Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • 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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• • 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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
• • 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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
  • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
• • 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
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/10—Cells modified by introduction of foreign genetic material
• • 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.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides
• • 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
  • C12P19/44—Preparation of O-glycosides, e.g. glucosides

Definitions

• the present disclosure relates to cytidine diphosphate choline glycosides, compositions, methods for producing glycosides of cytidine residue-containing compounds, genetically modified microorganisms, methods for producing cytidine residue-containing compounds, and methods for inhibiting the production of glycosides of cytidine residue-containing compounds.
• Cytidine residue-containing compounds refer to compounds that have a cytidine residue in their structure, and specific examples include cytidine, cytidylic acid, cytidine diphosphate (CDP), and cytidine diphosphate choline (CDP-choline).
• CDP-choline is a biosynthetic precursor of phosphatidylcholine, a component of phospholipids in higher organisms, and is known to have neuroprotective properties (Non-Patent Document 1).
• CDP-choline It is difficult to obtain large quantities of CDP-choline through extraction from nature or chemical synthesis; it is primarily synthesized using enzymatic reactions present in living organisms, starting from cytidine monophosphate (CMP) and orotic acid (Patent Documents 1 and 2, Non-Patent Documents 2 and 3). Additionally, methods for producing cytidylic acid and/or cytidine are known, including those described in Japanese Patent Publication Nos. 36-19749, 57-018872, and 36-21499 (Patent Documents 3, 4, and 5).
• osmoregulated periplasmic glucans biosynthesis protein H (hereinafter referred to as opgH) is known to work in cooperation with OpgG (glucans biosynthesis protein G) in response to osmotic pressure, contributing to glucan synthesis in the periplasm.
• OpgG glucans biosynthesis protein G
• opgH is known to be functionally similar to UgtP (processive diacylglycerol beta-glucosyltransferase) in Bacillus subtilis, function as a Moon Lighting Protein, and be involved in regulating cell size (Non-Patent Document 4).
• the present invention aims to provide compounds converted from cytidine residue-containing compounds such as CDP-choline, methods for producing the same, and means for inhibiting the conversion of cytidine residue-containing compounds such as CDP-choline into other compounds.
• the inventors surprisingly discovered a new activity possessed by proteins or microorganisms: the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, more specifically, the activity of transferring glucose from UDP-glucose to CDP-choline. They also discovered that this activity can be used to convert a cytidine residue-containing compound into a novel glycoside of the cytidine residue-containing compound, more specifically, to convert CDP-choline to CDP-choline glycoside, leading to the completion of the present invention.
• the present disclosure includes the following: ⁇ 1> Cytidine diphosphate choline glycoside represented by the following general formula (1), a salt thereof, an N-oxide thereof, or a solvate thereof:
• R is a sugar residue.
• ⁇ 3> A composition containing at least one of the cytidine diphosphate choline glycoside according to ⁇ 1> or ⁇ 2> above, a salt thereof, an N-oxide thereof, and a solvate thereof.
• ⁇ 4> A method for producing a glycoside of a cytidine residue-containing compound, using a protein having an activity of transferring a sugar from a sugar nucleotide to the cytidine residue-containing compound.
• ⁇ 5> The method for producing a glycoside of a cytidine residue-containing compound according to the above ⁇ 4>, wherein the glycoside of the cytidine residue-containing compound is the cytidine diphosphate choline glycoside according to the above ⁇ 1> or ⁇ 2>.
• ⁇ 6> The method for producing a glycoside of a cytidine residue-containing compound according to the above ⁇ 4>, wherein the protein having the activity of transferring a sugar from a sugar nucleotide to the cytidine residue-containing compound is an enzyme classified as at least one of a GT2 enzyme and a GT28 enzyme in the CAZy classification.
• ⁇ 7> The method for producing a glycoside of a cytidine residue-containing compound according to the above ⁇ 4>, wherein the protein having the activity of transferring a sugar from a sugar nucleotide to the cytidine residue-containing compound is at least one selected from the following [1] to [3]: [1] A protein comprising the amino acid sequence represented by SEQ ID NO: 8 or 10. [2] A mutant protein comprising an amino acid sequence represented by SEQ ID NO: 8 or 10 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having the activity of transferring a sugar from a sugar nucleotide to a compound containing a cytidine residue.
• a homologous protein comprising an amino acid sequence having 60% or more identity with the amino acid sequence represented by SEQ ID NO: 8 or 10, and having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• (B) A genetically modified microorganism in which the activity of a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or absent compared to the activity of the parent strain.
• the cytidine residue-containing compound is cytidine diphosphate choline.
• the protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is at least one selected from the following [1] to [3]: [1] A protein comprising the amino acid sequence represented by SEQ ID NO: 8 or 10. [2] A mutant protein comprising an amino acid sequence represented by SEQ ID NO: 8 or 10 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having the activity of transferring a sugar from a sugar nucleotide to a compound containing a cytidine residue.
• a homologous protein comprising an amino acid sequence having 60% or more identity with the amino acid sequence represented by SEQ ID NO: 8 or 10, and having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• the genetically modified microorganism according to ⁇ 8> above, wherein the protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is an enzyme classified as at least one of a GT2 enzyme and a GT28 enzyme in the CAZy classification.
• ⁇ 12> The genetically modified microorganism according to ⁇ 8> above, wherein the protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is Glucans biosynthesis glucosyltransferase H.
• ⁇ 13> The genetically modified microorganism according to ⁇ 8> above, wherein the genetically modified microorganism is Escherichia coli.
• ⁇ 14> A method for producing a cytidine residue-containing compound, comprising preparing the genetically modified microorganism according to any one of ⁇ 8> to ⁇ 13> above, and producing the cytidine residue-containing compound in at least one of a culture supernatant and intracellular space using the genetically modified microorganism.
• ⁇ 15> The method for producing a cytidine residue-containing compound according to the above ⁇ 14>, wherein the cytidine residue-containing compound is cytidine diphosphate choline.
• ⁇ 16> A method for inhibiting production of glycosides of a cytidine residue-containing compound, comprising preparing the genetically modified microorganism according to any one of ⁇ 8> to ⁇ 13> above.
• ⁇ 17> The method for inhibiting production of a glycoside of a cytidine residue-containing compound according to the above ⁇ 16>, wherein the glycoside of the cytidine residue-containing compound is a cytidine diphosphate choline glycoside represented by the following general formula (1):
• R is a sugar residue.
• a method comprising preparing a genetically modified microorganism according to any one of the above items ⁇ 8> to ⁇ 13>, and producing a composition using the microorganism, In the composition, the ratio of cytidine diphosphate choline glycoside to cytidine diphosphate choline is 35% or less. Method for producing the composition.
• composition according to ⁇ 18> wherein the ratio of the amount of cytidine diphosphate choline glycoside produced to the amount of cytidine diphosphate choline produced is 0.0005% or more and 35% or less.
• This disclosure provides novel glycosides of cytidine residue-containing compounds and methods for producing the same. This disclosure also provides methods for producing cytidine residue-containing compounds and methods for inhibiting the production of glycosides of cytidine residue-containing compounds.
• the present invention will be described in detail below, but these are examples of preferred embodiments and the present invention is not limited to these details.
• the "to” in a numerical range means a range that includes the numerical values before and after it.
• "0% by mass to 100% by mass” means a range that is 0% by mass or more and 100% by mass or less.
• the cytidine residue-containing compound of the present disclosure refers to a compound having a cytidine residue in the compound structure, and specific examples thereof include cytidine, cytidylic acid, cytidine diphosphate (hereinafter also referred to as CDP), CDP-choline, etc., with CDP-choline being preferred.
• glycosides of cytidine residue-containing compounds The present disclosure provides a glycoside of a cytidine residue-containing compound.
• the glycoside of a cytidine residue-containing compound is produced by transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• the sugar nucleotide is used as a sugar donor.
• sugar nucleotides examples include UDP-glucose (hereinafter also referred to as UDP-Glc), UDP-galactose (hereinafter also referred to as UDP-Gal), GDP-mannose (hereinafter also referred to as GDP-Man), UDP-N-acetylglucosamine (hereinafter also referred to as UDP-GlcNAc), UDP-N-acetylgalactosamine (hereinafter also referred to as UDP-GalNAc), GDP-fucose (hereinafter also referred to as GDP-Fuc), and UDP-glucuronic acid (hereinafter also referred to as UDP-GlcA), of which UDP-Glc or UDP-Gal are preferred, and UDP-Glc is more preferred.
• UDP-Glc UDP-glucose
• UDP-Gal UDP-galactose
• GDP-Man GDP-mannose
• glycosides of cytidine residue-containing compounds include CDP-choline glycosides represented by the following general formula (1) or (1') (hereinafter also referred to as "CDP-choline glycosides of the present disclosure” or “CDP-choline glycosides”), which are produced by transferring a sugar from a sugar nucleotide to CDP-choline.
• R is a sugar residue.
• R is preferably a glucose residue, a galactose residue, a mannose residue, an N-acetylglucosamine (also referred to as GlcNAc) residue, an N-acetylgalactosamine (also referred to as GalNAc) residue, a fucose residue, a glucuronic acid (also referred to as GlcA) residue, or another hexose residue (e.g., a fructose residue).
• a hexose refers to a monosaccharide having six carbon atoms, and includes, in addition to fructose, allose, talose, idose, gulose, altrose, glucose, and galactose. Glucose or galactose is preferred, and glucose is more preferred.
• the glycoside of the cytidine residue-containing compound is preferably a CDP-choline glycoside represented by the above general formula (1) or general formula (1').
• examples of the CDP-choline glycoside represented by the above general formula (1) or general formula (1') include cytidine diphosphate choline glucose glycoside represented by the following general formula (2) or general formula (2') (general formula (3-1) below, also referred to as CDP-choline glucose glycoside hereinafter), or cytidine diphosphate choline galactose glycoside (general formula (3-2) below, also referred to as CDP-choline galactose glycoside hereinafter).
• the following general formula (3-1) is preferably a structure in which ⁇ -glucose is bound, as represented by the following general formula (4-1), and the following general formula (3-2) is preferably a structure in which ⁇ -galactose is bound, as represented by the following general formula (4-2).
• CDP-choline glucose glycoside represented by the following general formula (3-1) is more preferred, and a structure with ⁇ -glucose bonded represented by the following general formula (4-1) is preferred.
• the CDP-choline glycosides, salts thereof, N-oxides thereof, and solvates thereof disclosed herein, like CDP-choline, may exhibit neuroprotective effects by maintaining the structure of neuronal membranes and suppressing brain dysfunction during brain pathologies such as cerebral infarction and head trauma, and may also be involved in improving memory and attention.
• the above-mentioned CDP-choline glycosides and the like are also expected to have increased water solubility and reduced toxicity.
• glycosides, which are generally water-soluble, are orally administered, the carbohydrate residues are typically removed by intestinal bacteria and digestive enzymes, making them more easily absorbed in the intestinal tract.
• CDP-choline glycosides disclosed herein will exhibit slower or more sustained absorption due to their stabilization and hydrophilicity, compared to CDP-choline.
• composition of the present disclosure contains at least one of the CDP-choline glycoside of the present disclosure, its salt, its N-oxide, and its solvate.
• a composition is also referred to as composition A containing CDP-choline glycoside.
