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WO2025083041A1 - New fucosyltransferases for in vivo synthesis of complex fucosylated human milk oligosaccharides mixtures comprising lnfp-vi or lnfp-v - Google Patents

New fucosyltransferases for in vivo synthesis of complex fucosylated human milk oligosaccharides mixtures comprising lnfp-vi or lnfp-v Download PDF

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WO2025083041A1
WO2025083041A1 PCT/EP2024/079175 EP2024079175W WO2025083041A1 WO 2025083041 A1 WO2025083041 A1 WO 2025083041A1 EP 2024079175 W EP2024079175 W EP 2024079175W WO 2025083041 A1 WO2025083041 A1 WO 2025083041A1
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lnfp
acid sequence
hmo
cell
seq
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Manos PAPADAKIS
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DSM IP Assets BV
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    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
    • C07H5/06—Aminosugars
    • 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
    • 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.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • 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
    • 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
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01065—3-Galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase (2.4.1.65), i.e. alpha-1-3 fucosyltransferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01152—4-Galactosyl-N-acetylglucosaminide 3-alpha-L-fucosyltransferase (2.4.1.152)

Definitions

  • the present disclosure relates to the production of complex fucosylated Human Milk Oligosaccharides (HMOs) and in particular to the production of the complex fucosylated HMO LNFP-VI or LNFP-V, with five or more monosaccharide units, where said production leads to a product which is essentially free of LNFP-111 and LNDFH-111 or LNFP-II and LNDFH-II, respectively.
  • HMOs Human Milk Oligosaccharides
  • the present disclosure also relates to genetically engineered cells and a-1 ,3- fucosyltransferases suitable for use in said production, as well as to methods for producing said fucosylated HMOs.
  • HMOs Human Milk Oligosaccharides
  • Dumon et al., 2004 (Biotechnol. Prog. 2004, 20, 412-419) further describes two a-1 , 3- fucosyltransferases, FutA and FutB, which are also suggested to produce mixtures of LNFP-VI, LNDFH-III and 3FL or LNnT, LNFP-III, LNFP-VI and LNDFH-I II, respectively.
  • WO2023/110995 discloses fucosyltransferases having alpha-1 , 3-fucosyltransferase activity on the N-acetylglucosamine (GIcNAc) and/or the glucose (Glc) on various saccharide structures including lactose, LNT and LNnT to produce mixtures of fucosylated oligosaccharides such as for example LNFP-III, LNFP-VI and LNDFH-III or LNFP-II, LNFP-V and LNDFH-II.
  • GIcNAc N-acetylglucosamine
  • Glc glucose
  • W02016/040531 discloses a number of a-1 , 3-fucosyltransferase, including CafC and CafF, which are capable of producing 3FL.
  • production of fucosylated HMOs especially specific complex fucosylated HMOs, such as LNFP-VI and LNFP-V, is challenging due to the lack of fucosyltransferases with the desired substrate specificity, as well as low production yield of the desired fucosylated HMOs as compared to other HMO products present after fermentation, such as HMO precursor products and complex fucosylated HMO by-products, which may require laborious separation procedures.
  • the need for highly substrate specific a-1 ,3-fucosyltransferases is solved by the identification of a selection of a-1 ,3-fucosyltransferases which exhibit low or no specificity for the N- acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNnT or LNT as a substrate for fucosylation reactions, but which are highly substrate specific for the Glucose (Glc) moiety in LNnT or LNT.
  • GIcNAc N- acetylglucosamine
  • Gal Galactose
  • a first aspect of the present disclosure relates to methods for producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI) or lacto-N-fucopentaose V (LNFP-V), with less than 5 % of the total molar content of HMO being fucosylated by-product oligosaccharides with 5 or 6 monosaccharide units, comprising the steps of a) providing a genetically engineered cell with a recombinant nucleic acid sequence encoding an a-1 ,3- fucosyltransferase derived from Bacteroidales bacterium , and b) cultivating said genetically modified cell under conditions that allow for formation of LNFP-VI of LNFP-V, and c) optionally, purifying said LNFP-VI or LNFP-V by removing by-products such as 3FL and/or LNnT or 3FL and/or LNT, respectively.
  • the a-1 ,3-fucosyltransferase has high specificity for the glucose (Glc) moity in LNnT and/or LNT and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNnT.
  • Glc glucose
  • GIcNAc N-acetylglucosamine
  • Gal Galactose
  • a second aspect is a genetically engineered cell capable of producing the Human Milk Oligosaccharide (HMO) selected from lacto-N-neofucopentaose VI (LNFP-VI) and lacto-N- fucopentaose V (LNFP-V), comprising a recombinant nucleic acid sequence encoding an a-1 , 3- fucosyltransferase, Bacbac2, comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2.
  • HMO Human Milk Oligosaccharide
  • LNFP-VI lacto-N-neofucopentaose VI
  • LNFP-V lacto-N- fucopentaose V
  • the cell may further produce one or more HMOs selected from the group consisting of 3FL, LNT-II and LNT and it is preferred that essentially no LNFP-II and/or LNDFH- II is produced by said cell.
  • the cell may comprise additional modifications such as a substrate importer selected from a lactose importer, a lacto-N-triose-ll (LNT-II) importer and a LNT importer.
  • a third aspect is a genetically engineered cell capable of producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI), comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase, be selected from the group consisting of, a) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO:
  • HMO Human Milk Oligosaccharide
  • LNFP-VI lacto-N-neofucopentaose VI
  • Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO:
  • the genetically engineered further comprises one or more recombinant nucleic acid sequences needed to produce LNnT in said cell.
  • the cell may further produces one or more HMOs selected from the group consisting of 3FL, LNT-II and LNnT and it is preferred that essentially no LNFP-III and/or LNDFH-111 is produced by said cell.
  • the cell may comprise additional modifications such as a substrate importer selected from a lactose importer, a lacto-N-triose-ll (LNT-II) importer and a LNnT importer.
  • a fifth aspect of the present disclosure relates to a mixture of HMOs, preferably produced with a method according to the disclosure, wherein the mixture of HMOs consists essentially of a) LNFP-VI and 3-FL, or b) LNFP-VI or c) LNnT, or LNFP-V, 3FL and LNnT or d) LNFP-V, 3FL and LNT.
  • a sixth aspect of the disclosure relates to the use of a mixture or composition according to the disclosure, in an infant formula, a dietary supplement and/or medical nutrition.
  • Figure 1 Overview of the synthesis of complex fucosylated HMO with an LNnT-backbone.
  • Figure 2 llustrate the data from the strains of table 7, clearly showing the difference in LNFP-VI production from the strains with different enzymes.
  • LNFP-VI is the black bar
  • 3FL is the grey bar
  • LNnT is the diagonally striped bar
  • LNDFH-111 is the chequered bar
  • LNFP-111 is the horizontally striped bar
  • pLNnH is the dotted bar.
  • FIG. 3 Overview of the synthesis of complex fucosylated HMO with an LNT-backbone.
  • Figure 4 Shows the regeneration and viability of lyophilized Lactobacillus rhamnosus (DSM 33156), incubated for 3 h at pH 3.0 and plated in up to 4 dilutions 1 :1000 (E-3), 1 :10,000 (E-4), 1 :100,000 (E-5) and 1 :1 ,000,000 (E-6).
  • A) is the control without HMOs;
  • B) is Lactobacillus rhamnosus (DSM 33156) in combination with an HMO mixture containing 80% LNFP-VI and 10% 3FL and 10% LNnT (mix 1);
  • C) is Lactobacillus rhamnosus (DSM 33156) in combination with an HMO mixture containing 60% LNFP-VI and 40% 3FL (mix2).
  • the present disclosure approaches the biotechnological challenges of in vivo HMO production of, in particular, complex fucosylated HMOs which comprise at least five monosaccharide units, of which at least one monosaccharide unit is a fucosyl unit, such as e.g., LNFP-VI and LNFP-V.
  • complex fucosylated HMOs which comprise at least five monosaccharide units, of which at least one monosaccharide unit is a fucosyl unit, such as e.g., LNFP-VI and LNFP-V.
  • the present disclosure offers specific strain engineering solutions to produce specific complex fucosylated HMOs, in particular, LNFP-VI or LNFP-V, by exploiting the substrate specificity of the identified a-1 ,3-fucosyltransferases, Bacbad , Bacbac2, Paral and CafF, disclosed herein, in particular towards the glucose (Glc) moiety and not the N-acetylglucosamine (GIcNAc) or galactose (Gal) moieties in LNnT (or LNT).
  • Glc glucose
  • GIcNAc N-acetylglucosamine
  • Gal galactose
  • a genetically engineered cell of the present disclosure expresses genes encoding key enzymes for the biosynthesis of fucosylated HMOs.
  • the genetically engineered cell expresses the genes needed to produce LNnT or LNT either from lactose or LNT-II as the initial substrate.
  • the cell may be engineered to take up LNnT (see for example W02023/099680) only needing the 1 ,3-fucosyltransferase activity in the cell.
  • a genetically engineered cell of the present disclosure further expresses one or more of the de novo GDP-fucose pathway genes, manA, manB, manC, gmd and/or wcaG, responsible for the formation of GDP-fucose. It may be advantageous to overexpress one or more of these genes and/or to upregulate the colanic acid gene cluster (CA), including the genes gmd, wcaG, wcaH, weal, manC and manB from E.
  • CA colanic acid gene cluster
  • nucleic acid construct encoding the CA as shown in SEQ ID NO: 41 , allowing for formation of GDP- fucose, which enables the cell to produce a higher level of fucosylated oligosaccharide from one or more oligosaccharide substrates, such as lactose, LNT-II and/or LNnT.
  • one or more additional glycosyltransferases and pathways for producing nucleotide-activated sugars such as glucose-UDP-GIcNAc, CMP-N-acetylneuraminic acid, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and/or CMP-N-acetylneuraminic acid can also be present in the genetically engineered cell.
  • a-1 ,3-fucosyltransferases of the present disclosure in the present context is their ability to specifically recognize and fucosylate the Glc moiety in LNnT, to generate LNFP-VI.
  • the present disclosure describes enzymes with a-1 , 3- fucosyltransferase activity (a-1 ,3-fucosyltransferases) that are more active on the Glc moiety of LNnT than a-1 ,3-fucosyltransferases described in the prior art, such as FutA and FutB (see Dumon et al., 2004), CafC (WO2016/040531) and FucT109 (WO2019/008133).
  • the a-1 ,3-fucosyltransferases described herein have very low activity on the GIcNAc moieties of LNnT. If LNnT is available in sufficient amounts inside the genetically engineered cell, very little, if any, LNFP-111 and/or LNDFH- 111 is produced by the a-1 ,3-fucosyltransferases described in the present disclosure.
  • the traits of the a-1 ,3-fucosyltransferases described herein are therefore well-suited for high-level industrial production of LNFP-VI without production of alternatively fucosylated complex oligosaccharide by-products, such as LNFP-III and LNDFH-III.
  • the a-1 ,3-fucosyltransferases of the present disclosure primarily fucosylate the glucose (Glc) moiety of an acceptor oligosaccharide such as e.g., LNnT.
  • the term “primarily” is to be understood as less than 5%, such as less than 4%, such as less than 3%, or such as less than 2% of other moieties in LNnT are fucosylated by the a-1 ,3-fucosyltransferases of the present disclosure.
  • the a-1 ,3-fucosyltransferases of the present disclosure has low or no activity on the N-acetylglucosamine (GIcNAc) moiety in the acceptor molecule.
  • the acceptor molecule is an oligosaccharide such as example e.g., an HMO, such as LNT-II or LNnT, but also other oligosaccharides or HMOs.
  • LNnT is the preferred acceptor molecule for the a-1 ,3- fucosyltransferases of the present disclosure.
  • the term low or no activity is to be understood as less than 5%, such as less than 4%, such as less than 3%, or such as less than 2% of the GIcNAc moiety in the acceptor molecule is fucosylated by the a-1 ,3-fucosyltransferases of the present disclosure. Accordingly, in embodiments, the oligosaccharides produced by the cell expressing said a-1 ,3-fucosyltransferases is essentially free of N-acetylglucosamine (GIcNAc) fucosylated oligosaccharides.
  • GIcNAc N-acetylglucosamine
  • the oligosaccharides produced by the cell expressing said a-1 ,3-fucosyltransferases is essentially free of the N-acetylglucosamine (GIcNAc) fucosylated oligosaccharides LNFP-III and/or LNDFH-111.
  • GIcNAc N-acetylglucosamine
  • the genetically engineered cells of the present disclosure which express an a-1 ,3- fucosyltransferase with high specificity for the Glc moity in LNnT, enable the production of high titters of LNFP-VI.
  • LNFP-VI in absence of other complex fucosylated oligosaccharide byproducts with 5 or 6 monosaccharide units, such as LNFP-III and LNDFH-111.
  • the present disclosure enables a more efficient LNFP-VI production, which is highly beneficial in biotechnological production of more complex fucosylated HMOs, such as LNFP-VI.
  • the mixtures of HMOs produced by the cells and/or methods described herein contain a high percentage of LNFP-VI out of the total amount of HMOs produced, such as at least 25% of the total amount of HMOs, preferably at least 50%, such as at least 60%, such as alt least 70%, such as at least 80% of the total amount of HMO produced by the cell.
  • LNFP-VI ability to fermentatively produce LNFP-VI with no or very low amounts of fucosylated oligosaccharide by-products with 5 or 6 monosaccharide units is a benefit in purification since separation of fucosylated HMOs of similar length is very challenging, and the method and genetically engineered cells described herein therefore provide a benefit in terms of obtaining LNFP-VI with at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% purity of the finished product, in particular by applying one or more purification steps after fermentation to remove undesired by-products, in particular HMO by-products.
  • the genetically engineered cells of the present disclosure may also be engineered to produce fucosylated HMOs with an LNT backbone.
  • Such a cell is further engineered to express an a-1 ,3-fucosyltransferase with high specificity for the Glc moity in LNT, enable the production of high titters of LNFP-V.
  • the present disclosure also enables a more efficient LNFP-V production, which is highly beneficial in biotechnological production of more complex fucosylated HMOs, such as LNFP-V.
  • the a-1 ,3-fucosyltransferases of the present disclosure primarily fucosylates the glucose (Glc) moiety of an acceptor oligosaccharide such as LNT.
  • the term “primarily” is to be understood as less than 5%, such as less than 4%, such as less than 3%, such as less than 2% of other moieties in LNnT are fucosylated by the a-1 ,3-fucosyltransferases of the present disclosure.
  • the a-1 ,3-fucosyltransferases of the present disclosure does not possess any or very low a-1 ,4-fucosyltransferase activity which prevents it from fucosylating the GIcNAc) moiety of LNT, since this is bound to the terminal galactose via an a-1 ,3-linkage and therefore not available for fucosylation by an enzyme that only possess a-1 ,3-fucosyltransferase activity.
  • low or no activity is to be understood as less than 2%, such as less than 1 .5%, such as less than 1 %, such as less than 0.5%, such as less than 0.1% of the GIcNAc moiety in the acceptor molecule, such as LNT, is fucosylated by the a-1 ,3-fucosyltransferases of the present disclosure.
  • the oligosaccharides produced by the cell expressing said a-1 ,3-fucosyltransferases is essentially free of N-acetylglucosamine (GIcNAc) fucosylated oligosaccharides such as e.g., N- acetylglucosamine (GIcNAc) fucosylated oligosaccharides with an LNT and/or LNnT backbone.
  • GIcNAc N-acetylglucosamine
  • the genetically engineered cell of the present disclosure which express an a-1 , 3- fucosyltransferase with high specificity for the Glc moity in LNT, enables the production of LNFP-V mixtures with very small amounts of other fucosylated oligosaccharides.
  • the genetically engineered cell produces a mixture of LNFO-V and LNT with less than 5%, such as les then 2% of other fucosylated HMOs.
  • the LNFP-V mixtures do not contain other complex fucosylated HMOs, such as LNFP-II and LNDFH-II, which eases purification of LNFP-V.
  • the oligosaccharides produced by the cell expressing said a-1 ,3-fucosyltransferases is essentially free of the N-acetylglucosamine (GIcNAc) fucosylated oligosaccharides LNFP-II and/or LNDFH-II.
  • GIcNAc N-acetylglucosamine
  • the mixtures of HMOs produced by the cells and/or methods described herein may alternatively contain a high percentage of LNFP-V out of the total amount of HMOs produced, such as at least 40% of the total amount of HMOs, preferably at least 45%, such as at least 50%, such as alt least 55%, or such as at least 57% of the total amount of HMO produced by the cell and/or method.
  • LNFP-V ability to fermentatively produce LNFP-V with no or very low amounts of fucosylated oligosaccharide by-products with 5 or 6 monosaccharide units is a benefit in purification since separation of fucosylated HMOs of similar length is very challenging, and the method and genetically engineered cells described herein therefore provide a benefit in terms of obtaining LNFP-V with at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% purity of the finished product, in particular by applying one or more purification steps after fermentation to remove undesired by-products, in particular HMO by-products.
  • oligosaccharide means a sugar polymer containing at least three monosaccharide units, i.e., a tri-, tetra-, penta-, hexa- or higher oligosaccharide.
  • the oligosaccharide can have a linear or branched structure containing monosaccharide units that are linked to each other by interglycosidic linkages.
  • the oligosaccharide comprises a lactose residue at the reducing end and one or more naturally occurring monosaccharides of 5-9 carbon atoms selected from aldoses (e.g., glucose, galactose, ribose, arabinose, xylose, etc.), ketoses (e.g., fructose, sorbose, tagatose, etc.), deoxysugars (e.g. rhamnose, fucose, etc.), deoxy-aminosugars (e.g.
  • aldoses e.g., glucose, galactose, ribose, arabinose, xylose, etc.
  • ketoses e.g., fructose, sorbose, tagatose, etc.
  • deoxysugars e.g. rhamnose, fucose, etc.
  • deoxy-aminosugars e.g.
  • the oligosaccharide is an HMO.
  • HMO Human milk oligosaccharide
  • oligosaccharides of the disclosure are human milk oligosaccharides (HMOs).
  • human milk oligosaccharide in the present context means a complex carbohydrate found in human breast milk.
  • the HMOs have a core structure comprising a lactose unit at the reducing end that can be elongated by one or more beta-N-acetyl- lactosaminyl and/or one or more beta-lacto-N-biosyl unit, and this core structure can be substituted by an a-L-fucopyranosyl and/or an a-N-acetyl-neuraminyl (fucosyl) moiety.
  • HMO structures are e.g., disclosed by Xi Chen in Chapter 4 of Advances in Carbohydrate Chemistry and Biochemistry 2015 vol 72.
  • fucosylated HMOs examples include, 2'-fucosyllactose (2’FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3- fucosyllactose (3FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N- fucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-III), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose (LNFP-V), lacto-N-fucopentaose VI (LNFP-VI), lacto-N- difucohexao
  • complex fucosylated HMOs are fucosylated HMOs that comprises at least 5 monosaccharide units of which at least one monosaccharide unit is a fucosyl unit
  • non-limiting examples of complex fucosylated HMOs are the fucosylated HMOs consisting of 5 monosaccharide units e.g., LNFP-I, LNFP-II, LNFP-III, LNFP-V and LNFP-VI and complex fucosylated HMO with 6 monosaccharide units such as but not limited to the di- fucosylated HMOs LNDFH-I, LNDFH-II and LNDFH-III or the sialyl-fucosyl HMOs FLST-a, FLST-b, FLST-c and FLST-d.
  • a complex fucosylated HMO is one that requires at least three different glycosyltransferase activities to be produced from lactose as the initial substrate, e.g., the formation of LNFP-VI requires an Glc specific a-1 ,3-fucosyltransferase, a - 1 ,3-N-acetyl-glucosaminyl-transferase and a p-1 ,4-galactosyltransferase (see figure 1), and the formation of LNDFH-III requires at least one a-1 ,3-fucosyltransferase, a p-1 ,3-N-acetyl- glucosaminyl-transferase and a p-1 ,4-galactosyltransferase.
  • Enzymes described herein preferably has a preferred activity on only the Glc moiety of LNnT or LNT, thus being capable of producing LNFP-VI from LNnT (see figure 1), or LNFP-V from LNT ( Figure 3) with no or only very little production complex fucosylated by-product oligosaccharides, such as LNFP-III and/or LNDFH-I II or LNFP-II and/or LNDFH-I II.
  • complex fucosylated HMOs are fucosylated HMOs that comprises at least 5 monosaccharide units of which at least one monosaccharide unit is a fucosyl unit
  • non-limiting examples of complex fucosylated HMOs are the fucosylated HMOs consisting of 5 monosaccharide units e.g., LNFP-I, LNFP-II, LNFP-III, LNFP-V and LNFP-VI and fucosylated HMO with 6 monosaccharide units, such as but not limited to LNDFH-I, LNDFH-II and LNDFH-I 11.
  • complex fucosylated by-product oligosaccharide refers to a complex fucosylated oligosaccharide which is not the desired product.
  • complex fucosylated HMOs mentioned above can be considered as by-products, also interchangeably termed HMO by-products, if they are not desired in the final product, which in the current disclosure is LNFP-VI or LNFP-V.
  • fucosylated by-product oligosaccharides with 5 our 6 monosaccharide units are e.g., LNFP-III, LNFP-II, LNDFH-III and LNDFH-II.
  • the fucosylated human milk oligosaccharide (HMO) produced by the cell is LNFP-VI, such as primarily LNFP-VI.
  • at least 25 %, such as at least 30%, 50%, 55%, 60%, 70%, 75%, 80% or 82% of the molar content of the total HMOs produced by said cell is LNFP-VI.
  • at least 60 % of the molar content of the total HMOs produced by said cell is LNFP-VI.
  • at least 80 % of the molar content of the total HMOs produced by said cell is LNFP-VI and LNnT.
  • At least 80 %, such as at least 85%, 90%, 95% or such as at least 99% or such as 100% of the molar content of the total HMOs produced by said cell is LNFP-VI and 3FL.
  • less than 3%, such as 0.0%, or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5% or such as less than 2.99% of the total molar content of HMOs produced by the cell is LNFP-III.
  • less than less than 3% such as 0.0%, or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5% or such as less than 2.99% of the total molar content of HMOs produced by the cell is LNDFH-III.
  • less than 5% of the molar content of the total HMOs produced by the cell is an alternative complex fucosylated oligosaccharide by-product.
  • an alternative fucosylated oligosaccharide by-product is considered to be one or more fucosylated oligosaccarides which is not LNFP-VI.
  • fucosylated oligosaccharides such as fucosylated HMO(s) may be selected from the group consisting of 3-FL, DFL, LNFP-III and LNDFH-III.
  • fucosylated HMO may be selected from the group consisting of LNFP-III and LNDFH-III.
  • no LNFP-III or LNDFH-III is produced by the cell.
  • less than 2% such as 0.0%, or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1 .5%, 1 .75%, or such as less than 1 .99% of the total molar content of HMOs produced by the cell is LNFP-II.
  • less than less than 2% such as 0.0%, or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 1.75%, or such as less than 1.99% of the total molar content of HMOs produced by the cell is LNDFH-II.
  • an alternative fucosylated oligosaccharide by-product is considered one or more fucosylated oligosaccharides, such as HMO, which is not LNFP-V.
  • An alternative fucosylated HMO(s) may be selected from the group consisting of 3-FL, DFL, LNFP-II and LNDFH-II.
  • An alternative complex fucosylated HMO may be selected from the group consisting of LNFP-II and LNDFH-II.
  • Production of LNFP-V may require the presence of two or more glycosyltransferase activities, in particular, if starting from lactose as the acceptor oligosaccharide.
  • An acceptor oligosaccharide may require the presence of two or more glycosyltransferase activities, in particular, if starting from lactose as the acceptor oligosaccharide.
  • a genetically engineered cell according to the present disclosure comprises a recombinant nucleic acid sequence encoding a fucosyltransferase with a-1 ,3-fucosyltransferase activity capable of transferring fucose from an activated sugar to the glucose moiety of an acceptor oligosaccharide, preferably LNnT or LNT, in an a-1 ,3 linkage.
  • an acceptor oligosaccharide is an oligosaccharide that can act as a substrate for a glycosyltransferase capable of transferring a glycosyl moiety from a glycosyl donor to the acceptor oligosaccharide.
  • the glycosyl donor is preferably a nucleotide-activated sugar as described in the section on “Glycosyl-donor - nucleotide-activated sugar pathways”.
  • the acceptor oligosaccharide is a precursor for making a more complex HMO and can also be termed the precursor molecule.
  • the acceptor oligosaccharide can be either an intermediate product of the present fermentation process, an end-product of a separate fermentation process employing a separate genetically engineered cell, or an enzymatically or chemically produced molecule.
  • said acceptor oligosaccharide for the a-1 ,3-fucosyltransferase is preferably lacto- N-tetraose (LNT), which is produced from the precursor molecules lactose (e.g., acceptor for the P-1 ,3-N-acetyl-glucosaminyl-transferase), and/or lacto-N-triose II (LNT-II) (e.g., acceptor for the P-1 ,3-galactosyltransferase).
  • Lactose may also be identified as the initial substrate, if this is what is supplied to during cultivations. In cases where it is desired that the cell does not produce 3FL, the preferred initial precursor molecule (alternative initial substrate) supplied to the cultivation is LNT-II.
  • the precursor molecule is preferably supplied to the genetically engineered cell at the beginning of the cultivation or by continuous feeding or pulse feeding during the cultivation or a combination, allowing the genetically engineered cell to produce LNnT or LNT from the initial precursor.
  • the initial precursor is lactose, and the genetically engineered cell is capable of producing the intermediate precursors (acceptor oligosaccharides, e.g. LNT-II and LNnT or LNT) inside the cell.
  • the initial precursor may however also be LNT-II or LNnT or LNT if the cell is capable of importing at least one of these compounds.
  • the genetically engineered cell according to the present disclosure comprises at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase, e.g., a fucosyltransferase, capable of transferring a fucosyl residue from a fucosyl donor to an acceptor oligosaccharide to synthesize one or more fucosylated human milk oligosaccharide product, i.e., a fucosyltransferase.
  • a fucosyltransferase capable of transferring a fucosyl residue from a fucosyl donor to an acceptor oligosaccharide to synthesize one or more fucosylated human milk oligosaccharide product, i.e., a fucosyltransferase.
  • the genetically engineered cell according to the present disclosure may comprise one or more further recombinant nucleic acids encoding one or more recombinant and/or heterologous glycosyltransferases capable of transferring a glycosyl residue from a glycosyl donor to an acceptor oligosaccharide.
  • the additional glycosyltransferase(s) enables the genetically engineered cell to synthesize LNnT or LNT from a precursor molecule, such as lactose or LNT-II.
  • the genetically engineered cell described herein comprises one or more further recombinant nucleic acid encoding one or more recombinant and/or heterologous glycosyltransferase.
  • the fucosyltransferase in the genetically engineered cell of the present disclosure is an a-1 ,3-fucosyltransferase.
  • the a-1 ,3-fucosyltransferase is capable of transferring a fucose unit onto the Glc moiety of an LNnT or LNT molecule.
  • the a-1 ,3- fucosyltransferase is specific towards the Glc moiety of an LNnT or LNT molecule.
  • LNFP-III and LNDFH-111 allow for an easier purification of LNFP-VI, as the purification of LNFP-VI from a mixture of HMOs predominantly comprising LNFP-VI would be simpler, as it is easier to separate LNFP-VI from smaller HMOs than separating different fucosylated HMOs of the same or similar size from each other, e.g., LNFP-VI from LNFP-III or LNDFH-111.
  • a lower initial amount or complete absence of LNFP-III and/or LNDFH-111 is considered beneficial in the purification of LNFP-VI.
  • a-1 ,3-fucosyltransferase it is desired for the a-1 ,3-fucosyltransferase to have low activity on the glucose moiety of lactose to reduce the 3FL formation during fermentation where lactose is used as the initial substrate.
  • the use of an a-1 ,3-fucosyltransferase according to the present disclosure results in that at least 14 %, such as at least 25%, such as at least 50% of the molar content of the total HMOs produced by a cell according to the present disclosure is LNFP-VI.
  • the a-1 ,3-fucosyltransferase according to the present disclosure produce less than 5% of LNFP-III and/or LNDFH-III, preferably essentially no LNFP-III and/or LNDFH-III is produced.
  • the a-1 ,3-fucosyltransferase capable of transferring a fucosyl moiety from a fucosyl donor to the glucose (Glc) moity in lacto-N-neotetraose (LNnT) is an a-1 , 3- fucosyltransferase derived from Bacteroidales bacterium.
  • Examples of such a-1 ,3- fucosyltransferases are of Bacbad or Bacbac2 with an amino acid sequence according to SEQ ID NO: 1 or 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 or 2.
  • the a-1 ,3-fucosyltransferase is Bacbac2 with an amino acid sequence according to SEQ ID NO: 2 or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2.
  • Bacbac2 can be used to produce LNFP-VI above 50%, such as above 60% of the molar content of the total HMOs.
  • the a-1 ,3-fucosyltransferase Bacbac2 according to the present disclosure produce less than 2% of LNFP-III and/or LNDFH-III, such as essentially no LNFP-III and LNDFH-111 of the molar content of the total HMOs produced by a cell or method.
  • Bacbac2 has low affinity towards lactose and therefore produce less than 15% 3FL, such as less than 10% 3FL of the molar content of the total HMOs produced by a cell or method.
  • Conversion of the substrate LNnT to LNFP-VI is highly efficient in fermentation in that less than 15% LNnT remains at the end of fermentation.
  • the high LNnT conversion in fermentation results in essentially no pLNnH being produced in fermentation.
  • all LNT-II is converted into LNnT since the fermentations are essentially free of LNT-II.
  • the a-1 ,3-fucosyltransferase Bacbac2 (SEQ ID NO: 2) as disclosed herein or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, can be used to LNFP-V above 50 %, such as at least 55% of the molar content of the total HMOs.
  • the a-1 ,3-fucosyltransferase Bacbac2 according to the present disclosure produce less than 2% of LNFP-II and/or LNDFH-II, such as essentially no LNFP-II and LNDFH-II of the molar content of the total HMOs produced by a cell or method.
  • Bacbac2 has low affinity towards lactose and therefore produce less than 5% 3FL, such as less than 2% 3FL of the molar content of the total HMOs produced by a cell or method.
  • all LNT-II is converted into LNT.
  • LNnT Conversion of the substrate LNnT to LNFP-VI is highly efficient in fermentation in that less than 5% LNnT, such as less than 2% LNnT remains at the end of fermentation. In addition, all LNT-II is converted into LNnT since the fermentations are essentially free of LNT-II.
  • the a-1 ,3-fucosyltransferase enzyme capable of transferring a fucosyl moiety from a fucosyl donor to an acceptor oligosaccharide can be selected from the group consisting of Bacbad , Bacbac2, Paral and CafF with an amino acid sequence according to SEQ ID NO: 1 , 2, 3, or 43, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , 2, 3, or 43 (table 1).
  • a-1 ,3- fucosyltransferase enzymes have a particular high specificity for the glucose (Glc) moity in lacto-N-neotetraose (LNnT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or galactose (Gal) moieties in LNnT.
  • Glc glucose
  • GIcNAc N-acetylglucosamine
  • Gal galactose
  • LNFP-VI LNFP-VI with less than 5% of the total molar content of HMO being fucosylated byproducts oligosaccharides with 5 or 6 monosaccharide units.
  • the a-1 ,3-fucosyltransferase is Paral with an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 3.
  • Paral can be used to produce LNFP-VI above 25% of the molar content of the total HMOs, with less than 2% of LNFP-II and/or LNDFH-II, such as essentially no LNFP-111 and LNDFH-111 present in the mixture.
  • These enzymes can e.g., be used to produce LNFP-VI with minor or no production of alternative complex fucosylated by-product HMOs, in particular with less than 5%, such as less than 2% of the total molar content of HMO being fucosylated by-products oligosaccharides with 5 or 6 monosaccharide units, such as LNFP-III and/or LNDFH-III .
  • the a-1 ,3-fucosyltransferase of the present disclosure can be selected from an amino acid sequence with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to the amino acid sequence of any one of the a-1 ,3-fucosyltransferases listed in table 1.
  • Table 1 List of a-1 ,3-fucosyltransferase enzymes of the present disclosure capable of producing LNFP-VI, with little or no LNFP-III and/or LNDFH-III by-product formation. 1
  • GenBank IDs reflect the full-length enzymes, in the present disclosure truncated, elongated or mutated versions may have been used, these are represented by the sequences indicated by the SEQ ID NOs.
  • Example 1 of the present disclosure discloses the identification of the heterologous a-1 , 3- fucosyltransferases Bacbacl , Bacbac2, Paral and CafF (SEQ ID NO: 1 , 2, 3, and 43, respectively), which are capable of producing mixtures of HMOs with LNFP-VI being the predominant complex fucosylated HMO in the mixture (i.e., more than 10 fold, such as 50 fold over LNFP-111 and LNDFH-111) when introduced into an LNnT producing cell, compared to the previously known a-1 ,3-fucosyltransferase FutA, FutB, CafC and FucT109 (SEQ ID NO: 5, 6, 47 and 45 respectively).
  • the three novel enzymes Bacbacl , Bacbac2 and Paral which are novel with respect to HMO production and the CafF enzyme known to produce 3FL can specifically transfer a fucosyl unit onto the Glc moiety of LNnT in an a-1 ,3 linkage to form LNFP- VI at a level above 14%, such as above 25% of the total HMO, while not producing any LNFP-III or LNDFH-III, while the prior art enzymes FutA, FutB, CafC and FucT109 also produces LNDFH-III and/or LNFP-III, respectively, as by-products.
  • Example 1 The fact that the experiments performed in Example 1 show that the enzymes Bacbacl , Bacbac2, Paral and CafF do not produce any LNFP-III or LNDFH-III, indicates that these enzymes are highly substrate-specific for the Glc moiety in LNnT.
  • the expression of an a-1 ,3-fucosyltransferase according to the present disclosure in a genetically engineered cell is further combined with expression of one or more further recombinant nucleic acids encoding one or more heterologous glycosyltransferases.
  • the expression of an a-1 ,3-fucosyltransferase of the disclosure in a genetically engineered cell is combined with expression of a p-1 ,4-galactosyltransferase, such as galT from Helicobacter pylori to enable formation of LNnT from LNT-II as initial substrate.
  • a third enzyme is added, such as a p-1 ,3-N-acetyl-glucosaminyl- transferase, e.g., LgtA from Neisseria meningitidis to enable formation of LNnT from lactose as the initial substrate.
