome of the host cell (that is, chromosomally integrated).
Also preferably, if one or more recombinant genes involved in the biosynthetic pathway to the activated sugar type nucleotides, preferably to UDP-GIcNAc, UDP-Gal, GDP-Fuc and/or CMP- sialic acid, necessary for the production of the oligosaccharide, preferably the HMO, are present in the genetically modified cell of the invention, it is (they are) also inserted in the genome of the host cell.
In this regard, the most preferred oligosaccharide producing cells of the invention are plasmid-free.
The control sequences or promoters operably linked to heterologous DNA sequences encoding the glycosyl transferases necessary for making the oligosaccharide product or the proteins necessary for the sucrose uptake can be any suitable promoter. In some embodiments, a promoter of the glp operon is used, preferably glpF or its variant (WO 2019/123324, the content of which is incorporated herein by reference).
The carbohydrate acceptor to be glycosylated by the microorganism of the invention can be a mono- or disaccharide selected from galactose, N-acetyl-glucosamine, a galactosylated monosaccharide, an N-acetyl-glucosaminylated monosaccharide, a lactose, and glycosidic derivatives thereof defined above. All these carbohydrate derivatives can be easily taken up by a cell having a LacY permease by means of an active transport and accumulate in the cell before being glycosylated (WO 01/04341, Fort et al. J. Chem. Soc., Chem. Comm. 2558 (2005), EP-A-1911850, WO 2013/182206, WO 2014/048439). This is because the cell is able to transport these carbohydrate acceptors into the cell using its LacY permease, and the cell lacks any enzymes that could degrade these acceptors, especially LacZ, this can be achieved by deleting or mutating the lacz gene to render it incapable of expressing functional LacZ. Preferably the cell has a deleted or deficient lacA gene on the lac operon.
According to another preferred embodiment, the lad gene for the lac repressor is also deleted or deactivated in the microorganism. In the absence of the functioning repressor, no inducer is needed for expressing any gene under the control of the promoter Plac.
According to one embodiment, the hlyE gene, coding for the toxin cytolysin A, is deleted or deactivated in order to eliminate the risk of unintended expression of the toxin and thus to obtain a safer manufacturing strain.
In one embodiment, if a glp promoter is used to express one or more recombinant genes in the microorganism or cell according to the invention (see above), the regulatory repressor gene glpR may be deleted or deactivated (WO 2019/123324), which may result in a higher overall production of the product oligosaccharides, particularly HMOs.
It is believed that the microorganisms of invention described above are highly stable under the process conditions of this invention described above. As a result, it is believed that these microorganisms can be used to produce oligosaccharides using sucrose as their carbon and energy sources at least at the same production rate as, and in a more reliable and reproducible manner than microorganisms using glycerol and/or glucose as their carbon and/or energy sources. Beneficially, the genetically modified cells used in the process of the invention produce less carbohydrate by-products while the HMO titre and yield can be at least retained or even slightly increased, compared to the glucose or glycerol utilizing strains. The use of the sucrose strains is therefore beneficial as it facilitates the purification process. Furthermore, the HMOs made by the sucrose strains are extremely low in the sum of unspecified impurities. The reduced by-product formation provides a bigger upstream process optimisation potential, since the sucrose strain does not waste as much energy in the production. Additionally, the sucrose is more resistant to heat sterilization than e.g. monosaccharide carbon sources like glucose, and thus the HMOs produced by the sucrose strain do not contain such degradation by-products. In particular the HMO product is free of isomaltose, kojibiose, gentiobiose and nigerose.
The genetically modified cell of the invention can have more than one recombinant gene, encoding more than one glycosyl transferase enzyme which is able to transfer the glycosyl residue of an activated sugar nucleotide to an internalized acceptor molecule or the previously glycosylated acceptor made by the same cell, so the oligosaccharide product is formed from the internalized acceptor by multiple glycosyl transfer mediated by multiple glycosyl transferases expressed by the cell. Accordingly, the resulting oligosaccharide product can be a glycosylated lactose or a glycoside thereof. The glycosylated lactose is preferably an N-acetyl- glucosaminylated, galactosylated, fucosylated and/or sialylated lactose. In order to produce these derivatives the cell comprises one or more recombinant genes encoding an N-acetyl- glucosaminyl transferase, a galactosyl transferase, a sialyl transferase and/or a fucosyl transferase, and also comprise a biosynthetic pathway to the corresponding activated sugar type nucleotides, that is UDP-GIcNAc, UDP-Gal, GDP-Fuc and/or CMP-sialic acid.
According to a preferred embodiment, the genetically modified E. coli cell is suitable for producing a neutral HMO, preferably of 3-6 monosaccharide units, and comprises
(ia) - a recombinant gene encoding a N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to internalized lactose or to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-GIcNAc; and/or
- a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to internalized lactose or to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc,
(ib) - optionally a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-Gal, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc and/or GDP-Fuc, and optionally UDP-Gal by the cell, and as energy source for making said neutral HMO. The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence and particularly does not contain scrK.
According to a preferred embodiment, the genetically modified E. coli cell suitable for producing an N-acetylated HMO, preferably of 3-6 monosaccharide units, comprises: - a recombinant gene encoding a N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to internalized lactose or to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose,
- optionally a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to the N-acetyl-glucosaminylated lactose or to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose, and
- one or more genes encoding a biosynthetic pathway to UDP-GIcNAc and optionally to UDP-Gal, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc and optionally UDP-Gal by the cell, and as energy source for making said N- acetylated HMO. The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence, and particularly does not contain scrK.
If the N-acetyl-glucosaminyl transferase is a b-1,3-N-acetyl-glucosaminyl transferase and no recombinant gene encoding a galactosyl transferase is present in the cell, the product is preferably lacto-N-triose II, and if a b-1,3- or a b-1,4^3ΐ80^^I transferase is also present in the cell, the product is preferably LNT or LNnT, respectively.
According to another preferred embodiment, the genetically modified E. coli cell is suitable for producing a fucosylated N-acetylated HMO, preferably LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II or LNDFH-III, and comprises:
- a recombinant gene encoding an N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-GIcNAc;
- a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc, and
- a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-Gal, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc, GDP-Fuc and UDP-Gal by the cell, and as energy source for making said fucosylated N-acetylated HMO. The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence and particularly does not contain scrK.
According to yet another preferred embodiment, the genetically modified E. coli cell is suitable for producing a fucosylated HMO, preferably 2’-FL, 3-FL or DFL, and comprises:
- a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to internalized lactose, and
- one or more genes encoding a biosynthetic pathway to GDP-Fuc, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of GDP-Fuc by the cell, and as energy source for making said fucosylated HMO. The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence and particularly does not contain scrK.
If the fucosyl transferase is an a-1,2-fucosyl transferase, the product is preferably 2’- fucosyllactose, if the fucosyl transferase is an a-1,3-fucosyl transferase, the product is preferably 3-fucosyllactose, and if both a-1,2- and a-1,3-fucosyl transferases are expressed in the cell, the product is preferably difucosyllactose.
