Cytidine diphosphocholine glycoside, composition, method for producing glycoside of cytidine residue-containing compound, gene recombinant microorganism, method for producing cytidine residue-containing compound, and method for suppressing production of glycoside of cytidine residue-containing compound

Abstract

The present invention relates to a cytidine diphosphocholine glycoside represented by general formula (1), a salt thereof, an N-oxide thereof, a solvate thereof, or the like. (In formula (1), R is a sugar residue.)

Description

Cytidine diphosphate choline glycoside, composition, method for producing glycoside of cytidine residue-containing compound, genetically modified microorganism, method for producing cytidine residue-containing compound, and method for inhibiting production of glycoside of cytidine residue-containing compound

[0001] 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.

[0002] A cytidine residue-containing compound refers to a compound having a cytidine residue in its compound structure, and specific examples thereof include cytidine, cytidylic acid, cytidine diphosphate (hereinafter also referred to as CDP), and cytidine diphosphate choline (hereinafter also referred to as CDP-choline). Among these, CDP-choline is a biosynthetic precursor of phosphatidylcholine, a component of phospholipids in higher organisms, and is known to have neuroprotective effects (Non-Patent Document 1). CDP-choline is difficult to obtain in large quantities by extraction from nature or chemical synthesis, and is therefore mainly synthesized by enzymatic reactions in living organisms using cytidine monophosphate (hereinafter also referred to as CMP) or orotic acid as starting materials (Patent Documents 1 and 2, Non-Patent Documents 2 and 3). Furthermore, 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).

[0003] It is also known that osmoregulated periplasmic glucans biosynthesis protein H (hereinafter also referred to as opgH) cooperates with OpgG (Glucans biosynthesis protein G) in response to osmotic pressure and contributes to glucan synthesis in the periplasm. Furthermore, 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).

[0004] International Publication No. 2003 / 095660 International Publication No. 2007 / 023830 Japanese Patent Publication No. 19749 / 1981 Japanese Patent Publication No. 018872 / 1982 Japanese Patent Publication No. 21499 / 1981

[0005] Nutrients (2020) Vol.12 p.793Bull. Inst. Chem. Res. Kyoto U. (1976) Vol. 53 p.546-562Appl. Microbiol. Biotechnol. (2017) Vol.101 p.1409-1417PLoS Genet.(2013) Vol. 9: e1003663.

[0006] The further metabolic conversion of CDP-choline to other compounds in microorganisms is largely unknown, even in organisms that naturally produce CDP-choline, and there have been no reports of this happening in organisms that do not naturally produce CDP-choline, such as Escherichia coli. Furthermore, the relationship between opgH and CDP-choline and its ability to transfer glucose using CDP-choline as a substrate were unknown.

[0007] An object of the present invention is to provide a compound converted from a cytidine residue-containing compound such as CDP-choline and a method for producing the same, and to provide a means for inhibiting the conversion of a cytidine residue-containing compound such as CDP-choline into another compound.

[0008] The present inventors have surprisingly discovered a novel activity possessed by proteins or microorganisms, namely, 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, and have found that this activity can convert a cytidine residue-containing compound to a novel glycoside of the cytidine residue-containing compound, more specifically, convert CDP-choline to CDP-choline glycoside, thereby completing the present invention. Furthermore, they have found that inhibiting the activity possessed by proteins or microorganisms of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound can inhibit the production of cytidine residue-containing compound glycosides during the production of the cytidine residue-containing compound.

[0009] The present disclosure includes the following: <1> A cytidine diphosphate choline glycoside represented by the following general formula (1), a salt thereof, an N-oxide thereof, or a solvate thereof:

[0010]

[0011] (In formula (1), R is a sugar residue.)

[0012] <2> The cytidine diphosphate choline glycoside, a salt thereof, an N-oxide thereof, or a solvate thereof according to <1> above, wherein the cytidine diphosphate choline glycoside is a cytidine diphosphate choline glucose glycoside represented by the following general formula (2):

[0013]

[0014] <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 the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound. <5> The method for producing a glycoside of a cytidine residue-containing compound according to <4> above, wherein the glycoside of the cytidine residue-containing compound is the cytidine diphosphate choline glycoside according to <1> or <2> above. <6> The method for producing a glycoside of a cytidine residue-containing compound according to <4> 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 GT2 enzyme and GT28 enzyme in the CAZy classification. <7> The method for producing a glycoside of a cytidine residue-containing compound according to <4> above, 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]: [1] A protein comprising the amino acid sequence represented by SEQ ID NO: 8 or 10. [2] A mutant protein comprising the 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 cytidine residue-containing compound. [3] A homologous protein comprising an amino acid sequence having 60% or more identity to 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. <8> A genetically modified microorganism of the following (A) or (B): (A) 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. (B) A genetically modified microorganism in which the activity of a protein having an 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. <9> The genetically modified microorganism according to <8> above, wherein the cytidine residue-containing compound is cytidine diphosphate choline.<10> 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 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 the 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 cytidine residue-containing compound. [3] A homologous protein comprising an amino acid sequence having 60% or more identity to 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. <11> 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> A method for producing a cytidine residue-containing compound according to <14> above, 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 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):

[0015]

[0016] (In formula (1), R is a sugar residue.)