• Composition A may contain at least one of CDP-choline glycoside, its salt, its N-oxide, and its solvate, and the amount contained is not limited.
• the CDP-choline glycosides of the present disclosure can be converted into salts using known methods.
• salts include acid addition salts, alkali metal salts, alkaline earth metal salts, ammonium salts, and amine salts.
• Acid addition salts include, for example, inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, and nitrate, and organic acid salts such as acetate, lactate, tartrate, benzoate, citrate, methanesulfonate, ethanesulfonate, trifluoroacetate, benzenesulfonate, toluenesulfonate, isethionate, glucuronate, and gluconate.
• inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, and nitrate
• organic acid salts such as acetate, lactate, tartrate, benzoate, citrate, methanesulfonate, ethanesulfonate, trifluoroacetate, benzenesulfonate, toluenesulfonate, iseth
• alkali metal salts examples include potassium and sodium.
• alkaline earth metal salts examples include calcium and magnesium.
• ammonium salts examples include tetramethylammonium.
• Amine salts include, for example, triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris(hydroxymethyl)aminomethane, lysine, arginine, and N-methyl-D-glucamine.
• the CDP-choline glycoside of the present disclosure can be converted to an N-oxide using known methods.
• An N-oxide refers to a CDP-choline glycoside of the present disclosure in which the nitrogen atom has been oxidized.
• the CDP-choline glycosides of the present disclosure can be converted into solvates using known methods.
• the solvates are preferably non-toxic and water-soluble. Suitable solvates include, for example, solvates with water or alcoholic solvents (e.g., ethanol, etc.).
• Glycosides of cytidine residue-containing compounds of the present disclosure are produced using proteins having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• the activity of transferring a sugar from a sugar nucleotide to a cytidine residue in a cytidine residue-containing compound refers to the activity of transferring a sugar from the sugar nucleotide to the cytidine residue in the cytidine residue-containing compound, more specifically, the activity of transferring a sugar from the sugar nucleotide to the 2-hydroxyl group of the ribose of the cytidine residue in the cytidine residue-containing compound, more specifically, the activity of transferring a sugar from the sugar nucleotide to CDP-choline, and even more specifically, the activity of transferring glucose from UDP-glucose to CDP-choline.
• Proteins having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound include, for example, proteins classified in EC 2.4.1. However, it has not been previously known that proteins classified in EC 2.4.1. include proteins having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• the CDP-choline glucose glycoside represented by the general formula (2) of the present disclosure is produced using a protein that has the activity of transferring glucose from UDP-glucose to CDP-choline.
• the CDP-choline glucose glycoside represented by the general formula (2) of the present disclosure is produced by transferring glucose from UDP-glucose to CDP-choline represented by the following general formula (3).
• the protein that contributes to the production of CDP-choline glucose glycoside represented by the general formula (2) by transferring glucose from UDP-glucose to CDP-choline represented by the general formula (3) above is a protein that has the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, and more specifically, is a "protein that has the activity of transferring glucose from UDP-glucose to CDP-choline.”
• compounds containing cytidine residues can be produced efficiently by using genetically modified microorganisms in which the activity of proteins having this activity has been reduced or deleted compared to the parent strain. Furthermore, by using genetically modified microorganisms in which the activity of proteins having this activity has been reduced or deleted compared to the parent strain, the production of glycosides of compounds containing cytidine residues can be suppressed.
• Examples of the protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound include enzymes classified into at least one of glycosyltransferases classified in glycosyltransferase Family 2 (also referred to herein as "GT2 enzymes") and glycosyltransferases classified in glycosyltransferase Family 28 (also referred to herein as "GT28 enzymes”) in the CAZy classification.
• a glycosyltransferase classified as a GT2 enzyme in the CAZy classification is particularly preferred.
• the CAZy classification refers to the classification of enzymes in the Carbohydrate-Active enZYmes (also referred to as "CAZy” in this specification), a database that compiles information on the classification of carbohydrate-related enzymes. Enzyme classifications can be confirmed on websites such as http://www.cazy.org/ (Nucleic Acids Res. 2009 Jan;37(Database issue):D233-8. doi: 10.1093/nar/gkn663. Epub 2008 Oct 5.).
• GT2 enzymes include, for example, UDP-Glc ⁇ -1,6-glucan synthase, UDP-Glc ⁇ -glucosyltransferase, UDP-Glc: bactoprenol ⁇ -glucosyltransferase, UDP-Glc: ⁇ -1,3-glucan synthase, and U Examples include DP-Glc: 1,2-diacylglycerol 3-glucosyltransferase and UDP-Glc ⁇ -1,2-glucan synthase, and among these, proteins with glucans biosynthesis glucosyltransferase activity are more preferred.
• osmoregulated periplasmic glucans biosynthesis protein H (hereinafter also referred to as opgH).
• opgH osmoregulated periplasmic glucans biosynthesis protein H from E. coli (Accession Nos. WP_001295445.1, AAC74133.1, KAB1960533.1, AMH24428.1, and NP_415567.1, etc.), Pseudomonas syringae pv.
• opgH from Escherichia coli (UniProt ID: P20401), Xanthomonas euvesicatoria (UniProt ID: Q83Z42), Caulobacter vibrioides (UniProt ID: B8GX72), Bradyrhizobium diazoefficiens (UniProt ID: Q89BU5), and Cupriavidus pinatubonensis (UniProt ID: Q46TZ4) are examples of opgH. More preferred is opgH derived from E. coli (also referred to herein as "E. coli").
• amino acid sequence of opgH derived from Escherichia coli is the amino acid sequence shown in SEQ ID NO: 8 (Accession No. WP_001295445.1).
• nucleotide sequence of the DNA encoding opgH is the nucleotide sequence shown in SEQ ID NO: 3 (Accession No. CP081489.1 2449812-2452352).
• opgH is also known as mdoH or Glucans biosynthesis glucosyltransferase H.
• GT28 enzymes include, for example, UDP-Glc: 1,2-diacylglycerol 3-glucosyltransferase, UDP-GlcNAc: undecaprenyldiphospho-muramoylpentapeptide ⁇ -1,4-N-acetylglucosaminyltrans Among them, UDP-Glc:1,2-diacylglycerol 3-glucosyltransferase is more preferred. Furthermore, an example of a protein having UDP-Glc:1,2-diacylglycerol 3-glucosyltransferase activity is UgtP.
• UgtP examples include UgtP derived from Bacillus subtilis, UgtP derived from Staphylococcus aureus, and UgtP derived from Bacillus mycoides. Of these, UgtP derived from Bacillus subtilis is more preferred, as it is a Moon Lighting Protein like E. coli opgH and is a functional homolog of E. coli opgH.
• amino acid sequence of UgtP derived from Bacillus subtilis is the amino acid sequence represented by SEQ ID NO: 10 (Accession No. NP_390075.1).
• nucleotide sequence of DNA encoding UgtP is the nucleotide sequence represented by SEQ ID NO: 9 (Accession No. NC_000964.3 2306514-2307662).
• proteins having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound include at least one selected from the following [1] to [3].
• a homologous protein comprising an amino acid sequence having 60% or more identity with the amino acid sequence represented by SEQ ID NO: 8 or 10, and having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• [1] to [3] may be the following [1'] to [3'], respectively.
• [1'] A protein consisting of the amino acid sequence represented by SEQ ID NO: 8 or 10.
• [2'] A mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 8 or 10 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having the activity of transferring a sugar from a sugar nucleotide to a compound containing a cytidine residue.
• a homologous protein consisting of an amino acid sequence having 60% or more identity with the amino acid sequence represented by SEQ ID NO: 8 or 10, and having the activity of transferring a sugar from a sugar nucleotide to a compound containing a cytidine residue.
• [1] As a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, [1] is preferred, and a protein containing the amino acid sequence represented by SEQ ID NO: 8 is more preferred.
• mutant protein refers to a protein obtained by artificially deleting or substituting amino acid residues in a parent protein, or by inserting or adding amino acid residues into the protein.
• amino acids deleted, substituted, inserted, or added means that 1 to 20 amino acids may be deleted, substituted, inserted, or added at any position within the same sequence.
• the number of amino acids deleted, substituted, inserted, or added is 1 to 20, preferably 1 to 10, more preferably 1 to 8, and most preferably 1 to 5.
• amino acids to be deleted, substituted, inserted, or added may be naturally occurring or non-naturally occurring.
• Naturally occurring amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.
• groups A to H are as follows: Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine; Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid; Group C: asparagine, glutamine; Group D: lysine, arginine, or
• a homologous protein refers to a protein found in organisms that exists in nature, whose structure and function are similar to those of the original protein, and whose encoding gene is thought to have the same evolutionary origin as the gene encoding the original protein.
• homologous proteins include amino acid sequences that have an identity of preferably 60% or more, more preferably 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence of the target protein.
• the percentage of sequence identity between two amino acid sequences or two nucleotide sequences is calculated as the ratio of matching residues when the two sequences are aligned so that the residues are most identical.
• the percentage of sequence identity can be determined using a mathematical algorithm.
• Such mathematical algorithms include the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, and the homology alignment algorithm of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.
• suitable mathematical algorithms include the similarity search method described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877, and the improved algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264.
• mathematical algorithms are not limited to the above examples.
• Alignment for determining the percentage of sequence identity can be performed using programs based on these mathematical algorithms.
• the programs can be executed by a computer as appropriate. Examples of such programs include, but are not limited to, the PC/Gene program CLUSTAL (Intelligenetics, Examples of such programs include BLAST, FASTA, and TFASTA. Examples of such programs include BLAST, ...
• CLUSTAL program is described in Higgins et al. (1988) Gene 73:237-244, Higgins et al. (1989) CABIOS 5:151-153, Corpet et al. (1988) Nucleic Acids Res. 16:10881-90, Huang et al. (1992) CABIOS 8:155-65, and Pearson et al.
• BLAST is described in Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). 215(3), 403-410., Mount D. W. (2007). CSH protocols, 2007, pdb.top17., etc.
• programs called BLASTP and BLASTN have been developed based on BLAST, and the percentage of sequence identity can be calculated using these programs with default settings.
• the activity of a protein to transfer a sugar from a sugar nucleotide to a compound containing a cytidine residue can be confirmed, for example, by the following method.
• recombinant DNA containing DNA encoding the protein and a tag sequence for enzyme purification is prepared using the method described below.
• a microorganism obtained by transforming the recombinant DNA is cultured, and the protein is prepared as a purified enzyme from the resulting culture.
• the purified enzyme is contacted with an appropriate substrate and sugar donor—for example, when confirming that the protein has the activity to transfer glucose from UDP-glucose to CDP-choline, CDP-choline and UDP-glucose are contacted to produce CDP-choline glucose glycoside.
• CDP-choline glucose glycoside in the reaction solution can be detected using a standard analytical method such as high-performance chromatography or gas chromatography, thereby confirming that the target protein has the activity to transfer a sugar from a sugar nucleotide to a compound containing a cytidine residue.
• tag sequences for enzyme purification are described in the prior art and are known to those skilled in the art. Examples include sequences that can be used to (affinity) purify the polypeptide chain (see, e.g., Kimple ME, Brill AL, Pasker RL. Overview of affinity tags for protein purification. Curr Protoc Protein Sci. 2013;73:9.9.1-9.9.23. Published 2013 Sep 24. doi:10.1002/0471140864.ps0909s73), calmodulin-binding peptides, His-tags such as 6His-tags, and/or maltose-binding protein sequences.
• a His tag is a polyhistidine amino acid motif in proteins that typically consists of at least six histidine (His) residues and is often located at the N- or C-terminus of the protein.
• Polyhistidine tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in Escherichia coli and other prokaryotic expression systems by incubation with affinity resins containing bound divalent nickel or cobalt ions, of which various types are commercially available.