  • a p-1 ,3-N-acetyl-glucosaminyl- transferase e.g., LgtA from Neisseria meningitidis to enable formation of LNnT from lactose as the initial substrate.
  • a genetically engineered cell is combined with expression of a p-1 ,3-galactosyltransferase, such as galTK from Helicobacter pylori to enable formation of LNT from LNT-II as initial substrate.
  • a third enzyme is added, such as a p-1 ,3-N-acetyl-glucosaminyl-transferase, e.g., LgtA from Neisseria meningitidis to enable formation of LNT from lactose as the initial substrate.
  • meningitidis or a functional homologue thereof with an amino acid sequence with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to SEQ ID NO: 14.
  • the LNT-II precursor is formed using a p- 1 ,3-N-acetylglucosaminyltransferase.
  • the genetically engineered cell comprises a p-1 ,3-N-acetylglucosaminyltransferase gene, or a functional homologue or fragment thereof, to produce the intermediate LNT-II from lactose as the initial substrate.
  • LgtA heterologous p-1 ,3-N-acetyl-glucosaminyl-transferase
  • a p-1 ,3-Galactosyltransferase is any protein that comprises the ability of transferring the galactose of UDP-Galactose to a N-acetyl-glucosaminyl moiety to an acceptor molecule in a beta-1 , 3-linkage.
  • a p-1 ,3-galactosyltransferase used herein does not originate in the species of the genetically engineered cell i.e., the gene encoding the p-1 ,3- galactosyltransferase is of heterologous origin.
  • Non-limiting examples of p-1 ,3-galactosyltransferases are given in table 12.
  • p-1 ,3- galactosyltransferases variants may also be useful, preferably such variants are at least 80%, such as at least 85%, such as at least 90, such as at least 95% identical, such as at least 96%, such as at least 97%, such as at least 98% or such as 99% identical to one of the p-1 ,3- galactosyltransferases in table 12.
  • acceptor molecule is an acceptor saccharide, e.g., LNT-II, or more complex HMO structures.
  • GalTK heterologous p-1 ,3-galactosyltransferase
  • LNFP-V heterologous p-1 ,3-galactosyltransferase
  • the cell of the present disclosure further comprises a recombinant nucleic acid sequence encoding a p-1 ,3-N-acetyl-glucosaminyltransferase.
  • the recombinant nucleic acid sequence encoding a p-1 ,3-galactosyltransferases comprises or consists of the amino acid sequence of SEQ ID NO: 42 (galTK from H.
  • the genetically modified cell comprises a p-1 ,3-galactosyltransferase gene, or a functional homologue or fragment thereof.
  • LgtA from Neisseria meningitidis is used in combination with galTK from Helicobacter pylori and Bacbac2 from Bacteroidales bacterium to produce LNFP-V starting from lactose as initial substrate.
  • galTK from Helicobacter pylori is used in combination with Bacbac2 from Bacteroidales bacterium to produce LNFP-V starting from LNT-II as initial substrate.
  • a p-1 ,4-galactosyltransferase is any protein that comprises the ability of transferring the galactose of UDP-Galactose to a N-acetyl-glucosaminyl moiety to an acceptor molecule in a p- 1 ,4-linkage (see figure 1).
  • a p-1 ,4-galactosyltransferase used herein does not originate in the species of the genetically engineered cell i.e., the gene encoding the p-1 ,4- galactosyltransferase is of heterologous origin.
  • the acceptor molecule is an acceptor saccharide, e.g., LNT-II, or more complex HMO structures.
  • the genetically engineered cell comprises one or more recombinant nucleic acid sequence(s) encoding a p-1 ,4- galactosyltransferase.
  • Non-limiting examples of p-1 ,4-galactosyltransferases are provided in table 2.
  • p-1 ,4- galactosyltransferases variants may also be useful, preferably such variants are at least 80%, such as at least 85%, such as at least 90, such as at least 95% identical, such as at least 96%, such as at least 97%, such as at least 98% or such as 99% identical to the amino acid sequence of any one of the p-1 ,4-galactosyltransferases in table 3.
  • p-1 ,4-glycosyltransferases In embodiments described herein the p-1 ,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis, and the p-1 ,3-galactosyltransferase from Helicobacter pylori, respectively.
  • the recombinant nucleic acid sequence encoding a p-1 ,4- galactosyltransferases comprises or consists of the amino acid sequence of SEQ ID NO: 15 (galT from H. pylori) or a functional homologue thereof with an amino acid sequence with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to SEQ ID NO: 15.
  • the genetically engineered cell comprises a p-1 ,4-galactosyltransferase gene, or a functional homologue or fragment thereof.
  • the p-1 ,3-N- acetylglucosaminyltransferase is from Neisseria meningitidis and the p-1 ,4- galactosyltransferase is from Helicobacter pylori.
  • the pi ,3-N- acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO: 14, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 14 and the p-1 ,4-galactosyltransferase has an amino acid sequence according to SEQ ID NO: 15, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 15.
  • a glycosyltransferase mediated glycosylation reaction takes place in which an activated sugar nucleotide serves as glycosyl- donor.
  • An activated sugar nucleotide generally has a phosphorylated glycosyl residue attached to a nucleoside.
  • a specific glycosyl transferase enzyme accepts only a specific sugar nucleotide.
  • activated sugar nucleotides are involved in the glycosyl transfer: glucose-UDP-GIcNAc, UDP-galactose, UDP-glucose, UDP-N- acetylglucosamine, UDP-N-acetylgalactosamine (GIcNAc) and CMP-N-acetylneuraminic acid.
  • the genetically engineered cell according to the present disclosure can comprise one or more pathways to produce a nucleotide-activated sugar selected from the group consisting of glucose-UDP-GIcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine (GIcNAc), UDP-N-acetylgalactosamine and CMP-N-acetylneuraminic acid.
  • a nucleotide-activated sugar selected from the group consisting of glucose-UDP-GIcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine (GIcNAc), UDP-N-acetylgalactosamine and CMP-N-acetylneuraminic acid.
  • the genetically engineered cell is capable of producing one or more activated sugar nucleotides mentioned above by a de novo pathway.
  • an activated sugar nucleotide is made by the cell under the action of enzymes involved in the de novo biosynthetic pathway of that respective sugar nucleotide in a stepwise reaction sequence starting from a simple carbon source like glycerol, sucrose, fructose or glucose (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)).
  • the enzymes involved in the de novo biosynthetic pathway of an activated sugar nucleotide can be naturally present in the cell or introduced into the cell by means of gene technology or recombinant DNA techniques, all of them are parts of the general knowledge of the skilled person.
  • the genetically engineered cell can utilize salvaged monosaccharides for sugar nucleotide.
  • monosaccharides derived from degraded oligosaccharides are phosphorylated by kinases, and converted to nucleotide sugars by pyrophosphorylases.
  • the enzymes involved in the procedure can be heterologous ones, or native ones of the host cell.
  • the colanic acid gene cluster of Escherichia coll encodes selected enzymes involved in the de novo synthesis of GDP-fucose (gmd, wcaG, wcaH, weal, manB, manC), whereas one or several of the genes downstream of GDP-L-fucose such as wcaJ, which are responsible for the production of the extracellular polysaccharide colanic acid, a major oligosaccharide of the bacterial cell wall, can be deleted to prevent conversion of GDP-fucose to colanic acid.
  • the promoter of the native colanic acid gene cluster may be exchanged with a stronger promoter, generating a recombinant colanic acid gene cluster, to drive additional production of GDP-fucose.
  • an extra copy of the colanic acid gene cluster or selected genes thereof can be introduced in the genetically engineered cells as described in the examples.
  • the colanic acid gene cluster may be expressed from its native genomic locus.
  • the expression may be actively modulated.
  • the expression can be modulated by swapping the native promoter with a promoter of interest, and/or increasing the copy number of the colanic acid genes coding said protein(s) by expressing the gene cluster from another genomic locus than the native, or episomally expressing the colanic acid gene cluster or specific genes thereof.
  • the term “native genomic locus”, in relation to the colanic acid gene cluster relates to the original and natural position of the gene cluster in the genome of the genetically engineered cell.
  • the de novo GDP-fucose pathway genes responsible for the formation of GDP-fucose comprises or consists of the following genes: i) manA which encodes the protein mannose-6 phosphate isomerase (EC 5.3.1 .8, UniProt accession nr. P00946), which facilitates the interconversion of fructose 6-phosphate (F6P) and mannose-6-phosphate; ii) manB which encodes the protein phosphomannomutase (EC 5.4.2.8, UniProt accession nr P24175), which is involved in the biosynthesis of GDP-mannose by catalyzing conversion mannose-6-phosphate into mannose-1-phosphate;
  • ManC which encodes the protein mannose-1 -phosphate guanylyltransferase guanylyltransferase (EC:2.7.7.13, UniProt accession nr P24174), which is involved in the biosynthesis of GDP-mannose through synthesis of GDP-mannose from GTP and a-D- mannose-1 -phosphate;
  • gmd which encodes the protein GDP-mannose-4,6-dehydratase (UniProt accession nr P0AC88), which catalyzes the conversion of GDP-mannose to GDP-4-dehydro-6-deoxy- D-mannose;
  • v) wcaG (fcl) which encodes the protein GDP-L-fucose synthase (EC 1 .1 .1 .271 , UniProt accession nr P32055) which catalyses the two-step NADP-dependent conversion of GDP-4-dehydro-6-deoxy-D-mannose to GDP-fu
  • the genetically engineered cell when producing one or more fucosylated heterologous products, overexpresses either the entire colonic acid gene cluster (e.g. as identified in SEQ ID NO: 41 or a functional variant thereof) and/or one or more genes of the de novo GDP-fucose pathway selected from the group consisting of manA, manB, manC, gmd and wcaG.
  • Lactose permease is a membrane protein which is a member of the major facilitator superfamily and can be classified as a symporter, which uses the proton gradient towards the cell to transport p-galactosides such as lactose in the same direction into the cell.
  • lactose is often the initial substrate being decorated to produce any HMO of interest in a bioconversion that happens in the cell interior.
  • HMOs human milk oligosaccharides
  • the lactose permease is as shown in SEQ ID NO: 16, or a functional homologue thereof having an amino acid sequence which is at least 80 % identical, such as at least 85 %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 16.
  • the expression of the lactose permease is regulated by a promoter according to the present disclosure.
  • a host cell suitable for HMO production may comprise an endogenous
  • E. coli comprises an endogenous lacZ gene (e.g., GenBank Accession Number V00296 (GI:41901)).
  • the genetically engineered cell does not express a functional p-galactosidase to avoid the degradation of lactose if lactose is used as the initial substrate for producing the complex fucosylated HMO.
  • the lacZ gene may be inactivated by a complete or partial deletion of the corresponding nucleic acid sequence from the bacterial genome, or the gene sequence is mutated in the way that it is not transcribed, or, if transcribed, the transcript is not translated or if translated to a protein (i.e., p-galactosidase), the protein does not have the corresponding enzymatic activity.
  • the HMO-producing bacterium accumulates an increased intracellular lactose pool which is beneficial for the production of HMOs.
  • HMO producing cells are genetically engineered to use lactose as the initial substrate since this is easily taken up by lactose permease as described above.
  • lactose it may be desired to use an initial substrate that will require the presence of fewer glycosyltransferases in the cell, since this will reduce the strain on the cell in terms of producing multiple enzymes and in addition it can reduce the by-product profile, e.g. if lactose is not used as initial substrate a cell comprising a fucosyltransferase will not produce 3FL as by-product allowing the fucose to be used to produce e.g. more LNFP-VI.
  • the cell may further comprise a substrate importer selected from a lactose importer, a lacto-N-triose-ll (LNT-II) importer and a LNnT importer.
  • a substrate importer selected from a lactose importer, a lacto-N-triose-ll (LNT-II) importer and a LNnT importer.
  • WQ2022/242860 suggests how it may be possible to identify LNT-II importers.
  • W02023/099680 also suggests a number of potential LNT and LNT-II importers.
  • LNT-II importers examples include e.g., LNT-II importers
  • Lactose permease (LacY) mutants such as LacY mutant Y236H or LacY mutant A177V+S306T, wherein the mutations are equivalent with the corresponding position in the sequence of SEQ ID NO: 16,
  • ABC transporter protein complexes such as ABC transporter from B. pseudocatenulatum JCM 1200 BBPC_1775, 1776, 1777, (NCBI accession Nrs BAR04453.1 , BAR04454.1 and BAR04455.1 , respectively) or ABC transporter from B. breve UCC2003 BBR_0527/lntP1 , BBR_0528/lntP2, BBR_0530/lntS and BBR_0531 (NCBI accession Nrs ABE95224.1 , ABE95225.1 , ABE95226.1 and ABE95228.1), and/or
  • MFS transporters such as but not limited to Blon_0962 (NCBI accession Nr ACJ52061.1).
  • nucleic acid or a cluster of nucleic acids encoding one of these transporters may be introduced into a genetically modified cell as described herein.
  • the expression of such transporters enables the production of complex fucosylated oligosaccharide with LNT-II as the initial substrate.
  • the oligosaccharide product such as the HMO produced by the cell
  • the product can be transported to the supernatant in a passive way, i.e., it diffuses outside across the cell membrane.
  • the more complex HMO products may remain in the cell, which is likely to eventually impair cellular growth, thereby affecting the possible total yield of the product from a single fermentation.
  • the HMO transport can be facilitated by major facilitator superfamily transporter proteins that promote the effluence of sugar derivatives from the cell to the supernatant.
  • the exporter can be present exogenously or endogenously and is overexpressed under the conditions of the fermentation to enhance the export of the oligosaccharide derivative (HMO) produced.
  • the specificity towards the oligosaccharide product to be secreted can be altered by mutation by means of known recombinant DNA techniques.
  • the genetically engineered cell according to the present disclosure can further comprise a nucleic acid sequence encoding an exporter protein capable of exporting the fucosylated human milk oligosaccharide product or products, such as transporter protein can for example be a member of the major facilitator superfamily transport proteins.
  • the genetically engineered cell according to the method described herein further comprises a gene product that acts as an LNFP-VI and/ or LNFP-V transporter.
  • the gene product that acts as LNFP-VI or LNFP-V transporter may be encoded by a recombinant nucleic acid sequence that is expressed in the genetically engineered cell.
  • the recombinant nucleic acid sequence encoding the LNFP-VI or LNFP-V transporter may be integrated into the genome of the genetically engineered cell, or expressed using a plasmid.
  • a genetically engineered cell and "a genetically modified cell” are used interchangeably.
  • a genetically engineered cell is a host cell whose genetic material has been altered by human intervention using a genetic engineering technique, such a technique is e.g., but not limited to transformation or transfection e.g., with a heterologous and/or recombinant polynucleotide sequence, Crisper/Cas editing and/or random mutagenesis.
  • the genetically engineered cell has been transformed or transfected with a recombinant nucleic acid sequence.
  • the genetic modifications can e.g., be selected from inclusion of glycosyltransferases, and/or metabolic pathway engineering deletion of repressors or undesired enzymes and inclusion of transporters as described in the above sections, which the skilled person will know how to combine into a genetically engineered cell capable of producing one or more fucosylated HMO’s.
  • the genetically engineered cell comprises a recombinant nucleic acid sequence encoding a fucosyltransferase with a-1 ,3-fucosyltransferase activity as disclosed in the section “a-1 ,3-fucosyltransferase” above.
  • a genetically modified cell is capable of producing at least 14%, such as at least 25% LNFP-VI of the total molar HMO content produced by the cell.
  • the total HMOs produced by said cell is essentially free of LNFP-111 and/or LNDFH-111 or contain less than 5%, such as less than 4%, 3%, or such as 2%, of each of LNFP-III and LNDFH-111.
  • the total HMOs produced by said cell is essentially free of LNFP-III and/or LNDFH-111.
  • essentially free of LNFP-III and/or LNDFH-111 is to be understood as a content of LNFP-III and/or LNDFH-111 of the total HMO produced by the cell that is less than 1%, such as less than 0.5%, such as less than 0.2 %, such as less than 0.1% of the total molar HMO content produced by the cell.
  • the cell of the present disclosure produces a mixture of HMOs comprising LNFP- VI, LNnT, 3FL and/or pLNnH.
  • the fucosyltransferases have a-1 ,3-fucosyltransferase activity, allowing fucosylation of an oligosaccharide at position 3 of the Glc moiety in LNnT or LNT, while showing limited or no fucosylation at position 2 or 3 of the Gal or GIcNAc moieties (See figures 1 and 3).
  • the reducing end Glc moiety is fucosylated, and more preferably only the reducing end Glc moiety of oligosaccharide is LNnT or LNT is fucosylated.
  • the genetically engineered cell of the present disclosure is capable of producing LNFP-VI or LNFP-V, wherein said cell comprises a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-neotetraose (LNnT) and/or in lacto-N-tetraose (LNT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNnT or LNT, and wherein the cell produces a) less 5 %, such as less than 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2% or such as less than 0.1% of the total molar content of HMO produced by said cell is LNDFH-III and/or LNFP- III, or b) less 2 %, such as less than
  • the genetically engineered cell of the present disclosure produces a) more than 14 %, such as more than 20%, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85% or such as more than 90% of the total molar content of HMO produced by said cell LNFP-VI or b) more than 50 %, such as more than 55%, 60%, 65%, 70%, 75%, 80%, 85% or such as more than 90% of the total molar content of HMO produced by said cell LNFP-V.
  • the genetically engineered cell of the present disclosure is capable of producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI), wherein said cell comprises a recombinant nucleic acid sequence encoding an a-1 ,3- fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-neotetraose (LNnT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNnT, and wherein the cell produces, a) less than 5 %, such as less than 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2% or such as less than 0.1% of the total molar content of HMO produced by said cell is LNDFH-III and/or LNFP-III, and b) more than 14 %
  • the genetically engineered cell of the present disclosure further produces one or more HMOs selected from the group consisting of 3FL, LNT-II, LNnT and pLNnH.
  • essentially no LNFP-III and/or LNDFH-III is produced by said cell.
  • the recombinant nucleic acid encodes an a-1 ,3-fucosyltransferase derived from Bacteroidales bacterium.
  • the recombinant nucleic acid encodes an a-1 ,3-fucosyltransferase is selected from the group consisting of, a) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO:
  • Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO:
  • the genetically engineered cell capable of producing LNFP-VI comprises a recombinant nucleic acid sequence encoding a fucosyltransferase with a- 1 ,3-fucosyltransferase activity, wherein said fucosyltransferase is Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional homologue thereof with an amino acid sequence that is at least 80 %, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to SEQ ID NO: 1.
  • said genetically engineered cells produce i.
  • the genetically engineered cell capable of producing LNFP-VI or LNFP-V comprises a recombinant nucleic acid sequence encoding a fucosyltransferase with a-1 ,3-fucosyltransferase activity, wherein said fucosyltransferase is Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 %, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to SEQ ID NO: 2.
  • said genetically engineered cells produce i. LNFP-VI in a molar content of at least at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, or such as at least 90% of the total HMO produced by said cell, and/or ii. LNFP-VI and LNnT produced by said cell is at least 85%, such as at least 90%, or such as at least 95% or such as at least 99%, or such as 100% of the total HMO produced by said cell, and/or
  • LNFP-VI Hi. 55-90 molar% of LNFP-VI, 0-15 molar% 3FL, 0-35 molar% LNnT, 0-10% pLNnH in total adding up to 100% molar content
  • the molar content of LNFP-111 and LNDFH-111 is less than 5 %, such as less than 4%, 3%, 2%, 1%, 0.5%, 0.3% or such as less than 0.1% of the total molar content of HMO produced by said cell or iv.
  • LNFP-V and LNT produced by said cell is at least at least 90%, or such as at least 95%or such as at least 99%, or such as 100% of the total HMO produced by said cell, and/or v.
  • LNFP-V 50-70 molar% of LNFP-V, 0-5 molar% 3FL, 30-50 molar% LNT, in total adding up to 100% molar content, wherein the molar content of LNFP-II and LNDFH-II is less than 2 %, such as less than 1 .5%, 1%, 0.5%, 0.3%, 0.2% or such as less than 0.1% of the total molar content of HMO produced by said cell
  • the genetically engineered cell capable of producing LNFP-VI comprises a recombinant nucleic acid sequence encoding a fucosyltransferase with a- 1 ,3-fucosyltransferase activity, wherein said fucosyltransferase is Paral comprising or consisting of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to SEQ ID NO: 3.
  • said genetically engineered cells produce i.
  • the molar content of LNFP-VI produced by said cell is at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 55%, such as at least 60%, or such as at least 65% of the total HMO produced by said cell, and/or ii.
  • the molar content of LNFP-VI and LNnT produced by said cell is at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, or such as at least 90% of the total HMO produced by said cell, and/or
  • the genetically engineered cell capable of producing LNFP-VI comprises a recombinant nucleic acid sequence encoding a fucosyltransferase with a- 1 ,3-fucosyltransferase activity, wherein said fucosyltransferase is CafF comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a functional homologue thereof with an amino acid sequence that is at least 80 %, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to SEQ ID NO: 43.
  • this genetically engineered cell also comprises a recombinant nucleic acid sequence encoding a p-1 ,4-galactosyltransferase and a recombinant nucleic acid sequence encoding an LNT-II importer.
  • said genetically engineered cells produce a) the molar content of LNFP-VI produced by said cell is at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, or such as at least 45% of the total HMO produced by said cell, and/or b) the molar content of LNFP-VI, 3FL and LNnT produced by said cell is at least at least 80%, such as at least 85%, or such as at least 90% of the total HMO produced by said cell, and/or c) 10-30 molar% of LNFP-VI, 35-60 molar% 3FL, 25-40 molar% LNnT, 0-10% pLNnH in total adding up to 100% molar content, and wherein the molar content of LNFP-111 and LNDFH-III is less than 5 %, such as less than 4%, 3%, 2%, 1%, 0.5%, 0.3% or such as less than
  • the present disclosure also relates to a genetically engineered cell capable of producing the Human Milk Oligosaccharide (HMO) lacto-N-fucopentaose V (LNFP-V), comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-tetraose (LNT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal), wherein the cell produces less than 2 %, such as less than 1 %, 0.5%, 0.3%, 0.2% or such as less than 0.1% of the total molar content of HMO produced by said cell is LNDFH-II and/or LNFP-II.
  • HMO Human Milk Oligosaccharide
  • LNFP-V Human Milk Oligosaccharide
  • LNFP-V Human Milk Oligos
  • said cell also produces more than 50 % of the total molar content of HMO of LNFP- V.
  • the cell capable of producing LNFP-V comprises one or two copies or multiple copies, preferably genomically integrated, of a nucleic acid sequence encoding Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 %, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% identical to SEQ ID NO: 2.
  • the genetically engineered cell described herein preferably expresses genes encoding key enzymes for the biosynthesis of fucosylated HMOs.
  • the genetically engineered cell expresses the genes needed to produce LNnT or LNT, either from lactose or LNT-II as the initial substrate (see figure 1 and figure 3), and/or alternatively the cell expresses importers for LNT-II or LNnT or LNT.
  • the genetically engineered cell comprises one or more additional glycosyltransferases.
  • the additional one or more glycosyltransferases are preferably selected from the group consisting of, galactosyltransferases, glucosaminyltransferases, fucosyltransferases and N-acetylglucosaminyl transferases.
  • the genetically engineered cell comprises one or more recombinant nucleic acid sequence(s) encoding a p-1 ,4-galactosyltransferase, and optionally a p-1 ,3-N- acetylglucosaminyltransferase.
  • the p-1 ,3-N-acetyl- glucosaminyltransferase is from Neisseria meningitidis
  • the p-1 ,4-galactosyltransferase is from Helicobacter pylori.
  • the genetically engineered cell comprises one or more recombinant nucleic acid sequence(s) encoding a p-1 ,3-galactosyltransferase, and optionally a p-1 ,3-N- acetylglucosaminyltransferase.
  • the p-1 ,3-N-acetyl- glucosaminyltransferase is from Neisseria meningitidis
  • the p-1 ,3-galactosyltransferase is from Helicobacter pylori.
  • nucleic acid construct encoding the CA as shown in SEQ ID NO: 41 or equivalents thereof, allowing for formation of GDP-fucose, which enables the cell to produce a higher level of fucosylated oligosaccharides from one or more intermediate oligosaccharide substrates, such as lactose or LNnT, and/or LNT.
  • one or more additional glycosyltransferases and pathways for producing nucleotide-activated sugars such as glucose-UDP-GIcNAc, CMP-N-acetylneuraminic acid, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and/or CMP-N-acetylneuraminic acid can also be present in the genetically engineered cell.
  • the genetically engineered cell described herein may further comprise any of the modifications described above, e.g., additional glycosyltransferases, suitable importer proteins such as overexpression of lactose permease, LNT-II or LNT importers, p-galactosidase inactivation in particular if lactose is used as the initial substrate, as well suitable exporter proteins for the complex fucosylated HMOs produced by the cell.
  • suitable importer proteins such as overexpression of lactose permease, LNT-II or LNT importers, p-galactosidase inactivation in particular if lactose is used as the initial substrate, as well suitable exporter proteins for the complex fucosylated HMOs produced by the cell.
  • the engineered cell is a microorganism.
  • the genetically engineered cell is preferably a microbial cell, such as a prokaryotic cell or eukaryotic cell.
  • Appropriate microbial cells that may function as a host cell include bacterial cells, archaebacterial cells, algae cells and fungal cells.
  • the genetically engineered cell may be e.g., a bacterial or yeast cell. In one preferred embodiment, the genetically engineered cell is a bacterial cell.
  • the bacterial host cells there are, in principle, no limitations; they may be eubacteria (gram-positive or gram-negative) or archaebacteria, as long as they allow genetic manipulation for insertion of a gene of interest and can be cultivated on a manufacturing scale.
  • the host cell has the property to allow cultivation to high cell densities.
  • Non-limiting examples of bacterial host cells that are suitable for recombinant industrial production of an HMO(s) according to the disclosure could be member of the Enterobacterales order, preferably of the genus Escherichia, more preferably of the species E. coll.
  • suitable host cell Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Campylobacter sp, Corynebacterium sp., Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris.
  • Bacteria of the genus Bacillus may also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans.
  • bacteria of the genera Lactobacillus and Lactococcus may be engineered using the methods of this disclosure, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus easel, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis.
  • Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species.
  • strains engineered as described here, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium long urn, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa).
  • Enterococcus e.g., Enterococcus faecium and Enterococcus thermophiles
  • Bifidobacterium e.g., Bifidobacterium long urn, Bifidobacterium infantis, and B
  • Non-limiting examples of fungal host cells that are suitable for recombinant industrial production of a heterologous product are e.g., yeast cells, such as Komagataella, Kluyveromyces, Yarrowia, Pichia, Saccaromyces, Schizosaccharomyces or Hansenula or from a filamentous fungus of the genera Aspargillus, Fusarium or Thricoderma.
  • yeast cells such as Komagataella, Kluyveromyces, Yarrowia, Pichia, Saccaromyces, Schizosaccharomyces or Hansenula or from a filamentous fungus of the genera Aspargillus, Fusarium or Thricoderma.
  • the genetically engineered cell is selected from the group consisting of Escherichia sp., Bacillus sp., lactobacillus sp., Corynebacterium sp. and Campylobacter sp.
  • the genetically engineered cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris and Saccharomyces cerevisiae.
  • the genetically engineered cell is B. subtilis.
  • the genetically engineered cell is S. Cerevisiae or P pastoris.
  • the genetically engineered cell is Escherichia coli.
  • the present disclosure relates to a genetically engineered cell, wherein the cell is derived from the E. coli K-12 strain or E. coli DE3 strain.
  • the present disclosure relates to a genetically engineered cell comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with Glc specific a-1 ,3- fucosyltransferase activity, such as an enzyme selected from the group consisting of Bacbad , Bacbac2 and Paral , wherein said cell produces Human Milk Oligosaccharides (HMO).
  • HMO Human Milk Oligosaccharides
  • at least one fucosylated HMO and preferably with a molar % content of LNFP-VI above 25 %, such as above 50% of the total HMO produced.
  • nucleic acid sequence “recombinant gene/nucleic acid/nucleotide sequence/DNA encoding” or “coding nucleic acid sequence” is used interchangeably and intended to mean an artificial nucleic acid sequence (i.e. produced in vitro using standard laboratory methods for making nucleic acid sequences) that comprises a set of consecutive, non-overlapping triplets (codons) which is transcribed into mRNA and translated into a protein when under the control of the appropriate control sequences, i.e., a promoter sequence.
  • the boundaries of the coding sequence are generally determined by a ribosome binding site located just upstream of the open reading frame at the 5’end of the mRNA, a transcriptional start codon (AUG, GUG or UUG), and a translational stop codon (UAA, UGA or UAG).
  • a coding sequence can include, but is not limited to, genomic DNA, cDNA, synthetic, and recombinant nucleic acid sequences.
  • nucleic acid includes RNA, DNA and cDNA molecules. It is understood that, as a result of the degeneracy of the genetic code, a multitude of nucleic acid sequences encoding a given protein may be produced.
  • the recombinant nucleic acid sequence may be a coding DNA sequence e.g., a gene, or noncoding DNA sequence e.g., a regulatory DNA, such as a promoter sequence or other noncoding regulatory sequences.
  • heterologous refers to a polypeptide, amino acid sequence, nucleic acid sequence or nucleotide sequence that is foreign to a cell or organism, i.e., to a polypeptide, amino acid sequence, nucleic acid molecule or nucleotide sequence that does not naturally occurs in said cell or organism.
  • the disclosure also relates to a nucleic acid construct comprising a coding nucleic sequence, i.e. recombinant DNA sequence of a gene of interest, e.g., an a-1 ,3-fucosyltransferase gene as described herein, and a non-coding regulatory DNA sequence, e.g., a promoter DNA sequence, e.g., a recombinant promoter sequence derived from the promoter sequence of the lac operon or the glp operon, or a promoter sequence derived from another genomic promoter DNA sequence, or a synthetic promoter sequence, wherein the coding and promoter sequences are operably linked.
  • a coding nucleic sequence i.e. recombinant DNA sequence of a gene of interest, e.g., an a-1 ,3-fucosyltransferase gene as described herein
  • a non-coding regulatory DNA sequence e.g., a promoter DNA sequence, e.
  • operably linked refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. It refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
  • a promoter sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
  • the nucleic acid construct of the present disclosure may be a part of the vector DNA, in another embodiment, the construct it is an expression cassette/cartridge that is integrated in the genome of a host cell.
  • nucleic acid construct means an artificially constructed segment of nucleic acids, in particular a DNA segment, which is intended to be inserted into a target cell, e.g., a bacterial cell, to modify expression of a gene of the genome or expression of a gene/coding DNA sequence which may be included in the construct.
  • the present disclosure relates to a nucleic acid construct comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase, wherein said recombinant nucleic acid sequence is selected from the group consisting of nucleic acid sequences encoding Bacbad , Bacbac2, Paral , or CafF, such as a nucleic acid sequence according to SEQ ID NO: 7, 8, 9, or 44, or functional variants thereof.
  • the genetically engineered cell according to the present disclosure may also comprise multiple copies of the recombinant nucleic acid sequence encoding a a-1 ,3-fucosyltransferase. Enhancing the copy number of the fucosyltransferase was shown in Example 1 to change the ratio of the produced HMOs. In specific it was shown that increasing the copy number of Paral by introduction of a high copy-number plasmid (pBB-B9-Para1-PglpF) resulted in an increased LNFP-VI production, while reducing the strains production of LNnT and pLNnH. Furthermore, increasing the copy number of Bacbac2 to two genomic copies in Example 2, slightly increased the relative amount of LNFP-VI produced, combined with a decrease in the amount of LNnT produced.
  • the copy number variation of the glycosyltransferase(s) may be used in the production to optimize the HMO production, in this case optimizing the production of LNFP-VI.
  • the genetically engineered cell of the present disclosure comprises one, two, three or more genomic copies of the recombinant nucleic acid sequence encoding the glycosyltransferase selected from the group consisting of a) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , b) Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, c) Paral comprising or consisting of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 3 and d) CafF comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to
  • the plasmid is a high copy number plasmid, preferably, a pUC57 or pBB-B9 plasmid.
  • nucleic acid construct comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase, wherein said recombinant nucleic acid sequence is selected from the group consisting of a) a nucleic acid encoding Bacbad , comprising or consisting of a nucleic acid sequence according to SEQ ID NO: 7, or a functional homologue thereof with a nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 7, b) a nucleic acid encoding Bacbac2, comprising or consisting of a nucleic acid sequence according to SEQ ID NO: 7, or a functional homologue thereof with a nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 7,
  • the a-1 ,3-fucosyltransferase encoding sequence is under the control of a promoter sequence selected from promotor sequences with a nucleic acid sequence as identified in Table
  • the nucleic acid construct is suitable for genomic integration in a desired host cell.
  • the promoter may be of heterologous origin, native to the genetically engineered cell or it may be a recombinant promoter, combining heterologous and/or native elements.
  • One way to increase the production of a product may be to regulate the production of the desired enzyme activity used to produce the product, such as the glycosyltransferases or enzymes involved in the biosynthetic pathway of the glycosyl donor.
  • Increasing the promoter strength driving the expression of the desired enzyme may be one way of doing this.
  • the strength of a promoter can be assessed using a lacZ enzyme assay where
  • the expression of said nucleic acid sequences are under control of a strong promoter selected from the group consisting of SEQ ID NOs 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27 and 28.
  • the expression of said nucleic acid sequences described herein is under control of a PglpF (SEQ ID NO: 29) or Plac (SEQ ID NO: 38) promoter or PmglB_UTR70 (SEQ ID NO: 26) or PglpA_70UTR (SEQ ID NO: 27) or PglpT_70UTR (SEQ ID NO: 28) or variants thereof such as promoters identified in Table 4, in particular the PglpF_SD4 variant of SEQ ID NO: 24 or Plac_70UTR variant of SEQ ID NO: 20, or PmglB_70UTR variants of SEQ ID NO: 17, 18, 19, 21 , 22, 23, 25 and 26.
  • PglpF, PglpA_70UTR, PglpT_70UTR and PmglB_70UTR promoter sequences are described in or WO2019/123324 and W02020/255054 respectively (hereby incorporated by reference).
  • the recombinant nucleic acid sequences individually are under the control of one or more promoters selected from the group consisting of PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 29, 38, 26, 27 and 28, respectively) and variants thereof.
  • clones that carry the expression cassette can be selected e.g., by means of a marker gene, or loss or gain of gene function.