[0017] <18> A composition comprising cytidine diphosphate choline produced using the genetically modified microorganism according to any one of <8> to <13> above, wherein the abundance ratio of the amount of cytidine diphosphate choline glycoside produced relative to the amount of cytidine diphosphate choline produced is 35% or less. <19> A method for producing a composition, comprising: preparing the genetically modified microorganism according to any one of <8> to <13> above; and producing a composition using the microorganism, wherein the abundance ratio of cytidine diphosphate choline glycoside relative to the amount of cytidine diphosphate choline produced in the composition is 35% or less. <20> The composition according to <18>, wherein the abundance ratio of the amount of cytidine diphosphate choline glycoside produced relative to the amount of cytidine diphosphate choline produced is 0.0005% or more and 35% or less. <21> A method for producing the composition according to <19>, wherein the abundance ratio of cytidine diphosphate choline glycoside relative to the amount of cytidine diphosphate choline in the composition is 0.0005% or more and 35% or less.

[0018] The present disclosure provides novel glycosides of cytidine residue-containing compounds and methods for producing the same. The present disclosure also provides methods for producing cytidine residue-containing compounds and methods for inhibiting the production of glycosides of cytidine residue-containing compounds.

[0019] The present invention will be described in detail below, but these are merely examples of desirable embodiments and are not intended to limit the scope of the present invention. The "to" range in a numerical value indicates a range that includes both the preceding and following numerical values. For example, "0% by mass to 100% by mass" means a range that is 0% by mass or more and 100% by mass or less.

[0020] [Cytidine Residue-Containing Compound] 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.

[0021] [Glycoside of a cytidine residue-containing compound] 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. Examples of sugar nucleotides 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.

[0022] Specific examples of 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 glycoside of the present disclosure" or "CDP-choline glycoside"), which are produced by transferring a sugar from a sugar nucleotide to CDP-choline.

[0023]

[0024]

[0025] (In formulas (1) and (1'), R is a sugar residue.)

[0026] Preferred examples of R include glucose residues, galactose residues, mannose residues, N-acetylglucosamine (also referred to as GlcNAc) residues, N-acetylgalactosamine (also referred to as GalNAc) residues, fucose residues, glucuronic acid (also referred to as GlcA) residues, and other hexose residues (e.g., fructose residues). 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.

[0027] 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'). Further, 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') (the following general formula (3-1), hereinafter also referred to as CDP-choline glucose glycoside) or cytidine diphosphate choline galactose glycoside (the following general formula (3-2), hereinafter also referred to as CDP-choline galactose glycoside).

[0028] The following general formula (3-1) is preferably a structure with β-glucose bound thereto represented by the following general formula (4-1), and the following general formula (3-2) is preferably a structure with β-galactose bound thereto represented by the following general formula (4-2). Furthermore, the cytidine diphosphate choline glucose glycoside represented by the following general formula (2) or (2') is more preferably CDP-choline glucose glycoside represented by the following general formula (3-1), and a structure with β-glucose bound thereto represented by the following general formula (4-1) is preferred.

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035] The CDP-choline glycosides, salts thereof, N-oxides thereof, and solvates thereof of the present disclosure, 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. Compared to CDP-choline, the CDP-choline glycosides and the like are also expected to have effects such as increased water solubility and reduced toxicity. Furthermore, it is known that when glycosides, which are generally water-soluble, are orally administered, the carbohydrate residues are usually removed by intestinal bacteria or digestive enzymes, making them more easily absorbed in the intestinal tract. Therefore, it is known that the rate and location of absorption and metabolism differ from those of unglycosylated glycosides, and that they exhibit different functions. Therefore, the CDP-choline glycosides of the present disclosure are expected to exhibit slower or more sustained absorption due to stabilization and hydrophilization, compared to CDP-choline.

[0036] The composition of the present disclosure contains at least one of the CDP-choline glycoside of the present disclosure, a salt thereof, an N-oxide thereof, and a solvate thereof. In this specification, such a composition is also referred to as composition A containing CDP-choline glycoside. Composition A may contain at least one of CDP-choline glycoside, a salt thereof, an N-oxide thereof, and a solvate thereof, and the amount contained is not limited.

[0037] The CDP-choline glycosides of the present disclosure can be converted into salts by known methods, such as acid addition salts, alkali metal salts, alkaline earth metal salts, ammonium salts, or amine salts.

[0038] Acid addition salts include, for example, inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, nitrate, or organic acid salts such as acetate, lactate, tartrate, benzoate, citrate, methanesulfonate, ethanesulfonate, trifluoroacetate, benzenesulfonate, toluenesulfonate, isethionate, glucuronate, or gluconate.

[0039] Examples of alkali metal salts include potassium and sodium.

[0040] Alkaline earth metal salts include, for example, calcium and magnesium.