According to still another preferred embodiment, the genetically modified E. coli cell suitable for producing a sialylated HMO, preferably of 3-5 monosaccharide units, more preferably a sialylated lactose, comprises:
- a recombinant gene encoding a sialyl transferase enzyme which is able to transfer the sialyl residue of CMP-sialic acid to internalized lactose or to an intermediate in the biosynthetic pathway to said sialylated HMO from lactose, and
- one or more genes encoding a biosynthetic pathway to CMP-sialic acid, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of CMP-sialic acid by the cell, and as energy source for making said sialylated HMO. The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, which encode the proteins of SEQ ID NO: 1, 2, 3 and 4, respectively or homologues thereof with at least 85%, such as 90%, such as 95%, such as 98% identity to the respective sequence, and particularly does not contain scrK.
If the sialyl transferase is an a-2,3-sialyl transferase, the product is preferably 3’-sialyllactose, and if the sialyl transferase is an a-2,6-sialyl transferase, the product is preferably 6’- sialyllactose.
According to another preferred embodiment, the genetically modified E. coli cell suitable for producing a fucosylated and sialylated HMO, preferably of 3-5 monosaccharide units, more preferably a sialyl-fucosyl-lactose, comprises:
- a recombinant gene encoding a sialyl transferase enzyme which is able to transfer the sialyl residue of CMP-sialic acid to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated and sialylated HMO from lactose,
- a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated and sialylated HMO from lactose, and
- one or more genes encoding a biosynthetic pathway to CMP-sialic acid and GDP-Fuc, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of CMP-sialic acid and GDP-Fuc by the cell, and as energy source for making said fucosylated and sialylated HMO. The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, and particularly does not contain scrK.
If the sialyl transferase is an a-2,3-sialyl transferase and the fucosyl transferase is an a-1,3- fucosyl transferase, the product is preferably 3’-sialyl-3-fucosyllactose.
According to another preferred embodiment, the genetically modified E. coli cell is suitable for producing a fucosylated LNT or LNnT and comprises:
- a recombinant gene encoding a N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to the internalized lactose to make lacto-N-triose II,
- a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to the lacto-N-triose II to make LNT or LNnT,
- a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to the LNT or LNnT,
- one or more genes encoding a biosynthetic pathway to GDP-Fuc
- one or more genes encoding a biosynthetic pathway to UDP-GIcNAc and to UDP-Gal, characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc, UDP-Gal and GDP-Fuc by the cell, and as energy source for making said fucosylated LNT or LNnT. The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, and particularly does not contain scrK.
EMBODIMENTS
The present invention specifically discloses the following embodiments:
1. A genetically modified microorganism for making an oligosaccharide or a glycoside of said oligosaccharide, comprising: a. a recombinant gene encoding a glycosyl transferase which can transfer a glycosyl residue of an activated sugar nucleotide to a carbohydrate acceptor or a glycoside of said acceptor, b. a biosynthetic pathway for making said activated sugar nucleotide from sucrose, and c. nucleic acid sequences which encode PTS-dependent sucrose utilization system, where the nucleic acid sequences encode: i) sucrose porin scrY of SEQ ID NO: 1 or a homologue thereof which is at least 85% identical to SEQ ID NO: 1, such as 90%, 95% or 98% identical to SEQ ID NO: 1; and ii) PTS permease scrA of SEQ ID NO: 2 or a homologue thereof which is at least 85% identical to SEQ ID NO: 2, such as 90%, 95% or 98% identical to SEQ ID NO: 2; and iii) sucrose-6-phosphate hydrolase scrB of SEQ ID NO: 3 or a homologue thereof which is at least 85% identical to SEQ ID NO: 3, such as 90%, 95% or 98% identical to SEQ ID NO: 3; and iv) repressor protein scrR of SEQ ID NO: 4 or a homologue thereof which is at least 85% identical to SEQ ID NO: 4 such as 90%, 95% or 98% identical to SEQ ID NO: 4.
2. A genetically modified microorganism for making an oligosaccharide or a glycoside of said oligosaccharide, comprising:
- a recombinant gene encoding a glycosyl transferase which can transfer a glycosyl residue of an activated sugar nucleotide to a carbohydrate acceptor or a glycoside of said acceptor,
- a biosynthetic pathway for making said activated sugar nucleotide from sucrose, and
- one or more genes encoding a heterologous PTS-dependent sucrose utilization system, so that said cell is capable to use sucrose as a carbon source for making said activated sugar nucleotide and as energy source for making said oligosaccharide, wherein the carbohydrate acceptor is not sucrose and said heterologous PTS-dependent sucrose utilization system comprises proteins encoded by scr genes scrY for a sucrose porin, scrA for a PTS permease, scrB for a sucrose-6-phosphate hydrolase and scrR for a repressor protein.
3. The genetically modified microorganism of embodiment 1 or 2, wherein the glycosyl transferase can transfer a glycosyl residue to a carbohydrate acceptor internalized by the microorganism.
4. The genetically modified microorganism of any one of embodiment 1 to 3, wherein the glycosyl transferase can transfer a glycosyl residue of an activated sugar nucleotide to an intermediate in the biosynthetic pathway to said oligosaccharide from said acceptor.
5. The genetically modified microorganism of any one of embodiment 1 to 4, wherein an aglycon in the glycoside of a recombinant oligosaccharide or a carbohydrate acceptor is selected from the group consisting of:
- -ORA, wherein RA IS a linear or branched hydrocarbon chain having, when saturated, 1-24, preferably 1-6 carbon atoms or, when unsaturated, 2-24, preferably 2-6 carbon atoms, or RA means an aryl moiety, or RA means a group removable by hydrogenolysis,
- -X-RB, wherein X is N or S, and RB means linear or branched hydrocarbon chain having, when saturated, 1-24, preferably 1-6 carbon atoms or, when unsaturated, 2-24, preferably 2-6 carbon atoms, or RB means an aryl moiety, or RB means a benzyl group,
- a group that links an anomeric carbon atom and an adjacent carbon atom to each other by a
-NH-C(=0)-0-bridge, thus forming a fused 5-membered ringA as depicted in case of an aldose:
Figure imgf000042_0001
- azide,
- -NH-C(R")=C(R')2, wherein each R' independently is an electron withdrawing group selected from -CN, -COOH, -COO-alkyl, -CO-alkyl, -CONH2, -CONH-alkyl and - CON(alkyl)2, or wherein the two R'-groups are linked together and form -CO-(CH2)2-4-CO- and thus form with the carbon atom, to which they are attached, a 5-7 membered cycloalkan-1,3-dione, in which dione any of its methylene groups is optionally substituted with 1 or 2 alkyl groups, and wherein R" is H or alkyl,
- a residue of an amino acid, - a polyethylene glycol residue,
- a polyvinyl alcohol residue, and
- an a,b-unsaturated amido group of formula A or an a,b-unsaturated carbonyl group of formula B
Figure imgf000043_0001
wherein Q2, Q3 and Q4 are, independently, H and Ci-C6-alkyl, which alkyl is optionally substituted with halogen, OH, nitro or phenyl groups.