  • the present disclosure relates to one or more recombinant nucleic acid sequences as illustrated in SEQ ID NOs: 7, 8 and 9 [nucleic acid sequence encoding Bacbad , Bacbac2 and Paral],
  • the present disclosure relates to one or more of a recombinant nucleic acid sequence and/or to a functional homologue thereof having a sequence which is at least 70% identical to SEQ ID NOs: 7, 8 or 9 [nucleic acid sequence encoding Bacbad , Bacbac2 and Paral], such as at least 75% identical, at least 80 % identical, at least 85 % identical, at least 90 % identical, at least, at least 95 % identical, at least 98 % identical, or 100 % identical.
  • sequence identity describes the relatedness between two amino acid sequences or between two nucleotide sequences, i.e., a candidate sequence (e.g., a sequence of the disclosure) and a reference sequence (such as a prior art sequence) based on their pairwise alignment.
  • sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.
  • sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1 970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), 10 preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, -endopen 10.0, -endextend 0.5 and the DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labelled " identity" (obtained using the -nobrief option) is used as the percent identity.
  • sequence identity may be calculated as follows: (Identical nucleotide residues x 100)/(aligned region).
  • a functional homologue or functional variant of a protein/nucleic acid sequence as described herein is a protein/nucleic acid sequence with alterations in the genetic code, which retain its original functionality.
  • a functional homologue may be obtained by mutagenesis or may be natural occurring variants from the same or other species.
  • the functional homologue should have a remaining functionality of at least 50%, such as at least 60%, 70%, 80 %, 90% or 100% compared to the functionality of the protein/nucleic acid sequence.
  • a functional homologue of any one of the disclosed amino acid or nucleic acid sequences can also have a higher functionality.
  • a functional homologue of any one of the a-1 ,3- fucosyltransferase amino acid sequences shown in table 1 or a recombinant nucleic acid encoding any one of the sequences of SEQ ID NO: 7, 8, 9, or 44, should ideally be able to participate in the production of fucosylated HMOs, in terms of increased HMO yield, export of HMO product out of the cell or import of substrate for the HMO production, such as a acceptor oligosaccharide of at least three monosaccharide units, improved purity/by-product formation, reduction in biomass formation, viability of the genetically engineered cell, robustness of the genetically engineered cell according to the disclosure, or reduction in consumables needed for the production.
  • a functional homologue of any one of the a-1 ,3-fucosyltransferase disclosed herein is capable of producing lacto-N-neofucopentaose VI (LNFP-VI), with less than 5 %, such as less than 2% of the total molar content of HMO being fucosylated by-product oligosaccharides with 5 or 6 monosaccharide units, such as essentially no LNFP-III and LNDFH-I when expressed in a suitable genetically engineered cell as described herein.
  • LNFP-VI lacto-N-neofucopentaose VI
  • the present disclosure also relates to any commercial use of the enzyme(s), genetically engineered cell(s) or the nucleic acid construct(s) disclosed herein, such as, but not limited to, in a method for producing one or more fucosylated human milk oligosaccharide (HMO), preferably, LNFP-VI.
  • HMO fucosylated human milk oligosaccharide
  • the present disclosure also relates to the use of an a-1 ,3-fucosyltransferase in the production of one or more fucosylated HMOs, wherein the a-1 ,3-fucosyltransferase is selected from the group consisting of Bacbad , Bacbac2, Paral and CafF comprising or consisting of the amino acid sequence of SEQ ID NO: 1 , 2, 3, or 43, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , 2, 3, or 43.
  • the a-1 ,3-fucosyltransferase of the present disclosure can also be used in the manufacturing of a fucosylated product, wherein the fucosylated product contains one or more oligosaccharides, such as one or more HMOs, and wherein the product contains LNFP-VI or LNFP-V.
  • the genetically engineered cell and/or the nucleic acid construct according to the disclosure is used in the manufacturing of HMOs.
  • the molar % content of LNFP-VI produced by the genetically engineered cell is above 14%, such as above 25% of the total amount of HMO produced.
  • the molar % content of LNFP-VI produced by the genetically engineered cell is above 70% such as above 75%, such as above 80%, such as above 90% of the total amount of HMO produced.
  • the genetically engineered cell and/or the nucleic acid construct according to the disclosure is used in the manufacturing of mixtures of HMOs, wherein the molar % content of LNFP-V produced by the genetically engineered cell is above 40% of the total amount of HMO produced.
  • the molar % content of LNFP-V produced by the genetically engineered cell is above 50% such as above 55%, such as above 57%, such as above 58% of the total amount of HMO produced.
  • the a-1 ,3-fucosyltransferase for use in production of LNFP-VI is selected from the group consisting of Bacbacl , Bacbac2, Paral and CafF with an amino acid sequence according to SEQ ID NO: 1 , 2, 3, or 43, or a functional homologue thereof which amino acid sequence is at least 80 % identical to SEQ ID NO: 1 , 2, 3, or 43.
  • the use can be in vivo (as described herein) or alternatively in an in vitro cell free process.
  • the genetically engineered cell and/or the nucleic acid construct according to the disclosure is used in the manufacturing of LNFP-VI or LNFP-V.
  • Production of these HMO’s may require the presence of two or more glycosyltransferase activities.
  • HMOs fucosylated human milk oligosaccharides
  • the present disclosure also relates to a method for producing LNFP-VI or LNFP-V, said method comprises culturing a genetically engineered cell according to the present disclosure under conditions suitable for production of HMOs.
  • the present disclosure thus also relates to a method for producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI), with less than 5 % of the total molar content of HMO being fucosylated by-products with 5 or 6 monosaccharide units, comprising the steps of: a) providing a genetically engineered cell with a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-neotetraose (LNnT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNnT, and b) cultivating said genetically modified cell under conditions that allow for formation of LNFP-VI, and c) Optionally, purifying said LNFP-VI to remove
  • the present disclosure also relates to a method for producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI), with less than 5 % of the total molar content of HMO being fucosylated by-product oligosaccharides with 5 or 6 monosaccharide units, comprising the steps of a) Providing a genetically engineered cell with a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase derived from Bacteroidales bacterium, and b) Cultivating said genetically modified cell under conditions that allow for formation of LNFP-VI, and c) Optionally, purifying said LNFP-VI to remove by-products.
  • HMO Human Milk Oligosaccharide
  • LNFP-VI Human Milk Oligosaccharide
  • LNFP-VI Human Milk Oligosaccharide
  • the method produces more than 25% such as more than 30 % of the total molar content of HMO of LNFP-VI.
  • the present disclosure thus also relates to a method for producing the HMO LNFP-VI, comprising providing and culturing a genetically engineered cell comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase, where a) less than 2 %, such as less than 1%, such as less than 0.5%, such as less than 0.2% of the total molar content of HMO produced by said method is LNDFH-111 , b) less than 2 % such as less than 1%, such as less than 0.5%, such as less than 0.2% of the total molar content of HMO produced by said method is LNFP-111 , and c) more than 25 % of the total molar content of HMO produced by said method LNFP-VI.
  • the recombinant nucleic acid encodes an a-1 ,3- fucosyltransferase selected from the group consisting of, a) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO:
  • Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO:
  • a further embodiment of the disclosure is a method for producing LNFP-VI, said method comprising culturing a genetically engineered cell comprising a) a recombinant nucleic acid sequence encoding an enzyme with p-1 ,3-N-acetyl- glucosaminyltransferase activity; and b) a recombinant nucleic acid sequence encoding an enzyme with a p-1 ,4- galactosyltransferase activity; and c) a recombinant nucleic acid sequence encoding a fucosyltransferase with a-1 ,3- fucosyltransferase activity, wherein said enzyme is selected from the group consisting of: i. Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO:
  • Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO:
  • a further embodiment of the disclosure is a method for producing LNFP-VI, said method comprising culturing a genetically engineered cell comprising a) a recombinant nucleic acid sequence encoding an enzyme with a p-1 ,4- galactosyltransferase activity; and b) a recombinant nucleic acid sequence encoding a fucosyltransferase with a-1 ,3- fucosyltransferase activity, wherein said enzyme is selected from the group consisting of: i. Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO:
  • Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO:
  • less 5 % of the total molar content of HMO produced by said method is LNDFH-III and/or LNFP-III. preferably, less than 2% LNDFH-III and less than 2% LNFP-III, such as e.g., less than 1% LNDFH-III and less than 1% LNFP-III, or between 0-5% LNDFH-III and/or LNFP-III. Accordingly, in embodiments, less than less than 5% of the molar content of the total HMOs produced in the culturing step is LNFP-III and less than 5% of the molar content of the total HMOs produced in the culturing step is LNDFH-111. In embodiments, essentially no LNFP-III and/or LNDFH-111 is produced in the culturing step.
  • the method particularly comprises culturing a genetically engineered cell that produces a fucosylated HMO, wherein the LNFP-VI content produced in said method is at least 25 % of the total HMO content produced by the method, and wherein the less than 5% of the molar content of the total HMOs produced is LNDFH-I II and/or LNFP-III.
  • the present disclosure also relates to a method for producing the Human Milk Oligosaccharide (HMO) lacto-N-fucopentaose (LNFP-V), with less than 2 % of the total molar content of HMO being fucosylated by-products with 5 or 6 monosaccharide units, comprising the steps of: a) providing a genetically engineered cell with a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-tetraose (LNT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNT, and b) cultivating said genetically modified cell under conditions that allow for formation of LNFP- VI, and c) optionally, purifying said LNFP-VI to remove by-products, such as 3
  • less than 2 % of the total molar content of HMO produced by said method is LNDFH-II and/or LNFP-II. More preferably, more than 50 % of the total molar content of HMO produced by said method LNFP-V.
  • the method for producing LNFP-V comprises culturing a genetically engineered cell comprising a) a recombinant nucleic acid sequence encoding an enzyme with a p-1 ,3- galactosyltransferase activity and b) a recombinant nucleic acid sequence encoding a fucosyltransferase with a-1 ,3- fucosyltransferase activity, being Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, and wherein LNT-II is used as initial substrate in the cultivation.
  • a further embodiment the method for producing LNFP-V comprises culturing a genetically engineered cell comprising a) a recombinant nucleic acid sequence encoding an enzyme with p-1 ,3-N-acetyl- glucosaminyltransferase activity; and b) a recombinant nucleic acid sequence encoding an enzyme with a p-1 ,3- galactosyltransferase activity; and c) at least one copy such as 1 , 2, 3, 4, 5, 10, 20 or 50 copies, or more than 50 copies of a recombinant nucleic acid sequence encoding the fucosyltransferase Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, and wherein lactose is used as initial substrate in the cultivation.
  • Culturing, cultivating, or fermenting or fermentation in a controlled bioreactor typically comprises (a) a first phase of exponential cell growth in a culture medium ensured by a carbon-source, and (b) a second phase of cell growth in a culture medium run under carbon limitation, where the carbon-source is added continuously together with the acceptor oligosaccharide, such as lactose, allowing formation of the HMO product in this phase.
  • carbon (sugar) limitation is meant the stage in the fermentation where the growth rate is kinetically controlled by the concentration of the carbon source (sugar) in the culture broth, which in turn is determined by the rate of carbon addition (sugar feed-rate) to the fermenter.
  • the method described herein further comprises providing an acceptor saccharide as initial substrate for the HMO formation, the acceptor substrate comprising at least two monosaccharide units, which is exogenously added to the culture medium and/or has been produced by a separate microbial fermentation.
  • the genetically modified cell may be further engineered to produce the initial substrate inside the cell (see for example WO2015/150328).
  • the genetically engineered cell is cultivated in the presence of an initial acceptor substrate selected from the group consisting of lactose, LNT-II and LNnT.
  • the initial acceptor substrate such as lactose, LNT-II and LNnT
  • lactose LNT-II and LNnT
  • the initial substrate for HMO formation is lactose which is fed to the culture during the fermentation of the genetically engineered cell.
  • the method of the present disclosure comprises providing a glycosyl donor, for the glycosylation of the acceptor substrate.
  • the glucosyl donor is produced by an endogenous or recombinant de novo pathway in the genetically engineered cell.
  • the genetically engineered cell comprises an upregulated biosynthetic pathway for making a fucose sugar nucleotide, such a GDP-fucose.
  • the glycosyl donor can be synthesized separately by one or more genetically engineered cells and/or can be exogenously added to the culture medium from an alternative source.
  • a “manufacturing” or “manufacturing scale” or “large-scale production” or “large-scale fermentation”, are used interchangeably and in the meaning of the disclosure defines a fermentation with a minimum volume of 100 L, such as WOOL, such as 10.000L, such as 100.000L, such as 200.000L culture broth.
  • a “manufacturing scale” process is defined by being capable of processing large volumes yielding amounts of the HMO product of interest that meet, e.g., in the case of a therapeutic compound or composition, the demands for toxicity tests, clinical trials as well as for market supply.
  • a manufacturing scale method is characterized by the use of the technical system of a bioreactor (fermenter) which is equipped with devices for agitation, aeration, nutrient feeding, monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.).
  • a bioreactor which is equipped with devices for agitation, aeration, nutrient feeding, monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.).
  • process parameters pH, temperature, dissolved oxygen tension, back pressure, etc.
  • Fucosyltransferases of the prior art such as FutA or FutB from Dumon et al., 2004, may not be suitable for large scale manufacturing of complex fucosylated HMOs, such as LNFP-VI, since the yield obtained from cell expressing such fucosyltransferases is either lower than the yield obtained from the strains disclosed herein, or the specificity of the enzymes is more promiscuous i.e., they produces one or more addition side products, such as LNFP-III and/or LNDFH-111 , which complicates the purification of LNFP-VI.
  • a suitable fucosyltransferase is one which enables large scale production of LNFP-VI or LNFP-V.
  • the fucosyltransferases disclosed herein, being Bacbad , Bacbac2 and Paral are especially suited for production of HMOs when introduced into a suitable production strain.
  • the genetically engineered cell of the present disclosure is suitable for large scale production of HMOs.
  • the method of the present disclosure is suitable for large scale manufacturing.
  • the culture medium may be semi-defined, i.e., containing complex media compounds (e.g., yeast extract, soy peptone, casamino acids, etc.), or it may be chemically defined, without any complex compounds.
  • the carbon-source can be selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.
  • the culturing media is supplemented with one or more energy and carbon sources selected form the group containing glycerol, sucrose and glucose.
  • lactose is added during the cultivation of the genetically engineered cells as a substrate for the HMO formation.
  • the method comprising culturing a genetically engineered cell that produces LNFP-VI or LNFP- further comprises culturing said genetically engineered cell in in the presence of an energy source (carbon source) selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.
  • an energy source selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.
  • the culturing media contains sucrose as the sole carbon and energy source.
  • the genetically engineered cell comprises one or more heterologous nucleic acid sequence encoding one or more heterologous polypeptide(s) which enables utilization of sucrose as sole carbon and energy source of said genetically engineered cell.
  • the genetically engineered cell comprises a PTS- dependent sucrose utilization system, further comprising the scrYA and scrBR operons as described in WO2015/197082 (hereby incorporated by reference).
  • the LNFP-VI or LNFP-V can be collected from the cell culture or fermentation broth in a conventional manner.
  • the LNFP-VI or LNFP-V can be retrieved from the culture, either from the culture medium and/or the genetically engineered cell.
  • the fucosylated human milk oligosaccharide is retrieved from the culture medium and/or the genetically engineered cell.
  • the term “retrieving” is used interchangeably with the term “harvesting”. Both “retrieving” and “harvesting” in the context relate to collecting the produced HMO(s) from the culture/broth following the termination of fermentation. In one or more exemplary embodiments it may include collecting the HMO(s) included in both the biomass (i.e., the host cells) and cultivation media, i.e., before/without separation of the fermentation broth from the biomass. In other embodiments, the produced HMOs may be collected separately from the biomass and fermentation broth, i.e., after/following the separation of biomass from cultivation media (i.e., fermentation broth).
  • the separation of cells from the medium can be carried out with any of the methods well known to the skilled person in the art, such as any suitable type of centrifugation or filtration.
  • the separation of cells from the medium can follow immediately after harvesting the fermentation broth or be carried out at a later stage after storing the fermentation broth at appropriate conditions.
  • Recovery of the produced HMO(s) from the remaining biomass (or total fermentation broth) include extraction thereof from the biomass (i.e., the production cells).
  • HMO(s) are available for further processing and purification.
  • several steps of filtration may be required, in example, the separation of the by-product LNDFH-III from LNFP-VI requires a step of size dependent purification, while separation of LNFP-III from LNFP-VI is very tedious since the charge and mass of the two species is identical. Accordingly, it is highly preferable that no LNFP-III or LNDFH-III is produced in the fermentation process leading to LNFP-VI.
  • the HMOs can be purified according to the procedures known in the art, e.g., such as described in WO2017/152918, WO2017/182965 or WO2015/188834, wherein the latter describes purification of fucosylated HMOs.
  • the purified HMOs can be used as nutraceuticals, pharmaceuticals, or for any other purpose, e.g., for research.
  • the oligosaccharide as product can be accumulated both in the intra- and the extracellular matrix.
  • the method according to the present disclosure comprises cultivating the genetically engineered microbial cell in a culture medium which is designed to support the growth of microorganisms, and which contains one or more carbohydrate sources or just carbon-source, such as selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.
  • the culturing media is supplemented with one or more energy and carbon sources selected form the group containing glycerol, sucrose and glucose.
  • the term “manufactured product” refers to the one or more HMOs intended as the one or more product HMO(s), or composition of a mixture of HMOs.
  • the product HMOs or composition is produced by a method described herein using a genetically engineered cell described herein. From the data presented in example 2, it can be seen that the Bacbac2 fucosyltransferase produced LNFP-VI of high purity showing the ability and suitability of Bacbac2 to produce highly pure LNFP-VI in large scale manufacturing.
  • the methods disclosed herein provide both a decreased ratio of by-product to product and an increased overall yield of the product (and/or HMOs in total). This, less byproduct formation in relation to product formation, facilitates an elevated product production and increases efficiency of both the production and product recovery process, providing superior manufacturing procedure of HMOs.
  • One embodiment relates to a composition comprising HMOs, such as e.g., a nutritional product comprising LNFP-VI, wherein said LNFP-VI is produced by a method of the present disclosure.
  • the manufactured product may be a powder, a composition, a suspension, or a gel comprising one or more HMOs.
  • the genetically engineered cell capable of producing one or more HMOs, preferably LNFP-VI, described herein will generally produce a mixture of HMOs as a result of the multistep process towards the final HMO product.
  • LNFP-VI lactose as the initial substrate
  • 3-FL fucosylated lactose
  • LNT-II LNnT
  • pLNnH lactose
  • LNFP-III LNDFH-III
  • the molar % of individual HMO components are supported by experimental data from the Examples and shows exemplary HMO compositions, wherein the mixture of HMOs consists essentially of LNFP-VI and LNnT, 3-FL and/or pLNnH.
  • a mixture of HMOs may consist essentially of a) LNFP-VI and 3-FL, or b) LNFP- VI and LNnT, or c) LNFP-VI, 3FL and LNnT, or d) LNFP-VI, 3FL, LNnT and pLNnH.
  • the HMO mixture consist essentially of HMOs within the following ranges 25-90 molar% of LNFP-VI, 0-70 molar% 3FL, 0-65 molar % LNnT, 0-15 molar% pLNnH and below 1 % LNFP-III and below 1 % LNDFH-III, in total adding up to 100% molar content.
  • an embodiment of the present disclosure relates to a mixture of HMOs according consists essentially of 25-70 molar% of LNFP-VI, 30-70 molar% 3FL, 0-5 % LNnT, in total adding up to 100 molar% molar content.
  • a further embodiment of the present disclosure relates to a mixture of HMOs according consists essentially of 55-90 molar% of LNFP-VI, 0-15 molar% 3FL, 0-35 % LNnT and 0-10 molar% pLNnH in total adding up to 100 molar% molar content.
  • a further embodiment of the present disclosure relates to a mixture of HMOs according consists essentially of 25-70 molar% of LNFP-VI, 0-25 molar% 3FL, 15-65 % LNnT and 0-15 molar% in total adding up to 100 molar% molar content.
  • a further embodiment of the present disclosure relates to a mixture of HMOs according consists essentially of 80 molar% of LNFP-VI, 10 molar% 3FL, 10 % LNnT.
  • a further embodiment of the present disclosure relates to a mixture of HMOs according consists essentially of 60 molar% of LNFP-VI and 40 molar%.
  • the genetically engineered cell capable of producing LNFP-V, described herein will generally produce a mixture of HMOs as a result of the multistep process towards the final HMO product.
  • 3-FL fucosylated lactose
  • LNT-II LNT and pLNnH
  • LNFP-II LNDFH-II
  • Example 3 shows exemplary HMO compositions, wherein the mixture of HMOs consists essentially of LNFP-V, 3FL and LNT.
  • an embodiment of the present disclosure relates to a mixture of HMOs according consists essentially of 50-70 molar% of LNFP-V, 0-5 molar% 3FL, 30-50 molar% LNT, in total adding up to 100% molar content. Specifically, the by-products LNFP-II and LNDFH-II is below 1 molar%.
  • Clinical data in infants indicate that Human Milk Oligosaccharide supplements may help to develop the desired microbiota by serving as a food source for the good bacteria in the intestine.
  • HMOs Naturally occurring in breast milk, HMOs have evolved over thousands of years, with HMO research (clinical and preclinical) now suggesting that specific HMOs at the correct level of supplementation can provide us with unique health benefits.
  • Human Milk Oligosaccharide supplements may help support immunity and gut health, with a potential role in cognitive development, which may open future innovation opportunities.
  • HMOs as described herein may also form part of a composition comprising additional parts, such as active pharmaceutical ingredients, food supplements, probiotics, excipients, surfactants etc.
  • additional parts such as active pharmaceutical ingredients, food supplements, probiotics, excipients, surfactants etc.
  • HMOs Naturally occurring in breast milk, HMOs have evolved over thousands of years, with HMO research (clinical and preclinical) now suggesting that specific HMOs at the correct level of supplementation can provide unique health benefits.
  • fucosylated HMOs constitute more than 60% of the total HMOs in human milk, mixtures with a high content of fucosylated HMOs are more desirable.
  • LNFP-VI and mixtures of HMOs comprising LNFP-VI are highly relevant as either a nutritional supplement or as a therapeutic.
  • Human Milk Oligosaccharide supplements may help to develop the desired microbiota by serving as a food source for the beneficial bacteria in the intestine.
  • Human Milk Oligosaccharide supplements may help support immunity and gut health, with a potential role in cognitive development, which may open future innovation opportunities.
  • the mixtures or composition of HMOs may be used to enhance the beneficial bacteria in the gut microbiome.
  • beneficial bacteria are for example bacteria of the Bifidobacterium sp., lactobacillus sp. or Barnesiella sp.
  • SCFAs short chain fatty acids
  • acetate, propionate and butyrate which have been shown to have many benefits in infants and young children, such as inhibition of pathogen bacteria, prevention of infection and diarrhoea, reduced risk of allergy and metabolic disorders (see for example W02006/130205, WO 2017/129644, WO2017/129649).
  • the mixtures or composition of HMOs produced according to the method described herein may be used to reduce the abundance of undesirable viruses and bacteria in the gut microbiome.
  • pathogenic bacteria and viruses that may be reduced by the HMO mixtures described herein are including Candida albicans, Clostridium difficile, Enterococcus faecium, Escherichia coll, Helicobacter pylori, Streptococcus agalactiae, Shigella dysenteriae, Staphylococcus aureus, nora virus and rota virus.
  • Each composition described herein can also be used to treat and/or reduce the risk of a broad range of bacterial infections of a human.
  • the mixtures or composition of HMOs produced according to the method described herein may be used to increase the regeneration and viability of lyophilized probiotics, including probiotics of Bifidobacterium sp, lactobacillus sp., in particular increased regeneration and/or viability and/or shelf-life in an acidic environment, such as the stomach or acidic food products, is an advantage using the HMO mixtures described herein.
  • probiotics of Bifidobacterium sp, lactobacillus sp. in particular increased regeneration and/or viability and/or shelf-life in an acidic environment, such as the stomach or acidic food products, is an advantage using the HMO mixtures described herein.
  • Bifidobacterium sp examples of Bifidobacterium sp.
  • Bifidobacterium animals lactis BB12 DSM 32269, Bifidobacterium animals lactis BIF6, Bifidobacterium longum DSM 32946, Bifidobacterium longum BB536, Bifidobacterium bifidum DSMZ 32403, Bifidobacterium infantis, Bifidobacterium breve DSM 33789, Bifidobacterium infantis SP37 DSM 32687, Bifidobacterium adolescentis DSM 34065 and/or Bifidobacterium animalis ssp. animalis DSM 16284.
  • lactobacillus sp which may have increased regeneration and viability are Lactobacillus rhamnosus GG DSM 32550, Lactobacillus rhamnosus 19070-2 DSM 26357, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus LBrGG, Lactobacillus reuteri DSM 12246, Lactobacillus plantarum TIFN101, Lactobacillus gasseri Lg-36 200B FloraFit Danisco, Lactobacillus casei DSM 32382, Lactobacillus paracasei, Lactobacillus plantarum PS 128, Lactobacillus plantarum (Sacco) DSM 32383, Lactococcus lactis PAREVE, Lactobacillus paracasei ssp. Paracasei and/or Lactobacillus Probio-Tec®LGG®, Limosilactobacillus reuteri S12 DSM 33752.
  • Regeneration means the process of regaining/ restoring a dried bacteria’s viability (i.e., “reviving” the bacterial cells by rehydration, wherein “rehydration” means restoring fluid). This process is also sometimes referred to as “reconstitution”.
  • “Viability” is the ability of a bacterial cell to live and function as a living cell.
  • One way of determining the viability of bacterial cells is by spreading them on an agar plate with suitable growth medium and counting the number of colonies formed after incubation for a predefined time (plate counting). Alternatively, FACS analysis may be used.
  • “Improving the regeneration” of Bifidobacterium sp. and/or Lactobacillus sp bacteria means to increase the amount (number) of Bifidobacterium sp. and/or Lactobacillus sp. bacteria successfully regenerating/ reviving compared to the respective control (i.e., the amount/ number of Bifidobacterium sp. and/or Lactobacillus sp. bacteria without the addition of HMO).
  • “Improving the viability” of Bifidobacterium sp. and/or Lactobacillus sp. bacteria means to increase the amount (number) of viable Bifidobacterium sp. and/or Lactobacillus sp. bacteria compared to the respective control (i.e., the amount/ number of Bifidobacterium sp. and/or Lactobacillus sp. bacteria without the addition of HMO).
  • acidic means having a pH below 7.0 (for example, having a pH ⁇ 6.0, or ⁇ 5.0, or ⁇ 4.0, or ⁇ 3.0, or in the range of 1 .0-6.0, such as from 2.0 to 5.0).
  • the pH measured in the stomach is in the range of about 1.5-3.5.
  • the pH measured in a healthy vagina is in the range of about 3.8-5.0.
  • the pH of fruit juices is in the range of about 2.0-
  • the mixtures or composition of HMOs produced according to the method described herein may be used to extend the shelf life of probiotics, such as Bifidobacterium sp. and/or lactobacillus sp.
  • probiotics such as Bifidobacterium sp. and/or lactobacillus sp.
  • An embodiment of the present invention is a composition comprising a mixture of HMOs as described herein, in particular in the section “Mixtures of HMOs”, and one or more probiotics.
  • the probiotic is a Bifidobacterium sp. and/or lactobacillus sp. such as any of the specific species mentioned above.
  • the mixtures or composition of HMOs produced according to the method described herein may be used to improve the flowability of a powder or decrease the viscosity of a liquid.
  • compositions and mixtures of HMOs described in the sections “Manufactured product” and “HMO mixtures” may also form part of a composition comprising additional parts, such as active pharmaceutical ingredients, food supplements, probiotics, excipients, carriers etc..
  • Nutritional compositions are for example, an infant formula, a rehydration solution, or a dietary maintenance, medical nutrition or supplement for elderly individuals or immunocompromised individuals.
  • Macronutrients such as edible fats, carbohydrates and proteins can also be included in such anti-infective compositions.
  • Edible fats include, for example, coconut oil, soy oil and monoglycerides and diglycerides.
  • Carbohydrates include, for example, glucose, edible lactose and hydrolysed cornstarch.
  • Proteins include, for example, soy protein, whey, and skim milk. Vitamins and minerals (e. g.
  • Vitamins A, E, D, C, and B complex can also be included in such anti-infective compositions.
  • embodiments described herein relate to the use of a mixture of HMOs or a composition comprising a mixture of HMOs, such as a mixture of HMOs produced according to the present disclosure, in an infant formula, as a dietary supplement or medical nutrition.
  • a dietary supplement or medical nutrition comprises a) LNFP-VI and 3-FL, b) LNFP-VI and LNnT, or c) LNFP-V, 3FL and LNnT.
  • Such infant formula, dietary supplement or medical nutrition may be obtained using methods disclosed herein.
  • the composition comprising a mixture of HMOs described herein, e.g., produced according to the present disclosure is a pharmaceutical composition.
  • the present disclosure also relates to the use of a mixture or composition according to the present disclosure as a dietary supplement and/or medical nutrition.
  • the disclosure relates to the use of a mixture or composition according to the present disclosure in infant nutrition.
  • a method for producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI comprising the steps of: a) Providing a genetically engineered cell with a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-neotetraose (LNnT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNnT, and b) Cultivating said genetically modified cell under conditions that allow for formation of LNFP-VI, and c) Optionally, purifying said LNFP-VI to remove by-products.
  • HMO Human Milk Oligosaccharide
  • LNnT lacto-N-neofucopentaose VI
  • a-1 ,3-fucosyltransferase is selected from the group consisting of: a) Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, b) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , c) Paral comprising or consisting of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 3, and d) CafF comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 43.
  • by-products may be one or more HMO by-products selected from 3FL, LNT-II and LNnT, LNFP-III, LNDFH-III and pLNnH.
  • the cell further comprises a substrate importer selected from a lactose importer, a lacto-N-triose-l I (LNT-II) importer or a LNnT importer.
  • a substrate importer selected from a lactose importer, a lacto-N-triose-l I (LNT-II) importer or a LNnT importer.
  • LNFP-VI conditions that allow for formation of LNFP-VI include the presence of a culture medium with an energy source that is preferably selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.
  • the genetically engineered further comprises a recombinant nucleic acid sequence encoding a [3-1 ,4- galactosyltransferase. 18. The method according to any one of the preceding items, wherein the genetically engineered, further comprises a recombinant nucleic acid sequence encoding a [3-1 ,3-N- acetyl-glucosaminyltransferase.
  • the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding a p-1 ,3-N-acetyl- glucosaminyltransferase and a recombinant nucleic acid sequence encoding a [3-1 ,4- galactosyltransferase and lactose is added during the cultivation of the genetically engineered cells as a acceptor substrate for the HMO formation.
  • the genetically engineered cell comprises one or more pathways to produce a nucleotide-activated sugar selected from the group consisting of UDP-GIcNAc, GDP-fucose, UDP-galactose and UDP- glucose.
  • the one or more enzyme is selected from the group consisting of mannose-6 phosphate isomerase (manA), phosphomannomutase (manB), mannose-1 -phosphate guanylyltransferase guanylyltransferase (manC), GDP- mannose-4,6-dehydratase (gmd) and GDP-L-fucose synthase (wcaG).
  • manA mannose-6 phosphate isomerase
  • manB mannose-1 -phosphate guanylyltransferase guanylyltransferase
  • gmd GDP- mannose-4,6-dehydratase
  • wcaG GDP-L-fucose synthase
  • a genetically engineered cell capable of producing the Human Milk Oligosaccharide (HMO) selected from lacto-N-neofucopentaose VI (LNFP-VI) or lacto-N-fucopentaose V (LNFP-V), comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase, Bacbac2, comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2.
  • HMO Human Milk Oligosaccharide
  • LNFP-VI lacto-N-neofucopentaose VI
  • LNFP-V lacto-N-fucopentaose V
  • the genetically engineered cell according to item 25 wherein the genetically engineered cell comprises one or more further recombinant nucleic acids encoding one or more heterologous glycosyltransferases.
  • 27 The genetically engineered cell according to item 25 or 26, wherein the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding a [3-1 ,3-N- acetyl-glucosaminyltransferase and a p-1 ,4-galactosyltransferase or a p-1 ,3- galactosyltransferase.
  • 25 % such as more than 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,
  • the genetically engineered cell according to any one of items 25 to 29, wherein the cell further produces one or more HMOs selected from the group consisting of 3FL, LNT-II and LNT.
  • a genetically engineered cell capable of producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI), comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase, wherein less than 5 % of the total molar content of HMO produced by said cell are fucosylated by-products with 5 or 6 monosaccharide units.
  • HMO Human Milk Oligosaccharide
  • LNFP-VI lacto-N-neofucopentaose VI
  • the genetically engineered cell according to item 34 wherein more than 25%, such as more than 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, or more than 83% of the total molar content of HMO produced by said cell is LNFP-VI.
  • Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO:
  • the genetically engineered cell according to item 36 wherein the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding a p-1 ,3-N-acetyl- glucosaminyltransferase and a p-1 ,4-galactosyltransferase.
  • the genetically engineered cell according to item 41 wherein the cell produces 3FL and/or LNnT and less than 12% of pLNnH and LNT-II, preferably less than 5% pLNnH and no LNT- II.
  • the genetically engineered cell according to any of one of items 25 to 41 wherein the cell further comprises a substrate importer selected from a lactose importer, a lacto-N-triose-l I (LNT-II) importer, an LNT and an LNnT importer.
  • the genetically engineered cell according to any of the items 25 to 43, wherein the cell comprises pathways to produce a nucleotide-activated sugar selected from the group consisting of UDP-GIcNAc, GDP-fucose, UDP-galactose and UDP-glucose.
  • the genetically engineered cell according to item 44 wherein the one or more enzyme is selected from the group consisting of mannose-6 phosphate isomerase (manA), phosphomannomutase (manB), mannose-1 -phosphate guanylyltransferase guanylyltransferase (manC), GDP-mannose-4,6-dehydratase (gmd) and GDP-L-fucose synthase (wcaG).
  • the cell further comprises a recombinant nucleic acid sequence according to SEQ ID NO: 41 encoding the colanic acid (CA) gene cluster.
  • the genetically engineered cell according to any of items 25 to 50, wherein said engineered cell is a bacterium or a fungus.