[0041] The ammonium salt includes, for example, tetramethylammonium.

[0042] Examples of amine salts include triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris(hydroxymethyl)aminomethane, lysine, arginine, and N-methyl-D-glucamine.

[0043] The CDP-choline glycoside of the present disclosure can be converted to an N-oxide by a known method. The N-oxide refers to a CDP-choline glycoside of the present disclosure in which the nitrogen atom is oxidized.

[0044] The CDP-choline glycosides of the present disclosure can be converted into solvates by 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).

[0045] [Protein Having Activity to Transfer a Sugar from a Sugar Nucleotide to a Cytidine Residue-Containing Compound and DNA Encoding the Protein] Glycosides of cytidine residue-containing compounds of the present disclosure, for example, CDP-choline glycosides represented by general formula (1), are produced using proteins having activity to transfer a sugar from a sugar nucleotide to a cytidine residue-containing compound. Here, the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound refers to the activity of transferring a sugar from the sugar nucleotide to the cytidine residue of 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 of the cytidine residue-containing compound, even more specifically, the activity of transferring a sugar from the sugar nucleotide to CDP-choline, and even more specifically, includes the activity of transferring glucose from UDP-glucose to CDP-choline. Proteins having activity to transfer a sugar from a sugar nucleotide to a cytidine residue-containing compound include, for example, proteins classified in EC 2.4.1. It has not been known until now that the proteins classified as (1) include proteins having the activity of transferring sugars from sugar nucleotides to compounds containing cytidine residues.

[0046] Specifically, the CDP-choline glucose glycoside represented by the general formula (2) of the present disclosure is produced using a protein having the activity of transferring glucose from UDP-glucose to CDP-choline. Specifically, 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):

[0047]

[0048] In this specification, the protein that contributes to the production of CDP-choline glucose glycoside represented by the above general formula (2) by transferring glucose from UDP-glucose to CDP-choline represented by the above general formula (3) is a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, and more specifically, is a "protein having the activity of transferring glucose from UDP-glucose to CDP-choline."

[0049] As will be described later, a cytidine residue-containing compound can be efficiently produced by using a genetically modified microorganism in which the activity of a protein having this activity has been reduced or deleted compared to that of a parent strain, and the production of glycosides of a cytidine residue-containing compound can be inhibited by using a genetically modified microorganism in which the activity of a protein having this activity has been reduced or deleted compared to that of a parent strain.

[0050] ((Proteins Having the Activity of Transferring a Sugar from a Sugar Nucleotide to a Cytidine Residue-Containing Compound)) Examples of proteins 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 as glycosyltransferase Family 2 (also referred to herein as "GT2 enzymes") and glycosyltransferases classified as glycosyltransferase Family 28 (also referred to herein as "GT28 enzymes") in the CAZy classification. As proteins having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, glycosyltransferases classified as GT2 enzymes in the CAZy classification are particularly preferred.

[0051] The CAZy classification refers to the classification of enzymes in the Carbohydrate-Active enZYmes (also referred to as "CAZy" in this specification) database, which is a database that compiles information on the classification of carbohydrate-related enzymes. The classification of enzymes can be confirmed on the website (http: / / www.cazy.org / ) or the like (Nucleic Acids Res. 2009 Jan;37(Database issue):D233-8. doi: 10.1093 / nar / gkn663. Epub 2008 Oct 5.).

[0052] Examples of GT2 enzymes include UDP-Glc β-1,6-glucan synthase, UDP-Glc β-glucosyltransferase, UDP-Glc: bactoprenol β-glucosyltransferase, UDP-Glc: β-1,3-glucan synthase, UDP-Glc: 1,2-diacylglycerol 3-glucosyltransferase, and UDP-Glc β-1,2-glucan synthase. A protein having glucosyltransferase activity is more preferred.

[0053] Examples of proteins having glucans biosynthesis glucosyltransferase activity include osmoregulated periplasmic glucans biosynthesis protein H (hereinafter also referred to as opgH). Examples of opgH include opgH derived from Escherichia coli (Accession Nos. WP_001295445.1, AAC74133.1, KAB1960533.1, AMH24428.1, and NP_415567.1, etc.), and opgH derived from 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").

[0054] An example of the amino acid sequence of opgH derived from Escherichia coli is the amino acid sequence represented by SEQ ID NO: 8 (Accession No. WP_001295445.1). An example of the nucleotide sequence of DNA encoding opgH is the nucleotide sequence represented by SEQ ID NO: 3 (Accession No. CP081489.1 2449812-2452352). opgH is also known as mdoH or Glucans biosynthesis glucosyltransferase H.

[0055] Examples of GT28 enzymes include UDP-Glc: 1,2-diacylglycerol 3-glucosyltransferase, UDP-GlcNAc: undecaprenyldiphospho-muramoylpentapeptide β-1,4-N-acetylglucosaminyltransferase, digalactosyldiacylglycerol synthase, and monogalactosyldiacylglycerol synthase. 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. Examples of UgtP include UgtP derived from Bacillus subtilis, UgtP derived from Staphylococcus aureus, and UgtP derived from Bacillus mycoides. Among these, UgtP derived from Bacillus subtilis is more preferred, as it is a Moon Lighting Protein like E. coli-derived opgH and is a functional homolog of E. coli-derived opgH.