• These resins are generally Sepharose/agarose functionalized with chelators such as iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel and carboxymethylaspartic acid (Co-CMA) for cobalt, to which polyhistidine tags bind with micromolar affinity.
• chelators such as iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel and carboxymethylaspartic acid (Co-CMA) for cobalt, to which polyhistidine tags bind with micromolar affinity.
• the resin is then typically washed with phosphate buffer to remove proteins that do not specifically interact with cobalt or nickel ions.
• the addition of 20 mM imidazole can improve wash efficiency (proteins are typically eluted with 150-300 mM imidazole).
• Examples of DNA encoding E. coli-derived opgH, an example of a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, include Accession Nos. CP081489.1 2449812-2452352 and X64197.1 1951-4494.
• Examples of DNA encoding Bacillus subtilis-derived UgtP include Accession Nos. NC_000964.3 2306514-2307662.
• examples of DNA encoding a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound in this embodiment include at least one selected from the following [4] to [9].
• a DNA encoding a homologous protein comprising an amino acid sequence having 60% or more identity with the amino acid sequence represented by SEQ ID NO: 8 or 10 and having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• DNA encoding a protein comprising a base sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to the base sequence represented by SEQ ID NO: 3 or 9, and having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• [5] to [9] may be the following [5'] to [9'], respectively.
• [5'] DNA encoding a mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 8 or 10 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having the activity of transferring a sugar from a sugar nucleotide to a compound containing a cytidine residue.
• [6'] DNA encoding a homologous protein consisting of an amino acid sequence having 60% or more identity with the amino acid sequence represented by SEQ ID NO: 8 or 10 and having the activity of transferring a sugar from a sugar nucleotide to a compound containing a cytidine residue.
• DNA consisting of the base sequence shown in SEQ ID NO: 3 or 9.
• DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 3 or 9 and encodes a protein having the activity of transferring a sugar from a sugar nucleotide to a compound containing a cytidine residue.
• DNA encoding a protein having a base sequence that is 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identical to the base sequence represented by SEQ ID NO: 3 or 9, and having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• To hybridize means that DNA hybridizes to DNA having a specific base sequence or a portion of that DNA. Therefore, DNA having that specific base sequence or a portion of that DNA can be used as a probe in Northern or Southern blot analysis, and can also be used as an oligonucleotide primer in PCR analysis.
• DNA used as a probe is DNA of at least 100 bases, preferably 200 bases or more, and more preferably 500 bases or more.
• DNA used as a primer is DNA of at least 10 bases, preferably 15 bases or more.
• DNA that hybridizes under stringent conditions can be obtained by following the instructions provided with a commercially available hybridization kit.
• commercially available hybridization kits include the Random Primed DNA Labeling Kit (manufactured by Roche Diagnostics), which prepares probes using the random prime method and performs hybridization under stringent conditions.
• An example of the stringent conditions described above is incubating a DNA-immobilized filter and probe DNA overnight at 42°C in a solution containing 50% formamide, 5x SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured salmon sperm DNA, followed by washing the filter in a 0.2x SSC solution at approximately 65°C.
• 5x SSC 750 mM sodium chloride, 75 mM sodium citrate
• 50 mM sodium phosphate pH 7.6
• 5x Denhardt's solution 10% dextran sulfate
• 20 ⁇ g/ml denatured salmon sperm DNA followed by washing the filter in a 0.2x SSC solution at approximately 65°C.
• the various conditions described above can also be set by adding or changing the blocking reagent used to suppress background in hybridization experiments.
• the addition of the blocking reagent described above may be accompanied by a change in the hybridization conditions to adapt the conditions.
• DNA that can hybridize under the above-mentioned stringent conditions includes DNA that has at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to DNA containing the base sequence represented by SEQ ID NO: 7 or 11, when calculated based on the above-mentioned parameters using, for example, BLAST or FASTA.
• DNAs encoding proteins having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound the DNA described in [4] or [7] above can be obtained, for example, by Southern hybridization to a microbial chromosomal DNA library using probe DNA that can be designed based on the amino acid sequence shown in SEQ ID NO: 8 or 10, or the nucleotide sequence shown in SEQ ID NO: 3 or 9, or by PCR using primer DNA that can be designed based on the nucleotide sequence and the microbial chromosomal DNA as a template [PCR Protocols, Academic Press (1990)].
• the origin of the microbial chromosomal DNA used in the above procedure is not particularly limited, but can be, for example, a prokaryote belonging to the genus Escherichia, Shewanella, Xanthomonas, or Pseudomonas, with a prokaryote belonging to the genus Escherichia (Escherichia coli) being preferred.
• DNAs encoding proteins having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound the DNA described in [6], [8], or [9] above can be obtained, for example, by searching various protein sequence databases for amino acid sequences that are 60% or more identical to the amino acid sequence represented by SEQ ID NO: 8 or 10, preferably 70% or more, 80% or more, 90% or more, 95% or more, more preferably 98% or more, and most preferably 99% or more identical, in that order; or by searching various gene sequence databases for nucleotide sequences that are 95% or more identical to the nucleotide sequence represented by SEQ ID NO: 3, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identical; and then using probe DNA or primer DNA that can be designed based on the amino acid sequence or nucleotide sequence obtained by the search, and a microorganism containing the DNA, using methods such as Southern hybridization or PCR, similar to the
• DNAs encoding proteins having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound the DNA described in [5] or [8] above can be obtained, for example, by subjecting a DNA containing a base sequence encoding the amino acid sequence represented by SEQ ID NO: 8 or 10, or a base sequence represented by SEQ ID NO: 3 or 9, as a template to error-prone PCR or the like.
• the DNA described in [5] or [8] above can also be obtained by PCR [Gene, 77, 51 (1989)] using a pair of PCR primers each having a base sequence at its 5' end designed to insert the desired mutation (deletion, substitution, insertion, or addition).
• PCR is performed using the DNA as a template with a sense primer corresponding to the 5' end of DNA containing a base sequence encoding the amino acid sequence represented by SEQ ID NO: 8 or 10, or a base sequence represented by SEQ ID NO: 3 or 9, and an antisense primer corresponding to the sequence immediately preceding (5' side) the mutation site, which has a sequence complementary to the mutation sequence at its 5' end, to amplify fragment A from the 5' end of the DNA to the mutation site (with the mutation introduced at the 3' end).
• PCR is performed using the DNA as a template with a sense primer corresponding to the sequence immediately following (3' side) the mutation site, which has the mutation sequence at its 5' end, and an antisense primer corresponding to the 3' end of the DNA, to amplify fragment B from the mutation site to the 3' end of the DNA with the mutation introduced at the 5' end.
• these amplified fragments are purified, mixed, and PCR is performed without adding a template or primer, the sense strand of amplified fragment A and the antisense strand of amplified fragment B hybridize because they share the same mutation site, and the PCR reaction proceeds using them as both a primer and template, amplifying the mutated DNA.
• the obtained DNA described in [4] to [9] above can be used as is or cleaved with an appropriate restriction enzyme or the like and inserted into a vector by standard methods.
• the resulting recombinant DNA can then be introduced into host cells, and the base sequence of the DNA can be determined by a commonly used base sequence analysis method, such as the dideoxy method [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)], or by analysis using a base sequence analyzer such as the Applied Biosystems 3500 Genetic Analyzer or the Applied Biosystems 3730 DNA Analyzer (both manufactured by Thermo Fisher Scientific).
• Examples of host cells that can be used to determine the base sequence of DNA include Escherichia coli DH5 ⁇ , Escherichia coli HST08 Premium, Escherichia coli HST02, Escherichia coli HST04 dam-/dcm-, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (all manufactured by Takara Bio Inc.), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli W3110, Escherichia coli MP347, Escherichia coli NM522,
• Examples of the above vectors include pBluescriptII KS(+), pPCR-Script Amp SK(+) (both manufactured by Agilent Technologies), pT7Blue (manufactured by Merck Millipore), pCRII (manufactured by Thermo Fisher Scientific), pCR-TRAP (manufactured by Gene Hunter), and pDIRECT [Nucleic Acids Res., 18, 6069 (1990)].
• full-length DNA can be obtained by Southern hybridization or other methods using the partial length DNA as a probe against a chromosomal DNA library.
• the desired DNA can be prepared by chemical synthesis using an NTS M series DNA synthesizer manufactured by Nippon Techno Service Co., Ltd., based on the determined DNA base sequence or the base sequence represented by SEQ ID NO: 3 or 9.
• the methods for producing glycosides of cytidine residue-containing compounds and methods for producing cytidine residue-containing compounds of the present disclosure may use genetically modified microorganisms.
• genetically modified microorganisms include genetically modified microorganisms in which the activity of a target protein is enhanced compared to that of a parent strain, such as genetically modified microorganisms in which the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is enhanced.
• the definition of such microorganisms and methods for producing such microorganisms are described.
• protein activity is enhanced may mean that the activity of the protein is enhanced compared to the parent strain.
• protein activity is enhanced may specifically mean that the activity of the protein per cell is enhanced compared to the parent strain.
• the term "parent strain” as used herein may refer to a type strain (i.e., the type strain of the species to which the microorganism belongs) that is the target of genetic recombination and transformation, or a strain that has already undergone recombination other than recombination that enhances the activity of the target protein relative to the type strain.
• types strain i.e., the type strain of the species to which the microorganism belongs
• examples of such strains include, but are not limited to, those exemplified in the description of (parent strain) below. That is, in one embodiment, the activity of the protein may be enhanced compared to the parent strain.
• enhanced protein activity may mean, more specifically, that the number of molecules of the protein per cell has increased compared to the parent strain, and/or that the function of the protein per molecule has been enhanced.
• activity in “enhanced protein activity” is not limited to the catalytic activity of the protein, but may also mean the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein.
• enhanced protein activity encompasses not only enhancing the activity of a target protein in a strain that originally has that activity, but also imparting the activity of that protein to a strain that does not originally have that activity. Furthermore, as long as the resulting protein activity is enhanced, the activity of the target protein originally possessed by the host may be reduced or eliminated before being imparted.
• the degree of enhancement of protein activity is not particularly limited, as long as the protein activity is enhanced compared to that of the parent strain.
• the protein activity may be increased, for example, by 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more compared to the activity of the protein in the parent strain.
• the protein may be produced by introducing a gene encoding the protein, and, for example, the protein may be produced to an extent that its activity can be measured.
• Enhanced protein activity can also be confirmed by confirming that the expression of the gene encoding the protein is increased compared to the parent strain. Increased gene expression can be confirmed by confirming that the transcription level of the gene is increased compared to the parent strain, and by confirming that the amount of protein expressed from the gene is increased compared to the parent strain.
• Modification that enhances protein activity can also be achieved by, for example, enhancing the specific activity of the protein. Enhancement of specific activity may also include desensitization to feedback inhibition. In other words, if a protein is subject to feedback inhibition by metabolites, the activity of the protein can be enhanced by having the host carry a gene encoding a mutant protein in which feedback inhibition is desensitized.