  • the genetically engineered cell according to item 51 wherein said fungus is selected from a yeast cell of the genera Komagataella sp., Kluyveromyces sp., Yarrowia sp., Pichia sp., Saccaromyces sp., Schizosaccharomyces sp. or Hansenula sp. or from a filamentous fungus of the genera Aspargillus sp., Fusarium sp. or Thricoderma sp..
  • the genetically engineered cell according to item 51 wherein said bacterium is selected from the group consisting of Escherichia sp., Bacillus sp., lactobacillus sp., Corynebacterium sp. and Campylobacter sp.
  • the genetically engineered cell according to any of one of items 25 to 53, wherein said engineered cell is selected from the group consisting of Escherichia Coll, Bacillus subtilis, lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris and Saccharomyces cerevisiae.
  • a-1 ,3-fucosyltransferase Bacbac2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2 in the production of one or more fucosylated HMOs.
  • a-1 ,3-fucosyltransferase in the production LNFP-VI, wherein the a-1 , 3- fucosyltransferase is selected from the group consisting of Bacbad , Bacbac2, Paral and CafF, comprising or consisting of the amino acid sequence of SEQ ID NO: 1 , 2, 3, or 43, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , 2, 3, or 43.
  • a mixture of HMOs consisting essentially of a) LNFP-VI and 3-FL, or b) LNFP-VI and LNnT, or c) LNFP-VI, 3FL and LNnT.
  • the mixture of HMOs according to item 59 consisting essentially of a) 25-70 molar% of LNFP-VI, 35-70 molar% 3FL, 0-5 % LNnT, or b) 55-90 molar% of LNFP-VI, 0-15 molar% 3FL, 0-35 % LNnT and 0-10 molar% pLNnH, or c) 25-70 molar% of LNFP-VI, 0-25 molar% 3FL, 15-65 % LNnT and 0-15 molar% pLNnH, in total adding up to 100% molar content.
  • the mixture of HMOs according to item 59 or 60 wherein the mixture is produced by the method according to any of items 1 to 24, or from a genetically engineered cell according to any of items 34 to 54.
  • a composition comprising a mixture of HMOs according to any of items 59 to 61 .
  • the composition according to item 62 wherein the mixture further comprises a one or more probiotics.
  • the composition according to item 63 wherein the probiotic is a Bifidobacterium sp and/or a lactobacillus sp.
  • a method for producing the Human Milk Oligosaccharide (HMO) lacto-N-fucopentaose V (LNFP-V), with less than 2% of the total molar content of HMO being fucosylated byproducts with 5 or 6 monosaccharide units comprising the steps of: a) Providing a genetically engineered cell with a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-tetraose (LNT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNT, and b) Cultivating said genetically modified cell under conditions that allow for formation of LNFP-V, and c) Optionally, purifying said LNFP-V to remove by-products.
  • HMO Human Milk Oligosaccharide
  • the method according to item 66 wherein the genetically engineered cell is a cell according to any of items 25 to 31 , and wherein less than 2 %, such as less than 1%, of the total molar content of HMO produced by said method is LNDFH-II and/or LNFP-II. 68.
  • the method according to any of items 66 or 67, wherein at least 50%, such as at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or such as at least 59% of the molar content of the total HMOs produced by said method is LNFP-V.
  • HMOs consisting essentially of LNFP-V, 3-FL and LNT
  • a composition comprising a mixture of HMOs according to any of items 59 to 61 , or any of items 73 to 75.
  • composition according to item 76 wherein the mixture further comprises one or more probiotics.
  • composition according to item 77, wherein the probiotic is a Bifidobacterium sp and/or a lactobacillus sp.
  • GenBank ID and origin of the 4 glucose-specific a-1 ,3-fucosyltransferases (Bacbacl , Bacbac2, Paral and CafF), a multi-specific a-1 ,3-fucosyltransferase (Prevl), as well as the prior art a-1 ,3-fucosyltransferases, FutA, FutB and FutT109/CafA, are provided in table 5.
  • sequences used in the present application may be truncated at the N- or C-terminal as compared to the GenBank sequence these are represented by the SEQ ID NO.
  • FutA has been shown to produce LNFP-VI, LNDFH-111 and 3FL mixtures and FutB has been shown to produce LNFP-VI, LNFP-III, LNDFH-111 and 3FL mixtures in Dumon et al., 2004 (a-1,3- fucosyltransferase, Biotechnol. Prog. 2004, 20, 412-419).
  • FucT109** has been shown to produce LNFP-V and LNFP-VI in WO2019/000133
  • the strains (genetically engineered cells) constructed in the present application were based on Escherichia coll K-12 DH1 with the genotype: F", A ⁇ , gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. Additional modifications were made to the E. coli K-12 DH1 strain to generate the MDO strain with the following modifications: lacZ: deletion of 1 .5 kbp, /acA: deletion of 0.5 kbp, nanKETA’. deletion of 3.3 kbp, melA'. deletion of 0.9 kbp, wcaJ deletion of 0.5 kbp, mdoH'. deletion of 0.5 kbp, and insertion of Plac promoter upstream of the gmd gene.
  • the MDO strain was further engineered by chromosomally integrating a p-1 , 3-GlcNAc transferase (LgtA from Neisseria meningitidis, homologous to NCBI Accession nr. WP_033911473.1 and shown as SEQ ID NO: 14) and a [3-1 ,4- galactosyltransferase (GalT from Helicobacter pylori, homologous to GenBank ID
  • the MDO strain was further engineered by chromosomally integrating a p-1 , 3-GlcNAc transferase (LgtA from Neisseria meningitidis, homologous to NCBI Accession nr. WP_033911473.1 and shown as SEQ ID NO: 14) and a p-1 ,3- galactosyltransferase (GalTK from Helicobacter pylori, homologous to GenBank Accession nr. BD182026.1 and as shown in SEQ ID NO: 42) both under the control of a PglpF promoter (SEQ ID NO: 29), this strain is named the LNT strain.
  • LgtA from Neisseria meningitidis, homologous to NCBI Accession nr. WP_033911473.1 and shown as SEQ ID NO: 14
  • GalTK from Helicobacter pylori, homologous to GenBank Accession nr. BD182026.1
  • Codon optimized DNA sequences encoding individual a-1 ,3-fucosyltransferases were genomically integrated into the LNnT or LNT strain.
  • the genotypes of the background strain (MDO), the LNnT strain and the a-1 ,3- fucosyltransferase expressing strains capable of producing LNFP-VI are provided in Table 6.
  • *1 ,3FT is an abbreviation of a-1 ,3-fucosyltransferase, and the DNA sequence is inserted into the genome of the host strain or integrated via. a plasmid.
  • 3 CA extra colanic acid gene cluster (gmd-wcaG-wcaH-wcal-manC-manB, SEQ ID NO: 41) under the control of a PglpF promoter at a locus that is different than the native locus.
  • 4 pUC57 is a high-copy number (>300) plasmid having the pUC origin of replication.
  • the antibiotic resistance marker on the pBB vector is ampicillin.
  • the indicated a-1 ,3FT is expressed from the plasmid.
  • Deep Well Assays in the current examples were performed as originally described by Lv et al (Bioprocess Biosyst Eng 20 (2016) 39:1737 — 1747) and optimized for the purposes of the current disclosure. More specifically, the strains disclosed in the present example were screened in 96 deep well plates using a 4-day protocol. During the first 24 hours, precultures were grown to high densities (OD600 up to 5) and subsequently transferred to a medium that allowed induction of gene expression and product formation.
  • Basal minimal medium BMM (pH 7,0) supplemented with magnesium sulphate (0.12 g/L), thiamine (0.004 g/L) and glucose (5.5 g/L).
  • Basal Minimal medium had the following composition: NaOH (1 g/L), KOH (2.5 g/L), KH2PO4 (7 g/L), NH4H2PO4 (7 g/L), Citric acid (0.5 g/l), trace mineral solution (5 mL/L).
  • the trace mineral stock solution contained; ZnSO ⁇ *7H ⁇ O 0.82 g/L, Citric acid 20 g/L, MnSO4*H2O 0.98 g/L, FeSO4*7H2O 3.925 g/L, CuSO4*5H2O 0.2 g/L.
  • the pH of the Basal Minimal Medium was adjusted to 7.0 with 5 N NaOH and autoclaved. The precultures were incubated for 24 hours at 34 °C and 1000 rpm shaking and then further transferred to 0.75 mL of a new BMM (pH 7,5) to start the main culture.
  • the new BMM was supplemented with magnesium sulphate (0.12 g/L), thiamine (0.02 g/L), a bolus of glucose solution (0.1-0.15 g/L) and a bolus of lactose solution (5-20 g/L) Moreover, a 20 % stock solution of sucrose (40-45 g/L) or maltodextrin (19-20 g/L) was provided as carbon source, accompanied by the addition of a specific hydrolytic enzyme, sucrose hydrolase or glycoamylase, respectively, so that glucose was released at a rate suitable for carbon-limited growth and similar to that of a typical fed- batch fermentation process. The main cultures were incubated for 72 hours at 28 °C and 1000 rpm shaking. For the analysis of total broth, the 96 well plates were boiled at 100°C, subsequently centrifuged, and finally the supernatants were analysed by HPLC. Fermentation
  • the E. coli strains were cultivated in 250 mL fermenters (Ambr250 HT Bioreactor system, Sartorius) starting with 100 mL of mineral culture medium consisting of 30 g/L glucose and a mineral medium comprised of NH 4 H 2 PO 4 , KH 2 PO 4 , MgSO 4 x 7H 2 O, KOH, NaOH, citric acid, trace element solution, antifoam and thiamine.
  • the dissolved oxygen level was kept at 20% by a cascade of first agitation and then airflow starting at 700 rpm (up to max 4500 rpm) and 1 WM (up to max 3 WM).
  • the pH was kept at 6.8 by titration with 8.5% NH4OH solution.
  • the cultivations were started with 2% (v/v) inoculums from pre-cultures comprised of 10 g/L glucose, (NH 4 ) 2 HPO 4 , KH 2 PO 4 , MgSO4 x 7H 2 O, KOH, NaOH, citric acid, trace element solution, antifoam and thiamine.
  • a feed solution containing glucose, MgSO 4 x 7H 2 O, H 3 PO 4 and trace mineral solution was continuously added to the fermenter at a rate that maintained carbon-limiting conditions.
  • the temperature was initially at 33°C but was dropped to 30°C with a 3-hour linear ramp initiated 12 hours after the start of the feed.
  • Lactose was added as bolus additions of 25% lactose monohydrate solution 36 hours after feed start and then every 19 hours to keep lactose from becoming a rate limiting factor.
  • the growth, metabolic activity and metabolic state of the cells was followed by on-line measurements of agitation, dissolved oxygen tension, reflectance, NH 4 OH base addition, O 2 uptake rate and CO 2 evolution rate. Throughout the fermentations, samples were taken to determine the concentration of HMO products, lactose and other minor by-products using HPLC.
  • Table 6 lists the genotype of the strains capable of producing LNFP-VI.
  • the molar content of individual HMOs produced by the strains was calculated from sample measurements performed by HPLC.
  • the results of the LNFP-VI producing cells are shown in table 7 as the fraction of the total molar HMO content (in percentage, %) produced by each strain.
  • Table 7 Content of individual HMO’s as % of total HMO molar (mM) content produced by each strain (results are as a minimum the average of 3 replicates).
  • the three novel enzymes Bacbad , Bacbac2 and Paral can transfer a fucosyl unit specifically onto the Glc moiety of LNnT in an a- 1 ,3 linkage to form LNFP-VI at a level above 25% of the total HMO, with no production of the complex fucosylated by-product HMOs, LNDFH-III or LNFP-111 , as compared to the prior art enzyme FutA and FucT109 and the enzyme Prevl which produce 65%, 18% and 30% LNFP-VI of the total HMO, respectively, along with major production of the unintended by-products LNDFH-III at 25%, 24% and 41%, respectively.
  • the enzymes FucT109 and Prevl also produced the unintended HMO by-product LNFP-III in an amount of 27 and15%, respectively.
  • FutB which was previously suggested to produce a mixture of LNDFH-III, LNFP-III, LNFP-VI and 3FL in Dumon et al., 2004, did not produce any LNDFH-III and only minor LNFP- III and LNFP-VI.
  • Absence of alternative fucosylated species in the produced mixture is highly advantageous and preferred if it is desired to purify the produced LNFP-VI.
  • the low levels of LNnT achieved by expressing the Bacbacl and Paral in high copy number is beneficial if pure LNFP-VI is desired.
  • the enzymes do not severely affect the overall HMO production capability of the cells, and even the lowest producing cell with Bacbac2 still produces about 87 % of the HMO that the FutA strain produces and is therefore still advantageous since none of the produced HMO is the unintended by-products LNFP-III and LNDFH-III, so the overall LNFP-VI yield will still be higher.
  • Both strains show the suitability of Bacbacl and Bacbac2 for the production of an LNFP-VI product with only low production of similar complex fucosylated by-product HMOs (LNFP-III and LNDFH-I II) in fermentation, in particular Bacbac2 has very low LNnT and 3FL production and no LNFP-III or LNDFH-I 11 production, allowing for the opportunity to obtain very pure LNFP-VI with low purification efforts.
  • HMOs including LNFP-V
  • LNFP-V LNFP-V
  • results of HMOs, including LNFP-V, produced by the LNT backbone cells with the different a-1 ,3-fucosyltransferases are shown in table 10 as the fraction of the total molar HMO content (in percentage, %) produced by each strain.
  • Table 10 Content of individual HMO’s as % of total HMO content produced by the LNT background strain From the data presented in table 10, it can be seen that the Bacbac2 enzyme has quite similar activity on LNT and LNnT in that it can transfer a fucosyl unit specifically onto the Glc moiety of LNT and not onto the GIcNAc moiety, therefore no complex fucosylated HMO by-products such as LNFP-II and LNDFH-II were formed. This is an indication that Bacbac2 does not have any alpha-1 , 4-fucosyltranferase activity.
  • the enzymes Bacbac 1 and Para 1 seem to have very low activity on any moiety in LNT, and essentially resembles how FutB acted in the LNnT strain. FutB on the other hand shows some fucosyltransferase activity and appears to have specificity towards the glucose moiety on LNT. CafF seems to have slightly higher activity on lactose in the LNT background strain and only produces low amounts of complex fucosylated HMOs.
  • the enzymes FutA, FucT109 and CafC known from Dumon et al., 2004, WO2019/008133 and WO2016/040531 , respectively, seem to have some a-1 ,4-fucosyltransferase activity and can therefore fucosylate the GIcNAc moiety in LNT resulting in some LNFP-II or LNDFH-II in the LNT background.
  • the novel enzyme Bacbac2 is the only enzyme that can transfer a fucosyl unit specifically onto the Glc moiety of LNT in an a-1 ,3 linkage to form LNFP-V at levels above 50% of the total HMO, with no production of LNDFH-II or LNFP-II as compared to the prior art enzyme FutA which produce 79% LNFP-V of the total HMO, respectively, along with production of the unintended products LNDFH-II at 13% respectively.
  • the total amount of HMO produced in the 2x Bacbac2 strain is higher (relative total production of 127 %) than the amount of HMO produced by the 2x FutA carrying strain, leading to a production of LNFP-V by the Bacbac2 expressing strain of 95% of the FutA strain.
  • the FutB strain seems to produce even more total HMO than the Bacbac2 strain, but still the amount of LNFP-V is lower than what is produced by the Bacbac2 strain.
  • Bacbac2 strain produces an almost identical amount of LNFP-V as the FutA strain, while not producing the by-product LNDFH-II.
  • Example 4 Regeneration and viability of lyophilized Lactobacillus rhamnosus
  • Probiotics may be consumed as live bacteria or as a dried (e.g. lyophilized) product.
  • rehydration involves an important step in the recovery of dehydrated bacteria; an inadequate rehydration/ regeneration step may lead to poor cell viability and a low final survival rate.
  • Rehydration is therefore a highly critical step in the revitalization of a lyophilized culture.
  • the survival of the bacteria under acidic conditions is critical since they need to pass through the acidic environment of the stomach and may also be faced with storage (shelf-life) in acidic food products.
  • the lyophilized probiotic was added to the tube (0.4 mg/ml), alone (control) or in combination with HMO mixtures (5% w/v) as indicated in table 13.
  • the tubes were incubated at 37 °C for 3 h.
  • the samples were further diluted and 100 l were spread in duplicates onto MRS agar plates which were incubated at 37 °C in anaerobic chambers.
  • Table 14 Average CFU/ml for the indicated strains after 3 h acid treatment followed by 48h subsequent incubation at 37°C
  • the lyophilized Lactobacillus strain dissolved with the HMO mixtures described herein showed an enhanced regeneration and survivability compared to control without the HMO mixtures.

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Abstract

The present disclosure relates to the production of complex fucosylated Human Milk Oligosaccharides (HMOs) and in particular to the production of the complex fucosylated HMO LNFP-VI or LNFP-V, with five or more monosaccharide units, where said production leads to a product which is essentially free of LNFP-III and LNDFH-III, or LNFP-II and LNADH-II, respectively using alpha-1,3-fucosyltransferases from Bacteroidales bacterium. The present disclosure also relates to genetically engineered cells and alpha-1,3-fucosyltransferases suitable for use in said production, as well as to methods for producing said fucosylated HMOs.

Description

NEW FUCOSYLTRANSFERASES FOR IN VIVO SYNTHESIS OF COMPLEX FUCOSYLATED
HUMAN MILK OLIGOSACCHARIDES MIXTURES COMPRISING LNFP-VI OR LNFP-V
FIELD
The present disclosure relates to the production of complex fucosylated Human Milk Oligosaccharides (HMOs) and in particular to the production of the complex fucosylated HMO LNFP-VI or LNFP-V, with five or more monosaccharide units, where said production leads to a product which is essentially free of LNFP-111 and LNDFH-111 or LNFP-II and LNDFH-II, respectively. The present disclosure also relates to genetically engineered cells and a-1 ,3- fucosyltransferases suitable for use in said production, as well as to methods for producing said fucosylated HMOs.
BACKGROUND
The design and construction of bacterial cell factories to produce fucosylated Human Milk Oligosaccharides (HMOs), especially for more complex fucosylated HMOs is of paramount importance to provide innovative and scalable solutions for the more complex products of tomorrow.
Production of complex fucosylated HMOs has e.g., been described in WO2019/008133, wherein it is reported that the a-1 ,3-fucosyltransferase FucT109 which appears to fucosylate both the glucose (Glc) and N-acetylglucosamine (GIcNAc) moiety of Lacto-N-neotetraose (LNnT), thus potentially generating a mixture containing all three of LNnT, LNFP-111 and LNFP- VI, while apparently also being capable of producing LNFP-V using LNT as the backbone.
Dumon et al., 2004 (Biotechnol. Prog. 2004, 20, 412-419) further describes two a-1 , 3- fucosyltransferases, FutA and FutB, which are also suggested to produce mixtures of LNFP-VI, LNDFH-III and 3FL or LNnT, LNFP-III, LNFP-VI and LNDFH-I II, respectively.
WO2023/110995 discloses fucosyltransferases having alpha-1 , 3-fucosyltransferase activity on the N-acetylglucosamine (GIcNAc) and/or the glucose (Glc) on various saccharide structures including lactose, LNT and LNnT to produce mixtures of fucosylated oligosaccharides such as for example LNFP-III, LNFP-VI and LNDFH-III or LNFP-II, LNFP-V and LNDFH-II.
Furthermore, W02016/040531 discloses a number of a-1 , 3-fucosyltransferase, including CafC and CafF, which are capable of producing 3FL. In summary, production of fucosylated HMOs, especially specific complex fucosylated HMOs, such as LNFP-VI and LNFP-V, is challenging due to the lack of fucosyltransferases with the desired substrate specificity, as well as low production yield of the desired fucosylated HMOs as compared to other HMO products present after fermentation, such as HMO precursor products and complex fucosylated HMO by-products, which may require laborious separation procedures.
SUMMARY
The need for highly substrate specific a-1 ,3-fucosyltransferases is solved by the identification of a selection of a-1 ,3-fucosyltransferases which exhibit low or no specificity for the N- acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNnT or LNT as a substrate for fucosylation reactions, but which are highly substrate specific for the Glucose (Glc) moiety in LNnT or LNT. Such a-1 ,3-fucosyltransferases can be used to produce high amounts of the complex fucosylated HMO LNFP-VI or LNFP-V, or mixtures of HMOs with LNFP-VI or LNFP-V, and very low levels of complex fucosylated HMO by-products such as LNFP-III and LNDFH-III or LNFP-II and LNDFH-II. Hence, provided herein are enzymes, mixtures, compositions, uses, genetically engineered cells and methods for the production of LNFP-VI or LNFP-V.
A first aspect of the present disclosure relates to methods for producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI) or lacto-N-fucopentaose V (LNFP-V), with less than 5 % of the total molar content of HMO being fucosylated by-product oligosaccharides with 5 or 6 monosaccharide units, comprising the steps of a) providing a genetically engineered cell with a recombinant nucleic acid sequence encoding an a-1 ,3- fucosyltransferase derived from Bacteroidales bacterium , and b) cultivating said genetically modified cell under conditions that allow for formation of LNFP-VI of LNFP-V, and c) optionally, purifying said LNFP-VI or LNFP-V by removing by-products such as 3FL and/or LNnT or 3FL and/or LNT, respectively.
Preferably, the a-1 ,3-fucosyltransferase has high specificity for the glucose (Glc) moity in LNnT and/or LNT and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNnT.
A second aspect is a genetically engineered cell capable of producing the Human Milk Oligosaccharide (HMO) selected from lacto-N-neofucopentaose VI (LNFP-VI) and lacto-N- fucopentaose V (LNFP-V), comprising a recombinant nucleic acid sequence encoding an a-1 , 3- fucosyltransferase, Bacbac2, comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2.
In addition to LNFP-V the cell may further produce one or more HMOs selected from the group consisting of 3FL, LNT-II and LNT and it is preferred that essentially no LNFP-II and/or LNDFH- II is produced by said cell. The cell may comprise additional modifications such as a substrate importer selected from a lactose importer, a lacto-N-triose-ll (LNT-II) importer and a LNT importer.
A third aspect is a genetically engineered cell capable of producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI), comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase, be selected from the group consisting of, a) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO:
1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , or b) Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO:
2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, wherein the genetically engineered further comprises one or more recombinant nucleic acid sequences needed to produce LNnT in said cell.
In addition to LNFP-VI the cell may further produces one or more HMOs selected from the group consisting of 3FL, LNT-II and LNnT and it is preferred that essentially no LNFP-III and/or LNDFH-111 is produced by said cell. The cell may comprise additional modifications such as a substrate importer selected from a lactose importer, a lacto-N-triose-ll (LNT-II) importer and a LNnT importer.
A fourth aspect relates to the use of an a-1 ,3-fucosyltransferase in the production of LNFP-VI, wherein the a-1 ,3-fucosyltransferase is selected from Bacbad , and Bacbac2 comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 2 or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 or2.
A fifth aspect of the present disclosure relates to a mixture of HMOs, preferably produced with a method according to the disclosure, wherein the mixture of HMOs consists essentially of a) LNFP-VI and 3-FL, or b) LNFP-VI or c) LNnT, or LNFP-V, 3FL and LNnT or d) LNFP-V, 3FL and LNT. A sixth aspect of the disclosure relates to the use of a mixture or composition according to the disclosure, in an infant formula, a dietary supplement and/or medical nutrition.
BRIEF DESCRIPTION OF FIGURES
Figure 1 Overview of the synthesis of complex fucosylated HMO with an LNnT-backbone.
Figure 2 llustrate the data from the strains of table 7, clearly showing the difference in LNFP-VI production from the strains with different enzymes. LNFP-VI is the black bar, 3FL is the grey bar, LNnT is the diagonally striped bar, LNDFH-111 is the chequered bar, LNFP-111 is the horizontally striped bar, and pLNnH is the dotted bar.
Figure 3 Overview of the synthesis of complex fucosylated HMO with an LNT-backbone.
Figure 4: Shows the regeneration and viability of lyophilized Lactobacillus rhamnosus (DSM 33156), incubated for 3 h at pH 3.0 and plated in up to 4 dilutions 1 :1000 (E-3), 1 :10,000 (E-4), 1 :100,000 (E-5) and 1 :1 ,000,000 (E-6). A) is the control without HMOs; B) is Lactobacillus rhamnosus (DSM 33156) in combination with an HMO mixture containing 80% LNFP-VI and 10% 3FL and 10% LNnT (mix 1); C) is Lactobacillus rhamnosus (DSM 33156) in combination with an HMO mixture containing 60% LNFP-VI and 40% 3FL (mix2).
DETAILED DESCRIPTION
The present disclosure approaches the biotechnological challenges of in vivo HMO production of, in particular, complex fucosylated HMOs which comprise at least five monosaccharide units, of which at least one monosaccharide unit is a fucosyl unit, such as e.g., LNFP-VI and LNFP-V. The present disclosure offers specific strain engineering solutions to produce specific complex fucosylated HMOs, in particular, LNFP-VI or LNFP-V, by exploiting the substrate specificity of the identified a-1 ,3-fucosyltransferases, Bacbad , Bacbac2, Paral and CafF, disclosed herein, in particular towards the glucose (Glc) moiety and not the N-acetylglucosamine (GIcNAc) or galactose (Gal) moieties in LNnT (or LNT).
A genetically engineered cell of the present disclosure expresses genes encoding key enzymes for the biosynthesis of fucosylated HMOs. In addition, it is advantageous if the genetically engineered cell expresses the genes needed to produce LNnT or LNT either from lactose or LNT-II as the initial substrate. Alternatively, the cell may be engineered to take up LNnT (see for example W02023/099680) only needing the 1 ,3-fucosyltransferase activity in the cell. In some embodiments, a genetically engineered cell of the present disclosure further expresses one or more of the de novo GDP-fucose pathway genes, manA, manB, manC, gmd and/or wcaG, responsible for the formation of GDP-fucose. It may be advantageous to overexpress one or more of these genes and/or to upregulate the colanic acid gene cluster (CA), including the genes gmd, wcaG, wcaH, weal, manC and manB from E. Coll, through introduction of a nucleic acid construct encoding the CA as shown in SEQ ID NO: 41 , allowing for formation of GDP- fucose, which enables the cell to produce a higher level of fucosylated oligosaccharide from one or more oligosaccharide substrates, such as lactose, LNT-II and/or LNnT. Depending on the intended use of substrate, one or more additional glycosyltransferases and pathways for producing nucleotide-activated sugars, such as glucose-UDP-GIcNAc, CMP-N-acetylneuraminic acid, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and/or CMP-N-acetylneuraminic acid can also be present in the genetically engineered cell.
Production of LNFP-VI
The advantage of using any one of the a-1 ,3-fucosyltransferases of the present disclosure in the present context is their ability to specifically recognize and fucosylate the Glc moiety in LNnT, to generate LNFP-VI. In particular, the present disclosure describes enzymes with a-1 , 3- fucosyltransferase activity (a-1 ,3-fucosyltransferases) that are more active on the Glc moiety of LNnT than a-1 ,3-fucosyltransferases described in the prior art, such as FutA and FutB (see Dumon et al., 2004), CafC (WO2016/040531) and FucT109 (WO2019/008133). Furthermore, the a-1 ,3-fucosyltransferases described herein have very low activity on the GIcNAc moieties of LNnT. If LNnT is available in sufficient amounts inside the genetically engineered cell, very little, if any, LNFP-111 and/or LNDFH- 111 is produced by the a-1 ,3-fucosyltransferases described in the present disclosure. The traits of the a-1 ,3-fucosyltransferases described herein are therefore well-suited for high-level industrial production of LNFP-VI without production of alternatively fucosylated complex oligosaccharide by-products, such as LNFP-III and LNDFH-III.
In embodiments the a-1 ,3-fucosyltransferases of the present disclosure primarily fucosylate the glucose (Glc) moiety of an acceptor oligosaccharide such as e.g., LNnT. The term “primarily” is to be understood as less than 5%, such as less than 4%, such as less than 3%, or such as less than 2% of other moieties in LNnT are fucosylated by the a-1 ,3-fucosyltransferases of the present disclosure.
In embodiments the a-1 ,3-fucosyltransferases of the present disclosure has low or no activity on the N-acetylglucosamine (GIcNAc) moiety in the acceptor molecule. The acceptor molecule is an oligosaccharide such as example e.g., an HMO, such as LNT-II or LNnT, but also other oligosaccharides or HMOs. LNnT is the preferred acceptor molecule for the a-1 ,3- fucosyltransferases of the present disclosure. The term low or no activity is to be understood as less than 5%, such as less than 4%, such as less than 3%, or such as less than 2% of the GIcNAc moiety in the acceptor molecule is fucosylated by the a-1 ,3-fucosyltransferases of the present disclosure. Accordingly, in embodiments, the oligosaccharides produced by the cell expressing said a-1 ,3-fucosyltransferases is essentially free of N-acetylglucosamine (GIcNAc) fucosylated oligosaccharides. In embodiments, the oligosaccharides produced by the cell expressing said a-1 ,3-fucosyltransferases is essentially free of the N-acetylglucosamine (GIcNAc) fucosylated oligosaccharides LNFP-III and/or LNDFH-111.
The genetically engineered cells of the present disclosure which express an a-1 ,3- fucosyltransferase with high specificity for the Glc moity in LNnT, enable the production of high titters of LNFP-VI. In particular, in absence of other complex fucosylated oligosaccharide byproducts with 5 or 6 monosaccharide units, such as LNFP-III and LNDFH-111. Thereby, the present disclosure enables a more efficient LNFP-VI production, which is highly beneficial in biotechnological production of more complex fucosylated HMOs, such as LNFP-VI.
Consequently, the mixtures of HMOs produced by the cells and/or methods described herein contain a high percentage of LNFP-VI out of the total amount of HMOs produced, such as at least 25% of the total amount of HMOs, preferably at least 50%, such as at least 60%, such as alt least 70%, such as at least 80% of the total amount of HMO produced by the cell.
The ability to fermentatively produce LNFP-VI with no or very low amounts of fucosylated oligosaccharide by-products with 5 or 6 monosaccharide units is a benefit in purification since separation of fucosylated HMOs of similar length is very challenging, and the method and genetically engineered cells described herein therefore provide a benefit in terms of obtaining LNFP-VI with at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% purity of the finished product, in particular by applying one or more purification steps after fermentation to remove undesired by-products, in particular HMO by-products.
Production of LNFP-V
Additionally, the genetically engineered cells of the present disclosure may also be engineered to produce fucosylated HMOs with an LNT backbone. Such a cell is further engineered to express an a-1 ,3-fucosyltransferase with high specificity for the Glc moity in LNT, enable the production of high titters of LNFP-V. In particular, in absence of other complex fucosylated HMO by-products, such as LNFP-II and LNDFH-II. Thereby, the present disclosure also enables a more efficient LNFP-V production, which is highly beneficial in biotechnological production of more complex fucosylated HMOs, such as LNFP-V. Furthermore, the a-1 ,3-fucosyltransferases described herein have very low activity on the GIcNAc moieties of LNT. If LNT is available in sufficient amounts inside the genetically engineered cell, very little, if any, LNFP-II and/or LNDFH-II is produced by the a-1 ,3- fucosyltransferases described herein. The traits of the a-1 ,3-fucosyltransferases described herein are therefore well-suited for high-level industrial production of LNFP-V without production of alternatively fucosylated complex oligosaccharide by-products, such as LNFP-II and LNDFH- II.
In embodiments the a-1 ,3-fucosyltransferases of the present disclosure primarily fucosylates the glucose (Glc) moiety of an acceptor oligosaccharide such as LNT. The term “primarily” is to be understood as less than 5%, such as less than 4%, such as less than 3%, such as less than 2% of other moieties in LNnT are fucosylated by the a-1 ,3-fucosyltransferases of the present disclosure. Accordingly, an a-1 ,3-fucosyltransferases of the present disclosure which primarily fucosylates the glucose (Glc) moiety of an acceptor oligosaccharide may also be referred to herein as having a high specificity for the glucose (Glc) moiety in said acceptor oligosaccharide, and it is therefore to be understood that such terminologies may be used interchangeably.
In embodiments the a-1 ,3-fucosyltransferases of the present disclosure has low or no activity on the N-acetylglucosamine (GIcNAc) moiety in the acceptor molecule. The acceptor molecule is an oligosaccharide such as for example an HMO such as LNT-II or LNT, but also other oligosaccharides or HMOs. LNT is the preferred acceptor molecule for the a-1 ,3- fucosyltransferases of the present disclosure. Particularly, the a-1 ,3-fucosyltransferases of the present disclosure does not possess any or very low a-1 ,4-fucosyltransferase activity which prevents it from fucosylating the GIcNAc) moiety of LNT, since this is bound to the terminal galactose via an a-1 ,3-linkage and therefore not available for fucosylation by an enzyme that only possess a-1 ,3-fucosyltransferase activity. The term low or no activity is to be understood as less than 2%, such as less than 1 .5%, such as less than 1 %, such as less than 0.5%, such as less than 0.1% of the GIcNAc moiety in the acceptor molecule, such as LNT, is fucosylated by the a-1 ,3-fucosyltransferases of the present disclosure. Accordingly, in embodiments, the oligosaccharides produced by the cell expressing said a-1 ,3-fucosyltransferases is essentially free of N-acetylglucosamine (GIcNAc) fucosylated oligosaccharides such as e.g., N- acetylglucosamine (GIcNAc) fucosylated oligosaccharides with an LNT and/or LNnT backbone.
The genetically engineered cell of the present disclosure which express an a-1 , 3- fucosyltransferase with high specificity for the Glc moity in LNT, enables the production of LNFP-V mixtures with very small amounts of other fucosylated oligosaccharides. In embodiments the genetically engineered cell produces a mixture of LNFO-V and LNT with less than 5%, such as les then 2% of other fucosylated HMOs. In particular the LNFP-V mixtures do not contain other complex fucosylated HMOs, such as LNFP-II and LNDFH-II, which eases purification of LNFP-V. Thus, in embodiments, the oligosaccharides produced by the cell expressing said a-1 ,3-fucosyltransferases is essentially free of the N-acetylglucosamine (GIcNAc) fucosylated oligosaccharides LNFP-II and/or LNDFH-II. Thereby, the present disclosure enables a more efficient LNFP-V production, which is highly beneficial in biotechnological production of more complex fucosylated HMOs, such as LNFP-V.
Consequently, the mixtures of HMOs produced by the cells and/or methods described herein may alternatively contain a high percentage of LNFP-V out of the total amount of HMOs produced, such as at least 40% of the total amount of HMOs, preferably at least 45%, such as at least 50%, such as alt least 55%, or such as at least 57% of the total amount of HMO produced by the cell and/or method.