6. The genetically modified microorganism of any one of embodiment 1 to 5, which is E. coli.
7. The genetically modified microorganism of any one of embodiment 1 to 6, wherein one or more of the following genes are deleted or deactivated: glpR, lacZ, lad, lacA and hlyE.
8. The genetically modified microorganism according to embodiment 7, wherein lacZ is deleted or deactivated, and optionally one or more of the following genes are deleted or deactivated: glpR, lacl, lacA and hlyE.
9. The genetically modified microorganism of any one of embodiment 1 to 7, wherein recombinant gene encoding a glycosyl transferase is chromosomally integrated in the microorganism.
10. The genetically modified microorganism according to embodiment 9, which is plasmid-free.
11. The genetically modified microorganism of any one of embodiment 1 to 10, wherein the glycosyl transferase(s) is operably linked to a glp, such as a glpF promoter, such as the glpF promoter of SEQ ID NO: 5.
12. The genetically modified microorganism of any one of embodiment 1 to 11, wherein the scr genes are chromosomally integrated in the genome of the microorganism.
13. The genetically modified microorganism of embodiment 12, wherein scrY and scrA are operably linked and scrB and scrR are operably linked.
14. The genetically modified microorganism of any one of embodiments 2 to 13, wherein the scrYA genes are present in SEQ ID NO: 6, or a homologous sequence encoding identical proteins.
15. The genetically modified microorganism of any one of embodiments 2 to 14, wherein the the scrBR genes present in SEQ ID NO: 7 or a codon homologous sequence encoding identical proteins. 16. The genetically modified microorganism of any one of embodiments 1 to 15, wherein the scr genes are operably linked to a glp, such as a glpF promoter, such as the glpF promoter of SEQ ID NO: 5.
17. The genetically modified microorganism of any one of embodiments 1 to 16, wherein the the scrYA and scrBR genes are integrated at two sites in the mixed-acid fermentation pathway.
18. The genetically modified microorganism of any one of embodiment 1 to 17, which is plasmid-free.
19. The genetically modified microorganism of any one of embodiment 1 to 18 for producing an oligosaccharide of formula 1
Figure imgf000044_0001
1 wherein:
Y is
- -OH,
- -ORA, wherein RA IS a linear or branched hydrocarbon chain having, when saturated, 1- 24, preferably 1-6 carbon atoms or, when unsaturated, 2-24, preferably 2-6 carbon atoms, or RA means an aryl moiety, or RA means a group removable by hydrogenolysis,
- -X-RB, wherein X is N or S, and RB means linear or branched hydrocarbon chain having, when saturated, 1-24, preferably 1-6 carbon atoms or, when unsaturated, 2-24, preferably 2-6 carbon atoms, or RB means an aryl moiety, or RB means a benzyl group,
- a group that links an anomeric carbon atom and an adjacent carbon atom to each other by a
-NH-C(=0)-0-bridge, thus forming a fused 5-membered ringA as depicted in case of an aldose:
Figure imgf000044_0002
- azide,
- -NH-C(R")=C(R')2, wherein each R' independently is an electron withdrawing group selected from -CN, -COOH, -COO-alkyl, -CO-alkyl, -CONH2, -CONH-alkyl and - CON(alkyl)2, or wherein the two R'-groups are linked together and form -CO-(CH2)2-4- CO- and thus form with the carbon atom, to which they are attached, a 5-7 membered cycloalkan-1,3-dione, in which dione any of its methylene groups is optionally substituted with 1 or 2 alkyl groups, and wherein R" is H or alkyl,
- a residue of an amino acid,
- a polyethylene glycol residue,
- a polyvinyl alcohol residue, or
- an a,b-unsaturated amido group of formula A or an a,b-unsaturated carbonyl group of formula B
Figure imgf000045_0001
wherein Q2, Q3 and Q4 are, independently, H and Ci-C6-alkyl, which alkyl is optionally substituted with halogen, OH, nitro or phenyl groups,
Ri is fucosyl or H,
R2 is fucosyl or H,
R3IS selected from H, sialyl, N-acetyl-lactosaminyl and lacto-N-biosyl groups, wherein the N- acetyl-lactosaminyl group optionally carries a glycosyl residue comprising one or more N- acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; each of the N-acetyl- lactosaminyl and lacto-N-biosyl groups is optionally substituted with one or more sialyl and/or fucosyl residue,
R4 is selected from H, sialyl and N-acetyl-lactosaminyl groups optionally substituted with a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N- biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups is optionally substituted with one or more sialyl and/or fucosyl residue, provided that at least one of the Ri, R2, Rsand R4 groups is different from H.
20. The genetically modified microorganism of embodiment 19, wherein Y is -OH.
21. The genetically modified microorganism of embodiment 19 or 20, wherein the oligosaccharide of formula 1 represents a human milk oligosaccharide (HMO).
22. The genetically modified microorganism of embodiment 21, wherein the microorganism is E. coli and the HMO is a neutral HMO, and the E. coli comprises: (i) - a recombinant gene encoding an N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to an internalized lactose or to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-GIcNAc; and/or - a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to an internalized lactose or to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc,
(ii) - optionally a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer a galactosyl residue of UDP-Gal to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-Gal.
23. The genetically modified microorganism of embodiment 21, wherein the microorganism is E. coli and the HMO is an N-acetylated HMO, and the E. coli comprises:
- a recombinant gene encoding an N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to an internalized lactose or to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose,
- optionally a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer a galactosyl residue of UDP-Gal to N-acetyl-glucosaminylated lactose or to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose, and
- one or more genes encoding a biosynthetic pathway to UDP-GIcNAc and optionally to UDP-Gal.
24. The genetically modified microorganism of embodiment 22 or 23, wherein:
- the N-acetyl-glucosaminyl transferase is a b-1,3-N-acetyl-glucosaminyl transferase, no recombinant gene encoding a galactosyl transferase is present in the cell, and the N- acetylated HMO is lacto-N-triose II,
- the N-acetyl-glucosaminyl transferase is a b-1,3-N-acetyl-glucosaminyl transferase, the galactosyl transferase is a b-1,3^3ΐ80^^I transferase, and the N-acetylated HMO is lacto- N-tetraose, or
- the N-acetyl-glucosaminyl transferase is a b-1,3-N-acetyl-glucosaminyl transferase, the galactosyl transferase is a b-1,4^3ΐ80^^I transferase, and the N-acetylated HMO is lacto- N-neotetraose.
25. The genetically modified microorganism of embodiment 21, wherein the microorganism is E. coli and the HMO is an N-acetylated HMO, and the E. coli comprises:
- a recombinant gene encoding an N-acetyl-glucosaminyl transferase enzyme which is able to transfer a GlcNAc of UDP-GIcNAc to an internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-GIcNAc; and
- a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer a fucosyl residue of GDP-Fuc to an internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc, and
- a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer a galactosyl residue of UDP-Gal to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-Gal.