• “desensitization to feedback inhibition” can include cases where feedback inhibition is completely removed, as well as cases where feedback inhibition is reduced. Furthermore, “feedback inhibition being desensitized” (i.e., feedback inhibition being reduced or removed) is also referred to as “resistance to feedback inhibition.”
• Proteins with enhanced specific activity can be obtained, for example, by searching for and locating various organisms. Highly active proteins can also be obtained by introducing mutations into existing proteins. The mutations introduced may be, for example, the substitution, deletion, insertion, or addition of one or several amino acids at one or several positions in the protein.
• Microorganisms with enhanced activity of a target protein can also be produced by transforming a parent strain of a microorganism with recombinant DNA containing DNA encoding the protein, thereby increasing the expression of the DNA encoding the protein compared to the parent strain.
• Increased DNA expression is also referred to as “increased gene expression.”
• gene expression increases may mean that the expression of the gene is enhanced compared to the parent strain. As used herein, “gene expression increases” may specifically mean that the expression level of the gene per cell is enhanced compared to the parent strain. As used herein, “gene expression increases” may more specifically mean that the transcription level (mRNA level) of the gene is enhanced and/or the translation level (protein level) of the gene is enhanced.
• increasing gene expression can also be referred to as "enhancing gene expression.”
• gene expression may increase, for example, by 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more compared to the expression of the gene in the parent strain.
• increasing gene expression not only includes increasing the expression level of a gene of interest in a strain that originally expresses the gene, but also includes expressing the gene in a strain that does not originally express the gene of interest.
• “increasing gene expression” can mean, for example, introducing a gene of interest into a strain that does not harbor the gene and expressing the gene.
• Increasing gene expression can be achieved, for example, by increasing the copy number of the gene, selecting a promoter with a high frequency of transcription initiation, or by disrupting or enhancing the expression of transcriptional regulatory factors involved in controlling the expression of the target gene. That is, in the case of transcriptional regulatory factors that contribute to suppressing the expression of the target gene, this can be achieved by disruption, and in the case of transcriptional regulatory factors that contribute to promoting the expression of the target gene, this can be achieved by enhancement.
• microorganisms in which the copy number of DNA encoding a protein is increased compared to the parent strain include microorganisms in which the copy number of DNA encoding the protein on the chromosomal DNA is increased by transforming the parent strain of the microorganism with recombinant DNA containing the gene encoding the protein, and microorganisms in which the DNA encoding the protein is carried outside the chromosomal DNA as plasmid DNA.
• Recombinant DNA refers to, for example, DNA that is capable of autonomous replication within a parent strain and in which the DNA encoding the target protein (hereinafter also referred to as the target DNA) has been incorporated into an expression vector containing a promoter at a position where the DNA can be transcribed.
• the vector is not particularly limited as long as it is a suitable DNA molecule for introducing, amplifying, and expressing the target DNA into a host cell.
• plasmids other vectors such as artificial chromosomes, transposon-based vectors, and cosmids may also be used.
• the target DNA itself is also recombinant DNA containing the target DNA. If the recombinant DNA can be integrated into the chromosomal DNA of the parent strain, it does not need to contain a promoter.
• Recombinant DNA capable of autonomous replication in prokaryotes such as bacteria is preferably recombinant DNA composed of a promoter, ribosome binding sequence, target DNA, and transcription termination sequence. A gene that controls the promoter may also be included. It is preferable to use recombinant DNA in which the distance between the Shine-Dalgarno sequence (ribosome binding sequence) and the start codon has been adjusted to an appropriate distance (e.g., 6 to 18 bases).
• a transcription termination sequence is not necessarily required for expression of the DNA, but it is preferable to place a transcription termination sequence immediately downstream of the structural gene.
• examples of expression vectors include pColdI, pSTV28, pSTV29, pUC118 (all manufactured by Takara Bio Inc.), pMW119 (manufactured by Nippon Gene Co., Ltd.), pET21a, pCOLADuet-1, pCDFDuet-1, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore), pMAL-c5x (manufactured by New England Biolabs), pGEX-4T-1, pTrc99A (all manufactured by pGEMEX-1 (Promega), pQE-30, pQE80L (Qiagen), pET-3, pBluescript II SK(+), pBluescript II KS(-) (Agilent Technologies), pKYP10 (JP 58-110600 A), pKYP200 [
• any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Escherichia.
• Examples include promoters of genes involved in amino acid biosynthesis, such as the trp promoter and ilv promoter, and promoters derived from Escherichia coli or phages, such as the uspA promoter, lac promoter, PL promoter, PR promoter, and PSE promoter.
• Artificially engineered promoters can also be used, such as a promoter with two trp promoters in tandem, the tac promoter, the trc promoter, the lacT7 promoter, and the letI promoter.
• examples of expression vectors include pCG1 (Japanese Patent Publication No. 57-134500), pCG2 (Japanese Patent Publication No. 58-35197), pCG4 (Japanese Patent Publication No. 57-183799), pCG11 (Japanese Patent Publication No. 57-134500), pCG116, pCE54, pCB101 (all Japanese Patent Publication No. 58-105999), pCE51, pCE52, and pCE53 [all Molecular and General Genetics, 196, 175 (1984)].
• any promoter that functions in the cells of coryneform bacteria may be used, but an example is the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, pp. 674-679 (2000)].
• examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.
• any promoter that functions in the cells of a yeast strain may be used, including, for example, the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MF ⁇ 1 promoter, and CUP1 promoter.
• Recombinant DNA can be produced, for example, using the In-FusionTM HD Cloning Kit (Takara Bio Inc.), or by treating a DNA fragment prepared to encode the desired enzyme with a restriction enzyme and inserting it downstream of the promoter of the appropriate expression vector described above.
• the expression level of the protein encoded by the DNA can be improved by substituting bases in the DNA sequence to optimize codons for expression in host cells.
• Information on codon usage frequencies in parent strains used in the production method of the present invention is available through public databases.
• a genetically modified microorganism with enhanced activity of a target protein contains DNA that encodes the target protein (target DNA), or is obtained by transforming a parent strain with recombinant DNA that contains the target DNA.
• target DNA DNA that encodes the target protein
• parent strain recombinant DNA that contains the target DNA.
• ⁇ Parent plant> The parent strains used to construct the microorganisms described herein are not particularly limited.
• the parent strain is preferably a prokaryote or yeast strain, more preferably a prokaryote belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, and of these, a prokaryote belonging to the genus Escherichia (E. coli) is preferred.
• parent strains include Escherichia coli BL21 codon plus, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli BL21(DE3)pLysS (manufactured by Merck Millipore), Escherichia coli BL21(DE3) (manufactured by Novagen), Escherichia coli B (ATCC23226), Escherichia coli B RC912, Escherichia coli BL21, Escherichia coli DH5 ⁇ , Escherichia coli HST08 Premium, Escherichia coli HST02, Escherichia coli HST04 dam-/dcm-, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, Escherichia coli
• ⁇ Incorporation of DNA into parent strain Any method for introducing recombinant DNA into a parent strain to obtain a genetically modified microorganism by incorporating the target DNA can be used, as long as it is a method for introducing DNA into a parent strain, such as a method using calcium ions (Proc. Natl. Acad. Sci., USA, 69, 2110, 1972), the protoplast method (JP 63-248394 A), the electroporation method (Nucleic Acids Res., 16, 6127, 1988), the spheroplast method [Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)], and the lithium acetate method [J. Bacteriol., 153, 163 (1983)].
• a method using calcium ions Proc. Natl. Acad. Sci., USA, 69, 2110, 1972
• the protoplast method JP 63-248394 A
• the electroporation method Nucleic Acids Res., 16, 6
• the target DNA or recombinant DNA may be inserted into the genome or may exist as an autonomously replicating plasmid, but the target DNA is contained in a transcriptional state.
• a single parent strain may contain only one type of DNA, or two or more types of DNA.
• a method such as homologous recombination can be used. That is, DNA containing a portion of the chromosomal region where the target DNA is to be introduced is introduced into a microorganism, and homologous recombination occurs in a portion of that chromosomal region, thereby integrating the target DNA into the genome.
• a method using homologous recombination frequently used in Escherichia coli is a method using the lambda phage homologous recombination system to introduce recombinant DNA [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)].
• the chromosomal region where introduction occurs is not particularly limited, but is preferably a non-essential gene region or a non-genic region upstream of a non-essential gene region.
• Any method for introducing DNA into a host cell can be used to introduce the DNA into a microorganism cell, including, for example, the calcium ion method, the protoplast method, and electroporation.
• Whether a microorganism has been obtained by introducing recombinant DNA containing DNA encoding a target protein into a parent strain in an expressible manner can be confirmed, for example, by comparing the amount of transcription of the DNA in the microorganism by Northern blotting, or the amount of production of the protein in the microorganism by Western blotting, with the amount of transcription of the DNA or the amount of production of the protein in the parent strain before the DNA was introduced.
• the following method can be used to confirm that a microorganism created by expressing recombinant DNA containing DNA encoding a target protein into a parent strain using the method described above is a genetically modified microorganism capable of producing the target protein.
• the parent strain before the DNA introduction and the genetically modified microorganism created are each cultured in medium, and a cell extract containing the target protein is prepared from the resulting culture.
• the cell extract is contacted with a substrate, and the target protein is allowed to act on the substrate to produce a reaction product.
• the reaction product in the reaction solution is detected using an analytical method appropriate for the reaction product, thereby confirming that the created microorganism is a genetically modified microorganism capable of producing the target protein.
• the detected target product is greater than that of the parent strain before the DNA introduction, it can be said that the activity of the target protein has been enhanced compared to the parent strain before the DNA introduction.
• the desired product is produced using a microorganism
• those skilled in the art will understand that it is produced by an enzymatic method or a fermentation method, but is not limited to these.
• the "enzyme method” is a method for producing a target product by using a culture obtained by culturing a microorganism or a processed product of the culture as an enzyme source, and by causing the enzyme source and substrate to exist in an aqueous medium and react.
• a reaction system that uses microbial cells in a dormant or stationary state without proliferation as a catalyst (also called an enzyme source) is called a "microbial cell reaction method.”
• Fermentation is a method of producing a desired product within microorganisms using growing or growing microorganisms. In this case, it is necessary to add medium components for the growth or development of the microorganisms.
• the method for producing a glycoside of a cytidine residue-containing compound of the present disclosure includes using a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• a sugar is transferred from the sugar nucleotide to the cytidine residue-containing compound by using a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• the glycoside of the cytidine residue-containing compound is CDP-choline glycoside will be described in detail below.
• the method for producing CDP-choline glycoside of the present disclosure includes using a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• a sugar is transferred from a sugar nucleotide to a cytidine residue-containing compound by using a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• a protein having the activity of transferring glucose from UDP-glucose to CDP-choline glucose from UDP-glucose is transferred to CDP-choline to produce CDP-choline glucose glycoside.