The ability to fermentatively produce LNFP-V with no or very low amounts of fucosylated oligosaccharide by-products with 5 or 6 monosaccharide units is a benefit in purification since separation of fucosylated HMOs of similar length is very challenging, and the method and genetically engineered cells described herein therefore provide a benefit in terms of obtaining LNFP-V with at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% purity of the finished product, in particular by applying one or more purification steps after fermentation to remove undesired by-products, in particular HMO by-products.
In the following sections, individual elements of the disclosure, and in particular of the genetically engineered cell are described. It is understood that these elements can be combined across the individual sections.
Oligosaccharides
In the present context, the term “oligosaccharide” means a sugar polymer containing at least three monosaccharide units, i.e., a tri-, tetra-, penta-, hexa- or higher oligosaccharide. The oligosaccharide can have a linear or branched structure containing monosaccharide units that are linked to each other by interglycosidic linkages. Particularly, the oligosaccharide comprises a lactose residue at the reducing end and one or more naturally occurring monosaccharides of 5-9 carbon atoms selected from aldoses (e.g., glucose, galactose, ribose, arabinose, xylose, etc.), ketoses (e.g., fructose, sorbose, tagatose, etc.), deoxysugars (e.g. rhamnose, fucose, etc.), deoxy-aminosugars (e.g. N-acetyl-glucosamine, N-acetyl-mannosamine, N-acetyl- galactosamine, etc.), uronic acids and ketoaldonic acids (e.g. N-acetylneuraminic acid). Preferably, the oligosaccharide is an HMO.
Human milk oligosaccharide (HMO)
Preferred oligosaccharides of the disclosure are human milk oligosaccharides (HMOs).
The term “human milk oligosaccharide" or "HMO" in the present context means a complex carbohydrate found in human breast milk. The HMOs have a core structure comprising a lactose unit at the reducing end that can be elongated by one or more beta-N-acetyl- lactosaminyl and/or one or more beta-lacto-N-biosyl unit, and this core structure can be substituted by an a-L-fucopyranosyl and/or an a-N-acetyl-neuraminyl (fucosyl) moiety. HMO structures are e.g., disclosed by Xi Chen in Chapter 4 of Advances in Carbohydrate Chemistry and Biochemistry 2015 vol 72.
The present disclosure focuses on fucosylated HMO’s. Examples of fucosylated HMOs include, 2'-fucosyllactose (2’FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3- fucosyllactose (3FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N- fucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-III), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose (LNFP-V), lacto-N-fucopentaose VI (LNFP-VI), lacto-N- difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-para-lacto-N-hexaose I (FpLNH-l), fucosyl-para-lacto-N-neohexaose II (F-pLNnH II), fucosyl-lacto-N-neohexaose (FLNnH), 3-fucosyl-3’-fucosyllactose (FSL), fucosyl-LST-a (FLST-a), fucosyl-LST b (FLST b), fucosyl-LST-c (FLST-c), fucosyl-LST d (FLST-d) and fucosyl-lacto-N-hexaose (SLNH).
In the context described herein, complex fucosylated HMOs are fucosylated HMOs that comprises at least 5 monosaccharide units of which at least one monosaccharide unit is a fucosyl unit, non-limiting examples of complex fucosylated HMOs are the fucosylated HMOs consisting of 5 monosaccharide units e.g., LNFP-I, LNFP-II, LNFP-III, LNFP-V and LNFP-VI and complex fucosylated HMO with 6 monosaccharide units such as but not limited to the di- fucosylated HMOs LNDFH-I, LNDFH-II and LNDFH-III or the sialyl-fucosyl HMOs FLST-a, FLST-b, FLST-c and FLST-d. Preferably, a complex fucosylated HMO is one that requires at least three different glycosyltransferase activities to be produced from lactose as the initial substrate, e.g., the formation of LNFP-VI requires an Glc specific a-1 ,3-fucosyltransferase, a - 1 ,3-N-acetyl-glucosaminyl-transferase and a p-1 ,4-galactosyltransferase (see figure 1), and the formation of LNDFH-III requires at least one a-1 ,3-fucosyltransferase, a p-1 ,3-N-acetyl- glucosaminyl-transferase and a p-1 ,4-galactosyltransferase. Enzymes described herein preferably has a preferred activity on only the Glc moiety of LNnT or LNT, thus being capable of producing LNFP-VI from LNnT (see figure 1), or LNFP-V from LNT (Figure 3) with no or only very little production complex fucosylated by-product oligosaccharides, such as LNFP-III and/or LNDFH-I II or LNFP-II and/or LNDFH-I II.
In the context of the present disclosure, complex fucosylated HMOs are fucosylated HMOs that comprises at least 5 monosaccharide units of which at least one monosaccharide unit is a fucosyl unit, non-limiting examples of complex fucosylated HMOs are the fucosylated HMOs consisting of 5 monosaccharide units e.g., LNFP-I, LNFP-II, LNFP-III, LNFP-V and LNFP-VI and fucosylated HMO with 6 monosaccharide units, such as but not limited to LNDFH-I, LNDFH-II and LNDFH-I 11. Preferably, a complex fucosylated HMO is one that require at least three different glycosyltransferase activities to be produced from lactose as the initial substrate, e.g., the formation of LNFP-VI requires an Glc specific a-1 ,3-fucosyltransferase, a p-1 ,3-N-acetyl- glucosaminyl-transferase and a p-1 ,4-galactosyltransferase.
When used in the present disclosure the term “complex fucosylated by-product oligosaccharide”, refers to a complex fucosylated oligosaccharide which is not the desired product. This means that the complex fucosylated HMOs mentioned above can be considered as by-products, also interchangeably termed HMO by-products, if they are not desired in the final product, which in the current disclosure is LNFP-VI or LNFP-V. Examples of fucosylated by-product oligosaccharides with 5 our 6 monosaccharide units are e.g., LNFP-III, LNFP-II, LNDFH-III and LNDFH-II.
In embodiments of the present disclosure, the fucosylated human milk oligosaccharide (HMO) produced by the cell is LNFP-VI, such as primarily LNFP-VI. In a further embodiment of the present disclosure, at least 25 %, such as at least 30%, 50%, 55%, 60%, 70%, 75%, 80% or 82% of the molar content of the total HMOs produced by said cell is LNFP-VI. Preferably, at least 60 % of the molar content of the total HMOs produced by said cell is LNFP-VI. In further embodiments, at least 80 % of the molar content of the total HMOs produced by said cell is LNFP-VI and LNnT. In additional embodiments, at least 80 %, such as at least 85%, 90%, 95% or such as at least 99% or such as 100% of the molar content of the total HMOs produced by said cell is LNFP-VI and 3FL. In additional embodiments of the disclosure, less than 3%, such as 0.0%, or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5% or such as less than 2.99% of the total molar content of HMOs produced by the cell is LNFP-III. In additional embodiments of the disclosure, less than less than 3%, such as 0.0%, or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5% or such as less than 2.99% of the total molar content of HMOs produced by the cell is LNDFH-III. In additional embodiments less than 5% of the molar content of the total HMOs produced by the cell is an alternative complex fucosylated oligosaccharide by-product. In the present context, an alternative fucosylated oligosaccharide by-product is considered to be one or more fucosylated oligosaccarides which is not LNFP-VI. An alternative fucosylated oligosaccharides, such as fucosylated HMO(s), may be selected from the group consisting of 3-FL, DFL, LNFP-III and LNDFH-III. An alternative complex fucosylated HMO may be selected from the group consisting of LNFP-III and LNDFH-III.
Preferably, no LNFP-III or LNDFH-III is produced by the cell.
Production of LNFP-VI may require the presence of two or more glycosyltransferase activities, in particular, if starting from lactose as the initial acceptor oligosaccharide.
In alternative embodiments of the present disclosure, the fucosylated human milk oligosaccharide (HMO) produced by the cell is LNFP-V, such as primarily LNFP-V. In a further alternative embodiment of the present disclosure, at least 25 %, such as at least 30%, 50%, 55%, 56%, 57%, 58%, or 59% of the molar content of the total HMOs produced by said cell is LNFP-V. Preferably, at least 50% of the molar content of the total HMOs produced by said cell is LNFP-V. In further embodiments, at least 80 % of the molar content of the total HMOs produced by said cell is LNFP-V and LNT. In additional alternative embodiments of the disclosure, less than 2%, such as 0.0%, or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1 .5%, 1 .75%, or such as less than 1 .99% of the total molar content of HMOs produced by the cell is LNFP-II. In additional alternative embodiments of the disclosure, less than less than 2%, such as 0.0%, or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 1.75%, or such as less than 1.99% of the total molar content of HMOs produced by the cell is LNDFH-II. In additional alternative embodiments less than 5% of the molar content of the total HMOs produced by the cell is an alternative complex fucosylated oligosaccharide by-product. In the present context, an alternative fucosylated oligosaccharide by-product to LNFP-V, is considered one or more fucosylated oligosaccharides, such as HMO, which is not LNFP-V. An alternative fucosylated HMO(s) may be selected from the group consisting of 3-FL, DFL, LNFP-II and LNDFH-II. An alternative complex fucosylated HMO may be selected from the group consisting of LNFP-II and LNDFH-II.
Preferably, no LNFP-II or LNDFH-II is produced by the cell.
Production of LNFP-V may require the presence of two or more glycosyltransferase activities, in particular, if starting from lactose as the acceptor oligosaccharide. An acceptor oligosaccharide
A genetically engineered cell according to the present disclosure comprises a recombinant nucleic acid sequence encoding a fucosyltransferase with a-1 ,3-fucosyltransferase activity capable of transferring fucose from an activated sugar to the glucose moiety of an acceptor oligosaccharide, preferably LNnT or LNT, in an a-1 ,3 linkage.
As described herein, an acceptor oligosaccharide is an oligosaccharide that can act as a substrate for a glycosyltransferase capable of transferring a glycosyl moiety from a glycosyl donor to the acceptor oligosaccharide. The glycosyl donor is preferably a nucleotide-activated sugar as described in the section on “Glycosyl-donor - nucleotide-activated sugar pathways”. Preferably, the acceptor oligosaccharide is a precursor for making a more complex HMO and can also be termed the precursor molecule.
The acceptor oligosaccharide can be either an intermediate product of the present fermentation process, an end-product of a separate fermentation process employing a separate genetically engineered cell, or an enzymatically or chemically produced molecule.
In the present context, said acceptor oligosaccharide for the a-1 ,3-fucosyltransferase is preferably lacto-N-neotetraose (LNnT), which is produced from the precursor molecules lactose (e.g., acceptor for the p-1 ,3-N-acetyl-glucosaminyl-transferase) and/or lacto-N-triose II (LNT-II) (e.g., acceptor for the p-1 ,4-galactosyltransferase). Lactose may also be identified as the initial substrate, if this is what is supplied to during cultivations. In cases where it is desired that the cell does not produce 3FL, the preferred precursor molecule (alternative initial substrate) supplied to the cultivation is LNT-II.
Alternatively, said acceptor oligosaccharide for the a-1 ,3-fucosyltransferase is preferably lacto- N-tetraose (LNT), which is produced from the precursor molecules lactose (e.g., acceptor for the P-1 ,3-N-acetyl-glucosaminyl-transferase), and/or lacto-N-triose II (LNT-II) (e.g., acceptor for the P-1 ,3-galactosyltransferase). Lactose may also be identified as the initial substrate, if this is what is supplied to during cultivations. In cases where it is desired that the cell does not produce 3FL, the preferred initial precursor molecule (alternative initial substrate) supplied to the cultivation is LNT-II.
The precursor molecule is preferably supplied to the genetically engineered cell at the beginning of the cultivation or by continuous feeding or pulse feeding during the cultivation or a combination, allowing the genetically engineered cell to produce LNnT or LNT from the initial precursor. Most often the initial precursor is lactose, and the genetically engineered cell is capable of producing the intermediate precursors (acceptor oligosaccharides, e.g. LNT-II and LNnT or LNT) inside the cell. The initial precursor may however also be LNT-II or LNnT or LNT if the cell is capable of importing at least one of these compounds.
Glycosyltransferases
The genetically engineered cell according to the present disclosure comprises at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase, e.g., a fucosyltransferase, capable of transferring a fucosyl residue from a fucosyl donor to an acceptor oligosaccharide to synthesize one or more fucosylated human milk oligosaccharide product, i.e., a fucosyltransferase.
The genetically engineered cell according to the present disclosure may comprise one or more further recombinant nucleic acids encoding one or more recombinant and/or heterologous glycosyltransferases capable of transferring a glycosyl residue from a glycosyl donor to an acceptor oligosaccharide. Preferably, the additional glycosyltransferase(s) enables the genetically engineered cell to synthesize LNnT or LNT from a precursor molecule, such as lactose or LNT-II. In embodiments, the genetically engineered cell described herein, comprises one or more further recombinant nucleic acid encoding one or more recombinant and/or heterologous glycosyltransferase.
The additional glycosyltransferase is preferably selected from the group consisting of, galactosyltransferases, glucosaminyltransferases, fucosyltransferases N-acetylglucosaminyl transferases and sialyltransferases. a-1 ,3-fucosyltransferase
The term “a-1 ,3-fucosyltransferase” refers to a glycosyltransferase that catalyzes the transfer of fucosyl from a donor substrate, such as GDP-fucose, to an acceptor molecule, preferably the Glc moiety in LNnT or LNT, in an a-1 ,3-linkage (see figure 1). Preferably, an a-1 , 3- fucosyltransferase used in the present disclosure does not originate in the species of the genetically engineered cell, i.e., the gene encoding the a-1 ,3-fucosyltransferase is of heterologous origin and is selected from an a-1 ,3-fucosyltransferase identified in table 1 . In the context described herein, the acceptor molecule for the a-1 ,3-fucosyltransferase is preferably an acceptor oligosaccharide of at least four monosaccharide units with a GIcNAc moiety, e.g., LNnT or LNT. Heterologous a-1 ,3-fucosyltransferases that are capable of transferring a fucosyl moiety onto LNnT or LNT are known in the art, specifically FutA has been shown to produce a mixture of LNFP-VI and LNDFH-III (Dumon et al 2004 Biotechnol. Prog. 20:412-419). In one aspect, the fucosyltransferase in the genetically engineered cell of the present disclosure is an a-1 ,3-fucosyltransferase. Preferably, the a-1 ,3-fucosyltransferase is capable of transferring a fucose unit onto the Glc moiety of an LNnT or LNT molecule. More preferably, the a-1 ,3- fucosyltransferase is specific towards the Glc moiety of an LNnT or LNT molecule.
Where the desired HMO product is LNFP-VI, an a-1 ,3-fucosyltransferase with a higher substrate-specificity for the Glc moiety in LNnT compared to the substrate-specificity for the GIcNAc or Gal moieties in LNnT is advantageous, since such an a-1 ,3-fucosyltransferase would in theory produce less or no by-product oligosaccharides of 5 or 6 monosaccharide units, such as LNFP-III and LNDFH-111. A lower amount of LNFP-III and LNDFH-111 allow for an easier purification of LNFP-VI, as the purification of LNFP-VI from a mixture of HMOs predominantly comprising LNFP-VI would be simpler, as it is easier to separate LNFP-VI from smaller HMOs than separating different fucosylated HMOs of the same or similar size from each other, e.g., LNFP-VI from LNFP-III or LNDFH-111. Hence, a lower initial amount or complete absence of LNFP-III and/or LNDFH-111 is considered beneficial in the purification of LNFP-VI.
In a further embodiment it is desired for the a-1 ,3-fucosyltransferase to have low activity on the glucose moiety of lactose to reduce the 3FL formation during fermentation where lactose is used as the initial substrate.
In preferred embodiments, the use of an a-1 ,3-fucosyltransferase according to the present disclosure results in that at least 14 %, such as at least 25%, such as at least 50% of the molar content of the total HMOs produced by a cell according to the present disclosure is LNFP-VI. In particular, the a-1 ,3-fucosyltransferase according to the present disclosure produce less than 5% of LNFP-III and/or LNDFH-III, preferably essentially no LNFP-III and/or LNDFH-III is produced.
In the present disclosure, the a-1 ,3-fucosyltransferase capable of transferring a fucosyl moiety from a fucosyl donor to the glucose (Glc) moity in lacto-N-neotetraose (LNnT) is an a-1 , 3- fucosyltransferase derived from Bacteroidales bacterium. Examples of such a-1 ,3- fucosyltransferases are of Bacbad or Bacbac2 with an amino acid sequence according to SEQ ID NO: 1 or 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 or 2.
In a preferred embodiment, the a-1 ,3-fucosyltransferase is Bacbac2 with an amino acid sequence according to SEQ ID NO: 2 or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2. Bacbac2 can be used to produce LNFP-VI above 50%, such as above 60% of the molar content of the total HMOs. In particular the a-1 ,3-fucosyltransferase Bacbac2 according to the present disclosure produce less than 2% of LNFP-III and/or LNDFH-III, such as essentially no LNFP-III and LNDFH-111 of the molar content of the total HMOs produced by a cell or method. Furthermore, Bacbac2 has low affinity towards lactose and therefore produce less than 15% 3FL, such as less than 10% 3FL of the molar content of the total HMOs produced by a cell or method. Conversion of the substrate LNnT to LNFP-VI is highly efficient in fermentation in that less than 15% LNnT remains at the end of fermentation. The high LNnT conversion in fermentation results in essentially no pLNnH being produced in fermentation. In addition, all LNT-II is converted into LNnT since the fermentations are essentially free of LNT-II.
In an alternative embodiment, the a-1 ,3-fucosyltransferase Bacbac2 (SEQ ID NO: 2) as disclosed herein or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, can be used to LNFP-V above 50 %, such as at least 55% of the molar content of the total HMOs. In particular the a-1 ,3-fucosyltransferase Bacbac2 according to the present disclosure produce less than 2% of LNFP-II and/or LNDFH-II, such as essentially no LNFP-II and LNDFH-II of the molar content of the total HMOs produced by a cell or method. Furthermore, Bacbac2 has low affinity towards lactose and therefore produce less than 5% 3FL, such as less than 2% 3FL of the molar content of the total HMOs produced by a cell or method. In addition, all LNT-II is converted into LNT.
In a further embodiment, the a-1 ,3-fucosyltransferase is Bacbad with an amino acid sequence according to SEQ ID NO: 1 or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 . Bacbad can be used to produce LNFP-VI above 15%, such as above 30% of the molar content of the total HMOs. In particular the a-1 , 3- fucosyltransferase Bacbad according to the present disclosure produce less than 2% of LNFP- III and/or LNDFH-III, such as essentially no LNFP-III and LNDFH-III of the molar content of the total HMOs produced by a cell or method. Conversion of the substrate LNnT to LNFP-VI is highly efficient in fermentation in that less than 5% LNnT, such as less than 2% LNnT remains at the end of fermentation. In addition, all LNT-II is converted into LNnT since the fermentations are essentially free of LNT-II.
In an alternative embodiment, the a-1 ,3-fucosyltransferase enzyme capable of transferring a fucosyl moiety from a fucosyl donor to an acceptor oligosaccharide can be selected from the group consisting of Bacbad , Bacbac2, Paral and CafF with an amino acid sequence according to SEQ ID NO: 1 , 2, 3, or 43, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , 2, 3, or 43 (table 1). These a-1 ,3- fucosyltransferase enzymes have a particular high specificity for the glucose (Glc) moity in lacto-N-neotetraose (LNnT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or galactose (Gal) moieties in LNnT. These enzymes can e.g., be used to produce LNFP-VI. In particular LNFP-VI with less than 5% of the total molar content of HMO being fucosylated byproducts oligosaccharides with 5 or 6 monosaccharide units.
In a further embodiment, the a-1 ,3-fucosyltransferase is Paral with an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 3. Paral can be used to produce LNFP-VI above 25% of the molar content of the total HMOs, with less than 2% of LNFP-II and/or LNDFH-II, such as essentially no LNFP-111 and LNDFH-111 present in the mixture.
In a further embodiment, the a-1 ,3-fucosyltransferase is CafF with an amino acid sequence according to SEQ ID NO: 43, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 43. CafF can be used to produce LNFP-VI above 14% of the molar content of the total HMOs, with essentially no LNFP-III and LNDFH-III present in the mixture. It is highly likely that the yield of LNFP-VI when using CafF, can be increased by for example using LNT-II as initial substrate since this will prevent the fucosylation of lactose, which CafF is known to do quite efficiently (WO2019/008133).
These enzymes can e.g., be used to produce LNFP-VI with minor or no production of alternative complex fucosylated by-product HMOs, in particular with less than 5%, such as less than 2% of the total molar content of HMO being fucosylated by-products oligosaccharides with 5 or 6 monosaccharide units, such as LNFP-III and/or LNDFH-III .
The a-1 ,3-fucosyltransferase of the present disclosure can be selected from an amino acid sequence with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to the amino acid sequence of any one of the a-1 ,3-fucosyltransferases listed in table 1.
Table 1. List of a-1 ,3-fucosyltransferase enzymes of the present disclosure capable of producing LNFP-VI, with little or no LNFP-III and/or LNDFH-III by-product formation.
Figure imgf000018_0001
1The GenBank IDs reflect the full-length enzymes, in the present disclosure truncated, elongated or mutated versions may have been used, these are represented by the sequences indicated by the SEQ ID NOs.
Example 1 of the present disclosure discloses the identification of the heterologous a-1 , 3- fucosyltransferases Bacbacl , Bacbac2, Paral and CafF (SEQ ID NO: 1 , 2, 3, and 43, respectively), which are capable of producing mixtures of HMOs with LNFP-VI being the predominant complex fucosylated HMO in the mixture (i.e., more than 10 fold, such as 50 fold over LNFP-111 and LNDFH-111) when introduced into an LNnT producing cell, compared to the previously known a-1 ,3-fucosyltransferase FutA, FutB, CafC and FucT109 (SEQ ID NO: 5, 6, 47 and 45 respectively). Specifically, the three novel enzymes Bacbacl , Bacbac2 and Paral which are novel with respect to HMO production and the CafF enzyme known to produce 3FL, can specifically transfer a fucosyl unit onto the Glc moiety of LNnT in an a-1 ,3 linkage to form LNFP- VI at a level above 14%, such as above 25% of the total HMO, while not producing any LNFP-III or LNDFH-III, while the prior art enzymes FutA, FutB, CafC and FucT109 also produces LNDFH-III and/or LNFP-III, respectively, as by-products.
The fact that the experiments performed in Example 1 show that the enzymes Bacbacl , Bacbac2, Paral and CafF do not produce any LNFP-III or LNDFH-III, indicates that these enzymes are highly substrate-specific for the Glc moiety in LNnT.
In embodiments, the expression of an a-1 ,3-fucosyltransferase according to the present disclosure in a genetically engineered cell is further combined with expression of one or more further recombinant nucleic acids encoding one or more heterologous glycosyltransferases. In preferred embodiments, the expression of an a-1 ,3-fucosyltransferase of the disclosure in a genetically engineered cell is combined with expression of a p-1 ,4-galactosyltransferase, such as galT from Helicobacter pylori to enable formation of LNnT from LNT-II as initial substrate. In a further embodiment, a third enzyme is added, such as a p-1 ,3-N-acetyl-glucosaminyl- transferase, e.g., LgtA from Neisseria meningitidis to enable formation of LNnT from lactose as the initial substrate.
In alternative embodiments, the expression of an a-1 ,3-fucosyltransferase, Bacbac2 (SEQ ID NO: 2) or a functional homologue thereof, a genetically engineered cell is combined with expression of a p-1 ,3-galactosyltransferase, such as galTK from Helicobacter pylori to enable formation of LNT from LNT-II as initial substrate. In a further embodiment, a third enzyme is added, such as a p-1 ,3-N-acetyl-glucosaminyl-transferase, e.g., LgtA from Neisseria meningitidis to enable formation of LNT from lactose as the initial substrate. P-1 ,3-N-acetyl-glucosaminyl-transferase
A p-1 ,3-N-acetyl-glucosaminyl-transferase is any protein which comprises the ability of transferring the N-acetyl-glucosamine of UDP-N-acetyl-glucosamine to lactose or another acceptor molecule, in a beta-1 , 3-linkage (see figure 1). Preferably the p-1 ,3-N-acetyl- glucosaminyl-transferase used herein does not originate in the species of the genetically engineered cell, i.e., the gene encoding the p-1 ,3-N-acetyl-glucosaminyl-transferase is of heterologous origin.
Accordingly, in embodiments, the genetically engineered cell further comprises one or more recombinant nucleic acid sequence(s) encoding a p-1 ,3-N-acetyl-glucosaminyltransferase.
Non-limiting examples of p-1 ,3-N-acetyl-glucosaminyltransferases are given in table 2. p-1 ,3-N- acetyl-glucosaminyltransferase variants may also be useful, preferably such variants are at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identical, such as at least 96%, such as at least 97%, such as at least 98% or such as 99% identical to the amino acid sequence of any one of the p-1 ,3-N-acetyl-glucosaminyltransferase in table 2.
Table 2. List of p-1 ,3-N-acetyl-glucosaminyltransferase
Figure imgf000020_0001
n embodiments, the genetically engineered cell comprises a recombinant nucleic acid sequence encoding a p-1 ,3-N-acetyl-glucosaminyltransferase. In one embodiment, the recombinant nucleic acid sequence encoding a p-1 ,3-N-acetylglucosaminyltransferase comprises or consists of the amino acid sequence of SEQ ID NO: 14 (LgtA from N. meningitidis) or a functional homologue thereof with an amino acid sequence with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to SEQ ID NO: 14.
For the production of LNnT from lactose as substrate, the LNT-II precursor is formed using a p- 1 ,3-N-acetylglucosaminyltransferase. In one embodiment the genetically engineered cell comprises a p-1 ,3-N-acetylglucosaminyltransferase gene, or a functional homologue or fragment thereof, to produce the intermediate LNT-II from lactose as the initial substrate.
Some of the examples below use the heterologous p-1 ,3-N-acetyl-glucosaminyl-transferase named LgtA from Neisseria meningitidis or a variant thereof.
P-1 ,3-galactosyltransferase
A p-1 ,3-Galactosyltransferase is any protein that comprises the ability of transferring the galactose of UDP-Galactose to a N-acetyl-glucosaminyl moiety to an acceptor molecule in a beta-1 , 3-linkage. Preferably, a p-1 ,3-galactosyltransferase used herein does not originate in the species of the genetically engineered cell i.e., the gene encoding the p-1 ,3- galactosyltransferase is of heterologous origin.
Non-limiting examples of p-1 ,3-galactosyltransferases are given in table 12. p-1 ,3- galactosyltransferases variants may also be useful, preferably such variants are at least 80%, such as at least 85%, such as at least 90, such as at least 95% identical, such as at least 96%, such as at least 97%, such as at least 98% or such as 99% identical to one of the p-1 ,3- galactosyltransferases in table 12.
Table 12. List of p-1 ,3-glycosyltransferases
Figure imgf000021_0001
In the context of the present disclosure the acceptor molecule, is an acceptor saccharide, e.g., LNT-II, or more complex HMO structures.
The examples below use the heterologous p-1 ,3-galactosyltransferase named GalTK or a variant thereof, to produce e.g., LNFP-V.
In embodiments the cell of the present disclosure further comprises a recombinant nucleic acid sequence encoding a p-1 ,3-N-acetyl-glucosaminyltransferase. In one embodiment, the recombinant nucleic acid sequence encoding a p-1 ,3-galactosyltransferases comprises or consists of the amino acid sequence of SEQ ID NO: 42 (galTK from H. pylori) or a functional homologue thereof with an amino acid sequence with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% identity to SEQ ID NO: 42. To produce LNT form an LNT-II precursor, a p-1 ,3-galactosyltransferase is needed. In one embodiment, the genetically modified cell comprises a p-1 ,3-galactosyltransferase gene, or a functional homologue or fragment thereof.
Below are examples of genetically modified strains according to the present disclosure with specific combinations of glycosyl transferases that will lead to production of LNFP-V using lactose as initial substrate.
In one example, LgtA from Neisseria meningitidis is used in combination with galTK from Helicobacter pylori and Bacbac2 from Bacteroidales bacterium to produce LNFP-V starting from lactose as initial substrate.
In yet another example, galTK from Helicobacter pylori is used in combination with Bacbac2 from Bacteroidales bacterium to produce LNFP-V starting from LNT-II as initial substrate.
P-1 ,4-galactosyltransferase
A p-1 ,4-galactosyltransferase is any protein that comprises the ability of transferring the galactose of UDP-Galactose to a N-acetyl-glucosaminyl moiety to an acceptor molecule in a p- 1 ,4-linkage (see figure 1). Preferably, a p-1 ,4-galactosyltransferase used herein does not originate in the species of the genetically engineered cell i.e., the gene encoding the p-1 ,4- galactosyltransferase is of heterologous origin. In the context described herein the acceptor molecule, is an acceptor saccharide, e.g., LNT-II, or more complex HMO structures.
The examples below use the heterologous p-1 ,4-galactosyltransferase GalT, or a variant thereof, to produce LNnT e.g., and in in combination with a-1 ,3-fucosyltransferase described herein it can produce LNFP-VI. Accordingly, in embodiments, the genetically engineered cell comprises one or more recombinant nucleic acid sequence(s) encoding a p-1 ,4- galactosyltransferase.
Non-limiting examples of p-1 ,4-galactosyltransferases are provided in table 2. p-1 ,4- galactosyltransferases variants may also be useful, preferably such variants are at least 80%, such as at least 85%, such as at least 90, such as at least 95% identical, such as at least 96%, such as at least 97%, such as at least 98% or such as 99% identical to the amino acid sequence of any one of the p-1 ,4-galactosyltransferases in table 3.
Table 3. List of p-1 ,4-glycosyltransferases
Figure imgf000022_0001
In embodiments described herein the p-1 ,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis, and the p-1 ,3-galactosyltransferase from Helicobacter pylori, respectively.
In one embodiment, the recombinant nucleic acid sequence encoding a p-1 ,4- galactosyltransferases comprises or consists of the amino acid sequence of SEQ ID NO: 15 (galT from H. pylori) or a functional homologue thereof with an amino acid sequence with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to SEQ ID NO: 15.
To produce LNnT form an LNT-II precursor, a p-1 ,4-galactosyltransferase is needed. In one embodiment, the genetically engineered cell comprises a p-1 ,4-galactosyltransferase gene, or a functional homologue or fragment thereof. In embodiments, the p-1 ,3-N- acetylglucosaminyltransferase is from Neisseria meningitidis and the p-1 ,4- galactosyltransferase is from Helicobacter pylori. In further embodiments, the pi ,3-N- acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO: 14, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 14 and the p-1 ,4-galactosyltransferase has an amino acid sequence according to SEQ ID NO: 15, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 15.
Glycosyl-donor - nucleotide-activated sugar pathways
When carrying out the method of this disclosure, preferably a glycosyltransferase mediated glycosylation reaction takes place in which an activated sugar nucleotide serves as glycosyl- donor. An activated sugar nucleotide generally has a phosphorylated glycosyl residue attached to a nucleoside. A specific glycosyl transferase enzyme accepts only a specific sugar nucleotide. Thus, preferably the following activated sugar nucleotides are involved in the glycosyl transfer: glucose-UDP-GIcNAc, UDP-galactose, UDP-glucose, UDP-N- acetylglucosamine, UDP-N-acetylgalactosamine (GIcNAc) and CMP-N-acetylneuraminic acid.
The genetically engineered cell according to the present disclosure can comprise one or more pathways to produce a nucleotide-activated sugar selected from the group consisting of glucose-UDP-GIcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine (GIcNAc), UDP-N-acetylgalactosamine and CMP-N-acetylneuraminic acid.
In one embodiment of the current disclosure, the genetically engineered cell is capable of producing one or more activated sugar nucleotides mentioned above by a de novo pathway. In this regard, an activated sugar nucleotide is made by the cell under the action of enzymes involved in the de novo biosynthetic pathway of that respective sugar nucleotide in a stepwise reaction sequence starting from a simple carbon source like glycerol, sucrose, fructose or glucose (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)).
The enzymes involved in the de novo biosynthetic pathway of an activated sugar nucleotide can be naturally present in the cell or introduced into the cell by means of gene technology or recombinant DNA techniques, all of them are parts of the general knowledge of the skilled person.
In another embodiment, the genetically engineered cell can utilize salvaged monosaccharides for sugar nucleotide. In the salvage pathway, monosaccharides derived from degraded oligosaccharides are phosphorylated by kinases, and converted to nucleotide sugars by pyrophosphorylases. The enzymes involved in the procedure can be heterologous ones, or native ones of the host cell.
Colanic acid gene cluster
For the production of fucosylated HMOs, the de novo GDP-fucose pathway is important to ensure presence of sufficient GDP-fucose. The colanic acid gene cluster of Escherichia coll encodes selected enzymes involved in the de novo synthesis of GDP-fucose (gmd, wcaG, wcaH, weal, manB, manC), whereas one or several of the genes downstream of GDP-L-fucose such as wcaJ, which are responsible for the production of the extracellular polysaccharide colanic acid, a major oligosaccharide of the bacterial cell wall, can be deleted to prevent conversion of GDP-fucose to colanic acid.
To secure sufficient amounts of GDP-fucose the promoter of the native colanic acid gene cluster may be exchanged with a stronger promoter, generating a recombinant colanic acid gene cluster, to drive additional production of GDP-fucose. Furthermore, an extra copy of the colanic acid gene cluster or selected genes thereof can be introduced in the genetically engineered cells as described in the examples.
In embodiments, the colanic acid gene cluster may be expressed from its native genomic locus. The expression may be actively modulated. The expression can be modulated by swapping the native promoter with a promoter of interest, and/or increasing the copy number of the colanic acid genes coding said protein(s) by expressing the gene cluster from another genomic locus than the native, or episomally expressing the colanic acid gene cluster or specific genes thereof. In relation to the present disclosure, the term “native genomic locus”, in relation to the colanic acid gene cluster, relates to the original and natural position of the gene cluster in the genome of the genetically engineered cell.