26. The genetically modified microorganism of embodiment 21, wherein the microorganism is E. coli and the HMO is 2'-FL, 3-FL or DFL, and the E. coli comprises:
- a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer a fucosyl residue of GDP-Fuc to an internalized lactose, and
- one or more genes encoding a biosynthetic pathway to GDP-Fuc.
27. The genetically modified microorganism according to embodiment 26, wherein the DFL/2’- FL ratio is decreased by at least 15%, preferably at least 20%, when the genetically modified organism is grown on sucrose as compared to an organism grown on glucose.
28. The genetically modified microorganism of embodiment 21, wherein the microorganism is E. coli and the HMO is a sialylated HMO, and the E. coli comprises:
- a recombinant gene encoding a sialyl transferase enzyme which is able to transfer a sialyl residue of CMP-sialic acid to an internalized lactose or to an intermediate in the biosynthetic pathway to said sialylated HMO from lactose, and
- one or more genes encoding a biosynthetic pathway to CMP-sialic acid.
29. The genetically modified microorganism of embodiment 21, wherein the microorganism is E. coli and the HMO is a fucosylated and sialylated HMO, and the E. coli comprises:
- a recombinant gene encoding a sialyl transferase enzyme which is able to transfer a sialyl residue of CMP-sialic acid to an internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated and sialylated HMO from lactose, and
- a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer a fucosyl residue of GDP-Fuc to an internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated and sialylated HMO from lactose, and
- one or more genes encoding a biosynthetic pathway to CMP-sialic acid and GDP-Fuc.
30. The genetically modified microorganism of any one of embodiments 21 to 29, wherein the E. coli is selected from K12 or B21. 31. A process for making an oligosaccharide or a glycoside of said oligosaccharide by glycosylating a carbohydrate acceptor, or a glycoside of said acceptor, wherein the carbohydrate acceptor is not sucrose, comprising the steps of: a) providing of a genetically modified microorganism of any one of embodiments 1 to 30, b) culturing said cell in the presence of said acceptor and sucrose, and c) separating said oligosaccharide from said cell, from the culture medium or from both.
32. . The process of embodiment 31, wherein the genetically modified microorganism is E. coli , such as K12 or B21.
33. The process of embodiment 31 or 32, wherein the oligosaccharide is a human milk oligosaccharide (HMO).
34. The process of embodiment 31 , wherein the HMO is a neutral HMO.
35. The process of embodiment 34, wherein the neutral HMO is an N-acetylated HMO selected from the group consisting of lacto-N-neotetraose, para-lacto-N-hexaose, para-lacto-N- neohexaose, lacto-N-neohexaose, para-lacto-N-octaose, lacto-N-neooctaose, lacto-N-tetraose, lacto-N-hexaose, lacto-N-octaose, iso-lacto-N-octaose, lacto-N-decaose and lacto-N- neodecaose, more preferably lacto-N-neotetraose, lacto-N-tetraose, lacto-N-hexaose, para- lacto-N-hexaose, para-lacto-N-neohexaose and lacto-N-neohexaose.
36. The process of embodiment 34 or 35, where the HMO is selected from LNT or LNnT.
37. The process of embodiment 34, wherein the neutral HMO is a fucosylated HMO.
38. The process of embodiment 34 or 37, wherein the neutral HMO is a fucosylated lactose selected from 2'-fucosyllactose, 3- fucosyllactose or difucosyllactose.
39. The process of embodiment 34 or 38, wherein the neutral HMO is 2'-fucosyllactose.
40. The process of embodiment 36 or 39, wherein the amount of other HMO components are decreased by at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, when the genetically modified organism is grown on sucrose as compared to an organism grown on glucose.
41. The process of any one of embodiments 31 to 40, wherein the oligosaccharide separated from the broth and the cell is substantially free of isomaltose, kojibiose, gentiobiose and nigerose.
DESCRIPTION OF SEQUENCES
Figure imgf000048_0001
Figure imgf000049_0001
EXAMPLES
Example 1 : Comparative test for making LNnT by a glycerol or sucrose utilizing E. coli
Bacterial strains:
Both strains were constructed from Escherichia coli K12 strain DH1 which was obtained from the Deutsche Sammlung von Mikroorganismen (reference DSM 5346) by deleting the genes: lacZ nanKETA lacA melA wcaJ mdoH, by inserting a Plac promoter, and maintaining genes involved in the UDP-GIcNAc and UDP-Gal biosynthesis. The glycerol utilizing strain (strain I) contains a pBBR3 -IgtA-tet plasmid carrying N. meningitidis IgtA gene for b-1 ,3-N- acetylglucosaminyl transfearse and the tetracycline resistant gene, and a pBS-galT-amp plasmid carrying Helicobacter pylori galT gene for b-I-4-galactosyl transferase and the ampicillin resistant gene. The sucrose utilizing strain (strain II) contains the two following plasmids:
- pBS-scrBR-galT-amp which is a pUC derivative carrying the galT gene encoding an b-1,4- galactosyl transferase, the s crR gene encoding a sucrose repressor, the scrB gene encoding a sucrose-6-phosphate hydrolase and the ampicillin resistance gene;
- pBBR3-scrYA-lgtA-tet which is a pBBR1-MCS3 derivative carrying the IgtA gene encoding a b-I^-N-qobΐg^IuooeqGhίhgI transferase, the scrA gene encoding a PTS system sucrose- specific EIIBC component, the scrY gene encoding a sucrose porin and the tetracycline resistance gene.
Fermentation procedure:
Glucose, glycerol, sucrose and lactose were each sterilized at 120 °C. Isopropyl thio^-D- galactopyranoside (IPTG) was filter sterilized.
The culture was carried out in a 3 I fermenter containing «0.9 I of mineral culture medium (Samain et al. J. Biotechnol. 72, 33 (1999); does not contain antibiotics for the sucrose system). The temperature was kept at 33 °C and the pH regulated at 6.8 with 28% NH4OH. The inoculum of the producing strain consisted in a LB medium (20 ml) supplemented with ampicillin and tetracycline for strain I or M9 medium (20 ml) supplemented with sucrose for strain II was added to the fermenter. The exponential growth phase started with the inoculation and stopped until exhaustion of the carbon source (glucose for strain I or sucrose for strain II, «17.5 g/l) initially added to the medium. A lactose solution (70 g of lactose/500 ml of water) was then added before starting the feeding with the carbon source (500 g/l solution, 4.5 g/h of glycerol for strain I and 3 g/h of sucrose for strain II). The inducer (isopropyl thio^-D-galactopyranoside, IPTG, 1-2 ml of a 50 mg/ml solution) was also added. The glycerol-fed fermentation (strain I) lasted for 90 hours after which the cells died (LNnT titre: 45 g/l). The sucrose-fed fermentation (strain II) produced an LNnT concentration of 56 g/l after 116 hours.