• Methods for producing the CDP-choline glycoside disclosed herein include (I) a method for producing CDP-choline glycoside by fermentation, and (II) a method for producing CDP-choline glycoside by enzymatic methods, using a microorganism capable of producing a protein that has the activity of transferring a sugar from a sugar nucleotide to the above-mentioned cytidine residue-containing compound. Each production method is described below.
• the method for producing the CDP-choline glycoside of the present disclosure by fermentation includes a method for producing CDP-choline glycoside by culturing a microorganism described below in a medium and producing CDP-choline glycoside in the culture.
• the production method may include, for example, producing CDP-choline glycoside in the culture, accumulating it, and collecting CDP-choline glycoside from the culture.
• the microorganism used in producing the CDP-choline glycoside of the present disclosure by fermentation (hereinafter also referred to as "CDP-choline glycoside-producing microorganism (I)") is a microorganism that has a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound and produces CDP-choline glycoside.
• the CDP-choline glycoside-producing microorganism (I) may be a microorganism in which the activity of the protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is enhanced compared to the parent strain, and in which CDP-choline glycoside productivity is improved.
• a microorganism having a protein with the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound may be a microorganism that originally has a protein with the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, or, if the type strain used is a microorganism that does not originally have a protein with the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, it may be a genetically modified microorganism to which a protein with the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound has been artificially added.
• An example of a microorganism (I) for producing CDP-choline glycoside is a microorganism in which the "target protein" in the aforementioned "genetically modified microorganism with enhanced activity of the target protein” has the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, and preferably the "target protein” is an enzyme classified as at least one of the GT2 enzyme and GT28 enzyme in the CAZy classification described above, or at least one selected from the above [1] to [3].
• the microorganism can be produced by the method described above as a method for producing a "genetically modified microorganism with enhanced activity of a target protein" by using, as the "target DNA,” DNA encoding an enzyme classified as at least one of GT2 enzymes and GT28 enzymes in the CAZy classification, specifically, DNA exemplified above as DNA encoding E. coli-derived opgH, or DNA exemplified above as DNA encoding E. coli-derived opgH.
• the microorganism can be produced by the above-mentioned method for producing a "genetically modified microorganism with enhanced activity of a target protein" by using at least one selected from the above-mentioned [4] to [9] as the "target DNA.”
• Microorganisms can be cultured according to conventional methods.
• the medium for culturing the microorganisms may be either a natural or synthetic medium, as long as it contains a substrate for a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, or the starting material for the substrate, a carbon source, a nitrogen source, inorganic salts, etc. that can be utilized by the microorganism, and allows efficient cultivation of the microorganism.
• Substrates for proteins that have the activity of transferring sugars from sugar nucleotides to cytidine residue-containing compounds include, for example, sugar nucleotides and CDP-choline.
• sugar nucleotides include UDP-Glc, UDP-Gal, GDP-Man, UDP-GlcNAc, UDP-GalNAc, GDP-Fuc, and UDP-GlcA, with UDP-Glc being more preferred.
• These sugar nucleotides are also referred to as "sugar nucleotides such as UDP-glucose.”
• Starting materials for the substrate include, for example, orotic acid, CMP, choline chloride, and glucose.
• the carbon source may be anything that can be assimilated by the microorganism, and examples include carbohydrates such as glucose, fructose, sucrose, and molasses containing these, starch, and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.
• carbohydrates such as glucose, fructose, sucrose, and molasses containing these, starch, and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.
• Nitrogen sources include, for example, ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation bacteria, and their digested products.
• ammonia ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation bacteria, and their digested products.
• inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
• the microorganism used may be a microorganism that has the ability to produce (also referred to as "production ability") sugar nucleotides such as UDP-glucose and/or CDP-choline, which serve as substrates for proteins that have the activity of transferring sugars from sugar nucleotides to cytidine residue-containing compounds.
• a microorganism that has the ability to produce sugar nucleotides such as UDP-glucose and/or CDP-choline may be a type strain, or if the type strain does not have the ability to produce sugar nucleotides such as UDP-glucose and/or CDP-choline, it may be a strain to which the ability to produce sugar nucleotides such as UDP-glucose and/or CDP-choline has been artificially imparted.
• a microorganism capable of producing sugar nucleotides such as UDP-glucose and/or CDP-choline may be co-cultured with the CDP-choline glycoside-producing microorganism (I) to supply sugar nucleotides such as UDP-glucose and/or CDP-choline to the microorganism.
• Methods for artificially imparting or enhancing the ability to produce sugar nucleotides such as UDP-glucose and/or CDP-choline to a microorganism used as a parent strain include the following methods (a) to (e), and the above-mentioned known methods can be used alone or in combination.
• sugar nucleotides such as UDP-glucose and/or CDP-choline may be added to the medium.
• UDP-glucose is available, for example, as UDP-glucose (product code: 15602) manufactured by Funakoshi Co., Ltd.
• CDP-choline is available, for example, as CDP-choline (product code: C3438) manufactured by Tokyo Chemical Industry Co., Ltd.
• these microorganisms When culturing two or more types of microorganisms in the same medium, these microorganisms may be cultured simultaneously, or the remaining microorganisms may be cultured in the medium while one microorganism is being cultured or after the other microorganism has been cultured.
• a compound that serves as a substrate for CDP-choline may be added.
• a microorganism only has a partial CDP-choline-producing activity as described in WO 2007/023830 two or more types of microorganisms may be appropriately combined and used as a biocatalyst with CDP-choline-producing activity so that CDP-choline-producing activity can be obtained.
• two or more types of microorganisms can be combined.
• R in the CDP-choline glycoside represented by the general formula (1) is a hexose residue
• the R structure of the CDP-choline glycoside can be converted to a hexose residue by the action of an endogenous enzyme in the microorganism, thereby enabling the CDP-choline glycoside represented by the general formula (1) to be produced.
• R is a fructose residue
• glucose is transferred from UDP-Glc to CDP-choline to produce CDP-choline glucose glycoside, and then the glucose residue portion of CDP-choline glucose glycoside is converted to a fructose residue by the action of an enzyme such as isomerase, thereby producing the CDP-choline glycoside represented by the general formula (1).
• Cultivation is preferably carried out under aerobic conditions, such as shaking culture or submerged aeration and stirring culture.
• the culture temperature is preferably 15 to 40°C, and the culture time is usually 5 hours to 7 days.
• the pH during cultivation is preferably maintained at 3.0 to 9.0.
• the pH can be adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.
• antibiotics such as ampicillin or tetracycline may be added to the medium during cultivation as needed.
• an inducer may be added to the medium as needed.
• isopropyl- ⁇ -D-thiogalactopyranoside or the like may be added to the medium
• indoleacrylic acid or the like may be added to the medium.
• CDP-choline glycoside can be produced by culturing as described above and producing it in the culture.
• the amount of CDP-choline glycoside produced can be quantified using HPLC (e.g., Shimadzu Corporation's SPD-M20A analytical instrument) according to the method described in the "Analysis Examples" section below.
• CDP-choline glycoside can be collected from the culture by combining the usual ion exchange resin method, precipitation method, and other known methods. Furthermore, if CDP-choline glycoside accumulates within the bacterial cells, the cells can be disrupted, for example, by ultrasonication, and the cells can be removed by centrifugation. From the resulting supernatant, CDP-choline glycoside can be collected by the ion exchange resin method, for example.
• the method for producing CDP-choline glycoside of the present disclosure by an enzymatic method includes using, as an enzyme source, a culture of a microorganism capable of producing a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, or a processed product of the culture, and bringing the culture, together with a substrate or the enzyme source, into an aqueous medium to produce CDP-choline glycoside in the aqueous medium.
• the production method may include, for example, producing CDP-choline glycoside in the aqueous medium, accumulating the produced CDP-choline glycoside, and collecting the CDP-choline glycoside from the aqueous medium.
• CDP-choline glycoside-producing microorganism is a microorganism capable of producing a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• the CDP-choline glycoside-producing microorganism (II) may be a microorganism that originally has a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, as long as it is a microorganism capable of producing a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, or the type strain used may be a microorganism capable of producing a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• the microorganism may be a genetically modified microorganism in which a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound has been artificially imparted with the protein by enhancing the activity of the protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• the microorganism may also be a genetically modified microorganism in which the activity of the protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound has been further enhanced in a microorganism that originally has a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• the CDP-choline glycoside-producing microorganism (II) is a microorganism in which, in the aforementioned "genetically modified microorganism with enhanced activity of a target protein," the "target protein” is a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, and preferably the "target protein” is an enzyme classified as at least one of the GT2 enzyme and the GT28 enzyme in the CAZy classification described above, or at least one selected from the above [1] to [3].
• the microorganism can be produced by the method described above as a method for producing a "genetically modified microorganism with enhanced activity of a target protein" by using, as the "target DNA,” DNA encoding at least one of the GT2 enzyme and GT28 enzyme in the CAZy classification, specifically, DNA exemplified above as DNA encoding E. coli-derived opgH, DNA exemplified above as DNA encoding E. coli-derived opgH, etc.
• the microorganism can be produced by the above-mentioned method for producing a "genetically modified microorganism with enhanced activity of a target protein" by using at least one selected from the above-mentioned [4] to [9] as the "target DNA.”
• the microorganism may be capable of producing not only a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, but also CDP-choline, which is a substrate for CDP-choline glycoside, and enzymes (e.g., CCT, pyrG, etc.) required to produce CDP-choline from its starting substrates, such as choline, phosphorylcholine, uridine-5'-triphosphate (also referred to as CTP), and UTP.
• CDP-choline which is a substrate for CDP-choline glycoside
• enzymes e.g., CCT, pyrG, etc.
• a specific example of a recombinant microorganism capable of producing a protein that has the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is BL21(DE3)opgH::kan/pET21a-opgH, which is described below in the Examples.
• the method and medium for culturing the microorganism are the same as those described above in "(I) Method for producing CDP-choline glycoside by fermentation.”
• the "enzyme source” refers to a culture obtained by culturing the genetically modified microorganism of this embodiment described above, in which the activity of a protein that has the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound has been enhanced, or a processed product of the culture.