The de novo GDP-fucose pathway genes responsible for the formation of GDP-fucose comprises or consists of the following genes: i) manA which encodes the protein mannose-6 phosphate isomerase (EC 5.3.1 .8, UniProt accession nr. P00946), which facilitates the interconversion of fructose 6-phosphate (F6P) and mannose-6-phosphate; ii) manB which encodes the protein phosphomannomutase (EC 5.4.2.8, UniProt accession nr P24175), which is involved in the biosynthesis of GDP-mannose by catalyzing conversion mannose-6-phosphate into mannose-1-phosphate;
Hi) manC which encodes the protein mannose-1 -phosphate guanylyltransferase guanylyltransferase (EC:2.7.7.13, UniProt accession nr P24174), which is involved in the biosynthesis of GDP-mannose through synthesis of GDP-mannose from GTP and a-D- mannose-1 -phosphate; iv) gmd which encodes the protein GDP-mannose-4,6-dehydratase (UniProt accession nr P0AC88), which catalyzes the conversion of GDP-mannose to GDP-4-dehydro-6-deoxy- D-mannose; v) wcaG (fcl) which encodes the protein GDP-L-fucose synthase (EC 1 .1 .1 .271 , UniProt accession nr P32055) which catalyses the two-step NADP-dependent conversion of GDP-4-dehydro-6-deoxy-D-mannose to GDP-fucose.
Accordingly, it is preferred that the genetically engineered cell, when producing one or more fucosylated heterologous products, overexpresses either the entire colonic acid gene cluster (e.g. as identified in SEQ ID NO: 41 or a functional variant thereof) and/or one or more genes of the de novo GDP-fucose pathway selected from the group consisting of manA, manB, manC, gmd and wcaG.
Lactose permease
Lactose permease is a membrane protein which is a member of the major facilitator superfamily and can be classified as a symporter, which uses the proton gradient towards the cell to transport p-galactosides such as lactose in the same direction into the cell. In oligosaccharide- production, especially in the production of human milk oligosaccharides (HMOs), lactose is often the initial substrate being decorated to produce any HMO of interest in a bioconversion that happens in the cell interior. Thus, in the production of HMOs, there is a desire to be able to import lactose into the cell, e.g., by expression and/or overexpression of a lactose permease such as lacY of E. coli.
In embodiments, the lactose permease is as shown in SEQ ID NO: 16, or a functional homologue thereof having an amino acid sequence which is at least 80 % identical, such as at least 85 %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 16.
In embodiments, the expression of the lactose permease is regulated by a promoter according to the present disclosure.
P-galactosidase
A host cell suitable for HMO production, e.g., E. coli, may comprise an endogenous |3- galactosidase gene or an exogenous p-galactosidase gene, e.g., E. coli comprises an endogenous lacZ gene (e.g., GenBank Accession Number V00296 (GI:41901)). For the purposes of the disclosure, when producing an HMO, it is preferred that the genetically engineered cell does not express a functional p-galactosidase to avoid the degradation of lactose if lactose is used as the initial substrate for producing the complex fucosylated HMO. In embodiments the lacZ gene may be inactivated by a complete or partial deletion of the corresponding nucleic acid sequence from the bacterial genome, or the gene sequence is mutated in the way that it is not transcribed, or, if transcribed, the transcript is not translated or if translated to a protein (i.e., p-galactosidase), the protein does not have the corresponding enzymatic activity. In this way the HMO-producing bacterium accumulates an increased intracellular lactose pool which is beneficial for the production of HMOs.
Importer proteins
Most commonly HMO producing cells are genetically engineered to use lactose as the initial substrate since this is easily taken up by lactose permease as described above. However, it may be desired to use an initial substrate that will require the presence of fewer glycosyltransferases in the cell, since this will reduce the strain on the cell in terms of producing multiple enzymes and in addition it can reduce the by-product profile, e.g. if lactose is not used as initial substrate a cell comprising a fucosyltransferase will not produce 3FL as by-product allowing the fucose to be used to produce e.g. more LNFP-VI.
Accordingly, in embodiments, the cell may further comprise a substrate importer selected from a lactose importer, a lacto-N-triose-ll (LNT-II) importer and a LNnT importer. WQ2022/242860 suggests how it may be possible to identify LNT-II importers. W02023/099680 also suggests a number of potential LNT and LNT-II importers.
Examples of suitable LNT-II importers are e.g.,
Lactose permease (LacY) mutants, such as LacY mutant Y236H or LacY mutant A177V+S306T, wherein the mutations are equivalent with the corresponding position in the sequence of SEQ ID NO: 16,
- ABC transporter protein complexes, such as ABC transporter from B. pseudocatenulatum JCM 1200 BBPC_1775, 1776, 1777, (NCBI accession Nrs BAR04453.1 , BAR04454.1 and BAR04455.1 , respectively) or ABC transporter from B. breve UCC2003 BBR_0527/lntP1 , BBR_0528/lntP2, BBR_0530/lntS and BBR_0531 (NCBI accession Nrs ABE95224.1 , ABE95225.1 , ABE95226.1 and ABE95228.1), and/or
MFS transporters, such as but not limited to Blon_0962 (NCBI accession Nr ACJ52061.1).
In additional embodiments a nucleic acid or a cluster of nucleic acids encoding one of these transporters may be introduced into a genetically modified cell as described herein. The expression of such transporters enables the production of complex fucosylated oligosaccharide with LNT-II as the initial substrate.
Exporter proteins
The oligosaccharide product, such as the HMO produced by the cell, can be accumulated both in the intra- and the extracellular matrix. The product can be transported to the supernatant in a passive way, i.e., it diffuses outside across the cell membrane. The more complex HMO products may remain in the cell, which is likely to eventually impair cellular growth, thereby affecting the possible total yield of the product from a single fermentation. The HMO transport can be facilitated by major facilitator superfamily transporter proteins that promote the effluence of sugar derivatives from the cell to the supernatant. The exporter can be present exogenously or endogenously and is overexpressed under the conditions of the fermentation to enhance the export of the oligosaccharide derivative (HMO) produced. The specificity towards the oligosaccharide product to be secreted can be altered by mutation by means of known recombinant DNA techniques.
Thus, the genetically engineered cell according to the present disclosure can further comprise a nucleic acid sequence encoding an exporter protein capable of exporting the fucosylated human milk oligosaccharide product or products, such as transporter protein can for example be a member of the major facilitator superfamily transport proteins.
In the resent years, several new and efficient major facilitator superfamily transporter proteins have been identified as exporters of HMOs, each having specificity for different recombinantly produced HMOs and development of recombinant cells expressing said proteins are advantageous for high scale industrial HMO manufacturing (See for example WO2010/142305, WO2017/042382, WO2021/148615, WO2021/148614, WO2021 /148611 , and WO2021/148620).
Thus, in one or more exemplary embodiments, the genetically engineered cell according to the method described herein further comprises a gene product that acts as an LNFP-VI and/ or LNFP-V transporter. The gene product that acts as LNFP-VI or LNFP-V transporter may be encoded by a recombinant nucleic acid sequence that is expressed in the genetically engineered cell. The recombinant nucleic acid sequence encoding the LNFP-VI or LNFP-V transporter, may be integrated into the genome of the genetically engineered cell, or expressed using a plasmid.
The genetically engineered cell
In the present context, the terms “a genetically engineered cell” and "a genetically modified cell” are used interchangeably. As used herein “a genetically engineered cell” is a host cell whose genetic material has been altered by human intervention using a genetic engineering technique, such a technique is e.g., but not limited to transformation or transfection e.g., with a heterologous and/or recombinant polynucleotide sequence, Crisper/Cas editing and/or random mutagenesis. In one embodiment the genetically engineered cell has been transformed or transfected with a recombinant nucleic acid sequence.
The genetic modifications can e.g., be selected from inclusion of glycosyltransferases, and/or metabolic pathway engineering deletion of repressors or undesired enzymes and inclusion of transporters as described in the above sections, which the skilled person will know how to combine into a genetically engineered cell capable of producing one or more fucosylated HMO’s.
In one aspect of the disclosure, the genetically engineered cell comprises a recombinant nucleic acid sequence encoding a fucosyltransferase with a-1 ,3-fucosyltransferase activity as disclosed in the section “a-1 ,3-fucosyltransferase” above. Such a genetically modified cell is capable of producing at least 14%, such as at least 25% LNFP-VI of the total molar HMO content produced by the cell. In embodiments, the total HMOs produced by said cell is essentially free of LNFP-111 and/or LNDFH-111 or contain less than 5%, such as less than 4%, 3%, or such as 2%, of each of LNFP-III and LNDFH-111. In embodiments, the total HMOs produced by said cell is essentially free of LNFP-III and/or LNDFH-111. In the present disclosure, essentially free of LNFP-III and/or LNDFH-111 , is to be understood as a content of LNFP-III and/or LNDFH-111 of the total HMO produced by the cell that is less than 1%, such as less than 0.5%, such as less than 0.2 %, such as less than 0.1% of the total molar HMO content produced by the cell. In additional embodiments, the cell of the present disclosure produces a mixture of HMOs comprising LNFP- VI, LNnT, 3FL and/or pLNnH.
Preferably, the fucosyltransferases have a-1 ,3-fucosyltransferase activity, allowing fucosylation of an oligosaccharide at position 3 of the Glc moiety in LNnT or LNT, while showing limited or no fucosylation at position 2 or 3 of the Gal or GIcNAc moieties (See figures 1 and 3). Preferably, only the reducing end Glc moiety is fucosylated, and more preferably only the reducing end Glc moiety of oligosaccharide is LNnT or LNT is fucosylated.
In embodiments, the genetically engineered cell of the present disclosure is capable of producing LNFP-VI or LNFP-V, wherein said cell comprises a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-neotetraose (LNnT) and/or in lacto-N-tetraose (LNT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNnT or LNT, and wherein the cell produces a) less 5 %, such as less than 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2% or such as less than 0.1% of the total molar content of HMO produced by said cell is LNDFH-III and/or LNFP- III, or b) less 2 %, such as less than 1 %, 0.5%, 0.3%, 0.2% or such as less than 0.1% of the total molar content of HMO produced by said cell is LNDFH-II and/or LNFP-II
In further embodiments the genetically engineered cell of the present disclosure produces a) more than 14 %, such as more than 20%, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85% or such as more than 90% of the total molar content of HMO produced by said cell LNFP-VI or b) more than 50 %, such as more than 55%, 60%, 65%, 70%, 75%, 80%, 85% or such as more than 90% of the total molar content of HMO produced by said cell LNFP-V.
In preferred embodiments, the genetically engineered cell of the present disclosure is capable of producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI), wherein said cell comprises a recombinant nucleic acid sequence encoding an a-1 ,3- fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-neotetraose (LNnT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNnT, and wherein the cell produces, a) less than 5 %, such as less than 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2% or such as less than 0.1% of the total molar content of HMO produced by said cell is LNDFH-III and/or LNFP-III, and b) more than 14 %, such as more than 20%, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85% or such as more than 90% of the total molar content of HMO produced by said cell LNFP-VI.
In further embodiments, the genetically engineered cell of the present disclosure further produces one or more HMOs selected from the group consisting of 3FL, LNT-II, LNnT and pLNnH.
In preferred embodiments, essentially no LNFP-III and/or LNDFH-III is produced by said cell.
In preferred embodiments the recombinant nucleic acid encodes an a-1 ,3-fucosyltransferase derived from Bacteroidales bacterium.
In alternative embodiments, the recombinant nucleic acid encodes an a-1 ,3-fucosyltransferase is selected from the group consisting of, a) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO:
1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , b) Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO:
2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, c) Paral comprising or consisting of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 3 and d) CafF comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 43.
In presently preferred embodiments, the genetically engineered cell capable of producing LNFP-VI, comprises a recombinant nucleic acid sequence encoding a fucosyltransferase with a- 1 ,3-fucosyltransferase activity, wherein said fucosyltransferase is Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional homologue thereof with an amino acid sequence that is at least 80 %, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to SEQ ID NO: 1. In embodiments said genetically engineered cells produce i. LNFP-VI in a molar content of at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50% or such as at least 55% of the total HMO produced by said cell, and/or ii. LNFP-VI and 3FL in a molar content of at least 90%, such as at least 95%, or such as at least 99%, or such as 100% of the total HMO produced by said cell, and/or
Hi. 25-70 molar% of LNFP-VI, 30-70 molar% 3FL, 0-5 molar% LNnT, in total adding up to 100% molar content, and wherein the molar content of LNnT, LNFP-111 and LNDFH-111 is less than 5 %, such as less than 4%, 3%, 2%, 1%, 0.5%, 0.3% or such as less than 0.1% of the total molar content of HMO produced by said cell.
In presently preferred embodiments, the genetically engineered cell capable of producing LNFP-VI or LNFP-V, comprises a recombinant nucleic acid sequence encoding a fucosyltransferase with a-1 ,3-fucosyltransferase activity, wherein said fucosyltransferase is Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 %, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to SEQ ID NO: 2. In embodiments said genetically engineered cells produce i. LNFP-VI in a molar content of at least at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, or such as at least 90% of the total HMO produced by said cell, and/or ii. LNFP-VI and LNnT produced by said cell is at least 85%, such as at least 90%, or such as at least 95% or such as at least 99%, or such as 100% of the total HMO produced by said cell, and/or
Hi. 55-90 molar% of LNFP-VI, 0-15 molar% 3FL, 0-35 molar% LNnT, 0-10% pLNnH in total adding up to 100% molar content, and wherein the molar content of LNFP-111 and LNDFH-111 is less than 5 %, such as less than 4%, 3%, 2%, 1%, 0.5%, 0.3% or such as less than 0.1% of the total molar content of HMO produced by said cell or iv. LNFP-V and LNT produced by said cell is at least at least 90%, or such as at least 95%or such as at least 99%, or such as 100% of the total HMO produced by said cell, and/or v. 50-70 molar% of LNFP-V, 0-5 molar% 3FL, 30-50 molar% LNT, in total adding up to 100% molar content, wherein the molar content of LNFP-II and LNDFH-II is less than 2 %, such as less than 1 .5%, 1%, 0.5%, 0.3%, 0.2% or such as less than 0.1% of the total molar content of HMO produced by said cell
In presently preferred embodiments, the genetically engineered cell capable of producing LNFP-VI, comprises a recombinant nucleic acid sequence encoding a fucosyltransferase with a- 1 ,3-fucosyltransferase activity, wherein said fucosyltransferase is Paral comprising or consisting of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to SEQ ID NO: 3. In embodiments said genetically engineered cells produce i. the molar content of LNFP-VI produced by said cell is at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 55%, such as at least 60%, or such as at least 65% of the total HMO produced by said cell, and/or ii. the molar content of LNFP-VI and LNnT produced by said cell is at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, or such as at least 90% of the total HMO produced by said cell, and/or
Hi. 25-70 molar% of LNFP-VI, 0-25 molar% 3FL, 15-65 molar% LNnT, 0-15% pLNnH in total adding up to 100% molar content, and wherein the molar content of LNFP-111 and LNDFH-III is less than 5 %, such as less than 4%, 3%, 2%, 1%, 0.5%, 0.3% or such as less than 0.1% of the total molar content of HMO produced by said cell.
In presently preferred embodiments, the genetically engineered cell capable of producing LNFP-VI, comprises a recombinant nucleic acid sequence encoding a fucosyltransferase with a- 1 ,3-fucosyltransferase activity, wherein said fucosyltransferase is CafF comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a functional homologue thereof with an amino acid sequence that is at least 80 %, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to SEQ ID NO: 43. Preferably, this genetically engineered cell also comprises a recombinant nucleic acid sequence encoding a p-1 ,4-galactosyltransferase and a recombinant nucleic acid sequence encoding an LNT-II importer. In embodiments said genetically engineered cells produce a) the molar content of LNFP-VI produced by said cell is at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, or such as at least 45% of the total HMO produced by said cell, and/or b) the molar content of LNFP-VI, 3FL and LNnT produced by said cell is at least at least 80%, such as at least 85%, or such as at least 90% of the total HMO produced by said cell, and/or c) 10-30 molar% of LNFP-VI, 35-60 molar% 3FL, 25-40 molar% LNnT, 0-10% pLNnH in total adding up to 100% molar content, and wherein the molar content of LNFP-111 and LNDFH-III is less than 5 %, such as less than 4%, 3%, 2%, 1%, 0.5%, 0.3% or such as less than 0.1% of the total molar content of HMO produced by said cell.
In a further aspect the present disclosure also relates to a genetically engineered cell capable of producing the Human Milk Oligosaccharide (HMO) lacto-N-fucopentaose V (LNFP-V), comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-tetraose (LNT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal), wherein the cell produces less than 2 %, such as less than 1 %, 0.5%, 0.3%, 0.2% or such as less than 0.1% of the total molar content of HMO produced by said cell is LNDFH-II and/or LNFP-II.
Preferably, said cell also produces more than 50 % of the total molar content of HMO of LNFP- V.
In embodiments, the cell capable of producing LNFP-V, comprises one or two copies or multiple copies, preferably genomically integrated, of a nucleic acid sequence encoding Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 %, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% identical to SEQ ID NO: 2.
The genetically engineered cell described herein preferably expresses genes encoding key enzymes for the biosynthesis of fucosylated HMOs. In addition, it is advantageous if the genetically engineered cell expresses the genes needed to produce LNnT or LNT, either from lactose or LNT-II as the initial substrate (see figure 1 and figure 3), and/or alternatively the cell expresses importers for LNT-II or LNnT or LNT.
In embodiments the genetically engineered cell comprises one or more additional glycosyltransferases. The additional one or more glycosyltransferases are preferably selected from the group consisting of, galactosyltransferases, glucosaminyltransferases, fucosyltransferases and N-acetylglucosaminyl transferases.
In some embodiments the genetically engineered cell comprises one or more recombinant nucleic acid sequence(s) encoding a p-1 ,4-galactosyltransferase, and optionally a p-1 ,3-N- acetylglucosaminyltransferase. In some embodiments the p-1 ,3-N-acetyl- glucosaminyltransferase is from Neisseria meningitidis, and the p-1 ,4-galactosyltransferase is from Helicobacter pylori.
In some embodiments the genetically engineered cell comprises one or more recombinant nucleic acid sequence(s) encoding a p-1 ,3-galactosyltransferase, and optionally a p-1 ,3-N- acetylglucosaminyltransferase. In some embodiments the p-1 ,3-N-acetyl- glucosaminyltransferase is from Neisseria meningitidis, and the p-1 ,3-galactosyltransferase is from Helicobacter pylori.
In embodiments the genetically engineered cell disclosed herein comprises one or more pathways, needed to produce a nucleotide-activated sugar selected from the group consisting of UDP-GIcNAc, GDP-fucose, UDP-galactose and UDP-glucose. Preferably the cell comprises all the pathways needed to produce UDP-GIcNAc, GDP-fucose, UDP-galactose and UDP- glucose. Alternatively, one or more activated nucleotide(s) can be supplied to the cultivation medium.
In some embodiments, a genetically engineered cell described herein expresses the de novo GDP-fucose pathway genes responsible for the formation of GDP-fucose manA, manB, manC, gmd and wcaG. It may be advantageous to overexpress one or more of these genes and/or to upregulate the colanic acid gene cluster (CA), including the genes gmd, wcaG, wcaH, weal, manC and manB from E. Coll, through introduction of a nucleic acid construct encoding the CA as shown in SEQ ID NO: 41 or equivalents thereof, allowing for formation of GDP-fucose, which enables the cell to produce a higher level of fucosylated oligosaccharides from one or more intermediate oligosaccharide substrates, such as lactose or LNnT, and/or LNT. Depending on the intended use of substrate, one or more additional glycosyltransferases and pathways for producing nucleotide-activated sugars, such as glucose-UDP-GIcNAc, CMP-N-acetylneuraminic acid, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and/or CMP-N-acetylneuraminic acid can also be present in the genetically engineered cell.
It is further understood that the genetically engineered cell described herein may further comprise any of the modifications described above, e.g., additional glycosyltransferases, suitable importer proteins such as overexpression of lactose permease, LNT-II or LNT importers, p-galactosidase inactivation in particular if lactose is used as the initial substrate, as well suitable exporter proteins for the complex fucosylated HMOs produced by the cell.
Host cells
In embodiments, the engineered cell is a microorganism. The genetically engineered cell is preferably a microbial cell, such as a prokaryotic cell or eukaryotic cell. Appropriate microbial cells that may function as a host cell include bacterial cells, archaebacterial cells, algae cells and fungal cells.
The genetically engineered cell may be e.g., a bacterial or yeast cell. In one preferred embodiment, the genetically engineered cell is a bacterial cell.
Regarding the bacterial host cells, there are, in principle, no limitations; they may be eubacteria (gram-positive or gram-negative) or archaebacteria, as long as they allow genetic manipulation for insertion of a gene of interest and can be cultivated on a manufacturing scale. Preferably, the host cell has the property to allow cultivation to high cell densities. Non-limiting examples of bacterial host cells that are suitable for recombinant industrial production of an HMO(s) according to the disclosure could be member of the Enterobacterales order, preferably of the genus Escherichia, more preferably of the species E. coll. Other examples of suitable host cell are Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Campylobacter sp, Corynebacterium sp., Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillus may also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans. Similarly, bacteria of the genera Lactobacillus and Lactococcus may be engineered using the methods of this disclosure, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus easel, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species. Also included as useful species are strains, engineered as described here, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium long urn, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa).
Non-limiting examples of fungal host cells that are suitable for recombinant industrial production of a heterologous product are e.g., yeast cells, such as Komagataella, Kluyveromyces, Yarrowia, Pichia, Saccaromyces, Schizosaccharomyces or Hansenula or from a filamentous fungus of the genera Aspargillus, Fusarium or Thricoderma.
In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of Escherichia sp., Bacillus sp., lactobacillus sp., Corynebacterium sp. and Campylobacter sp.
In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris and Saccharomyces cerevisiae.
In one or more exemplary embodiments, the genetically engineered cell is B. subtilis.
In one or more exemplary embodiments, the genetically engineered cell is S. Cerevisiae or P pastoris.
In one or more exemplary embodiments, the genetically engineered cell is Escherichia coli.
In one or more exemplary embodiments, the present disclosure relates to a genetically engineered cell, wherein the cell is derived from the E. coli K-12 strain or E. coli DE3 strain.
A recombinant nucleic acid sequence
The present disclosure relates to a genetically engineered cell comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with Glc specific a-1 ,3- fucosyltransferase activity, such as an enzyme selected from the group consisting of Bacbad , Bacbac2 and Paral , wherein said cell produces Human Milk Oligosaccharides (HMO). In particular, at least one fucosylated HMO, and preferably with a molar % content of LNFP-VI above 25 %, such as above 50% of the total HMO produced.
In the present context, the term “recombinant nucleic acid sequence”, “recombinant gene/nucleic acid/nucleotide sequence/DNA encoding” or "coding nucleic acid sequence" is used interchangeably and intended to mean an artificial nucleic acid sequence (i.e. produced in vitro using standard laboratory methods for making nucleic acid sequences) that comprises a set of consecutive, non-overlapping triplets (codons) which is transcribed into mRNA and translated into a protein when under the control of the appropriate control sequences, i.e., a promoter sequence.
The boundaries of the coding sequence are generally determined by a ribosome binding site located just upstream of the open reading frame at the 5’end of the mRNA, a transcriptional start codon (AUG, GUG or UUG), and a translational stop codon (UAA, UGA or UAG). A coding sequence can include, but is not limited to, genomic DNA, cDNA, synthetic, and recombinant nucleic acid sequences.
The term "nucleic acid" includes RNA, DNA and cDNA molecules. It is understood that, as a result of the degeneracy of the genetic code, a multitude of nucleic acid sequences encoding a given protein may be produced.
The recombinant nucleic acid sequence may be a coding DNA sequence e.g., a gene, or noncoding DNA sequence e.g., a regulatory DNA, such as a promoter sequence or other noncoding regulatory sequences.
The recombinant nucleic acid sequence may in addition be heterologous. As used herein "heterologous" refers to a polypeptide, amino acid sequence, nucleic acid sequence or nucleotide sequence that is foreign to a cell or organism, i.e., to a polypeptide, amino acid sequence, nucleic acid molecule or nucleotide sequence that does not naturally occurs in said cell or organism.
The disclosure also relates to a nucleic acid construct comprising a coding nucleic sequence, i.e. recombinant DNA sequence of a gene of interest, e.g., an a-1 ,3-fucosyltransferase gene as described herein, and a non-coding regulatory DNA sequence, e.g., a promoter DNA sequence, e.g., a recombinant promoter sequence derived from the promoter sequence of the lac operon or the glp operon, or a promoter sequence derived from another genomic promoter DNA sequence, or a synthetic promoter sequence, wherein the coding and promoter sequences are operably linked.
The term “operably linked” refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. It refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. E.g., a promoter sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
In one exemplified embodiment, the nucleic acid construct of the present disclosure may be a part of the vector DNA, in another embodiment, the construct it is an expression cassette/cartridge that is integrated in the genome of a host cell.
Accordingly, the term “nucleic acid construct” means an artificially constructed segment of nucleic acids, in particular a DNA segment, which is intended to be inserted into a target cell, e.g., a bacterial cell, to modify expression of a gene of the genome or expression of a gene/coding DNA sequence which may be included in the construct. Thus, in embodiments, the present disclosure relates to a nucleic acid construct comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase, wherein said recombinant nucleic acid sequence is selected from the group consisting of nucleic acid sequences encoding Bacbad , Bacbac2, Paral , or CafF, such as a nucleic acid sequence according to SEQ ID NO: 7, 8, 9, or 44, or functional variants thereof.
The genetically engineered cell according to the present disclosure may also comprise multiple copies of the recombinant nucleic acid sequence encoding a a-1 ,3-fucosyltransferase. Enhancing the copy number of the fucosyltransferase was shown in Example 1 to change the ratio of the produced HMOs. In specific it was shown that increasing the copy number of Paral by introduction of a high copy-number plasmid (pBB-B9-Para1-PglpF) resulted in an increased LNFP-VI production, while reducing the strains production of LNnT and pLNnH. Furthermore, increasing the copy number of Bacbac2 to two genomic copies in Example 2, slightly increased the relative amount of LNFP-VI produced, combined with a decrease in the amount of LNnT produced.
Accordingly, the copy number variation of the glycosyltransferase(s) may be used in the production to optimize the HMO production, in this case optimizing the production of LNFP-VI.
Accordingly, in embodiments, the genetically engineered cell of the present disclosure comprises one, two, three or more genomic copies of the recombinant nucleic acid sequence encoding the glycosyltransferase selected from the group consisting of a) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , b) Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, c) Paral comprising or consisting of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 3 and d) CafF comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a functional homologue thereof with an amino acid sequence that is at least 80% identical to SEQ ID NO: 43.
In further embodiments, the recombinant nucleic acid sequence encoding the glycosyltransferase selected from the group consisting of a) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , b) Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, c) Paral comprising or consisting of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 3 and d) CafF comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a functional homologue thereof with an amino acid sequence that is at least 80% identical to SEQ ID NO: 43, is encoded on a plasmid.
In additional embodiments, the plasmid is a high copy number plasmid, preferably, a pUC57 or pBB-B9 plasmid.
One embodiment of the disclosure relates to a nucleic acid construct comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase, wherein said recombinant nucleic acid sequence is selected from the group consisting of a) a nucleic acid encoding Bacbad , comprising or consisting of a nucleic acid sequence according to SEQ ID NO: 7, or a functional homologue thereof with a nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 7, b) a nucleic acid encoding Bacbac2, comprising or consisting of a nucleic acid sequence according to SEQ ID NO: 7, or a functional homologue thereof with a nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 8 and c) a nucleic acid encoding Paral , comprising or consisting of a nucleic acid sequence according to SEQ ID NO: 9, or a functional homologue thereof with a nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 9.
Preferably, the a-1 ,3-fucosyltransferase encoding sequence is under the control of a promoter sequence selected from promotor sequences with a nucleic acid sequence as identified in Table
4. Preferably, the nucleic acid construct is suitable for genomic integration in a desired host cell.
Table 4 - Selected promoter sequences
Figure imgf000040_0001
*The promoter activity is assessed in the LacZ assay described below with the PglpF promoter run as positive reference in the same assay. To compare across assays the activity is calculated relative to the PglpF promoter, a range indicates results from multiple assays.
The promoter may be of heterologous origin, native to the genetically engineered cell or it may be a recombinant promoter, combining heterologous and/or native elements.
One way to increase the production of a product may be to regulate the production of the desired enzyme activity used to produce the product, such as the glycosyltransferases or enzymes involved in the biosynthetic pathway of the glycosyl donor.
Increasing the promoter strength driving the expression of the desired enzyme may be one way of doing this. The strength of a promoter can be assessed using a lacZ enzyme assay where |3- galactosidase activity is assayed as described previously (see e.g., Miller J. H. Experiments in molecular genetics, Cold spring Harbor Laboratory Press, NY, 1972). Briefly the cells are diluted in Z-buffer and permeabilized with sodium dodecyl sulfate (0.1%) and chloroform. The LacZ assay is performed at 30°C. Samples are preheated, the assay initiated by addition of 200 pl ortho-nitro-phenyl-p-galactosidase (4 mg/ml) and stopped by addition of 500 pl of 1 M Na2COs when the sample had turned slightly yellow. The release of ortho-nitrophenol is subsequently determined as the change in optical density at 420 nm. The specific activities are reported in Miller Units (MU) [A420/(min*ml*A600)]. A regulatory element with an activity above 10,000 MU is considered strong and a regulatory element with an activity below 3,000 MU is considered weak, what is in between has intermediate strength. An example of a strong regulatory element is the PglpF promoter with an activity of approximately 14.000 MU and an example of a weak promoter is Plac which when induced with IPTG has an activity of approximately 2300 MU.
In preferred embodiments, the expression of said nucleic acid sequences are under control of a strong promoter selected from the group consisting of SEQ ID NOs 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27 and 28.
In embodiments the expression of said nucleic acid sequences described herein is under control of a PglpF (SEQ ID NO: 29) or Plac (SEQ ID NO: 38) promoter or PmglB_UTR70 (SEQ ID NO: 26) or PglpA_70UTR (SEQ ID NO: 27) or PglpT_70UTR (SEQ ID NO: 28) or variants thereof such as promoters identified in Table 4, in particular the PglpF_SD4 variant of SEQ ID NO: 24 or Plac_70UTR variant of SEQ ID NO: 20, or PmglB_70UTR variants of SEQ ID NO: 17, 18, 19, 21 , 22, 23, 25 and 26. Further suitable variants of PglpF, PglpA_70UTR, PglpT_70UTR and PmglB_70UTR promoter sequences are described in or WO2019/123324 and W02020/255054 respectively (hereby incorporated by reference). In preferred embodiments, the recombinant nucleic acid sequences individually are under the control of one or more promoters selected from the group consisting of PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 29, 38, 26, 27 and 28, respectively) and variants thereof.
Integration of the nucleic acid construct of interest comprised in the construct (expression cassette) into the bacterial genome can be achieved by conventional methods, e.g. by using linear cartridges that contain flanking sequences homologous to a specific site on the chromosome, as described for the attTn7-site (Waddell C.S. and Craig N.L., Genes Dev. (1988) Feb;2(2): 137-49.); methods for genomic integration of nucleic acid sequences in which recombination is mediated by the Red recombinase function of the phage A or the RecE/RecT recombinase function of the Rac prophage (Murphy, J Bacteriol. (1998);180(8):2063-7; Zhang et al., Nature Genetics (1998) 20: 123-128 Muyrers et al., EMBO Rep. (2000) 1 (3): 239-243); methods based on Red/ET recombination (Wenzel et al., Chem Biol. (2005), 12(3):349-56.;
Vetcher et al., Appl Environ Microbiol. (2005) ;71 (4): 1829-35); or positive clones, i.e., clones that carry the expression cassette, can be selected e.g., by means of a marker gene, or loss or gain of gene function.
In one or more exemplary embodiments, the present disclosure relates to one or more recombinant nucleic acid sequences as illustrated in SEQ ID NOs: 7, 8 and 9 [nucleic acid sequence encoding Bacbad , Bacbac2 and Paral],
In particular, the present disclosure relates to one or more of a recombinant nucleic acid sequence and/or to a functional homologue thereof having a sequence which is at least 70% identical to SEQ ID NOs: 7, 8 or 9 [nucleic acid sequence encoding Bacbad , Bacbac2 and Paral], such as at least 75% identical, at least 80 % identical, at least 85 % identical, at least 90 % identical, at least, at least 95 % identical, at least 98 % identical, or 100 % identical.
Sequence identity
The term "sequence identity" as used herein describes the relatedness between two amino acid sequences or between two nucleotide sequences, i.e., a candidate sequence (e.g., a sequence of the disclosure) and a reference sequence (such as a prior art sequence) based on their pairwise alignment. For purposes disclosed herein, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later (available at https://www.ebi.ac.uk/Tools/psa/emboss_needle/). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, -endopen 10.0, -endextend 0.5 and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. The output of Needle labelled " identity" (obtained using the -nobrief option) is used as the percent identity. Generally sequence identity may be calculated as follows: (Identical Residues x 100)/(aligned region).
For purposes disclosed herein, the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1 970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), 10 preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, -endopen 10.0, -endextend 0.5 and the DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labelled " identity" (obtained using the -nobrief option) is used as the percent identity. Generally sequence identity may be calculated as follows: (Identical nucleotide residues x 100)/(aligned region).
Functional homologue
A functional homologue or functional variant of a protein/nucleic acid sequence as described herein is a protein/nucleic acid sequence with alterations in the genetic code, which retain its original functionality. A functional homologue may be obtained by mutagenesis or may be natural occurring variants from the same or other species. The functional homologue should have a remaining functionality of at least 50%, such as at least 60%, 70%, 80 %, 90% or 100% compared to the functionality of the protein/nucleic acid sequence.
A functional homologue of any one of the disclosed amino acid or nucleic acid sequences can also have a higher functionality. A functional homologue of any one of the a-1 ,3- fucosyltransferase amino acid sequences shown in table 1 or a recombinant nucleic acid encoding any one of the sequences of SEQ ID NO: 7, 8, 9, or 44, should ideally be able to participate in the production of fucosylated HMOs, in terms of increased HMO yield, export of HMO product out of the cell or import of substrate for the HMO production, such as a acceptor oligosaccharide of at least three monosaccharide units, improved purity/by-product formation, reduction in biomass formation, viability of the genetically engineered cell, robustness of the genetically engineered cell according to the disclosure, or reduction in consumables needed for the production. Specifically a functional homologue of any one of the a-1 ,3-fucosyltransferase disclosed herein is capable of producing lacto-N-neofucopentaose VI (LNFP-VI), with less than 5 %, such as less than 2% of the total molar content of HMO being fucosylated by-product oligosaccharides with 5 or 6 monosaccharide units, such as essentially no LNFP-III and LNDFH-I when expressed in a suitable genetically engineered cell as described herein.