Example 2 production of 2’-FL by a sucrose utilizing E. coli
Bacterial strain:
The strain was constructed from Escherichia coli K12 strain DH1 which was obtained from the Deutsche Sammlung von Mikroorganismen (reference DSM 5346) by deleting the genes: lacZ nanKETA lacA melA wcaJ mdoH and by inserting a Plac promoter to upstream the gmd gene. In addition the strain contains the following plasmids:
- pBS-futC-scrBR-amp which is a pUC derivative carrying the futC gene encoding an a-1,2- fucosyl transferase, the s crR gene encoding a sucrose repressor, the scrB gene encoding a sucrose-6-phosphate hydrolase and the ampicillin resistance gene;
- pBBR3-GMAB-scrYA-tet which is a pBBR1-MCS3 derivative carrying the manB, manC, gmd and wcaG genes involved in the GDP-Fuc biosynthesis, the scrA gene encoding a PTS system sucrose-specific EIIBC component, the scrY gene encoding a sucrose porin and the tetracycline resistance gene.
Fermentation procedure:
Sucrose and lactose were each sterilized at 120 °C. Isopropyl thio^-D-galactopyranoside (IPTG) was filter sterilized.
The culture was carried out in a 2 I fermenter containing «0.9 I of mineral culture medium (Samain et al. J. Biotechnol. 72, 33 (1999); does not contain antibiotics). The temperature was kept at 33 °C and the pH regulated at 6.8 with 28% NH4OH. The inoculum of the producing strain consisted in a M9 medium supplemented with sucrose (20 ml) was added to the fermenter. The exponential growth phase started with the inoculation and stopped until exhaustion of sucrose («22 g/l) initially added to the medium. The inducer (isopropyl thio^-D- galactopyranoside, IPTG, 1-2 ml of a 50 mg/ml solution) was then added. A feeding with a lactose + sucrose solution (160 g of lactose + 500 g of sucrose/I in water) started with a rate of 9 ml/h for 6 hours, then was continued for 115 hours at a rate of 6 ml/h. The concentration of 21- FL was around 100 g/l in the supernatant at the end of the fermentation. Example 3: Comparative test for making 2’-FL by a glucose or sucrose utilizing E. coli
Gene targeting in the chromosomal DNA was done using standard DNA manipulation techniques, e.g. as disclosed in Warming et al. Nucleic Acids Res. 33, e36 (2005). Insertion of genetic cassettes in the chromosomal DNA was done by gene Gorging as described by Herring et al. Gene 311, 153 (2003).
The strains were derived from an E. coli platform strain that was constructed from E. coli K12 DH1 (genotype: F~, A , gyrA96, recA1, relA1, endA1, thi-1, hsdRU, supE44, obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), www.dsmz.de, reference DSM 4235) by disrupting (deletions of) the genes lacZ, nanKETA , lacA, melA, wcaJ, mdoH and by inserting a P lac promoter upstream the gmd gene. In addition, two copies of codon optimized futC encoding an a1 ,2-fucosyl transferase under the control of the glpF promoter were integrated into the genome (chromosome) of the E. coli platform strain in loci related to sugar metabolism and expressed under the control of the glpF promoter (see WO 2019/123324); a single copy of the colanic acid cluster ( gmd-wcaG-wcaH-wcal-manC-manB ) was integrated in a locus involved in the utilization of alternative carbon sources and expressed under the control of the glpF promoter; lad was deleted from the lac operon ( Alacl ); hlyE gene, coding for the toxin cytolysin A, was deleted (in order to obtain a safer manufacturing strain, eliminating the risk of unintended expression of the toxin); and the functional regulatory gene glpR, which is a repressor of the glpF promoter, were deleted (see WO 2019/123324). The resulting strain was able to produce 2’-FL when grown on glucose, however, was not able to grow on sucrose (strain 1).
To make it able to grow on sucrose, strain 1 was further modified by inserting a) scrYA from Klebsiella pneumoniae (GeneBank accession ID: X57401.1 or SEQ ID NO: 6, herein) in another locus involved in sugar metabolism and expressed under the control of a glpF promoter with a modified SD sequence (PglpF_SD1 , see WO 2019/123324 and SEQ ID NO: 5 herein), b) scrBR from Salmonella thyphimurium (GeneBank accession ID: X67750.1, or SEQ ID NO: 7 herein) in one of the loci that enables sugar metabolism under the control of the Pscr promoter (strain 2). The strains are plasmid-free.
The fermentations were carried out in 250 ml fermenters (DASBOX Mini Bioreactor system, Eppendorf) starting with 100 ml of defined mineral culture medium, consisting of 25 g/kg carbon source (glucose or sucrose), (NH^HPO^ KH2PO4, MgS04 x 7H2O, KOH, NaOH, trace element solution, citric acid, antifoam and thiamine. The trace element solution contained Mn, Cu, Fe, Zn as sulfate salts and citric acid. Lactose was added pre-inoculation at 80 g/kg. The pH throughout fermentation was controlled at 6.8 by titration with 14 % NH4OH solution. Aeration was at 1 vvm using air and dissolved oxygen was controlled above 20 % of air saturation. Fermentations were started by inoculation with 2 % (v/v) of pre-cultures grown in a similar medium. After depletion of the carbon source contained in the batch medium, a sterile feed solution containing glucose or sucrose, MgSCL x 7H2O, trace metal solution and lactose was fed continuously at a constant feed rate in a carbon-limited manner. The lactose concentration in the broth was maintained above 15 g/kg throughout and until the end of the fermentations. End- of-fermentation was at approximately 120 hours.
Throughout the fermentation, samples were taken in order to determine the concentration of 2’-FL, DFL, lactose and other minor by-products using HPLC. Total broth samples were diluted three-fold in deionized water and boiled for 20 minutes. This was followed by centrifugation at 17000g for 3 minutes, where after the resulting supernatant was analysed by HPLC. The above measurements were used to accurately calculate the 2’-FL titre, the ratio of DFL/2’-FL and the yield of 2’-FL on the carbon source.
While the only change between the two tested strains was the introduction of the sucrose operon genes on the chromosome, the fermentation data in table 2 below show clear evidence that by-product formation in the form of DFL was significantly reduced by approximately 25 % at the end-of-fermentation, while 2’-FL titre and yield can be at least retained or even slightly increased, compared to the glucose reference strain. The use of the sucrose technology is therefore beneficial as it facilitates the purification process. The reduced DFL formation will furthermore provide a bigger upstream process optimisation potential, since the sucrose strain does not waste as much energy on the production of the GDP-fucose precursor.
Table 2
Figure imgf000052_0001
*EoF = end-of-fermentation 119.3 h (±1.4 h) **SD = standard deviation
Example 4: Comparative test for making 2’-FL by a glucose or sucrose utilizing E. coli
This test comprised strain 1, disclosed in Example 3 above, and a modified strain 2, referred to as strain 2’, which differs from strain 2 disclosed in Example 3 above in that the regulatory gene glpR was not deleted and thus remained functional.