• examples of processed culture products include concentrates of the above-mentioned cultures, dried products of the cultures, bacterial cells obtained by centrifuging or filtering the cultures, dried products of the bacterial cells, freeze-dried products of the bacterial cells, surfactant-treated products of the bacterial cells, solvent-treated products of the bacterial cells, enzyme-treated products of the bacterial cells, and immobilized products of the bacterial cells, which contain live bacterial cells that retain the same functions as the culture as an enzyme source, as well as ultrasonicated products of the bacterial cells, mechanically ground products of the bacterial cells, crude enzyme extracts obtained from the treated bacterial cells, and purified enzymes obtained from the treated bacterial cells.
• concentrates of the above-mentioned cultures dried products of the cultures, bacterial cells obtained by centrifuging or filtering the cultures, dried products of the bacterial cells, freeze-dried products of the bacterial cells, surfactant-treated products of the bacterial cells, solvent-treated products of the bacterial cells, enzyme-treated products of the bacterial cells, and immobilized products of the bacterial cells, which contain live bacterial cells that retain the same functions as the cultures as an enzyme source, as well as ultrasonicated products of the bacterial cells and mechanically ground products of the bacterial cells.
• concentrates of the above-mentioned cultures dried products of the cultures, bacterial cells obtained by centrifuging or filtering the cultures, dried products of the bacterial cells, freeze-dried products of the bacterial cells, surfactant-treated products of the bacterial cells, solvent-treated products of the bacterial cells, enzyme-treated products of the bacterial cells, and immobilized products of the bacterial cells, which contain live bacterial cells that retain the same functions as the cultures as an enzyme source.
• the amount of the protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound as an enzyme source is 0.01 mg/L to 10 g/L, preferably 0.1 mg/L to 1 g/L.
• the substrate concentration is preferably 0.1 mM to 10 M, and more preferably 1 mM to 1 M.
• substrates include sugar nucleotides and CDP-choline.
• the starting materials for the substrates may be the substances described in "(I) Method for producing CDP-choline glycoside by fermentation.”
• sugar nucleotides include UDP-Glc, UDP-Gal, GDP-Man, UDP-GlcNAc, UDP-GalNAc, GDP-Fuc, and UDP-GlcA, with UDP-Glc being preferred.
• Aqueous media include, for example, water, buffer solutions such as phosphate, carbonate, acetate, borate, citrate, and Tris, alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, and amides such as acetamide.
• buffer solutions such as phosphate, carbonate, acetate, borate, citrate, and Tris
• alcohols such as methanol and ethanol
• esters such as ethyl acetate
• ketones such as acetone
• amides such as acetamide.
• the culture medium of the microorganism used as the enzyme source can also be used as an aqueous medium.
• a chelating agent such as phytic acid, a surfactant, or an organic solvent may be added as needed.
• Surfactants include nonionic surfactants such as polyoxyethylene octadecylamine (e.g., Nymeen S-215, manufactured by NOF Corporation), cationic surfactants such as cetyltrimethylammonium bromide or alkyldimethyl benzylammonium chloride (e.g., Cationic F2-40E, manufactured by NOF Corporation), anionic surfactants such as lauroyl sarcosinate, and tertiary amines such as alkyldimethylamine (e.g., Tertiary Amine FB, manufactured by NOF Corporation), and any surfactant that promotes the production of CDP-choline may be used.
• One or more surfactants may be used in combination. Surfactants are typically used at a concentration of 0.1 to 50 g/L.
• Organic solvents include xylene, toluene, aliphatic alcohols, acetone, and ethyl acetate, and are typically used at a concentration of 0.1 to 50 ml/l.
• the CDP-choline production reaction is carried out in an aqueous medium at a pH of 5 to 10, preferably 6 to 8, and at a temperature of 20 to 50°C for 1 to 96 hours.
• adenine, adenosine-5'-monophosphate (AMP), adenosine-5'-triphosphate (ATP), magnesium sulfate, magnesium chloride, etc. may be added.
• Adenine, AMP, and ATP are typically used at concentrations of 0.01 to 100 mM.
• the CDP-choline glycoside produced in the aqueous medium can be quantified and collected by the method described above in "(I) Method for producing CDP-choline glycoside by fermentation.”
• the disclosed methods for producing a cytidine residue-containing compound and for inhibiting the production of glycosides of a cytidine residue-containing compound may use genetically modified microorganisms.
• genetically modified microorganisms include genetically modified microorganisms in which the activity of a target protein is reduced or eliminated compared to the parent strain, i.e., genetically modified microorganisms in which the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or eliminated.
• the definition of such microorganisms and methods for producing such microorganisms are described.
• Examples of the target protein in a "genetically modified microorganism in which the activity of the target protein is reduced or deleted compared to the parent strain" include proteins described in "Proteins having the activity of transferring sugars from sugar nucleotides to cytidine residue-containing compounds and DNA encoding said proteins.”
• protein activity is reduced or absent may mean that the activity of the protein is reduced or absent compared to the parent strain.
• protein activity is reduced or absent specifically means that the activity of the protein per cell is reduced or absent compared to the parent strain.
• the activity of the protein may be reduced to 80% or less, preferably 50% or less, more preferably 30% or less, even more preferably 20% or less, particularly preferably 10% or less, and most preferably 0% compared to the parent strain.
• the term "parent strain” as used herein may refer to a type strain (i.e., a type strain of the species to which the microorganism belongs) that is the target of genetic recombination, transformation, etc., or a strain to which a modification other than a modification that reduces or deletes the activity of the target protein has already been added to the type strain, and the strains exemplified in the above description of ⁇ parent strain> can be used, but are not limited to these. That is, in one embodiment, the activity of the protein may be reduced or absent compared to the parent strain.
• “activity” does not necessarily mean the catalytic activity of the protein, but may also mean the transcription level (mRNA level) or translation level (protein level) of the gene encoding the protein.
• the expression level of the gene may be reduced to 80% or less, preferably 50% or less, more preferably 30% or less, even more preferably 20% or less, particularly preferably 10% or less, and most preferably 0%.
• Recombination that reduces the activity of a protein can be achieved, for example, by disrupting part or all of the gene encoding the protein, introducing a stop codon, modifying expression regulatory sequences such as promoters or Shine-Dalgarno (SD) sequences, manipulating factors involved in expression control, introducing mutations into the coding region, mutagenesis, etc. to reduce the expression of the gene.
• modifying expression regulatory sequences such as promoters or Shine-Dalgarno (SD) sequences
• manipulating factors involved in expression control introducing mutations into the coding region, mutagenesis, etc.
• Gene disruption can be achieved, for example, by deleting (deleting) a gene on a chromosome.
• Examples include a method using PCR to knock out a specific gene [Baba T. et al., Mol Systems Biol (2006)] and a method using the lambda phage homologous recombination system [Proc. Natl. Acad. Sci. USA, 97, 6640-6645 (2000)].
• a decrease in protein activity can be confirmed by measuring the activity or protein amount per cell of the protein.
• a decrease in protein activity can also be confirmed by confirming a decrease in expression of the gene encoding the protein.
• a decrease in gene expression can be confirmed by confirming a decrease in the transcription level of the gene or a decrease in the amount of protein expressed from the gene.
• the reduction in the transcription level of a gene can be confirmed by comparing the amount of mRNA transcribed from the gene with that of the parent strain. Methods for evaluating the amount of mRNA include Northern hybridization and RT-PCR (Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)).
• the amount of mRNA may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of the parent strain.
• the reduction in protein amount can be confirmed by Western blotting using antibodies (Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)).
• the amount of protein (e.g., number of molecules per cell) may be reduced to, for example, less than 50%, less than 20%, less than 10%, less than 5%, or 0% of that of the parent strain.
• gene disruption can be confirmed by determining the base sequence, restriction enzyme map, or full length of part or all of the gene.
• the above-mentioned methods for reducing protein activity can be used to reduce the activity of any protein (e.g., by-product-producing enzymes) or the expression of any gene (e.g., a gene encoding that protein).
• any protein e.g., by-product-producing enzymes
• any gene e.g., a gene encoding that protein
• the method for producing a cytidine residue-containing compound of the present disclosure includes preparing a genetically modified microorganism in which the activity of a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or eliminated compared to that of a parent strain, and producing the cytidine residue-containing compound in at least one of a culture supernatant and intracellular space using the genetically modified microorganism.
• the genetically modified microorganism may be a genetically modified microorganism in which the activity of a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or eliminated compared to that of a parent strain and which has the ability to produce a cytidine residue-containing compound.
• the method for producing CDP-choline of the present disclosure includes preparing a genetically modified microorganism in which the activity of a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or eliminated compared to the activity of a parent strain, and producing CDP-choline in at least one of a culture supernatant and intracellular space using the genetically modified microorganism.
• methods for producing CDP-choline include (III) a method for producing CDP-choline by fermentation, and (IV) a method for producing CDP-choline by an enzymatic method.
• Methods for producing CDP-choline by fermentation include a method for producing CDP-choline by culturing a genetically modified microorganism described below in a medium and producing CDP-choline in the culture.
• the production method may include, for example, producing CDP-choline in the culture, accumulating it, and collecting CDP-choline from the culture.
• the microorganism used to produce CDP-choline by fermentation (hereinafter also referred to as "recombinant microorganism for producing CDP-choline (III)") is a genetically modified microorganism in which the activity of a protein that transfers a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or absent compared to the activity of the parent strain, and which has the ability to produce CDP-choline.
• CDP-choline glycoside by transferring glucose from UDP-glucose to CDP-choline can be suppressed, making it possible to efficiently produce CDP-choline.
• An example of a recombinant microorganism (III) for producing CDP-choline is a microorganism in which, in the aforementioned "genetically modified microorganism in which the activity of a target protein is reduced or deleted," the "target protein” is a protein that has the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, and preferably the "target protein” is an enzyme classified as at least one of the GT2 enzyme and GT28 enzyme in the CAZy classification described above, or at least one selected from the above [1] to [3], and is capable of producing CDP-choline.
• Such a microorganism can be produced by the method described above as a method for producing a "genetically modified microorganism in which the activity of a target protein is reduced or deleted.”
• the recombinant microorganism (III) for producing CDP-choline is capable of producing CDP-choline
• a microorganism capable of producing CDP-choline is used as the parent strain.
• the microorganism capable of producing CDP-choline may be a type strain, or if the type strain does not have the ability to produce CDP-choline, it may be a strain to which the ability to produce CDP-choline has been artificially imparted.
• the ability to produce CDP-choline may also be imparted to a microorganism that originally has the ability to produce CDP-choline.
• CDP-choline may be produced by combining two or more types of microorganisms.
• CDP-choline is synthesized from CTP and phosphorylcholine by choline phosphate cytidyltransferase [EC2.7.7.15] (hereafter abbreviated as CCT).
• CCT choline phosphate cytidyltransferase
• Microorganisms capable of producing CDP-choline are required to have the activity to synthesize CTP and phosphorylcholine, as well as CCT activity.
• Phosphorylcholine is produced from choline and ATP by choline kinase [EC2.7.1.32] (hereafter abbreviated as CKI). Therefore, microorganisms capable of producing phosphorylcholine are required to have CKI activity.
• CTP is produced according to the pyrimidine nucleic acid biosynthetic pathway, where orotic acid is produced from aspartic acid and carbamoyl phosphate, followed by orotidine-5'-monophosphate (OMP), uridine-5'-monophosphate (UMP), uridine-5'-diphosphate (UDP), and uridine-5'-triphosphate (UTP) from UDP, via which CTP is produced by cytidine-5'-triphosphate synthetase [EC 6.3.4.2] (PyrG).