[0056] An example of the 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). An example of the nucleotide sequence of DNA encoding UgtP is the nucleotide sequence represented by SEQ ID NO: 9 (Accession No. NC_000964.3 2306514-2307662).

[0057] 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]: [1] A protein comprising the amino acid sequence represented by SEQ ID NO: 8 or 10. [2] A mutant protein comprising the 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 cytidine residue-containing compound. [3] A homologous protein comprising an amino acid sequence having 60% or more identity to 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.

[0058] The above [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 the 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. [3'] A homologous protein consisting of an amino acid sequence having 60% or more identity to 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.

[0059] Among the above [1] to [3], the protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is preferably [1], and more preferably a protein comprising the amino acid sequence represented by SEQ ID NO: 8.

[0060] As used herein, a mutant protein refers to a protein obtained by artificially deleting or substituting amino acid residues in an original protein, or by inserting or adding amino acid residues into the protein.

[0061] In the mutant protein of [2] above, the deletion, substitution, insertion, or addition of amino acids may mean the deletion, substitution, insertion, or addition of 1 to 20 amino acids 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.

[0062] The 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.

[0063] Examples of amino acids that can be substituted for each other are shown below. Amino acids in the same group can be substituted for each other. ・Group A: Hydrophobic amino acids ・Group B: Acidic amino acids ・Group C: Polar amino acids ・Group D: Basic amino acids ・Group E: Secondary amino acids ・Group F: Amino acids with a hydroxyl group ・Group G: Aromatic amino acids ・Group H: Sulfur-containing amino acids More specifically, 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, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid ・Group E: proline, 3-hydroxyproline, 4-hydroxyproline ・Group F: serine, threonine, homoserine ・Group G: phenylalanine, tyrosine

[0064] As used herein, a homologous protein refers to a protein that is found in organisms in nature and is similar in structure and function to the original protein, such that the gene encoding the protein is considered to have the same evolutionary origin as the gene encoding the original protein.

[0065] Examples of 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.

[0066] 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 closely matched. For example, the percentage of sequence identity can be determined using a mathematical algorithm. Examples of 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 algorithm of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. Examples of suitable mathematical algorithms include the similarity search method of Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 85:2444-2448, and a modified version of the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877, but are not limited to these examples.

[0067] Alignment to determine 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 (available from Intelligenetics, Mountain View, Calif.), MAFFT (Katoh, K., Misawa, K., Kuma, K., & Miyata, T. (2002), 30(14), 3059-3066., http: / / mafft.cbrc.jp / alignment / server / ), MUSCLE (Edgar RC (2004). Nucleic acids research, 32(5), 1792-1797., http: / / www.ebi.ac.uk / Tools / msa / muscle / ), BLAST, FASTA, and TFASTA. Alignment using these programs can be performed, for example, using default parameters. The 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. (1994) Meth. Mol. Biol. 24:307-331. 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. Specifically, 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.

[0068] 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. First, a recombinant DNA containing DNA encoding the protein and a tag sequence for enzyme purification is prepared by the method described below. Next, a microorganism obtained by transforming the recombinant DNA is cultured, and the protein is prepared as a purified enzyme from the resulting culture. Next, the purified enzyme is contacted with an appropriate substrate and a sugar donor, for example, CDP-choline and UDP-glucose to produce CDP-choline glucose glycoside when confirming that the protein has the activity to transfer glucose from UDP-glucose to CDP-choline. Finally, CDP-choline glucose glycoside in the reaction solution can be detected by a general 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.

[0069] Tag sequences for enzyme purification are described in the prior art and are known to those skilled in the art, such as 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.

[0070] For example, a His tag (polyhistidine 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.

[0071] 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. The resin is then typically washed with phosphate buffer to remove proteins that do not specifically interact with cobalt or nickel ions. For Ni-based methods, the addition of 20 mM imidazole can improve wash efficiency (proteins are typically eluted at 150-300 mM imidazole).

[0072] ((DNA)) Examples of DNA encoding opgH from E. coli, which is 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. Furthermore, examples of DNA encoding UgtP from Bacillus subtilis include Accession Nos. NC_000964.3 2306514-2307662.

[0073] In this embodiment, the DNA encoding a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound can be at least one selected from the following [4] to [9]. [4] DNA encoding the amino acid sequence represented by SEQ ID NO: 8 or 10. [5] DNA encoding a mutant protein comprising the 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 cytidine residue-containing compound. [6] DNA encoding a homologous protein comprising an amino acid sequence having 60% or more identity to 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. [7] DNA comprising the nucleotide sequence represented by SEQ ID NO: 3 or 9. [8] DNA hybridizing under stringent conditions to DNA comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 or 9, and encoding a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound. [9] 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.