The strains were fermented separately, under equivalent industrialized conditions based on the conditions disclosed in Example 3, with glucose for strain 1 and sucrose for strain 21. The product 2’-FL was purified and isolated from the broths using identical unit step operations, without crystallization. Analysis of the purified samples showed substantial difference in the presence of specific carbohydrate impurities. Product of strain 1 (fermented with glucose) had more than 2 w/w% of glucose degradation products, containing (amongst other products) isomaltose, kojibiose, gentiobiose and nigerose, while the product of strain 2’ (fermented with sucrose) was totally free of these impurities. Furthermore, the sucrose-manufactured product is extremely low in the sum of unspecified impurities compared to the glucose-manufactured product, i.e. 0.05 w/w% vs 0.26 w/w%. The overall advantage of the sucrose technology is thereby two-fold, firstly to obtain a product with higher purity (thereby alleviating the need for costly crystallisation) and secondly, a 2 % higher carbon efficiency, since the carbon source upon heat sterilisation was not converted into non-metabolisable carbohydrate impurities.
Example 5 comparative test for making LNT
A strain was derived from an E. coli platform strain that was constructed from E. coli K12 DH1 (genotype: F~, A , gyrA96, recA1, relA1, endA1, thi-1, hsdRU, supE44, obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), www.dsmz.de, reference DSM 4235) by disrupting (deletions of) the genes lacZ, nanKETA , lacA, melA, wcaJ, mdoH and by inserting a P lac promoter upstream the gmd gene. In addition, three copies of codon optimized IgtA coding sequence for b1,3-N-acetyl glucosaminyl transferase were integrated into the genome (chromosome) of the E. coli platform strain in loci related to sugar metabolism and expressed under the control of the glpF promoter; two copies of codon optimized galTK encoding b1,3-galactosyl transferase (WO 2020/115671) was integrated into the genome of the E. coli platform strain in another loci involved in sugar metabolism and expressed under the control of the glpF promoter. The integration of the genes of the sucrose operon scrYABR (while split into scrYA and scrBR) was done at two sites in the mixed-acid fermentation pathway, thereby deleting genes that cause formation of undesired fermentative metabolites under anaerobic conditions, but not under normal aerobic fermentation conditions. In addition, an expression cassette containing the glpF promoter linked to a codon optimized DNA encoding a putative MFS transporter protein from Rosenbergiella nectarea (GenBank accession ID: WP_092672081.1), which facilitates the export of LNT from the cell, was inserted in a locus involved in the utilization of alternative carbon sources. Moreover, lad and hlyE were deleted. The so-constructed strain is plasmid-free and able to grow on glucose and sucrose.
The fermentations were carried out in 250 ml fermenters (DASBOX Mini Bioreactor system, Eppendorf) starting with 100 ml of defined mineral culture medium, consisting of 30 g/kg carbon source (glucose or sucrose), (NFU^HPCL, KH2PO4, MgSCL x TFhO, KOH, NaOH, trace element solution, citric acid, antifoam and thiamine. The trace element solution contained Mn, Cu, Fe, Zn as sulfate salts and citric acid. Lactose was provided by bolus addition of 80 g/kg approximately 5 hours after the end of the batch phase, i.e. at approximately 18-19 h elapsed fermentation time (ELT). The pH throughout fermentation was controlled at 6.8 by titration with 14 % NH4OH- solution. Aeration was at 1 vvm using air and dissolved oxygen was controlled above 23 % of air saturation. Fermentations were started by inoculation with 2 % (v/v) of pre-cultures grown in a similar medium. After depletion of the carbon source contained in the batch medium, a sterile feed solution containing glucose or sucrose, (NFU^SCL, trace metal solution and anti-foam was fed continuously at a constant feed rate in a carbon-limited manner. Lactose was almost depleted at 72.8 h ELT in the sucrose fermentations, while it was still at around 8-10 g/l in the glucose fermentation, indicating a higher lactose utilisation rate and hence product formation. End-of-fermentation samples were taken and analysed at 72.8 h.
Throughout the fermentation, samples were taken in order to determine the concentration of LNT and lactose. Total broth samples were diluted three-fold in deionized water and boiled for 20 minutes. This was followed by centrifugation at 17000g for 3 minutes, where after the resulting supernatant was analysed by HPLC. The HPLC measurements were used to accurately calculate the LNT titre, by-product ratios (not shown) and the accumulated yield of LNT on the carbon source. The latter takes also smaller variations in feed rates and dilutions into account and is therefore an important parameter for direct comparison.
This study describes a direct comparison of a sucrose-utilising strain in fermentations with sucrose versus glucose as carbon source. The processes were identical except for the type of carbon source used. The data demonstrate clearly superiority of sucrose in terms of LNT productivity and carbon yield. The table 3 below shows that the average carbon yield was increased by 17% when using sucrose instead of glucose. This is also reflected by the 19 % higher final LNT titre at end-of-fermentation and the increased lactose consumption rate (lactose was only added by bolus addition and not in the feed solution). Overall, the use of the sucrose technology is therefore beneficial as it not only facilitates the purification process due to the higher final titre, but also leads to a more efficient carbon source conversion to product.
Table 3: LNT production from strains with and without heterologous PTS-dependent sucrose utilization system.
Figure imgf000054_0001
*EoF = end-of-fermentation 72.8 h
Example 6 Strain without scr genes cannot grow on sucrose
This example illustrates that the background strain used for constructing the above strains are not capable of growing on sucrose. The background strain, MDO, was constructed from Escherichia coli K12 DH1. The E. coli K12 DH1 genotype is: F , k, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. In addition to the E. coli K12 DH1 genotype MDO has the following modifications: lacZ deletion of 1,5kbp, iacA\ deletion of 0,5kbp, nanKETA : deletion of 3,3kbp, melA\ deletion of 0,9kbp, wcaJ\ deletion of 0,5kbp, mdoH. deletion of 0,5kbp, and insertion of Plac promoter upstream of the gmd gene.
The strain MPLNnT can produce the tetrasaccharide HMO, LNnT, while the MDO strain does not form a product of biotechnological interest. The strain MPLNnT bears the genes that are responsible for the biosynthesis of LNnT, namely IgtA (encoding a beta-1, 3-N- acetyloglucosamine transferase) from N. meningitidis and galT (encoding a beta-1, 4- galactosyltransferase) from H. pylori, as well as four additional genes conferring the ability to grow on sucrose as the sole carbon and energy source. These four genes are organized in 2 operons, scrBR and scrYA, which are controlled by a wild-type sucrose promoter (Pscr) and a synthetic dual promoter (PglpF_SD1-Pscr), respectively and integrated into the genome.