Use of a genetically engineered cell or enzyme
The present disclosure also relates to any commercial use of the enzyme(s), genetically engineered cell(s) or the nucleic acid construct(s) disclosed herein, such as, but not limited to, in a method for producing one or more fucosylated human milk oligosaccharide (HMO), preferably, LNFP-VI.
Accordingly, the present disclosure also relates to the use of an a-1 ,3-fucosyltransferase in the production of one or more fucosylated HMOs, wherein the a-1 ,3-fucosyltransferase is selected from the group consisting of Bacbad , Bacbac2, Paral and CafF comprising or consisting of the amino acid sequence of SEQ ID NO: 1 , 2, 3, or 43, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , 2, 3, or 43.
In embodiments, the a-1 ,3-fucosyltransferase of the present disclosure can also be used in the manufacturing of a fucosylated product, wherein the fucosylated product contains one or more oligosaccharides, such as one or more HMOs, and wherein the product contains LNFP-VI or LNFP-V.
In an exemplified embodiment, the genetically engineered cell and/or the nucleic acid construct according to the disclosure is used in the manufacturing of HMOs. Preferably, in the manufacturing of mixtures of HMOs, wherein the molar % content of LNFP-VI produced by the genetically engineered cell is above 14%, such as above 25% of the total amount of HMO produced. Preferably, in the manufacturing of HMOs, wherein pure LNFP-VI is intended as the primary product, the molar % content of LNFP-VI produced by the genetically engineered cell is above 70% such as above 75%, such as above 80%, such as above 90% of the total amount of HMO produced.
In additional embodiments, the genetically engineered cell and/or the nucleic acid construct according to the disclosure is used in the manufacturing of mixtures of HMOs, wherein the molar % content of LNFP-V produced by the genetically engineered cell is above 40% of the total amount of HMO produced. Preferably, in the manufacturing of HMOs, wherein pure LNFP- V is intended as the primary product, the molar % content of LNFP-V produced by the genetically engineered cell is above 50% such as above 55%, such as above 57%, such as above 58% of the total amount of HMO produced. In further embodiments, the a-1 ,3-fucosyltransferase for use in production of LNFP-VI is selected from the group consisting of Bacbacl , Bacbac2, Paral and CafF with an amino acid sequence according to SEQ ID NO: 1 , 2, 3, or 43, or a functional homologue thereof which amino acid sequence is at least 80 % identical to SEQ ID NO: 1 , 2, 3, or 43. The use can be in vivo (as described herein) or alternatively in an in vitro cell free process.
In an exemplified embodiment, the genetically engineered cell and/or the nucleic acid construct according to the disclosure is used in the manufacturing of LNFP-VI or LNFP-V.
Production of these HMO’s may require the presence of two or more glycosyltransferase activities.
A method for producing fucosylated human milk oligosaccharides (HMOs)
The present disclosure also relates to a method for producing LNFP-VI or LNFP-V, said method comprises culturing a genetically engineered cell according to the present disclosure under conditions suitable for production of HMOs.
The present disclosure thus also relates to a method for producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI), with less than 5 % of the total molar content of HMO being fucosylated by-products with 5 or 6 monosaccharide units, comprising the steps of: a) providing a genetically engineered cell with a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-neotetraose (LNnT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNnT, and b) cultivating said genetically modified cell under conditions that allow for formation of LNFP-VI, and c) Optionally, purifying said LNFP-VI to remove by-products, such as 3FL and/or LNnT. Preferably, the method produces more than 14% such as more than 25 % of the total molar content of HMO of LNFP-VI.
The present disclosure also relates to a method for producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI), with less than 5 % of the total molar content of HMO being fucosylated by-product oligosaccharides with 5 or 6 monosaccharide units, comprising the steps of a) Providing a genetically engineered cell with a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase derived from Bacteroidales bacterium, and b) Cultivating said genetically modified cell under conditions that allow for formation of LNFP-VI, and c) Optionally, purifying said LNFP-VI to remove by-products.
Preferably, the method produces more than 25% such as more than 30 % of the total molar content of HMO of LNFP-VI.
The present disclosure thus also relates to a method for producing the HMO LNFP-VI, comprising providing and culturing a genetically engineered cell comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase, where a) less than 2 %, such as less than 1%, such as less than 0.5%, such as less than 0.2% of the total molar content of HMO produced by said method is LNDFH-111 , b) less than 2 % such as less than 1%, such as less than 0.5%, such as less than 0.2% of the total molar content of HMO produced by said method is LNFP-111 , and c) more than 25 % of the total molar content of HMO produced by said method LNFP-VI.
In embodiments of said method the recombinant nucleic acid encodes an a-1 ,3- fucosyltransferase selected from the group consisting of, a) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO:
1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , b) Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO:
2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, c) Paral comprising or consisting of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 3 and d) CafF comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 43.
A further embodiment of the disclosure is a method for producing LNFP-VI, said method comprising culturing a genetically engineered cell comprising a) a recombinant nucleic acid sequence encoding an enzyme with p-1 ,3-N-acetyl- glucosaminyltransferase activity; and b) a recombinant nucleic acid sequence encoding an enzyme with a p-1 ,4- galactosyltransferase activity; and c) a recombinant nucleic acid sequence encoding a fucosyltransferase with a-1 ,3- fucosyltransferase activity, wherein said enzyme is selected from the group consisting of: i. Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO:
1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , ii. Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO:
2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2 and
Hi. Paral comprising or consisting of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 3, and wherein lactose is used as initial substrate in the cultivation.
A further embodiment of the disclosure is a method for producing LNFP-VI, said method comprising culturing a genetically engineered cell comprising a) a recombinant nucleic acid sequence encoding an enzyme with a p-1 ,4- galactosyltransferase activity; and b) a recombinant nucleic acid sequence encoding a fucosyltransferase with a-1 ,3- fucosyltransferase activity, wherein said enzyme is selected from the group consisting of: i. Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO:
1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , ii. Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO:
2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2 and
Hi. Paral comprising or consisting of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 3, and wherein LNT-II is used as initial substrate in the cultivation.
In embodiments, less 5 % of the total molar content of HMO produced by said method is LNDFH-III and/or LNFP-III. preferably, less than 2% LNDFH-III and less than 2% LNFP-III, such as e.g., less than 1% LNDFH-III and less than 1% LNFP-III, or between 0-5% LNDFH-III and/or LNFP-III. Accordingly, in embodiments, less than less than 5% of the molar content of the total HMOs produced in the culturing step is LNFP-III and less than 5% of the molar content of the total HMOs produced in the culturing step is LNDFH-111. In embodiments, essentially no LNFP-III and/or LNDFH-111 is produced in the culturing step.
The method particularly comprises culturing a genetically engineered cell that produces a fucosylated HMO, wherein the LNFP-VI content produced in said method is at least 25 % of the total HMO content produced by the method, and wherein the less than 5% of the molar content of the total HMOs produced is LNDFH-I II and/or LNFP-III.
In embodiments, the present disclosure also relates to a method for producing the Human Milk Oligosaccharide (HMO) lacto-N-fucopentaose (LNFP-V), with less than 2 % of the total molar content of HMO being fucosylated by-products with 5 or 6 monosaccharide units, comprising the steps of: a) providing a genetically engineered cell with a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-tetraose (LNT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNT, and b) cultivating said genetically modified cell under conditions that allow for formation of LNFP- VI, and c) optionally, purifying said LNFP-VI to remove by-products, such as 3FL and/or LNT. Preferably, less than 2 % of the total molar content of HMO produced by said method is LNDFH-II and/or LNFP-II. More preferably, more than 50 % of the total molar content of HMO produced by said method LNFP-V.
In further embodiments, the method for producing LNFP-V, comprises culturing a genetically engineered cell comprising a) a recombinant nucleic acid sequence encoding an enzyme with a p-1 ,3- galactosyltransferase activity and b) a recombinant nucleic acid sequence encoding a fucosyltransferase with a-1 ,3- fucosyltransferase activity, being Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, and wherein LNT-II is used as initial substrate in the cultivation.
A further embodiment the method for producing LNFP-V, comprises culturing a genetically engineered cell comprising a) a recombinant nucleic acid sequence encoding an enzyme with p-1 ,3-N-acetyl- glucosaminyltransferase activity; and b) a recombinant nucleic acid sequence encoding an enzyme with a p-1 ,3- galactosyltransferase activity; and c) at least one copy such as 1 , 2, 3, 4, 5, 10, 20 or 50 copies, or more than 50 copies of a recombinant nucleic acid sequence encoding the fucosyltransferase Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, and wherein lactose is used as initial substrate in the cultivation.
Culturing, cultivating, or fermenting or fermentation (used interchangeably herein) in a controlled bioreactor typically comprises (a) a first phase of exponential cell growth in a culture medium ensured by a carbon-source, and (b) a second phase of cell growth in a culture medium run under carbon limitation, where the carbon-source is added continuously together with the acceptor oligosaccharide, such as lactose, allowing formation of the HMO product in this phase. By carbon (sugar) limitation is meant the stage in the fermentation where the growth rate is kinetically controlled by the concentration of the carbon source (sugar) in the culture broth, which in turn is determined by the rate of carbon addition (sugar feed-rate) to the fermenter.
The method described herein further comprises providing an acceptor saccharide as initial substrate for the HMO formation, the acceptor substrate comprising at least two monosaccharide units, which is exogenously added to the culture medium and/or has been produced by a separate microbial fermentation. As an alternative to adding the initial substrate for the production of the HMO to the fermentation medium, the genetically modified cell may be further engineered to produce the initial substrate inside the cell (see for example WO2015/150328).
In embodiments, the genetically engineered cell is cultivated in the presence of an initial acceptor substrate selected from the group consisting of lactose, LNT-II and LNnT.
The initial acceptor substrate, such as lactose, LNT-II and LNnT, can added prior to and/or during the cultivation of the genetically modified cell. In a preferred embodiment the initial substrate for HMO formation is lactose which is fed to the culture during the fermentation of the genetically engineered cell.
Furthermore, the method of the present disclosure comprises providing a glycosyl donor, for the glycosylation of the acceptor substrate. Preferably, the glucosyl donor is produced by an endogenous or recombinant de novo pathway in the genetically engineered cell. In preferred embodiments of the methods of the present invention, the genetically engineered cell comprises an upregulated biosynthetic pathway for making a fucose sugar nucleotide, such a GDP-fucose.
Alternatively, the glycosyl donor, can be synthesized separately by one or more genetically engineered cells and/or can be exogenously added to the culture medium from an alternative source.
The terms “manufacturing” or “manufacturing scale” or “large-scale production” or “large-scale fermentation”, are used interchangeably and in the meaning of the disclosure defines a fermentation with a minimum volume of 100 L, such as WOOL, such as 10.000L, such as 100.000L, such as 200.000L culture broth. Usually, a “manufacturing scale” process is defined by being capable of processing large volumes yielding amounts of the HMO product of interest that meet, e.g., in the case of a therapeutic compound or composition, the demands for toxicity tests, clinical trials as well as for market supply. In addition to the large volume, a manufacturing scale method, as opposed to simple lab scale methods like shake flask cultivation, is characterized by the use of the technical system of a bioreactor (fermenter) which is equipped with devices for agitation, aeration, nutrient feeding, monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.). To a large extent, the behaviour of an expression system in a lab scale method, such as shake flasks, benchtop bioreactors or the deep well format described in the examples of the disclosure, does allow to predict the behaviour of that system in the complex environment of a bioreactor.
Fucosyltransferases of the prior art such as FutA or FutB from Dumon et al., 2004, may not be suitable for large scale manufacturing of complex fucosylated HMOs, such as LNFP-VI, since the yield obtained from cell expressing such fucosyltransferases is either lower than the yield obtained from the strains disclosed herein, or the specificity of the enzymes is more promiscuous i.e., they produces one or more addition side products, such as LNFP-III and/or LNDFH-111 , which complicates the purification of LNFP-VI.
Accordingly, in the present disclosure, a suitable fucosyltransferase is one which enables large scale production of LNFP-VI or LNFP-V. Thus, the fucosyltransferases disclosed herein, being Bacbad , Bacbac2 and Paral , are especially suited for production of HMOs when introduced into a suitable production strain.
In embodiments, the genetically engineered cell of the present disclosure is suitable for large scale production of HMOs.
In embodiments, the method of the present disclosure is suitable for large scale manufacturing. With regards to the suitable cell medium used in the fermentation process, there are no limitations. The culture medium may be semi-defined, i.e., containing complex media compounds (e.g., yeast extract, soy peptone, casamino acids, etc.), or it may be chemically defined, without any complex compounds. The carbon-source can be selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol. In one or more exemplary embodiments, the culturing media is supplemented with one or more energy and carbon sources selected form the group containing glycerol, sucrose and glucose. In additional embodiments, lactose is added during the cultivation of the genetically engineered cells as a substrate for the HMO formation.
The method comprising culturing a genetically engineered cell that produces LNFP-VI or LNFP- further comprises culturing said genetically engineered cell in in the presence of an energy source (carbon source) selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.
In one or more exemplary embodiments, the culturing media contains sucrose as the sole carbon and energy source. In one or more exemplary embodiments, the genetically engineered cell comprises one or more heterologous nucleic acid sequence encoding one or more heterologous polypeptide(s) which enables utilization of sucrose as sole carbon and energy source of said genetically engineered cell.
In one or more exemplary embodiments, the genetically engineered cell comprises a PTS- dependent sucrose utilization system, further comprising the scrYA and scrBR operons as described in WO2015/197082 (hereby incorporated by reference).
After carrying out the method of this disclosure, the LNFP-VI or LNFP-V can be collected from the cell culture or fermentation broth in a conventional manner. The LNFP-VI or LNFP-V can be retrieved from the culture, either from the culture medium and/or the genetically engineered cell.
Retrieving/Harvesting
The fucosylated human milk oligosaccharide (HMO) is retrieved from the culture medium and/or the genetically engineered cell. In the present context, the term “retrieving” is used interchangeably with the term “harvesting”. Both “retrieving” and “harvesting” in the context relate to collecting the produced HMO(s) from the culture/broth following the termination of fermentation. In one or more exemplary embodiments it may include collecting the HMO(s) included in both the biomass (i.e., the host cells) and cultivation media, i.e., before/without separation of the fermentation broth from the biomass. In other embodiments, the produced HMOs may be collected separately from the biomass and fermentation broth, i.e., after/following the separation of biomass from cultivation media (i.e., fermentation broth).
The separation of cells from the medium can be carried out with any of the methods well known to the skilled person in the art, such as any suitable type of centrifugation or filtration. The separation of cells from the medium can follow immediately after harvesting the fermentation broth or be carried out at a later stage after storing the fermentation broth at appropriate conditions. Recovery of the produced HMO(s) from the remaining biomass (or total fermentation broth) include extraction thereof from the biomass (i.e., the production cells).
In example, after recovery from fermentation, HMO(s) are available for further processing and purification. During the purification, several steps of filtration may be required, in example, the separation of the by-product LNDFH-III from LNFP-VI requires a step of size dependent purification, while separation of LNFP-III from LNFP-VI is very tedious since the charge and mass of the two species is identical. Accordingly, it is highly preferable that no LNFP-III or LNDFH-III is produced in the fermentation process leading to LNFP-VI.
The HMOs can be purified according to the procedures known in the art, e.g., such as described in WO2017/152918, WO2017/182965 or WO2015/188834, wherein the latter describes purification of fucosylated HMOs. The purified HMOs can be used as nutraceuticals, pharmaceuticals, or for any other purpose, e.g., for research.
At the end of culturing, the oligosaccharide as product can be accumulated both in the intra- and the extracellular matrix.
The method according to the present disclosure comprises cultivating the genetically engineered microbial cell in a culture medium which is designed to support the growth of microorganisms, and which contains one or more carbohydrate sources or just carbon-source, such as selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol. In one or more exemplary embodiments, the culturing media is supplemented with one or more energy and carbon sources selected form the group containing glycerol, sucrose and glucose.
Manufactured product
The term “manufactured product” refers to the one or more HMOs intended as the one or more product HMO(s), or composition of a mixture of HMOs. Preferably, the product HMOs or composition is produced by a method described herein using a genetically engineered cell described herein. From the data presented in example 2, it can be seen that the Bacbac2 fucosyltransferase produced LNFP-VI of high purity showing the ability and suitability of Bacbac2 to produce highly pure LNFP-VI in large scale manufacturing.
Advantageously, the methods disclosed herein provide both a decreased ratio of by-product to product and an increased overall yield of the product (and/or HMOs in total). This, less byproduct formation in relation to product formation, facilitates an elevated product production and increases efficiency of both the production and product recovery process, providing superior manufacturing procedure of HMOs.
One embodiment relates to a composition comprising HMOs, such as e.g., a nutritional product comprising LNFP-VI, wherein said LNFP-VI is produced by a method of the present disclosure.
The manufactured product may be a powder, a composition, a suspension, or a gel comprising one or more HMOs.
HMO mixtures
The genetically engineered cell capable of producing one or more HMOs, preferably LNFP-VI, described herein will generally produce a mixture of HMOs as a result of the multistep process towards the final HMO product. In the production of LNFP-VI from lactose as the initial substrate, it is expected that 3-FL (fucosylated lactose), LNT-II, LNnT and pLNnH, and potentially also LNFP-III and LNDFH-III will be produced by the cell (See figure 1). Any of the below mentioned mixtures may be produced by the methods described herein.
The molar % of individual HMO components are supported by experimental data from the Examples and shows exemplary HMO compositions, wherein the mixture of HMOs consists essentially of LNFP-VI and LNnT, 3-FL and/or pLNnH.
In that regard, a mixture of HMOs may consist essentially of a) LNFP-VI and 3-FL, or b) LNFP- VI and LNnT, or c) LNFP-VI, 3FL and LNnT, or d) LNFP-VI, 3FL, LNnT and pLNnH. In embodiments the HMO mixture consist essentially of HMOs within the following ranges 25-90 molar% of LNFP-VI, 0-70 molar% 3FL, 0-65 molar % LNnT, 0-15 molar% pLNnH and below 1 % LNFP-III and below 1 % LNDFH-III, in total adding up to 100% molar content.
In that regard an embodiment of the present disclosure relates to a mixture of HMOs according consists essentially of 25-70 molar% of LNFP-VI, 30-70 molar% 3FL, 0-5 % LNnT, in total adding up to 100 molar% molar content. A further embodiment of the present disclosure relates to a mixture of HMOs according consists essentially of 55-90 molar% of LNFP-VI, 0-15 molar% 3FL, 0-35 % LNnT and 0-10 molar% pLNnH in total adding up to 100 molar% molar content.
A further embodiment of the present disclosure relates to a mixture of HMOs according consists essentially of 25-70 molar% of LNFP-VI, 0-25 molar% 3FL, 15-65 % LNnT and 0-15 molar% in total adding up to 100 molar% molar content.
A further embodiment of the present disclosure relates to a mixture of HMOs according consists essentially of 80 molar% of LNFP-VI, 10 molar% 3FL, 10 % LNnT.
A further embodiment of the present disclosure relates to a mixture of HMOs according consists essentially of 60 molar% of LNFP-VI and 40 molar%.
The genetically engineered cell capable of producing LNFP-V, described herein will generally produce a mixture of HMOs as a result of the multistep process towards the final HMO product. In the production of LNFP-V from lactose as the initial substrate, it is expected that 3-FL (fucosylated lactose), LNT-II, LNT and pLNnH, and potentially also LNFP-II and LNDFH-II will be produced by the cell (See figure 3). Any of the below mentioned mixtures may be produced by the methods described herein.
Example 3 shows exemplary HMO compositions, wherein the mixture of HMOs consists essentially of LNFP-V, 3FL and LNT.
In that regard an embodiment of the present disclosure relates to a mixture of HMOs according consists essentially of 50-70 molar% of LNFP-V, 0-5 molar% 3FL, 30-50 molar% LNT, in total adding up to 100% molar content. Specifically, the by-products LNFP-II and LNDFH-II is below 1 molar%. Clinical data in infants indicate that Human Milk Oligosaccharide supplements may help to develop the desired microbiota by serving as a food source for the good bacteria in the intestine. Naturally occurring in breast milk, HMOs have evolved over thousands of years, with HMO research (clinical and preclinical) now suggesting that specific HMOs at the correct level of supplementation can provide us with unique health benefits. In particular, Human Milk Oligosaccharide supplements may help support immunity and gut health, with a potential role in cognitive development, which may open future innovation opportunities.
Mixtures of HMOs as described herein may also form part of a composition comprising additional parts, such as active pharmaceutical ingredients, food supplements, probiotics, excipients, surfactants etc. Use of composition of or mixtures of HMOs
Naturally occurring in breast milk, HMOs have evolved over thousands of years, with HMO research (clinical and preclinical) now suggesting that specific HMOs at the correct level of supplementation can provide unique health benefits. As fucosylated HMOs constitute more than 60% of the total HMOs in human milk, mixtures with a high content of fucosylated HMOs are more desirable.
Accordingly, LNFP-VI and mixtures of HMOs comprising LNFP-VI are highly relevant as either a nutritional supplement or as a therapeutic.
Clinical data in infants indicate that Human Milk Oligosaccharide supplements may help to develop the desired microbiota by serving as a food source for the beneficial bacteria in the intestine. In particular, Human Milk Oligosaccharide supplements may help support immunity and gut health, with a potential role in cognitive development, which may open future innovation opportunities.
The mixtures or composition of HMOs may be used to enhance the beneficial bacteria in the gut microbiome. Beneficial bacteria are for example bacteria of the Bifidobacterium sp., lactobacillus sp. or Barnesiella sp. The enhancement of beneficial bacteria may in turn lead to increased production of short chain fatty acids (SCFAs) such as acetate, propionate and butyrate which have been shown to have many benefits in infants and young children, such as inhibition of pathogen bacteria, prevention of infection and diarrhoea, reduced risk of allergy and metabolic disorders (see for example W02006/130205, WO 2017/129644, WO2017/129649).
The mixtures or composition of HMOs produced according to the method described herein may be used to reduce the abundance of undesirable viruses and bacteria in the gut microbiome. Examples of pathogenic bacteria and viruses that may be reduced by the HMO mixtures described herein are including Candida albicans, Clostridium difficile, Enterococcus faecium, Escherichia coll, Helicobacter pylori, Streptococcus agalactiae, Shigella dysenteriae, Staphylococcus aureus, nora virus and rota virus. Each composition described herein can also be used to treat and/or reduce the risk of a broad range of bacterial infections of a human.
The mixtures or composition of HMOs produced according to the method described herein may be used to increase the regeneration and viability of lyophilized probiotics, including probiotics of Bifidobacterium sp, lactobacillus sp., in particular increased regeneration and/or viability and/or shelf-life in an acidic environment, such as the stomach or acidic food products, is an advantage using the HMO mixtures described herein. Examples of Bifidobacterium sp. which may have increased regeneration and viability are Bifidobacterium animals lactis BB12 DSM 32269, Bifidobacterium animals lactis BIF6, Bifidobacterium longum DSM 32946, Bifidobacterium longum BB536, Bifidobacterium bifidum DSMZ 32403, Bifidobacterium infantis, Bifidobacterium breve DSM 33789, Bifidobacterium infantis SP37 DSM 32687, Bifidobacterium adolescentis DSM 34065 and/or Bifidobacterium animalis ssp. animalis DSM 16284. Examples of lactobacillus sp which may have increased regeneration and viability are Lactobacillus rhamnosus GG DSM 32550, Lactobacillus rhamnosus 19070-2 DSM 26357, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus LBrGG, Lactobacillus reuteri DSM 12246, Lactobacillus plantarum TIFN101, Lactobacillus gasseri Lg-36 200B FloraFit Danisco, Lactobacillus casei DSM 32382, Lactobacillus paracasei, Lactobacillus plantarum PS 128, Lactobacillus plantarum (Sacco) DSM 32383, Lactococcus lactis PAREVE, Lactobacillus paracasei ssp. Paracasei and/or Lactobacillus Probio-Tec®LGG®, Limosilactobacillus reuteri S12 DSM 33752.
In the context of the present application “Regeneration” means the process of regaining/ restoring a dried bacteria’s viability (i.e., “reviving” the bacterial cells by rehydration, wherein “rehydration” means restoring fluid). This process is also sometimes referred to as “reconstitution”.
In the context of the present application “Viability” is the ability of a bacterial cell to live and function as a living cell. One way of determining the viability of bacterial cells is by spreading them on an agar plate with suitable growth medium and counting the number of colonies formed after incubation for a predefined time (plate counting). Alternatively, FACS analysis may be used.
In the context of the present application “Improving the regeneration” of Bifidobacterium sp. and/or Lactobacillus sp bacteria means to increase the amount (number) of Bifidobacterium sp. and/or Lactobacillus sp. bacteria successfully regenerating/ reviving compared to the respective control (i.e., the amount/ number of Bifidobacterium sp. and/or Lactobacillus sp. bacteria without the addition of HMO).
In the context of the present application “Improving the viability” of Bifidobacterium sp. and/or Lactobacillus sp. bacteria means to increase the amount (number) of viable Bifidobacterium sp. and/or Lactobacillus sp. bacteria compared to the respective control (i.e., the amount/ number of Bifidobacterium sp. and/or Lactobacillus sp. bacteria without the addition of HMO).
In the context of the present application “acidic” means having a pH below 7.0 (for example, having a pH < 6.0, or < 5.0, or < 4.0, or < 3.0, or in the range of 1 .0-6.0, such as from 2.0 to 5.0). The pH measured in the stomach is in the range of about 1.5-3.5. The pH measured in a healthy vagina is in the range of about 3.8-5.0. The pH of fruit juices is in the range of about 2.0-
4.5.
The mixtures or composition of HMOs produced according to the method described herein, may be used to extend the shelf life of probiotics, such as Bifidobacterium sp. and/or lactobacillus sp.
An embodiment of the present invention is a composition comprising a mixture of HMOs as described herein, in particular in the section “Mixtures of HMOs”, and one or more probiotics. Preferably, the probiotic is a Bifidobacterium sp. and/or lactobacillus sp. such as any of the specific species mentioned above.
The mixtures or composition of HMOs produced according to the method described herein, may be used to improve the flowability of a powder or decrease the viscosity of a liquid.
Composition and mixtures of HMOs described in the sections “Manufactured product” and “HMO mixtures” may also form part of a composition comprising additional parts, such as active pharmaceutical ingredients, food supplements, probiotics, excipients, carriers etc..
The mixtures or composition of HMOs described herein are used in a nutritional composition. Nutritional compositions are for example, an infant formula, a rehydration solution, or a dietary maintenance, medical nutrition or supplement for elderly individuals or immunocompromised individuals. Macronutrients such as edible fats, carbohydrates and proteins can also be included in such anti-infective compositions. Edible fats include, for example, coconut oil, soy oil and monoglycerides and diglycerides. Carbohydrates include, for example, glucose, edible lactose and hydrolysed cornstarch. Proteins include, for example, soy protein, whey, and skim milk. Vitamins and minerals (e. g. calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and B complex) can also be included in such anti-infective compositions.
Accordingly, embodiments described herein relate to the use of a mixture of HMOs or a composition comprising a mixture of HMOs, such as a mixture of HMOs produced according to the present disclosure, in an infant formula, as a dietary supplement or medical nutrition. In further embodiments the composition for use in an infant formula, a dietary supplement or medical nutrition comprises a) LNFP-VI and 3-FL, b) LNFP-VI and LNnT, or c) LNFP-V, 3FL and LNnT. Such infant formula, dietary supplement or medical nutrition may be obtained using methods disclosed herein.
In embodiments, the composition comprising a mixture of HMOs described herein, e.g., produced according to the present disclosure, is a pharmaceutical composition. The present disclosure also relates to the use of a mixture or composition according to the present disclosure as a dietary supplement and/or medical nutrition.
In embodiments, the disclosure relates to the use of a mixture or composition according to the present disclosure in infant nutrition. SEQUENCES
The current application contains a sequence listing in text format and electronical format which is hereby incorporated by reference.
An overview of the sequences described in the application can be found in the table below, and additionally promoter sequences used in the present application can be found in table 3 (promoter sequences SEQ ID NO: 17-40).
Sequences
Figure imgf000059_0001
ITEMS
1 . A method for producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI), comprising the steps of: a) Providing a genetically engineered cell with a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-neotetraose (LNnT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNnT, and b) Cultivating said genetically modified cell under conditions that allow for formation of LNFP-VI, and c) Optionally, purifying said LNFP-VI to remove by-products.
2. The method according to item 1 , wherein the a-1 ,3-fucosyltransferase is derived from Bacteroidales bacterium.
3. The method according to item 1 , wherein the a-1 ,3-fucosyltransferase is selected from the group consisting of: a) Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, b) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , c) Paral comprising or consisting of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 3, and d) CafF comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 43.
4. The method according to item 1 to 3, wherein the LNFP-VI produced by the cell expressing said a-1 ,3-fucosyltransferases is essentially free of any N-acetylglucosamine (GIcNAc) fucosylated oligosaccharides.
5. The method according to any one of the preceding items, wherein less than 5%, such as less than 2.5 %, such as less than 1 %, such as less than 0.2% of the total molar content of HMO produced by the method is fucosylated by-products with 5 or 6 monosaccharide units.
6. The method according to item 4 or 5, wherein the N-acetylglucosamine (GIcNAc) fucosylated oligosaccharides or fucosylated by-products with 5 or 6 monosaccharide units is LNDFH-III and/or LNFP-III.
7. The method according to according to any one of the preceding items, wherein less than 5%, such as less than 4%, 3%, 2%, 1% or such as less than 0.1% of the molar content of the total HMDs produced is LNFP-III and less than 5% such as less than 4%, 3%, 2%, 1% or such as less than 0.1% of the molar content of the total HMDs produced is LNDFH-III. 8. The method according to item 6 or 7, wherein essentially no fucosylated by-products with 5 or 6 monosaccharide units, such as LNFP-III and/or LNDFH-III, is produced in cultivation step (b).
9. The method according to any one of the preceding items, wherein the cell further produces one or more HMOs selected from the group consisting of 3FL, LNT-II and LNnT.
10. The method according to any one of the preceding items, wherein by-products may be one or more HMO by-products selected from 3FL, LNT-II and LNnT, LNFP-III, LNDFH-III and pLNnH.
11 . The method according to item 10, wherein the only HMO by-product are 3FL and/or LNnT and potentially less than 5% of pLNnH and/or LNT-II.
12. The method according to any one of the preceding items, wherein more than 25 %, such as more than 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,
56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,
72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, or more than 83% of the total molar content of HMO produced in the cultivation step (b) is LNFP-VI.
13. The method according to any one of the preceding items, wherein the genetically engineered cell is cultivated in the presence of an acceptor substrate selected from the group consisting of lactose, LNT-II and LNnT.
14. The method according to any one of the preceding items, wherein the cell further comprises a substrate importer selected from a lactose importer, a lacto-N-triose-l I (LNT-II) importer or a LNnT importer.
15. The method according to any one of the preceding items, wherein the cell comprises a lactose importer and lactose is added as substrate to the medium during the cultivation step.
16. The method according to any one of the preceding items, wherein the conditions that allow for formation of LNFP-VI include the presence of a culture medium with an energy source that is preferably selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.
17. The method according to any one of the preceding items, wherein the genetically engineered, further comprises a recombinant nucleic acid sequence encoding a [3-1 ,4- galactosyltransferase. 18. The method according to any one of the preceding items, wherein the genetically engineered, further comprises a recombinant nucleic acid sequence encoding a [3-1 ,3-N- acetyl-glucosaminyltransferase.
19. The method according to any one of items 13 to 18, wherein the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding a p-1 ,3-N-acetyl- glucosaminyltransferase and a recombinant nucleic acid sequence encoding a [3-1 ,4- galactosyltransferase and lactose is added during the cultivation of the genetically engineered cells as a acceptor substrate for the HMO formation.
20. The method according to any one of the preceding items, wherein the genetically engineered cell comprises one or more pathways to produce a nucleotide-activated sugar selected from the group consisting of UDP-GIcNAc, GDP-fucose, UDP-galactose and UDP- glucose.
21 . The method according to item 20, wherein the cell overexpresses at least one enzyme in the de novo GDP-fucose pathway responsible for the formation of GDP-fucose.
22. The method according to item 21 , wherein the one or more enzyme is selected from the group consisting of mannose-6 phosphate isomerase (manA), phosphomannomutase (manB), mannose-1 -phosphate guanylyltransferase guanylyltransferase (manC), GDP- mannose-4,6-dehydratase (gmd) and GDP-L-fucose synthase (wcaG).
23. The method according to any one of the preceding items, wherein the method produces LNFP-VI with at least 90% purity in the final product.
24. The method according to item 23, wherein purified LNFP-VI product contains less than 5% of other HMOs.
25. A genetically engineered cell capable of producing the Human Milk Oligosaccharide (HMO) selected from lacto-N-neofucopentaose VI (LNFP-VI) or lacto-N-fucopentaose V (LNFP-V), comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase, Bacbac2, comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2.
26. The genetically engineered cell according to item 25, wherein the genetically engineered cell comprises one or more further recombinant nucleic acids encoding one or more heterologous glycosyltransferases. 27. The genetically engineered cell according to item 25 or 26, wherein the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding a [3-1 ,3-N- acetyl-glucosaminyltransferase and a p-1 ,4-galactosyltransferase or a p-1 ,3- galactosyltransferase.
28. The genetically engineered cell according to item 25 to 27, wherein less than 2 %, such as less than 1.5 %, such as less than 1 %, such as less than 0.1 % of the total molar content of HMO produced by said cell are fucosylated by-products with 5 or 6 monosaccharide units, such as LNDFH-III and/or LNFP-III or LNDFH-II and/or LNFP-II.
29. The genetically engineered cell according to any one of items 25 to 28, wherein more than
25 %, such as more than 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,
53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,
69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, or more than 83% of the total molar content of HMO produced by said cell is LNFP-VI or LNFP-V.
30. The genetically engineered cell according to any one of items 25 to 29, wherein the cell further produces one or more HMOs selected from the group consisting of 3FL, LNT-II and LNT.
31 . The genetically engineered cell according to item 30, wherein the cell further produces LNT, and less than 2% of the total molar content of HMO produced by said cell is 3FL and/or LNT-II
32. The genetically engineered cell according to any one of items 25 to 29, wherein the cell further produces one or more HMOs selected from the group consisting of 3FL, LNT-II and LNnT and pLNnH.