[0074] The above [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 cytidine residue-containing compound. [6'] DNA encoding a homologous protein consisting of an amino acid sequence having 60% or more identity to 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. [7'] DNA consisting of the nucleotide sequence represented by SEQ ID NO: 3 or 9. [8'] DNA hybridizing under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 or 9, and encoding a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound. [9'] 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.

[0075] "Hybridizing" means that DNA hybridizes to DNA having a specific base sequence or a part of said DNA. Therefore, said DNA having a specific base sequence or a part of said DNA can be used as a probe in Northern or Southern blot analysis, or as an oligonucleotide primer in PCR analysis.

[0076] The DNA used as a probe is at least 100 bases long, preferably at least 200 bases long, more preferably at least 500 bases long. The DNA used as a primer is at least 10 bases long, preferably at least 15 bases long.

[0077] Methods for DNA hybridization experiments are well known, and hybridization conditions can be determined and experiments can be performed according to, for example, Molecular Cloning, 4th Edition (Cold Spring Harbor Laboratory Press (2012)), Methods for General and Molecular Bacteriology (ASM Press (1994)), Immunology Methods Manual (Academic Press (1997)), and many other standard textbooks.

[0078] Alternatively, DNA that hybridizes under stringent conditions can be obtained by following the instructions provided with a commercially available hybridization kit, such as the Random Primed DNA Labeling Kit (manufactured by Roche Diagnostics), which prepares a probe by the random prime method and hybridizes under stringent conditions.

[0079] Examples of stringent conditions include 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 µg / ml denatured salmon sperm DNA, followed by washing the filter in a 0.2x SSC solution at about 65°C.

[0080] The various conditions described above can also be achieved by adding or changing blocking reagents used to suppress background in hybridization experiments. The addition of blocking reagents described above may be accompanied by changes in hybridization conditions to suit the conditions.

[0081] Examples of DNA that can hybridize under the above-mentioned stringent conditions include 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.

[0082] Among 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 of 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 manipulation is not particularly limited, but may be, for example, a prokaryote belonging to the genus Escherichia, Shewanella, Xanthomonas, or Pseudomonas, and among these, a prokaryote belonging to the genus Escherichia (Escherichia coli) is preferred.

[0083] Among 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 have 60% or more identity, preferably 70% or more, 80% or more, 90% or more, 95% or more, more preferably 98% or more, and most preferably 99% or more identity to the amino acid sequence represented by SEQ ID NO: 8 or 10, in that order; or by searching various gene sequence databases for nucleotide sequences that have 95% or more identity, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to the nucleotide sequence represented by SEQ ID NO: 3, and then using a 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, by a method using Southern hybridization or PCR, similar to the method for obtaining the above-mentioned DNA.

[0084] Among 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 nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 8 or 10, or a nucleotide sequence represented by SEQ ID NO: 3 or 9, to error-prone PCR or the like as a template.

[0085] Alternatively, the DNA described in [5] or [8] above can be obtained by PCR [Gene, 77, 51 (1989)] using a pair of PCR primers each having a nucleotide sequence at its 5' end designed to insert a desired mutation (deletion, substitution, insertion, or addition). Specifically, PCR is first performed using the DNA as a template, with a sense primer corresponding to the 5' end of DNA containing a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 8 or 10, or a nucleotide sequence represented by SEQ ID NO: 3 or 9, and an antisense primer corresponding to the sequence immediately preceding (on the 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). Next, PCR is performed using the DNA as a template, with a sense primer corresponding to the sequence immediately following (on the 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 its 5' end. After purifying these amplified fragments, they are mixed and subjected to PCR without adding a template or primers. Since the sense strand of amplified fragment A and the antisense strand of amplified fragment B share the same mutation introduction site, they hybridize and act as both primer and template in the PCR reaction, amplifying the mutated DNA.

[0086] 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 then inserted into a vector by a standard method. The resulting recombinant DNA is then introduced into a host cell, and the DNA can be analyzed using a standard base sequence analysis method, such as the dideoxy method [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)], or a base sequence analyzer such as an Applied Biosystems 3500 Genetic Analyzer or an Applied Biosystems 3730 DNA Analyzer (both manufactured by Thermo Fisher Scientific), to determine the base sequence of the DNA.

[0087] Examples of host cells that can be used for determining the base sequence of DNA include Escherichia coli DH5α, 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 MV1184, and Escherichia coli TH2 (all manufactured by Takara Bio Inc.), and 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, and the like.

[0088] Examples of the vector 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)].

[0089] If the DNA obtained as a result of determining the base sequence is a partial length, full-length DNA can be obtained by Southern hybridization or the like against a chromosomal DNA library using the partial length DNA as a probe.

[0090] Furthermore, the desired DNA can also 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.

[0091] [Genetically Modified Microorganism with Enhanced Activity of Target Protein] The methods for producing a glycoside of a cytidine residue-containing compound and for producing a cytidine residue-containing compound of the present disclosure may use a genetically modified microorganism. Examples of 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. Here, the definition of such a microorganism and a method for producing the microorganism are described.