• OMP orotidine-5'-monophosphate
• UMP uridine-5'-monophosphate
• UDP uridine-5'-diphosphate
• UDP uridine-5'-triphosphate
• Efficient CTP synthesis requires the use of carbamoyl phosphate synthase [EC 6.3.5.5] to produce carbamoyl phosphate from glutamine, and aspartate carbamoyltransferase [EC 6.3.5.5] to synthesize carbamoyl phosphate from carbamoyl phosphate and aspartic acid, synthesizing N-carbamoyl aspartate from carbamoyl phosphate and aspartic acid.
• dihydroorotase [EC 3.5.2.3] that produces dihydroorotate from carbamoyl aspartate, dihydroorotate dehydrogenase [EC 1.3.5.2] that produces orotate from dihydroorotate, orotate phosphoribosyltransferase [EC 2.4.2.10] that has the activity of producing orotidine-5'-monophosphate (OMP) from orotate and PRPP, and orotidine-5'-monophosphate decarboxylase [EC 4.1.1.23] that has the activity of producing uridine-5'-monophosphate (UMP) from OMP.
• OMP orotidine-5'-monophosphate
• UMP uridine-5'-monophosphate
• the parent strain capable of producing CDP-choline used in the recombinant microorganism (III) for producing CDP-choline may be a genetically modified microorganism in which the activity of at least one of the enzymes required for the CTP synthetic pathway, including PyrG, and at least one of CCT and CKI, is enhanced.
• a microorganism is, for example, a microorganism described above as a "genetically modified microorganism with enhanced activity of a target protein," in which the "target protein” is at least one of the enzymes required for the CTP synthetic pathway, including PyrG, and at least one of CCT and CKI.
• Such a microorganism can be produced by the method described above as a "genetically modified microorganism with enhanced activity of a target protein" by using DNA encoding at least one of the enzymes required for the CTP synthesis pathway, including PyrG, and at least one of CCT and CKI, as the "target DNA.”
• Whether a microorganism is capable of producing CDP-choline can be confirmed by culturing the microorganism in a medium and detecting CDP-choline using the HPLC described below (for example, the SPD-M20A analytical device manufactured by Shimadzu Corporation).
• the culture conditions (aerobic conditions, temperature, time, pH), substances that may be added to the medium, and the method for collecting CDP-choline from the culture in "(III) Method for producing CDP-choline by fermentation” are the same as those described above in "(I) Method for producing CDP-choline by fermentation.”
• CDP-choline can be produced by culturing as described above and generating CDP-choline in the culture.
• the amount of CDP-choline produced can be quantified using HPLC (e.g., an SPD-M20A analyzer manufactured by Shimadzu Corporation) according to the method described in the "Analysis Examples" section below.
• CDP-choline can be collected from the culture by combining the usual ion exchange resin method, precipitation method, and other known methods. Furthermore, if CDP-choline accumulates within the bacterial cells, for example, the cells can be disrupted by ultrasonication or the like, and the cells removed by centrifugation. From the resulting supernatant, CDP-choline can be collected by the ion exchange resin method or the like.
• Method for Producing CDP-choline by an Enzymatic Method examples include a method for producing CDP-choline, which comprises causing a culture of a genetically modified microorganism described below or a treated product of the culture to be present in an aqueous medium together with a substrate or the enzyme source as an enzyme source for a reaction to produce CDP-choline, and producing CDP-choline in the aqueous medium.
• the microorganism used in the enzymatic production of CDP-choline (hereinafter also referred to as "recombinant microorganism for producing CDP-choline (IV)”) is a microorganism in which the activity of a protein that transfers a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or absent compared to the activity of the parent strain.
• a culture of a recombinant microorganism or a processed product of the culture is used as the enzyme source for the reaction that produces CDP-choline
• a genetically modified microorganism in which the activity of a protein that transfers a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or absent compared to the activity of the parent strain, the production of CDP-choline glycoside by transferring glucose from UDP-glucose to CDP-choline can be suppressed, and CDP-choline can be produced efficiently.
• An example of a recombinant microorganism (IV) for producing CDP-choline is a microorganism in which the "target protein" in the aforementioned "genetically modified microorganism having reduced or absent activity of the target protein" is a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, and preferably the "target protein” is an enzyme classified as at least one of the GT2 enzyme and GT28 enzyme in the CAZy classification described above, or at least one selected from the above [1] to [3].
• Such a microorganism can be produced by the method described above as a method for producing a "genetically modified microorganism having reduced or absent activity of the target protein.”
• the "enzyme source” refers to a culture obtained by culturing the genetically modified microorganism of the present embodiment described above, which has the activity of an enzyme involved in the CDP-choline biosynthetic pathway, or a processed product of the culture.
• enzymes involved in the CDP-choline biosynthetic pathway include pyrG, CCT, and CKI.
• a method for producing a recombinant microorganism (IV) for producing CDP-choline for example, when CDP-choline is produced using CTP and phosphorylcholine as substrates, if the recombinant microorganism capable of producing a protein with CCT activity as an enzyme source is a microorganism that originally has the activity of a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, the protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or deleted compared to the parent strain.
• a method for producing CDP-choline using an enzymatic method may include bringing a culture of this microorganism or a treated product of the culture and the substrates CTP and phosphorylcholine into an aqueous medium, producing CDP-choline, allowing it to accumulate, and collecting CDP-choline from the aqueous medium.
• a genetically modified microorganism that is a microorganism in which the activity of a protein that has the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or absent compared to the parent strain and that serves as an enzyme source that can be used in the CDP-choline production reaction may be a recombinant microorganism that has the ability to produce a protein with the above-mentioned CCT activity, or, when another substrate is used, may be a recombinant microorganism that has the ability to produce a protein with enzymatic activity that can react with that substrate.
• the method and medium for culturing the microorganism are the same as those described above in "(I) Method for producing CDP-choline glycoside by fermentation.”
• the treatment of the culture and the reaction conditions are the same as those described above in “(II) Method for producing CDP-choline glycoside by an enzymatic method.”
• the CDP-choline produced in the aqueous medium can be quantified and collected by the method described above in "(III) Method for producing CDP-choline by fermentation.”
• the method for inhibiting the production of glycosides of cytidine residue-containing compounds of the present disclosure comprises preparing a genetically modified microorganism in which the activity of a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or eliminated compared to the activity of a parent strain.
• the genetically modified microorganism may be a genetically modified microorganism in which the activity of a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or eliminated compared to the activity of a parent strain, and which has the ability to produce a cytidine residue-containing compound.
• a microorganism used to inhibit the production of glycosides of cytidine residue-containing compounds is also referred to as a "recombinant microorganism for inhibiting the production of glycosides of cytidine residue-containing compounds.”
• each microorganism is cultured and supplied with an appropriate substrate and a glycosyl donor, for example, CDP-choline and UDP-glucose to produce CDP-choline glucose glycoside when confirming the activity of a protein to transfer glucose from UDP-glucose to CDP-choline.
• a glycosyl donor for example, CDP-choline and UDP-glucose
• the amount of CDP-choline glucose glycoside produced in the reaction solution is analyzed by the analytical method described in the "Analysis Examples” below, and the amount of CDP-choline glucose glycoside produced in the genetically modified microorganism is compared with that in a control microorganism that has not been modified to reduce the activity, thereby confirming that the activity of transferring a sugar from a sugar nucleotide in the genetically modified microorganism is reduced or absent.
• An example of a "recombinant microorganism for inhibiting the production of glycosides of cytidine residue-containing compounds” is a microorganism similar to the aforementioned “genetically modified microorganism in which the activity of a target protein is reduced or deleted," in which the "target protein” is a protein that has the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, and preferably the "target protein” is an enzyme classified as at least one of the GT2 enzyme and GT28 enzyme in the CAZy classification described above, or at least one selected from the above [1] to [3].
• Such a microorganism can be produced by the method described above as a method for producing a "genetically modified microorganism in which the activity of a target protein is reduced or deleted.”
• inhibiting the production of glycosides of cytidine residue-containing compounds may also mean that the ratio of CDP-choline glycosides to the amount of CDP-choline contained in a composition containing CDP-choline, which is produced using a "genetically modified microorganism in which the activity of a target protein is reduced or deleted," is equal to or lower than a certain value.
• a composition produced using a recombinant microorganism for inhibiting the production of glycosides of cytidine residue-containing compounds is also referred to as composition B containing CDP-choline.
• composition B containing CDP-choline may be any composition containing CDP-choline and may further contain a CDP-choline glycoside.
• Composition B containing CDP-choline may be a culture produced by culturing a genetically modified microorganism in a medium to produce CDP-choline by the fermentation method described in "(III) Method for producing CDP-choline by fermentation” above, or a purified version of the culture. It may also be the aqueous medium produced by the enzymatic method described in "(IV) Method for producing CDP-choline by an enzymatic method," or a purified version of the aqueous medium.
• purification refers to a procedure of removing any components other than CDP-choline and CDP-choline glycoside from the culture or aqueous medium containing CDP-choline or CDP-choline and CDP-choline glycoside.
• the abundance ratio of the amount of CDP-choline glycoside produced relative to the amount of CDP-choline contained in Composition B is preferably 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 3% or less, or 1% or less, with the lower limit being not particularly limited, but preferably 0.0005%.
• the amount of CDP-choline glycoside produced depends on the amount of UDP-Glc as a substrate; when there is a sufficient amount of UDP-Glc, the amount of CDP-choline glycoside produced tends to be high, whereas when there is an insufficient amount of UDP-Glc, the amount of CDP-choline glycoside produced also tends to be low.
• the "recombinant microorganism for inhibiting the production of glycosides of cytidine residue-containing compounds” is not particularly limited, but preferably has the ability to produce cytidine residue-containing compounds.
• a microorganism capable of producing cytidine residue-containing compounds may be a type strain, or if the type strain does not have the ability to produce cytidine residue-containing compounds, it may be a strain to which the ability to produce cytidine residue-containing compounds has been artificially imparted.
• the ability to produce cytidine residue-containing compounds may also be imparted to a microorganism that originally has the ability to produce cytidine residue-containing compounds.
• the genetically modified microorganism described above in the "Method for producing cytidine diphosphate choline" can be used as a microorganism capable of producing the cytidine residue-containing compound, cytidine diphosphate choline.
• the inhibition of the production of glycosides of cytidine residue-containing compounds can be confirmed by culturing the microorganism in a medium, detecting and quantifying the glycosides of cytidine residue-containing compounds using the aforementioned HPLC (for example, the Shimadzu SPD-M20A analytical device), and comparing them with those of the parent strain.
• HPLC for example, the Shimadzu SPD-M20A analytical device
• the glycoside of the cytidine residue-containing compound is preferably CDP-choline glycoside, and more preferably CDP-choline glucose glycoside.
• a cytidine diphosphate choline glycoside represented by the following general formula (1), a salt thereof, an N-oxide thereof, or a solvate thereof:
• R is a sugar residue.
• ⁇ 3>> A composition containing at least one of the cytidine diphosphate choline glycoside, a salt thereof, an N-oxide thereof, and a solvate thereof according to ⁇ 1>> or ⁇ 2>> above.
• ⁇ 4>> A method for producing a glycoside of a cytidine residue-containing compound, using a protein having an activity of transferring a sugar from a sugar nucleotide to the cytidine residue-containing compound.
• ⁇ 5>> The method for producing a glycoside of a cytidine residue-containing compound according to the above ⁇ 4>, wherein the glycoside of the cytidine residue-containing compound is the cytidine diphosphate choline glycoside according to the above ⁇ 1>> or ⁇ 2>>.
• ⁇ 6>> The method for producing a glycoside of a cytidine residue-containing compound according to ⁇ 4>> or ⁇ 5>> above, wherein the protein having the activity of transferring a sugar from a sugar nucleotide to the cytidine residue-containing compound is an enzyme classified as at least one of a GT2 enzyme and a GT28 enzyme in the CAZy classification.
• ⁇ 7>> The method for producing a glycoside of a cytidine residue-containing compound according to ⁇ 4>> or ⁇ 5>> above, wherein the protein having the activity of transferring a sugar from a sugar nucleotide to the cytidine residue-containing compound is at least one selected from the following [1] to [3]: [1] A protein comprising the amino acid sequence represented by SEQ ID NO: 8 or 10. [2] A mutant protein comprising an amino acid sequence represented by SEQ ID NO: 8 or 10 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having the activity of transferring a sugar from a sugar nucleotide to a compound containing a cytidine residue.
• a homologous protein comprising an amino acid sequence having 60% or more identity with the amino acid sequence represented by SEQ ID NO: 8 or 10, and having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• a genetically modified microorganism that is either (A) or (B) below.
• (B) A genetically modified microorganism in which the activity of a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or absent compared to the activity of the parent strain.
• ⁇ 10>> The above-mentioned ⁇ 10>>, wherein the protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is at least one selected from the following [1] to [3]: A genetically modified microorganism according to ⁇ 8> or ⁇ 9>.
• a protein comprising the amino acid sequence represented by SEQ ID NO: 8 or 10.
• a mutant protein comprising an amino acid sequence represented by SEQ ID NO: 8 or 10 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having the activity of transferring a sugar from a sugar nucleotide to a compound containing a cytidine residue.
• a homologous protein comprising an amino acid sequence having 60% or more identity with the amino acid sequence represented by SEQ ID NO: 8 or 10, and having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.
• ⁇ 12>> The genetically modified microorganism according to any one of ⁇ 8>> to ⁇ 11>> above, wherein the protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is Glucans biosynthesis glucosyltransferase H.
• ⁇ 13>> The genetically modified microorganism according to any one of ⁇ 8>> to ⁇ 12>> above, wherein the genetically modified microorganism is Escherichia coli.
• ⁇ 14>> A method for producing a cytidine residue-containing compound, comprising preparing the genetically modified microorganism according to any one of the above ⁇ 8>> to ⁇ 13>>, and producing the cytidine residue-containing compound in at least one of a culture supernatant and intracellular space using the genetically modified microorganism.
• ⁇ 15>> The method for producing a cytidine residue-containing compound according to the above ⁇ 14>>, wherein the cytidine residue-containing compound is cytidine diphosphate choline.
• ⁇ 16>> A method for inhibiting production of a glycoside of a cytidine residue-containing compound, comprising preparing the genetically modified microorganism of any one of ⁇ 8>> to ⁇ 13>> above.
• ⁇ 17>> The method for inhibiting production of a glycoside of a cytidine residue-containing compound according to ⁇ 16>> above, wherein the glycoside of the cytidine residue-containing compound is a cytidine diphosphate choline glycoside represented by the following general formula (1):
• R is a sugar residue.
• composition comprising cytidine diphosphate choline produced using the genetically modified microorganism according to any one of ⁇ 8>> to ⁇ 13>> above, wherein the ratio of the amount of cytidine diphosphate choline glycoside produced to the amount of cytidine diphosphate choline produced is 35% or less.
• a method comprising preparing the genetically modified microorganism according to any one of the above items ⁇ 8>> to ⁇ 13>>, and producing a composition using the microorganism, In the composition, the ratio of cytidine diphosphate choline glycoside to cytidine diphosphate choline is 35% or less. Method for producing the composition.
• composition according to ⁇ 18> wherein the ratio of the amount of cytidine diphosphate choline glycoside produced to the amount of cytidine diphosphate choline produced is 0.0005% or more and 35% or less.
• ⁇ 21>> The method for producing a composition according to ⁇ 19>, wherein the ratio of cytidine diphosphate choline glycoside to cytidine diphosphate choline in the composition is 0.0005% or more and 35% or less.
• Example 1 Construction of an opgH-non-expressing strain (1) The opgH gene was disrupted using Escherichia coli BL21(DE3) strain (Novagen) as a host. For the disruption, a kanamycin resistance gene cassette containing the upstream and downstream regions of opgH was amplified using primers of SEQ ID NO: 1 and SEQ ID NO: 2, using genomic DNA from an opgH-disrupted strain from the KEIO collection (Baba T. et al. (2006) Mol Systems Biol, doi:10.1038/msb4100050.) as a template.
• This fragment was used to disrupt the opgH gene of strain BL21(DE3) using the Lambda-Red recombination system (Datsenko KA and Wanner BL (2000) Proc Natl Acad Sci USA 97:6640-6645).
• the pKD46 plasmid was introduced into strain BL21(DE3), and the strain was cultured in the presence of arabinose to express the ⁇ phage-derived recombinase on pKD46.
• a kanamycin resistance gene cassette containing the upper and lower regions of opgH was then introduced by electroporation, and an opgH-disrupted strain was obtained by selection in the presence of kanamycin.
• the resulting opgH-disrupted strain was cultured at 37°C to remove pKD46, resulting in a non-opgH expression strain (BL21 (DE3) opgH::kan) in which the opgH gene was deleted and opgH was not expressed.
• Example 2 Construction of opgH expression plasmid A plasmid that expresses the opgH gene (SEQ ID NO: 3) of E. coli was constructed by the following procedure. An opgH gene fragment was obtained by amplifying Escherichia coli genomic DNA as a template using primers of SEQ ID NO: 4 and SEQ ID NO: 5. Next, using the plasmid pET21a (Novagen) as a template, PCR was performed with the primers of SEQ ID NO: 6 and SEQ ID NO: 7. The resulting fragment and the E. coli opgH gene fragment prepared above were ligated using an In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pET21a-opgH.
• SEQ ID NO: 3 An opgH gene fragment was obtained by amplifying Escherichia coli genomic DNA as a template using primers of SEQ ID NO: 4 and SEQ ID NO: 5.
• Example 3 Construction of an opgH Non-Expressing Strain and an opgH Expressing Strain
• One strain of the opgH non-expressing strain (BL21 (DE3) opgH::kan) obtained in Example 1 was transformed by electroporation using the empty vector pET21a as a control and the other strain with the opgH expression plasmid (pET21a-opgH) constructed in Example 2, followed by selection with ampicillin to obtain an opgH non-expressing strain into which the empty vector had been introduced (BL21 (DE3) opgH::kan/pET21a) and an opgH expressing strain into which the opgH expression plasmid had been introduced (BL21 (DE3) opgH::kan/pET21a-opgH).
• 500 ⁇ L of the culture was inoculated into a baffled Erlenmeyer flask containing 50 ml of TB+Glc medium (Bactotryptone (Difco) 12 g/L, yeast extract (Difco) 24 g/L, glucose 10 g/L, potassium dihydrogen phosphate 2.31 g/L, dipotassium monohydrogen phosphate 12.54 g/L) containing 100 mg/ml ampicillin, and cultured at 30°C and 220 rpm for 24 hours. Two hours after the start of culture, IPTG was added to a final concentration of 0.1 mM. 50 ml of the culture was centrifuged to obtain wet cells.
• TB+Glc medium Bacillus subtilis
• the above-mentioned supernatant protein solution was added to a reaction solution containing a final concentration of 100 mM Tris-HCl (pH 8.0) (Fujifilm Wako Pure Chemical Industries, Ltd.), 10 mM CDP-choline (Kyowa Hakko Bio Co., Ltd.), and 10 mM UDP-glucose (Sigma-Aldrich) so that the total soluble protein concentration was 1 mg/ml, and the reaction was carried out by leaving it to stand at 30°C.
• Tris-HCl pH 8.0
• 10 mM CDP-choline Kerowa Hakko Bio Co., Ltd.
• UDP-glucose Sigma-Aldrich
• CDP-choline is glycosidized by transfer from UDP-glucose to produce CDP-choline glycoside, and that the production of CDP-choline glycoside is suppressed when microorganisms do not express opgH. This finding is thought to contribute to the production and suppression of CDP-choline glycoside.
• Example 5 Use of an opgH Non-Expressing Strain in CDP-Choline Production Using Escherichia coli as a host, a microorganism capable of producing CDP-choline is produced based on a known method described, for example, in Applied Microbiological Biotechnology, 101: 2017, 1409-1417. This microorganism capable of producing CDP-choline is an opgH-expressing strain. Using this microorganism as a host, an opgH non-expressing strain is constructed, which is a genetically modified microorganism in which opgH activity is reduced or deleted.
• This opgH non-expressing strain is cultured, and analysis of the culture medium after the completion of the culture confirms that the production of CDP-choline and the production of CDP-choline glycosides are suppressed.
• the ratio of CDP-choline glycoside to the amount of CDP-choline produced was 36%, whereas when the opgH-non-expressing strain was used, the ratio of CDP-choline glycoside to the amount of CDP-choline produced was 0%.
• the ratio of CDP-choline glycoside to the amount of CDP-choline produced is 4.3%, whereas when an opgH non-expressing strain is used, the ratio of CDP-choline glycoside to the amount of CDP-choline produced is 0%.
• a strain in which the activity of opgH, a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, is reduced or deleted compared to the activity of the parent strain is shown to be able to suppress the production of glycosides of the cytidine residue-containing compound.
• CDP-choline can be efficiently produced by using a strain in which the activity of a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or deleted compared to the activity of the parent strain.
• Example 6 Structural analysis of CDP-choline glycoside CDP-choline glycoside was produced with reference to the method described in Example 4, and separated and purified by HPLC according to the analytical method shown in the analytical examples. Based on the HPLC retention time, the compound to be analyzed was named RT22.
• DI-MS direct infusion mass spectrometry
• NMR nuclear magnetic resonance spectrometry
• Mass spectrometer Waters Synapt G2-S model Ionization method: Electrospray ionization Measurement mode: Positive mode and negative mode Measurement mass range: m/z 50 to 1000
• AVANCE 500 manufactured by Bruker Biospin Nuclide: 1 H, 13 C Measurement: 1D-TOCSY, COSY, HMQC, HMBC, DEPT Solvent: heavy water Standard: TSP (trimethylsilylpropanoic acid) was set to 0 ppm (internal standard) Frequency: 500 MHz
• SEQ ID NO: 1 Nucleotide sequence of primer Fw for amplifying a fragment for disrupting opgH
• SEQ ID NO: 2 Nucleotide sequence of primer Rv for amplifying a fragment for disrupting opgH
• SEQ ID NO: 3 Nucleotide sequence of opgH derived from Escherichia coli (Accession No.: CP081489.1 2449812-2452352)
• SEQ ID NO: 4 Nucleotide sequence of primer Fw for amplifying the opgH fragment
• SEQ ID NO: 5 Nucleotide sequence of primer Rv for amplifying the opgH fragment
• SEQ ID NO: 6 Nucleotide sequence of primer Fw for amplifying the pET21a fragment
• SEQ ID NO: 7 Nucleotide sequence of primer Rv for amplifying the pET21a fragment
• SEQ ID NO: 8 Amino acid sequence of opgH derived from Escher

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• General Health & Medical Sciences (AREA)
• Biomedical Technology (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Molecular Biology (AREA)
• Microbiology (AREA)
• Biophysics (AREA)
• Medicinal Chemistry (AREA)
• General Chemical & Material Sciences (AREA)
• Physics & Mathematics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Plant Pathology (AREA)
• Mycology (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Cell Biology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Neurosurgery (AREA)
• Gastroenterology & Hepatology (AREA)
• Neurology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Epidemiology (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Saccharide Compounds (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Priority Applications (1)

Applications Claiming Priority (2)

Publications (1)

Family

ID=97350078

Family Applications (1)

Country Status (2)

Citations (6)

• 2025
  • 2025-04-08 WO PCT/JP2025/014070 patent/WO2025216249A1/ja active Pending
  • 2025-04-08 JP JP2025550679A patent/JPWO2025216249A1/ja active Pending
  • 2025-08-29 JP JP2025143813A patent/JP7842291B2/ja active Active

Patent Citations (6)

Non-Patent Citations (5)

Also Published As

Similar Documents

Legal Events