The strains were grown in 96-well microtiter plates using a 4-day protocol. During the first 24 hours, cells were grown to high densities while in the next 72 hours cells were transferred to a medium containing sucrose as the sole carbon and energy source. Specifically, during day 1, fresh inoculums were prepared using a Luria-Bertani broth containing 20% glucose. After 24 hours of incubation of the prepared cultures at 34 °C, cells were transferred to a basal minimal medium (200 uL) supplemented with magnesium sulphate and thiamine to which an initial bolus of 20 % glucose solution and 15 g/L sucrose solution as carbon source was provided to the cells. After inoculation of the new medium, cells were shaken at 1200 rpm at 28 °C for 72 hours. The cells were grown in a batch mode of cultivation in microtiter plates that were compatible with the Varioskan LUX Multimode Microplate Reader from ThermoFisher Scientific.
In the present Example, it is demonstrated that the background strain, MDO, is not able to grow on sucrose whereas a strain with the chromosomally integrated scrBR and scrYA genes grows very efficiently on sucrose (Figure 2).
The MPLNnT stain has also been subjected to fermentation and has shown to be capable of producing between 83 and 85% LNnT relative to the total amount of HMO produced when grown on sucrose.
Example 7 Genomic integration vs plasmid expression of scrYA and scrBR
The benefit of integrating the genes necessary for HMO production into the genome as compared to expressing them from high-copy-number expression plasmids was analysed.
The plasmid containing strain of example 2 and the chromosomal strain 2’ of example 4 were compared in a head-to-head analysis. The chromosomal strain 2’ of example 4 has two copies of futC, which is necessary to achieve levels of futC which is similar to those obtained from the high expression plasmid pBS in example 2. Furthermore, the expression plasmids in the strain of example 2 have antibiotic markers (amp and tet) to secure that the plasmids are maintained within the cell when propagated, such markers are not needed in chromosomal strains, which is an advantage in production since antibiotics can be avoided in cell banking and during propagation. The strains were fermented separately, under equivalent industrialized conditions based on the conditions disclosed in Example 3, with sucrose for both strains. The product 2’-FL was purified and isolated from the broths using identical unit step operations, without crystallization.
The results are shown in table 4 below.
Table 4. 2’-FL expression from plasmid strain and chromosomal strain
Figure imgf000056_0001
From this it can be seen that the 2’FL yield is slightly higher (but statistically significant) for the chromosomal strain. In addition the amount of DFL by-product is reduced for the chromosomal strain in that the DFL/2’FL ratio is lower, this is a benefit in the down-stream processing since less by-product needs to be removed to obtain pure 2’FL.

Claims

1. A genetically modified microorganism for making an oligosaccharide or a glycoside of said oligosaccharide, comprising: a. a recombinant gene encoding a glycosyl transferase which can transfer a glycosyl residue of an activated sugar nucleotide i. to a carbohydrate acceptor or a glycoside of said acceptor, where said acceptor is internalized by the microorganism, and/or ii. to an intermediate in the biosynthetic pathway to said oligosaccharide from said acceptor, b. a biosynthetic pathway for making said activated sugar nucleotide from sucrose, and c. one or more genes encoding a heterologous PTS-dependent sucrose utilization system, so that said cell is capable to use sucrose as a carbon source for making said activated sugar nucleotide and as energy source for making said oligosaccharide, wherein the carbohydrate acceptor is not sucrose, or a glycoside of said acceptor, and said heterologous PTS-dependent sucrose utilization system comprises proteins encoded by scr genes scrY for a sucrose porin, scrA for a PTS permease, scrB for a sucrose-e- phosphate hydrolase and scrR for a repressor protein, and wherein the recombinant gene encoding a glycosyl transferase is inserted in the genome of the microorganism and/or the scr genes are inserted in the genome of the microorganism.
2. The genetically modified microorganism according to claim 1, which is plasmid-free.
3. The genetically modified microorganism according to claim 1 or 2, wherein the scr genes are operably linked to a glp promoter, such as a glpF promoter, such as the glpF promoter of SEQ ID NO: 5.
4. The genetically modified microorganism according to claim 1 to 3, wherein an aglycon in the glycoside of an oligosaccharide or a carbohydrate acceptor is selected from the group consisting of:
- -ORA, wherein RA IS a linear or branched hydrocarbon chain having, when saturated, 1-24, preferably 1-6 carbon atoms or, when unsaturated, 2-24, preferably 2-6 carbon atoms, or RA means an aryl moiety, or RA means a group removable by hydrogenolysis,
- -X-RB, wherein X is N or S, and RB means linear or branched hydrocarbon chain having, when saturated, 1-24, preferably 1-6 carbon atoms or, when unsaturated, 2-24, preferably 2-6 carbon atoms, or RB means an aryl moiety, or RB means a benzyl group,
- a group that links an anomeric carbon atom and an adjacent carbon atom to each other by a -NH-C(=0)-0-bridge, thus forming a fused 5-membered ringA as depicted in case of an aldose:
Figure imgf000058_0001
- azide,
- -NH-C(R")=C(R')2, wherein each R' independently is an electron withdrawing group selected from -CN, -COOH, -COO-alkyl, -CO-alkyl, -CONH2, -CONH-alkyl and - CON(alkyl)2, or wherein the two R'-groups are linked together and form -CO-(CH2)2-4-CO- and thus form with the carbon atom, to which they are attached, a 5-7 membered cycloalkan-1,3-dione, in which dione any of its methylene groups is optionally substituted with 1 or 2 alkyl groups, and wherein R" is H or alkyl,
- a residue of an amino acid,
- a polyethylene glycol residue,
- a polyvinyl alcohol residue, and
- an a,b-unsaturated amido group of formula A or an a,b-unsaturated carbonyl group of formula B
Figure imgf000058_0002
wherein Q2, Q3 and Q4 are, independently, H and CrC6-alkyl, which alkyl is optionally substituted with halogen, OH, nitro or phenyl groups.
5. The genetically modified microorganism according to any of the claims 1 to 4, which is E. coli.
6. The genetically modified microorganism according to any of the claims 1 to 5, wherein one or more of the following genes are deleted or deactivated: lacZ, lad, lacA and hlyE.
7. The genetically modified microorganism according to claim 6, wherein lacZ is deleted or deactivated, and optionally one or more of the following genes are deleted or deactivated: lacl, lacA and hlyE.