[0092] As used herein, "the activity of a protein is enhanced" may mean that the activity of the protein is enhanced compared to that of a parent strain. Specifically, as used herein, "the activity of a protein is enhanced" may mean that the activity of the protein per cell is enhanced compared to that of a parent strain.

[0093] In a genetically modified microorganism in which the activity of a protein of interest has been enhanced, 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, transformation, etc., or a strain that has already undergone recombination other than recombination that enhances the activity of the protein of interest relative to the type strain; the strains exemplified in the description of (parent strain) below can be used, but are not limited to these. That is, in one aspect, the activity of the protein may be enhanced compared to that of the parent strain.

[0094] As used herein, "enhanced protein activity" may more specifically mean that the number of molecules of the protein per cell is increased and / or the function of the protein per molecule is enhanced compared to that of the parent strain. That is, the "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.

[0095] Furthermore, "enhanced protein activity" encompasses not only enhancing the activity of a target protein in a strain that originally has the activity of that protein, but also imparting the activity of that protein to a strain that does not originally have the activity of that protein. Furthermore, as long as the resulting protein activity is enhanced, the activity of the target protein may be imparted after reducing or eliminating the activity of the target protein originally possessed by the host.

[0096] In this embodiment, 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. For example, the protein activity may be increased 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. Furthermore, if the parent strain does not have the activity of the target protein, the protein may be produced by introducing a gene encoding the protein, provided that the protein is produced to an extent that its activity can be measured.

[0097] 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, or by confirming that the amount of the protein expressed from the gene is increased compared to the parent strain.

[0098] Recombination 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. That is, when a protein is subject to feedback inhibition by metabolites, the activity of the protein can be enhanced by having the host harbor a gene encoding a mutant protein in which feedback inhibition is desensitized.

[0099] Unless otherwise specified, "desensitization to feedback inhibition" may include cases where feedback inhibition is completely released and cases where feedback inhibition is reduced. Furthermore, "desensitization to feedback inhibition" (i.e., feedback inhibition is reduced or released) is also referred to as "resistance to feedback inhibition."

[0100] Proteins with enhanced specific activity can be obtained, for example, by searching various organisms. Alternatively, highly active proteins can be obtained by introducing mutations into existing proteins. The mutations introduced may be, for example, substitutions, deletions, insertions, or additions of one or several amino acids at one or several positions in the protein.

[0101] Furthermore, when enhancing protein activity, efforts may be made to express the protein in a soluble fraction and ensure correct folding of the protein, etc. Specific examples include co-expression with a chaperone, investigation of gene inducers, refolding techniques, deletion of the N-terminus or C-terminus of the amino acid sequence of the protein, selection of a protein expression vector, optimization of purification conditions, and optimization of codons.

[0102] A microorganism 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."

[0103] As used herein, "gene expression is increased" may mean that the expression of the gene is enhanced compared to that of the parent strain. As used herein, "gene expression is increased" may specifically mean that the expression level of the gene per cell is enhanced compared to that of the parent strain. As used herein, "gene expression is increased" 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.

[0104] "Increasing gene expression" is also referred to as "enhancing gene expression." In this embodiment, gene expression 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 expression of the gene in the parent strain. Furthermore, "increasing gene expression" encompasses not only increasing the expression level of a gene of interest in a strain in which the gene is originally expressed, but also expressing the gene in a strain in which the gene of interest is not originally expressed. In other words, "increasing gene expression" may mean, for example, introducing the gene of interest into a strain that does not harbor the gene and expressing the gene.

[0105] Increased 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 disrupting or enhancing the expression of a transcriptional regulatory factor involved in controlling the expression of the target gene. That is, in the case of a transcriptional regulatory factor that contributes to suppressing the expression of the target gene, this can be achieved by disruption, and in the case of a transcriptional regulatory factor that contributes to promoting the expression of the target gene, this can be achieved by enhancement.

[0106] Examples of 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 a gene encoding the protein, and microorganisms in which the DNA encoding the protein is carried outside the chromosomal DNA as plasmid DNA.

[0107] <Recombinant DNA> Recombinant DNA refers to, for example, DNA that is capable of autonomous replication within a parent strain and in which DNA encoding a protein of interest (hereinafter also referred to as DNA of interest) has been incorporated into an expression vector containing a promoter at a position where the DNA can be transcribed.

[0108] The vector is not particularly limited as long as it is a suitable DNA molecule for introducing, propagating, and expressing the target DNA into a host cell. In addition to plasmids, for example, artificial chromosomes, vectors using transposons, and cosmids may also be used.

[0109] If the DNA can be integrated into the chromosome of the parent strain, the target DNA itself is also a 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.

[0110] Preferably, the recombinant DNA capable of autonomous replication in prokaryotes such as bacteria is recombinant DNA composed of a promoter, a ribosome binding sequence, a target DNA, and a 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, which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (e.g., 6 to 18 bases).

[0111] In an autonomously replicable recombinant DNA, 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.