33. The genetically engineered cell according to an 32, wherein the cell produces LNnT, and less than 15% of the total molar content of HMO produced by said cell is 3FL and/or pLNnH.
34. A genetically engineered cell capable of producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI), comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase, wherein less than 5 % of the total molar content of HMO produced by said cell are fucosylated by-products with 5 or 6 monosaccharide units.
35. The genetically engineered cell according to item 34, wherein more than 25%, such as more than 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, or more than 83% of the total molar content of HMO produced by said cell is LNFP-VI. The genetically engineered cell according to any one of items 34 and 35, wherein said recombinant nucleic acid encodes an a-1 ,3-fucosyltransferase selected from the group consisting of: a) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO:
1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , b) Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO:
2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, c) Paral comprising or consisting of an amino acid sequence according to SEQ ID NO: 3, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 3, and d) CafF comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 43. The genetically engineered cell according to item 36, wherein the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding a p-1 ,3-N-acetyl- glucosaminyltransferase and a p-1 ,4-galactosyltransferase. The genetically engineered cell according to any one of item 37, wherein the p-1 ,3-N- acetylglucosaminyltransferase is from Neisseria meningitidis, such as an amino acid sequence according to SEQ ID NO: 14, or a functional homologue thereof, and the p-1 ,4- galactosyltransferase is from Helicobacter pylori, such as an amino acid sequence according to SEQ ID NO: 15, or a functional homologue thereof. The genetically engineered cell according to any one of items 34 to 38, wherein the genetically engineered cell produces less than 5%, such as less than 2.5 %, such as less than 1 % of the total molar content of HMO of the fucosylated by-products LNDFH-111 and/or LNFP-III. The genetically engineered cell according to item 39, wherein essentially no LNFP-III and/or LNDFH-111 is produced by said cell. The genetically engineered cell according to any one of items 34 or 40, wherein the cell further produces one or more HMOs selected from the group consisting of 3FL, LNT-II, LNnT and pLNnH. The genetically engineered cell according to item 41 , wherein the cell produces 3FL and/or LNnT and less than 12% of pLNnH and LNT-II, preferably less than 5% pLNnH and no LNT- II. The genetically engineered cell according to any of one of items 25 to 41 , wherein the cell further comprises a substrate importer selected from a lactose importer, a lacto-N-triose-l I (LNT-II) importer, an LNT and an LNnT importer. The genetically engineered cell according to any of the items 25 to 43, wherein the cell comprises pathways to produce a nucleotide-activated sugar selected from the group consisting of UDP-GIcNAc, GDP-fucose, UDP-galactose and UDP-glucose. The genetically engineered cell according to any of the items 25 to 43, wherein the cell overexpresses at least one enzyme in the de novo GDP-fucose pathway responsible for the formation of GDP-fucose. The genetically engineered cell according to item 44, wherein the one or more enzyme is selected from the group consisting of mannose-6 phosphate isomerase (manA), phosphomannomutase (manB), mannose-1 -phosphate guanylyltransferase guanylyltransferase (manC), GDP-mannose-4,6-dehydratase (gmd) and GDP-L-fucose synthase (wcaG). The genetically engineered cell according to any of items 25 to 46, wherein the cell further comprises a recombinant nucleic acid sequence according to SEQ ID NO: 41 encoding the colanic acid (CA) gene cluster. The genetically engineered cell according to any one of items 25 to 47, wherein the cell further comprises a nucleic acid sequence encoding an MFS transporter protein capable of exporting LNFP-VI or LNFP-V into the extracellular medium. The genetically engineered cell according to any of one of items 25 to 48, wherein the recombinant nucleic acid sequences individually are under the control of one or more promoters selected from the group consisting of PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 29, 38, 26, 27 and 28) and variants thereof. 50. The genetically engineered cell according to any of items 25 to 49, wherein said engineered cell is a microorganism.
51 . The genetically engineered cell according to any of items 25 to 50, wherein said engineered cell is a bacterium or a fungus.
52. The genetically engineered cell according to item 51 , wherein said fungus is selected from a yeast cell of the genera Komagataella sp., Kluyveromyces sp., Yarrowia sp., Pichia sp., Saccaromyces sp., Schizosaccharomyces sp. or Hansenula sp. or from a filamentous fungus of the genera Aspargillus sp., Fusarium sp. or Thricoderma sp..
53. The genetically engineered cell according to item 51 , wherein said bacterium is selected from the group consisting of Escherichia sp., Bacillus sp., lactobacillus sp., Corynebacterium sp. and Campylobacter sp.
54. The genetically engineered cell according to any of one of items 25 to 53, wherein said engineered cell is selected from the group consisting of Escherichia Coll, Bacillus subtilis, lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris and Saccharomyces cerevisiae.
55. Use of the a-1 ,3-fucosyltransferase Bacbac2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2 in the production of one or more fucosylated HMOs.
56. The use according to item 55, wherein the fucosylated HMO is selected from LNFP-V and LNFP-VI.
57. The use according to item 55 or 56, wherein less than 15% of the total molar content of HMO produced is 3FL and essentially no LNFP-II, LNFP-III, LNDFH-II and/or LNDFH-III is produced.
58. Use of an a-1 ,3-fucosyltransferase in the production LNFP-VI, wherein the a-1 , 3- fucosyltransferase is selected from the group consisting of Bacbad , Bacbac2, Paral and CafF, comprising or consisting of the amino acid sequence of SEQ ID NO: 1 , 2, 3, or 43, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , 2, 3, or 43.
59. A mixture of HMOs, consisting essentially of a) LNFP-VI and 3-FL, or b) LNFP-VI and LNnT, or c) LNFP-VI, 3FL and LNnT. The mixture of HMOs according to item 59, consisting essentially of a) 25-70 molar% of LNFP-VI, 35-70 molar% 3FL, 0-5 % LNnT, or b) 55-90 molar% of LNFP-VI, 0-15 molar% 3FL, 0-35 % LNnT and 0-10 molar% pLNnH, or c) 25-70 molar% of LNFP-VI, 0-25 molar% 3FL, 15-65 % LNnT and 0-15 molar% pLNnH, in total adding up to 100% molar content. The mixture of HMOs according to item 59 or 60, wherein the mixture is produced by the method according to any of items 1 to 24, or from a genetically engineered cell according to any of items 34 to 54. A composition comprising a mixture of HMOs according to any of items 59 to 61 . The composition according to item 62, wherein the mixture further comprises a one or more probiotics. The composition according to item 63, wherein the probiotic is a Bifidobacterium sp and/or a lactobacillus sp. Use of a mixture according to any of items 59 to 61 , or a composition of HMOs according to any of items 62 to 64 in an infant formula, a dietary supplement and/or medical nutrition. A method for producing the Human Milk Oligosaccharide (HMO) lacto-N-fucopentaose V (LNFP-V), with less than 2% of the total molar content of HMO being fucosylated byproducts with 5 or 6 monosaccharide units, comprising the steps of: a) Providing a genetically engineered cell with a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase with high specificity for the glucose (Glc) moity in lacto-N-tetraose (LNT) and low or no specificity for the N-acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNT, and b) Cultivating said genetically modified cell under conditions that allow for formation of LNFP-V, and c) Optionally, purifying said LNFP-V to remove by-products. The method according to item 66, wherein the genetically engineered cell is a cell according to any of items 25 to 31 , and wherein less than 2 %, such as less than 1%, of the total molar content of HMO produced by said method is LNDFH-II and/or LNFP-II. 68. The method according to any of items 66 or 67, wherein at least 50%, such as at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or such as at least 59% of the molar content of the total HMOs produced by said method is LNFP-V.
69. The method according to any of items 66 to 68, wherein less than 4% of the molar content of the total HMOs produced in the cultivation is LNDFH-II.
70. The method according to any of items 66 to 69, wherein the essentially no LNFP-II and/or LNDFH-II is produced in the cultivation step.
71 . The method according to any one of items 66 to 70, wherein the genetically engineered cell is cultivated in the presence of an acceptor substrate selected from the group consisting of lactose, LNT-II and LNT.
72. The method according to any one of items 66 to 71 , wherein the conditions that allow for formation of LNFP-V include the presence of a culture medium with an energy source that is preferably selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.
73. A mixture of HMOs, consisting essentially of LNFP-V, 3-FL and LNT
74. The mixture of HMOs according to item 73, consisting essentially of 50-70 molar% of LNFP- V, 0-5 molar% 3FL, 30-50 molar% LNT, in total adding up to 100% molar content.
75. The mixture of HMOs according to item 73 or 74, wherein the mixture is produced by a method according to any of items 66 to 72, or by a genetically engineered cell according to any of items 25 to 31.
76. A composition comprising a mixture of HMOs according to any of items 59 to 61 , or any of items 73 to 75.
77. The composition according to item 76, wherein the mixture further comprises one or more probiotics.
78. The composition according to item 77, wherein the probiotic is a Bifidobacterium sp and/or a lactobacillus sp.
79. Use of a mixture or composition according to any of items 73 to 76 for improving the viability and/or regeneration of a Bifidobacterium sp. and/or Lactobacillus sp. bacteria for probiotic use in a human or animal. 80. Use of a mixture according to any of items 73 to 74, or a composition of HMOs according to any of items 76 to 78 in an infant formula, a dietary supplement and/or medical nutrition.
EXAMPLES
Methods
Unless stated otherwise, standard techniques, vectors, control sequence elements, and other expression system elements known in the field of molecular biology are used for nucleic acid manipulation, transformation, and expression. Such standard techniques, vectors, and elements can be found, e.g., in: Ausubel et al. (eds.), Current Protocols in Molecular Biology (1995) (John Wiley & Sons); Sambrook, Fritsch, & Maniatis (eds.), Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, NY); Berger & Kimmel, Methods in Enzymology 152: Guide to Molecular Cloning Techniques (1987) (Academic Press); Bukhari et al. (eds.), DNA Insertion Elements, Plasmids and Episomes (1977) (Cold Spring Harbor Laboratory Press, NY); Miller, J.H. Experiments in molecular genetics (1972.) (Cold spring Harbor Laboratory Press, NY)
The embodiments described below are selected to illustrate the invention and are not limiting the invention in any way.
Enzymes:
Screening of 50 enzymes with fucosyltransferase activity provided 4 enzymes that have not previously been shown to process glucose specific a-1 ,3-fucosyltransferase activity on LNnT, resulting in production of the complex fucosylated HMO LNFP-VI, without production of other complex fucosylated by-product oligosaccharides in the cultivation. The GenBank ID and origin of the 4 glucose-specific a-1 ,3-fucosyltransferases (Bacbacl , Bacbac2, Paral and CafF), a multi-specific a-1 ,3-fucosyltransferase (Prevl), as well as the prior art a-1 ,3-fucosyltransferases, FutA, FutB and FutT109/CafA, are provided in table 5.
Table 5. List of the enzymes tested in the framework of the present disclosure
Figure imgf000069_0001
Figure imgf000070_0001
Athe sequences used in the present application may be truncated at the N- or C-terminal as compared to the GenBank sequence these are represented by the SEQ ID NO.
AFutA has been shown to produce LNFP-VI, LNDFH-111 and 3FL mixtures and FutB has been shown to produce LNFP-VI, LNFP-III, LNDFH-111 and 3FL mixtures in Dumon et al., 2004 (a-1,3- fucosyltransferase, Biotechnol. Prog. 2004, 20, 412-419).
*CafC and CafF have been shown to produce 3FL in WO2016/040531 .
FucT109** has been shown to produce LNFP-V and LNFP-VI in WO2019/000133
Strains
The strains (genetically engineered cells) constructed in the present application were based on Escherichia coll K-12 DH1 with the genotype: F", A~, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. Additional modifications were made to the E. coli K-12 DH1 strain to generate the MDO strain with the following modifications: lacZ: deletion of 1 .5 kbp, /acA: deletion of 0.5 kbp, nanKETA’. deletion of 3.3 kbp, melA'. deletion of 0.9 kbp, wcaJ deletion of 0.5 kbp, mdoH'. deletion of 0.5 kbp, and insertion of Plac promoter upstream of the gmd gene.
Methods of inserting gene(s) of interest into the genome of E. coli are well known to the person skilled in the art. Insertion of genetic cassettes into the E. coll chromosome can be done using gene gorging (see e.g., Herring and Blattner 2004 J. Bacteriol. 186: 2673-81 and Warming et al 2005 Nucleic Acids Res. 33(4): e36) with specific selection marker genes and screening methods.
To obtain an LNnT producing strain the MDO strain was further engineered by chromosomally integrating a p-1 , 3-GlcNAc transferase (LgtA from Neisseria meningitidis, homologous to NCBI Accession nr. WP_033911473.1 and shown as SEQ ID NO: 14) and a [3-1 ,4- galactosyltransferase (GalT from Helicobacter pylori, homologous to GenBank ID
WP_001262061.1 and shown as SEQ ID NO: 15) both under the control of a PglpF promoter (SEQ ID NO: 29), this strain is named the LNnT strain.
To obtain an LNT producing strain the MDO strain was further engineered by chromosomally integrating a p-1 , 3-GlcNAc transferase (LgtA from Neisseria meningitidis, homologous to NCBI Accession nr. WP_033911473.1 and shown as SEQ ID NO: 14) and a p-1 ,3- galactosyltransferase (GalTK from Helicobacter pylori, homologous to GenBank Accession nr. BD182026.1 and as shown in SEQ ID NO: 42) both under the control of a PglpF promoter (SEQ ID NO: 29), this strain is named the LNT strain.
Codon optimized DNA sequences encoding individual a-1 ,3-fucosyltransferases were genomically integrated into the LNnT or LNT strain. The genotypes of the background strain (MDO), the LNnT strain and the a-1 ,3- fucosyltransferase expressing strains capable of producing LNFP-VI are provided in Table 6.
Table 6. Genotypes of the strains, capable of producing LNDFH-III, used in the present examples.
Figure imgf000071_0001
*1 ,3FT is an abbreviation of a-1 ,3-fucosyltransferase, and the DNA sequence is inserted into the genome of the host strain or integrated via. a plasmid.
1lgtA-PglpF - two genomically inserted copies of a gene encoding p-1 ,3-N-acetyl- glucosaminyltransferase (SEQ ID NO: 14) under control of a PglpF promoter.
2 galT-PglpF- one genomically inserted gene encoding p-1 ,4-Galactosyltransferase (SEQ ID NO: 15) under control of a PglpF promoter. 3CA = extra colanic acid gene cluster (gmd-wcaG-wcaH-wcal-manC-manB, SEQ ID NO: 41) under the control of a PglpF promoter at a locus that is different than the native locus.
4pUC57 is a high-copy number (>300) plasmid having the pUC origin of replication. The antibiotic resistance marker on the pBB vector is ampicillin. The indicated a-1 ,3FT is expressed from the plasmid.
4 galTK-PglpF - one genomically inserted gene encoding /3-1 ,3-Galactosyltransferase (SEQ ID NO: 42) under control of a PglpF promoter.
Deep well assay
Deep Well Assays in the current examples were performed as originally described by Lv et al (Bioprocess Biosyst Eng 20 (2016) 39:1737 — 1747) and optimized for the purposes of the current disclosure. More specifically, the strains disclosed in the present example were screened in 96 deep well plates using a 4-day protocol. During the first 24 hours, precultures were grown to high densities (OD600 up to 5) and subsequently transferred to a medium that allowed induction of gene expression and product formation.
More specifically, during day 1 , fresh precultures were prepared using a basal minimal medium (BMM) (pH 7,0) supplemented with magnesium sulphate (0.12 g/L), thiamine (0.004 g/L) and glucose (5.5 g/L). Basal Minimal medium had the following composition: NaOH (1 g/L), KOH (2.5 g/L), KH2PO4 (7 g/L), NH4H2PO4 (7 g/L), Citric acid (0.5 g/l), trace mineral solution (5 mL/L). The trace mineral stock solution contained; ZnSO~*7H~O 0.82 g/L, Citric acid 20 g/L, MnSO4*H2O 0.98 g/L, FeSO4*7H2O 3.925 g/L, CuSO4*5H2O 0.2 g/L. The pH of the Basal Minimal Medium was adjusted to 7.0 with 5 N NaOH and autoclaved. The precultures were incubated for 24 hours at 34 °C and 1000 rpm shaking and then further transferred to 0.75 mL of a new BMM (pH 7,5) to start the main culture. The new BMM was supplemented with magnesium sulphate (0.12 g/L), thiamine (0.02 g/L), a bolus of glucose solution (0.1-0.15 g/L) and a bolus of lactose solution (5-20 g/L) Moreover, a 20 % stock solution of sucrose (40-45 g/L) or maltodextrin (19-20 g/L) was provided as carbon source, accompanied by the addition of a specific hydrolytic enzyme, sucrose hydrolase or glycoamylase, respectively, so that glucose was released at a rate suitable for carbon-limited growth and similar to that of a typical fed- batch fermentation process. The main cultures were incubated for 72 hours at 28 °C and 1000 rpm shaking. For the analysis of total broth, the 96 well plates were boiled at 100°C, subsequently centrifuged, and finally the supernatants were analysed by HPLC. Fermentation
The E. coli strains were cultivated in 250 mL fermenters (Ambr250 HT Bioreactor system, Sartorius) starting with 100 mL of mineral culture medium consisting of 30 g/L glucose and a mineral medium comprised of NH4H2PO4, KH2PO4, MgSO4 x 7H2O, KOH, NaOH, citric acid, trace element solution, antifoam and thiamine. The dissolved oxygen level was kept at 20% by a cascade of first agitation and then airflow starting at 700 rpm (up to max 4500 rpm) and 1 WM (up to max 3 WM). The pH was kept at 6.8 by titration with 8.5% NH4OH solution. The cultivations were started with 2% (v/v) inoculums from pre-cultures comprised of 10 g/L glucose, (NH4)2HPO4, KH2PO4, MgSO4 x 7H2O, KOH, NaOH, citric acid, trace element solution, antifoam and thiamine. After depletion of the glucose contained in the basal minimal medium, a feed solution containing glucose, MgSO4 x 7H2O, H3PO4 and trace mineral solution was continuously added to the fermenter at a rate that maintained carbon-limiting conditions. The temperature was initially at 33°C but was dropped to 30°C with a 3-hour linear ramp initiated 12 hours after the start of the feed. Lactose was added as bolus additions of 25% lactose monohydrate solution 36 hours after feed start and then every 19 hours to keep lactose from becoming a rate limiting factor. The growth, metabolic activity and metabolic state of the cells was followed by on-line measurements of agitation, dissolved oxygen tension, reflectance, NH4OH base addition, O2 uptake rate and CO2 evolution rate. Throughout the fermentations, samples were taken to determine the concentration of HMO products, lactose and other minor by-products using HPLC.
Example 1 - in vivo LNFP-VI synthesis
Genetically modified cells expressing individual a-1 ,3-fucosyltransferase enzymes were screened for their ability to produce the fucosylated HMO LNFP-VI.
Four enzymes with low formation of complex fucosylated oligosaccharide by-products were identified and compared to enzymes with broader specificity to the monosaccharide units present in LNnT. Their ability to synthesize LNFP-VI when introduced into a genetically modified cells that produce LNnT and GDP-Fucose was tested (table 7).
Genetically modified strains expressing the four individual a-1 ,3-fucosyltransferases (table 5) were generated as described in the “Method” section. The cells were screened in the deep well assay setup as described in the “Method” section.
Table 6 lists the genotype of the strains capable of producing LNFP-VI. The molar content of individual HMOs produced by the strains was calculated from sample measurements performed by HPLC. The results of the LNFP-VI producing cells are shown in table 7 as the fraction of the total molar HMO content (in percentage, %) produced by each strain.
Table 7 Content of individual HMO’s as % of total HMO molar (mM) content produced by each strain (results are as a minimum the average of 3 replicates).
Figure imgf000074_0001
From the data presented in table 7 it can be seen that the four enzymes which have not previously been shown to produce LNFP-VI, namely Bacbad , Bacbac2, Paral and CafF can transfer a fucosyl unit specifically onto the Glc moiety of LNnT, while the enzymes FutA, CafC and FucT109 known from Dumon et al.2004, WO2016/040531 and WO2019/008133, respectively and Prevl , presented herein, transfer a fucosyl unit onto the Glc and GIcNAc moieties of LNnT in an a-1 ,3 linkage, thereby also producing LNFP-111 and/or LNDFH-III. FutB which is also described in Dumon et al. 2004 appear to be a quite inefficient alpha-1 , 3- fucosyltransferase when using LNnT as backbone.
From the data presented in table 7 it can be seen that the three novel enzymes Bacbad , Bacbac2 and Paral can transfer a fucosyl unit specifically onto the Glc moiety of LNnT in an a- 1 ,3 linkage to form LNFP-VI at a level above 25% of the total HMO, with no production of the complex fucosylated by-product HMOs, LNDFH-III or LNFP-111 , as compared to the prior art enzyme FutA and FucT109 and the enzyme Prevl which produce 65%, 18% and 30% LNFP-VI of the total HMO, respectively, along with major production of the unintended by-products LNDFH-III at 25%, 24% and 41%, respectively. In addition, the enzymes FucT109 and Prevl also produced the unintended HMO by-product LNFP-III in an amount of 27 and15%, respectively.
None of the four enzymes, Bacbad , Bacbac2, Paral and CafF produce any LNFP-III or LNDFH-III at all, clearly indicating the enzymes are highly specific for the Glc moiety of LNnT, without exercising any fucosyltransferase activity on the GIcNAc or Gal moieties in LNnT. Accordingly, the enzymes Bacbacl , Bacbac2, Paral and CafF allows for a simplified production of LNFP-VI, where unintended HMOs, such as LNDFH-III and LNFP-III are not produced, which overall simplifies the purification process, which is highly beneficial in a large-scale production.
Interestingly, FutB which was previously suggested to produce a mixture of LNDFH-III, LNFP-III, LNFP-VI and 3FL in Dumon et al., 2004, did not produce any LNDFH-III and only minor LNFP- III and LNFP-VI.
Furthermore, as shown in table 8, which demonstrates the overall amount of HMO produced in single copy strains, Bacbacl , Paral , CafF, CafC and FucT109 -expressing strains produce more total HMO than the FutA-expressing strain.
Table 8: total HMO produced by the strains relative to Put A
Figure imgf000075_0001
Absence of alternative fucosylated species in the produced mixture is highly advantageous and preferred if it is desired to purify the produced LNFP-VI. Also, the low levels of LNnT achieved by expressing the Bacbacl and Paral in high copy number is beneficial if pure LNFP-VI is desired. In addition, it is highly advantageous that the enzymes do not severely affect the overall HMO production capability of the cells, and even the lowest producing cell with Bacbac2 still produces about 87 % of the HMO that the FutA strain produces and is therefore still advantageous since none of the produced HMO is the unintended by-products LNFP-III and LNDFH-III, so the overall LNFP-VI yield will still be higher.
Example 2 - Fermentation using Bacbacl and Bacbac2 for production of LNFP-VI
To confirm the HMO profile observed in the deep well assays, and especially the content of LNFP-VI in the total HMO produced, the Bacbacl and Bacbac2 expressing strains of example 1 , containing a single genomic copy of Bacbacl , or a single or two genomic copies of Bacbac2, were fermented as described in the “Method” section above. The results are shown in table 9.
Table 9: Content of individual HMO’s as % of total HMO content produced by the strain
Figure imgf000075_0002
Figure imgf000076_0001
From the data presented table 9, it can be seen that the fraction of LNFP-VI for the Bacbacl was lower than the level produced in the deep well assay, while Bacbac2 was shown to produce a higher amount of LNFP-VI compared to the deep well assay.
Both strains show the suitability of Bacbacl and Bacbac2 for the production of an LNFP-VI product with only low production of similar complex fucosylated by-product HMOs (LNFP-III and LNDFH-I II) in fermentation, in particular Bacbac2 has very low LNnT and 3FL production and no LNFP-III or LNDFH-I 11 production, allowing for the opportunity to obtain very pure LNFP-VI with low purification efforts.
Example 3 - in vivo LNFP-V synthesis
Genetically modified cells expressing one or two genomic copies of the a-1 ,3- fucosyltransferases were screened for their ability to produce the fucosylated HMO LNFP-V (the genotypes are listed in table 6).
The cells and the ability of the enzymes to synthesize LNFP-V when introduced into a genetically modified cell that produce LNT and GDP-Fucose was assessed using the deep well assay setup as described in the “Method” section.
The results of HMOs, including LNFP-V, produced by the LNT backbone cells with the different a-1 ,3-fucosyltransferases are shown in table 10 as the fraction of the total molar HMO content (in percentage, %) produced by each strain.
Table 10: Content of individual HMO’s as % of total HMO content produced by the LNT background strain
Figure imgf000076_0002
From the data presented in table 10, it can be seen that the Bacbac2 enzyme has quite similar activity on LNT and LNnT in that it can transfer a fucosyl unit specifically onto the Glc moiety of LNT and not onto the GIcNAc moiety, therefore no complex fucosylated HMO by-products such as LNFP-II and LNDFH-II were formed. This is an indication that Bacbac2 does not have any alpha-1 , 4-fucosyltranferase activity. The enzymes Bacbac 1 and Para 1 , seem to have very low activity on any moiety in LNT, and essentially resembles how FutB acted in the LNnT strain. FutB on the other hand shows some fucosyltransferase activity and appears to have specificity towards the glucose moiety on LNT. CafF seems to have slightly higher activity on lactose in the LNT background strain and only produces low amounts of complex fucosylated HMOs. The enzymes FutA, FucT109 and CafC, known from Dumon et al., 2004, WO2019/008133 and WO2016/040531 , respectively, seem to have some a-1 ,4-fucosyltransferase activity and can therefore fucosylate the GIcNAc moiety in LNT resulting in some LNFP-II or LNDFH-II in the LNT background.
From the data presented in table 10, it can be seen that the novel enzyme Bacbac2 is the only enzyme that can transfer a fucosyl unit specifically onto the Glc moiety of LNT in an a-1 ,3 linkage to form LNFP-V at levels above 50% of the total HMO, with no production of LNDFH-II or LNFP-II as compared to the prior art enzyme FutA which produce 79% LNFP-V of the total HMO, respectively, along with production of the unintended products LNDFH-II at 13% respectively.
The data also shows that the specificity of enzymes changes with the backbone HMO produced by the strain they are expressed in. Only Bacbac2 seems to maintain similar activity both in the LNnT and LNT background strains.
The absence of alternative complex fucosylated by-products in the produced mixture when using the Bacbac2 enzyme in an LNT background strain is highly advantageous and preferred if it is desired to purify the produced LNFP-V, and accordingly, a lower % of LNFP-V out of the total HMO produced remains advantageous since no LNDFH-II or LNFP-II was produced. The other enzyme, FutB, which has the profile produces significantly less LNFP-V than the Bacbac2 strain.
Furthermore, as shown in table 11 , the total amount of HMO produced in the 2x Bacbac2 strain is higher (relative total production of 127 %) than the amount of HMO produced by the 2x FutA carrying strain, leading to a production of LNFP-V by the Bacbac2 expressing strain of 95% of the FutA strain. Interestingly the FutB strain seems to produce even more total HMO than the Bacbac2 strain, but still the amount of LNFP-V is lower than what is produced by the Bacbac2 strain.
Table 11. % of Total HMO and LNFP-V produced by the strains relative to the producing in the strain with 2x FutA.
Figure imgf000078_0001
Accordingly, it is highly advantageous that the Bacbac2 strain produces an almost identical amount of LNFP-V as the FutA strain, while not producing the by-product LNDFH-II.
Example 4 - Regeneration and viability of lyophilized Lactobacillus rhamnosus Probiotics may be consumed as live bacteria or as a dried (e.g. lyophilized) product. Independent of the drying method, rehydration involves an important step in the recovery of dehydrated bacteria; an inadequate rehydration/ regeneration step may lead to poor cell viability and a low final survival rate. Rehydration is therefore a highly critical step in the revitalization of a lyophilized culture. For both live and rehydrated bacteria, the survival of the bacteria under acidic conditions is critical since they need to pass through the acidic environment of the stomach and may also be faced with storage (shelf-life) in acidic food products.
In the present example it was tested whether the mixture of HMOs similar to the ones produced by the strains described in examples 1 and 2 can provide a benefit in the rehydration (regeneration) and viability of the probiotic strain, Lactobacillus rhamnosus Probio-Tec®LGG® - DSM 33156. The test was performed under acidic conditions to resemble the conditions bacteria have to survive when passing through the stomach or when dosed in an acidic beverage.
The lyophilized probiotic was added to the tube (0.4 mg/ml), alone (control) or in combination with HMO mixtures (5% w/v) as indicated in table 13. The composition was dissolved in sterile phosphate-buffered saline (PBS, pH = 3), warmed to 37 °C and vigorously mixed for about 30 sec until no visible clumps remained. The tubes were incubated at 37 °C for 3 h. The samples were further diluted and 100 l were spread in duplicates onto MRS agar plates which were incubated at 37 °C in anaerobic chambers.
Table 13: HMO compositions tested in the present example
Figure imgf000079_0001
The CFU/ml was calculated based on colonies counted 48 hours after incubation (average of two plates). Figure 4 shows pictures of the plates with the colonies of Lactobacillus rhamnosus DSM 33156 after 48 hours incubation. The results for all three strains are summarized in table 14.
Table 14: Average CFU/ml for the indicated strains after 3 h acid treatment followed by 48h subsequent incubation at 37°C
Figure imgf000079_0002
The lyophilized Lactobacillus strain dissolved with the HMO mixtures described herein showed an enhanced regeneration and survivability compared to control without the HMO mixtures. These data clearly show that the regeneration and viability of Lactobacillus rhamnosus strain after exposure to low pH conditions, such as in the stomach or in an acidic beverage, can be improved in the presence of any of the HMO mixtures. To our knowledge it has not previously been shown that the tested mixtures provide a benefit of improving the regeneration and survivability of a lactobacillus strain in an acidic environment.

Claims

1 . A method for producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI), with less than 5 % of the total molar content of HMO being fucosylated byproduct oligosaccharides with 5 or 6 monosaccharide units, comprising the steps of a) Providing a genetically engineered cell with a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase derived from Bacteroidales bacterium ,and b) Cultivating said genetically modified cell under conditions that allow for formation of LNFP-VI, and c) Optionally, purifying said LNFP-VI to remove by-products such as 3FL and/or LNnT.
2. The method according to claim 1 , wherein the a-1 ,3-fucosyltransferase has high specificity for the glucose (Glc) moity in lacto-N-neotetraose (LNnT) and low or no specificity for the N- acetylglucosamine (GIcNAc) or Galactose (Gal) moieties in LNnT.
3. The method according to claim 1 or 2, wherein the a-1 ,3-fucosyltransferase is selected from: a) Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, or b) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 .
4. The method according to any one of claims 1 to 3, wherein less than 5%, such as less than 2.5 %, such as less than 1 %, such as less than 0.2% of LNDFH-III and/or LNFP-III of the total molar content of HMO is produced in the cultivation step (b).
5. The method according to any one of the preceding claims, wherein the cell further produces one or more HMOs selected from the group consisting of 3FL, LNT-II, LNnT and pLNnH.
6. The method according to any one of the preceding claims, wherein more than 25 % of the total molar content of HMO produced in the cultivation step (b) is LNFP-VI.
7. The method according to any one of thy preceding claims, wherein the genetically engineered cell is cultivated in the presence of an acceptor substrate selected from the group consisting of lactose, LNT-II and LNnT.
8. The method according to any one of the preceding claims, wherein the genetically engineered, further comprises a recombinant nucleic acid sequence encoding a p-1 ,4- galactosyltransferase, and optionally further comprises a recombinant nucleic acid sequence encoding a p-1 ,3-N-acetyl-glucosaminyltransferase.
9. A genetically engineered cell capable of producing the Human Milk Oligosaccharide (HMO) selected from lacto-N-neofucopentaose VI (LNFP-VI) and lacto-N-fucopentaose V (LNFP- V), comprising a recombinant nucleic acid sequence encoding an a-1 ,3-fucosyltransferase, Bacbac2, comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2.
10. A genetically engineered capable of producing the Human Milk Oligosaccharide (HMO) lacto-N-neofucopentaose VI (LNFP-VI), comprising a recombinant nucleic acid encoding an a-1 ,3-fucosyltransferase selected from the group consisting of, a) Bacbac2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 2, b) Bacbad comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 , wherein the genetically engineered cell further comprises a recombinant nucleic acid sequence encoding a p-1 ,3-N-acetyl-glucosaminyltransferase and a [3-1 ,4- galactosyltransferase.
11 . The genetically engineered cell according to claim 9 or 10, wherein the genetically engineered cell produces less than 5%, such as less than 2.5 %, such as less than 1 % of the total molar content of HMO of fucosylated by-products with 5 or 6 monosaccharide units, such as LNDFH-III and/or LNFP-III.
12. The genetically engineered cell according to any one of claims 9 to 11 , wherein the cell further produces one or more HMOs selected from the group consisting of 3FL, LNT-II and LNnT.
13. The genetically engineered cell according to any one of claim 9 to 12, wherein the cell produces more than 25% LNFP-VI of the total molar content of HMO produced by said cell.
14. The genetically engineered cell according to any one of claims 9 to 13 claims, wherein said engineered cell is selected from the group consisting of Escherichia coll, Bacillus subtilis, lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris and Saccharomyces cerevisiae.
15. Use of an a-1 ,3-fucosyltransferase in the production of LNFP-VI which is essentially free of fucosylated by-products with 5 or 6 monosaccharide units, wherein the a-1 ,3- fucosyltransferase is selected from Bacbad or Bacbac2 comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 2, , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 or 2.
16. A mixture of HMOs, consisting essentially of a) LNFP-VI and 3-FL, or b) LNFP-VI and LNnT, or c) LNFP-VI, 3FL and LNnT, or d) LNFP-V, 3FL and LNT
17. The mixture of HMOs according to claim 16, consisting essentially of a) 25-70 molar% of LNFP-VI, 35-70 molar% 3FL, 0-5 % LNnT, or b) 55-90 molar% of LNFP-VI, 0-15 molar% 3FL, 0-35 % LNnT and 0-10 molar% pLNnH, or c) 80 molar% of LNFP-VI, 10 molar% 3FL, 10 % LNnT, or d) 60 molar% of LNFP-VI and 40 molar% 3FL in total adding up to 100% molar HMO content
PCT/EP2024/079175 2023-10-17 2024-10-16 New fucosyltransferases for in vivo synthesis of complex fucosylated human milk oligosaccharides mixtures comprising lnfp-vi or lnfp-v Pending WO2025083041A1 (en)

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