8. The genetically modified microorganism according to any of the claims 1 to 7 for producing an oligosaccharide of formula 1:
Figure imgf000059_0001
1 wherein:
Y is
- -OH,
- -ORA, wherein RA IS a linear or branched hydrocarbon chain having, when saturated, 1- 24, preferably 1-6 carbon atoms or, when unsaturated, 2-24, preferably 2-6 carbon atoms, or RA means an aryl moiety, or RA means a group removable by hydrogenolysis,
- -X-RB, wherein X is N or S, and RB means linear or branched hydrocarbon chain having, when saturated, 1-24, preferably 1-6 carbon atoms or, when unsaturated, 2-24, preferably 2-6 carbon atoms, or RB means an aryl moiety, or RB means a benzyl group,
- a group that links an anomeric carbon atom and an adjacent carbon atom to each other by a
-NH-C(=0)-0-bridge, thus forming a fused 5-membered ringA as depicted in case of an aldose:
Figure imgf000059_0002
- azide,
- -NH-C(R")=C(R')2, wherein each R' independently is an electron withdrawing group selected from -CN, -COOH, -COO-alkyl, -CO-alkyl, -CONH2, -CONH-alkyl and - CON(alkyl)2, or wherein the two R'-groups are linked together and form -CO-(CH2)2-4- CO- and thus form with the carbon atom, to which they are attached, a 5-7 membered cycloalkan-1,3-dione, in which dione any of its methylene groups is optionally substituted with 1 or 2 alkyl groups, and wherein R" is H or alkyl,
- a residue of an amino acid,
- a polyethylene glycol residue,
- a polyvinyl alcohol residue, or
- an a,b-unsaturated amido group of formula A or an a,b-unsaturated carbonyl group of formula B
Figure imgf000060_0001
wherein Q2, Q3 and Q4 are, independently, H and Ci-C6-alkyl, which alkyl is optionally substituted with halogen, OH, nitro or phenyl groups,
Ri is fucosyl or H,
R2 is fucosyl or H,
R3IS selected from H, sialyl, N-acetyl-lactosaminyl and lacto-N-biosyl groups, wherein the N- acetyl-lactosaminyl group optionally carries a glycosyl residue comprising one or more N- acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; each of the N-acetyl- lactosaminyl and lacto-N-biosyl groups is optionally substituted with one or more sialyl and/or fucosyl residue,
R4 is selected from H, sialyl and N-acetyl-lactosaminyl groups optionally substituted with a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N- biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups is optionally substituted with one or more sialyl and/or fucosyl residue, provided that at least one of the Ri, R2, Rsand R4 groups is different from H.
9. The genetically modified microorganism according to claim 8, wherein Y is -OH.
10. The genetically modified microorganism according to claim 9, wherein the oligosaccharide of formula 1 represents a human milk oligosaccharide (HMO).
11. The genetically modified microorganism according to claim 10, wherein the microorganism is E. coli and the HMO is a neutral HMO, and the E. coli comprises: i. a recombinant gene encoding an N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to the internalized lactose or to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-GIcNAc; and/or ii. a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to the internalized lactose or to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc, and iii. optionally a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to an intermediate in the biosynthetic pathway to said neutral HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-Gal.
12. The genetically modified microorganism according to claim 10, wherein the microorganism is
E. coli and the HMO is an N-acetylated HMO, and the E. coli comprises: i. a recombinant gene encoding an N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to the internalized lactose or to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose, and ii. optionally a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to the N-acetyl-glucosaminylated lactose or to an intermediate in the biosynthetic pathway to said N-acetylated HMO from lactose; and iii. one or more genes encoding a biosynthetic pathway to UDP-GIcNAc and optionally to UDP-Gal.
13. The genetically modified microorganism according to claim 12, wherein: i. the N-acetyl-glucosaminyl transferase is a b-1,3-N-acetyl-glucosaminyl transferase, no recombinant gene encoding a galactosyl transferase is present in the cell, and the N- acetylated HMO is lacto-N-triose II, or ii. the N-acetyl-glucosaminyl transferase is a b-1,3-N-acetyl-glucosaminyl transferase, the galactosyl transferase is a b-1,3^3ΐ80^^I transferase, and the N-acetylated HMO is lacto-N-tetraose, or iii. - the N-acetyl-glucosaminyl transferase is a b-1,3-N-acetyl-glucosaminyl transferase, the galactosyl transferase is a b-I,^qIqo^^I transferase, and the N-acetylated HMO is lacto-N-neotetraose.
14. The genetically modified microorganism according to claim 10, wherein the microorganism is
E. coli and the HMO is an N-acetylated HMO, and the E. coli comprises: i. a recombinant gene encoding an N-acetyl-glucosaminyl transferase enzyme which is able to transfer the GlcNAc of UDP-GIcNAc to the internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-GIcNAc; and ii. a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to the internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to GDP-Fuc, and iii. a recombinant gene encoding a galactosyl transferase enzyme which is able to transfer the galactosyl residue of UDP-Gal to an intermediate in the biosynthetic pathway to said fucosylated N-acetylated HMO from lactose, and one or more genes encoding a biosynthetic pathway to UDP-Gal.
15. The genetically modified microorganism according to claim 10, wherein the microorganism is E. coli and the HMO is 2'-FL, 3-FL or DFL, and the E. coli comprises: i. a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to the internalized lactose, and ii. one or more genes encoding a biosynthetic pathway to GDP-Fuc.
16. The genetically modified microorganism according to claim 15, wherein the DFL/2’-FL ratio is decreased by at least 15%, preferably at least 20%, when the genetically modified organism is grown on sucrose compared to a similar microorganism which does not contain the PTS-dependant sucrose system and is grown on glucose.
17. The genetically modified microorganism according to claim 10, wherein the microorganism is E. coli and the HMO is a sialylated HMO, and the E. coli comprises: i. a recombinant gene encoding a sialyl transferase enzyme which is able to transfer the sialyl residue of CMP-sialic acid to the internalized lactose or to an intermediate in the biosynthetic pathway to said sialylated HMO from lactose, and ii. one or more genes encoding a biosynthetic pathway to CMP-sialic acid.
18. The genetically modified microorganism according to claim 10, wherein the microorganism is E. coli and the HMO is a fucosylated and sialylated HMO, and the E. coli comprises: i. a recombinant gene encoding a sialyl transferase enzyme which is able to transfer the sialyl residue of CMP-sialic acid to the internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated and sialylated HMO from lactose, ii. a recombinant gene encoding a fucosyl transferase enzyme which is able to transfer the fucosyl residue of GDP-Fuc to the internalized lactose or to an intermediate in the biosynthetic pathway to said fucosylated and sialylated HMO from lactose, and iii. one or more genes encoding a biosynthetic pathway to CMP-sialic acid and GDP-Fuc.
19. A process for making an oligosaccharide or a glycoside of said oligosaccharide by glycosylating a carbohydrate acceptor, or a glycoside of said acceptor, wherein the carbohydrate acceptor is not sucrose, comprising the steps of: a. providing of a genetically modified microorganism according to any one of claims 1 to 18, b. culturing said cell in the presence of said acceptor and sucrose, and c. separating said oligosaccharide from said cell, from the culture medium or from both.
20. The process according to claim 19, wherein the genetically modified microorganism is E. coli.
21. The process according to claim 19 or 20, wherein the utilization of the sucrose carbon source is improved compared to when glucose is used as carbon source.
22. The process according to claim 19 to 21, wherein the oligosaccharide is a human milk oligosaccharide (HMO).
23. The process of any one of claims 19 to 22, wherein the oligosaccharide separated from the said cell, from the culture medium or from both is substantially free of isomaltose, kojibiose, gentiobiose and nigerose.
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