[0112] When a microorganism belonging to the genus Escherichia is used as the parent strain, examples of the expression vector 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 GE Healthcare Biosciences), pTrcHis, pSE280 (all manufactured by Thermo Fisher Scientific), pGEMEX-1 (manufactured by Promega), pQE-30, pQE80L (all manufactured by Qiagen), pET-3, and pBluescriptII. SK(+), pBluescriptII KS(-) (both manufactured by Agilent Technologies), pKYP10 (Japanese Patent Laid-Open Publication No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. , USA, 82, 4306 (1985)], pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, pp. 6378-6385], pPAC31 (WO 98 / 12343), pUC19 [Gene, 33, 103 (1985)], pPA1 (JP-A-63-233798), and the like.

[0113] When using the above-mentioned expression vector, any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Escherichia, and examples thereof include promoters of genes involved in amino acid biosynthesis, such as the trp promoter and the ilv promoter, and promoters derived from Escherichia coli or phages, such as the uspA promoter, the lac promoter, the PL promoter, the PR promoter, and the PSE promoter. Artificially designed and recombinant promoters, such as a promoter consisting of two trp promoters in tandem, the tac promoter, the trc promoter, the lacT7 promoter, and the letI promoter, can also be used.

[0114] When a coryneform bacterium is used as the parent strain, examples of the expression vector include pCG1 (Japanese Patent Application Laid-Open No. 57-134500), pCG2 (Japanese Patent Application Laid-Open No. 58-35197), pCG4 (Japanese Patent Application Laid-Open No. 57-183799), pCG11 (Japanese Patent Application Laid-Open No. 57-134500), pCG116, pCE54, pCB101 (all Japanese Patent Application Laid-Open No. 58-105999), pCE51, pCE52, and pCE53 [all of which are described in Molecular and General Genetics, 196, 175 (1984)].

[0115] When using the above expression vector, any promoter may be used as long as it functions in cells of coryneform bacteria, and an example thereof is the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, pp. 674-679 (2000)].

[0116] When a yeast strain is used as the parent strain, examples of the expression vector include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0117] When using the above expression vector, any promoter may be used as long as it functions in the cells of a yeast strain, and examples include the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0118] The recombinant DNA can be produced, for example, using In-Fusion™ HD Cloning Kit (Takara Bio Inc.), or by treating a DNA fragment prepared so as to encode the desired enzyme with a restriction enzyme and inserting it downstream of the promoter of the appropriate expression vector.

[0119] Here, the expression level of the protein encoded by the DNA can be improved by substituting bases in the base sequence of the DNA so that the codons are optimal for expression in the host cell. Information on codon usage frequencies in the parent strain used in the production method of the present invention is available through public databases.

[0120] A genetically modified microorganism with enhanced activity of a target protein is a genetically modified microorganism that contains DNA encoding the target protein (target DNA) or is obtained by transforming a parent strain with recombinant DNA containing the target DNA. Here, the parent strain will be described.

[0121] <Parent Strain> The parent strain used to construct the microorganisms described herein is not particularly limited.

[0122] The parent strain is preferably a prokaryote or a yeast strain, more preferably a prokaryote belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, and among these, a prokaryote belonging to the genus Escherichia (Escherichia coli) is preferred.

[0123] Specific examples of 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 DH5α, 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 MV1184, Escherichia coli TH2 (all manufactured by Takara Bio), Escherichia coli W (ATCC9637), Escherichia coli JM101, Escherichia coli W3110, Escherichia coli MG1655, Escherichia coli DH1, EscherichiaProkaryotes such as Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli MP347, Escherichia coli NM522, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Corynebacterium ammoniagenes, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, or Pseudomonas sp. D-0110, or yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvium, Pichia pastoris, or Candida utilis.

[0124] <Incorporation of DNA into Parent Strain> Any method for introducing recombinant DNA into a parent strain to incorporate the target DNA and obtain a genetically modified microorganism can be used as long as it is a method for introducing DNA into a parent strain. Examples include 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)].

[0125] In genetically modified microorganisms, 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 transcriptionally active state. One parent strain may contain only one type of DNA, or two or more types of DNA.

[0126] Furthermore, when inserting a recombinant DNA containing a target DNA into the genome of a parent strain, a method such as homologous recombination may be used. That is, DNA containing a portion of a chromosomal region that will induce the introduction of the target DNA is incorporated into a microorganism, and homologous recombination occurs in a portion of the chromosomal region, allowing the target DNA to be integrated into the genome. For example, a method utilizing homologous recombination frequently used in Escherichia coli is a method of introducing recombinant DNA using the lambda phage homologous recombination system [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 incorporate the DNA into a microorganism cell, including, for example, the calcium ion method, the protoplast method, and the electroporation method.

[0127] Furthermore, E. coli in which a target region on the chromosomal DNA of a host cell has been replaced with the target DNA or recombinant DNA can be obtained using a selection method that utilizes the fact that E. coli becomes sensitive to sucrose due to Bacillus subtilis levansucrase that has been incorporated into the chromosome together with the recombinant DNA, or a selection method that utilizes the fact that E. coli becomes sensitive to streptomycin by incorporating a wild-type rpsL gene into E. coli that has a mutant rpsL gene that is resistant to streptomycin [Mol. Microbiol., 55, 137 (2005), Biosci. Biotechnol. Biochem., 71, 2905 (2007)].

[0128] 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 transcription amount of the DNA in the microorganism by Northern blotting or the production amount of the protein in the microorganism by Western blotting with the transcription amount of the DNA or the production amount of the protein in the parent strain before the DNA was introduced.

[0129] For example, whether a microorganism constructed by expressing recombinant DNA containing DNA encoding a protein of interest into a parent strain using the above-described method is a genetically modified microorganism capable of producing the protein of interest can be confirmed by, for example, the following method. First, the parent strain before the introduction of the DNA and the constructed genetically modified microorganism are cultured in media, and a cell extract containing the protein of interest is prepared from the resulting culture. Next, the cell extract is contacted with a substrate, and the protein of interest is allowed to act on the substrate to produce a reaction product. Finally, the reaction product in the reaction solution is detected using an appropriate analytical method depending on the reaction product, thereby confirming that the constructed microorganism is a genetically modified microorganism capable of producing the protein of interest. In particular, if the detected amount of the target product is greater than that of the parent strain before the introduction of the DNA, it can be said that the activity of the protein of interest is enhanced compared to that of the parent strain before the introduction of the DNA.

[0130] [Manufacturing method of the target product]

[0131] When the target product is produced using a microorganism, those skilled in the art will understand that the production may be by an enzymatic method or a fermentation method, but is not limited to these.

[0132] In this embodiment, 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 allowing the enzyme source and a substrate to exist in an aqueous medium and react with each other. Among these methods, a reaction system that uses microbial cells in a dormant or stationary state without proliferation as a catalyst (also referred to as an enzyme source) is called a "microbial cell reaction method."

[0133] The term "fermentation method" refers to a method for producing a desired product within a microorganism by using a microorganism in a growing or growing state. In this case, it is necessary to add medium components for the growth or growth of the microorganism.

[0134] The enzymatic or fermentative methods for producing the desired product are described in more detail below, although the reaction between the microorganism and the substrate is not limited to the specific methods described below.

[0135] [Method for producing glycoside of cytidine residue-containing compound] The method for producing a glycoside of a cytidine residue-containing compound of the present disclosure comprises using a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound. In the method for producing a glycoside of a cytidine residue-containing compound of the present disclosure, a sugar is transferred from a 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 case where the glycoside of the cytidine residue-containing compound is CDP-choline glycoside is described in detail below.

[0136] [Method for Producing CDP-choline Glycoside] 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. In the method for producing CDP-choline glycoside of the present disclosure, 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. For example, by using 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.

[0137] Methods for producing CDP-choline glycoside according to the present disclosure include (I) a method for producing CDP-choline glycoside by a fermentation method, and (II) a method for producing CDP-choline glycoside by an enzymatic method, using a microorganism capable of producing a protein having the activity of transferring a sugar from a sugar nucleotide to the above-described cytidine residue-containing compound. Each production method will be described below.

[0138] (I) Method for Producing CDP-choline glycoside by fermentation 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.

[0139] 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 a parent strain, and in which CDP-choline glycoside productivity is improved.

[0140] The microorganism having a protein having 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 having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, or, when the type strain used is a microorganism that does not originally have a protein having 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 having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound has been artificially imparted. It may also 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 further enhanced in a microorganism originally having a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound.

[0141] An example of the microorganism (I) for producing CDP-choline glycoside is the aforementioned "genetically modified microorganism having enhanced activity of a target protein," wherein the "target protein" is a protein having 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 GT2 enzyme and GT28 enzyme in the CAZy classification described above, or at least one selected from the above-mentioned [1] to [3].

[0142] In the "genetically modified microorganism with enhanced activity of a target protein," when the "target protein" is an enzyme classified as at least one of GT2 enzymes and GT28 enzymes in the CAZy classification described above, 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," a DNA encoding an enzyme classified as at least one of GT2 enzymes and GT28 enzymes in the CAZy classification, specifically, a DNA exemplified above as a DNA encoding E. coli-derived opgH, or a DNA exemplified above as a DNA encoding E. coli-derived opgH.

[0143] In the "genetically modified microorganism with enhanced activity of a target protein," when the "target protein" is at least one selected from the above-mentioned [1] to [3], the microorganism can be produced by the above-mentioned method for producing the "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."

[0144] The microorganism can be cultured according to a conventional method. The medium for culturing the microorganism may be either a natural medium or a 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 a starting material for the substrate, a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the microorganism, and allows efficient cultivation of the microorganism.

[0145] Substrates for proteins that have the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound include, for example, sugar nucleotides and CDP-choline. Examples of sugar nucleotides include UDP-Glc, UDP-Gal, GDP-Man, UDP-GlcNAc, UDP-GalNAc, GDP-Fuc, and UDP-GlcA, with UDP-GlcA 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.

[0146] The carbon source may be any that can be assimilated by the microorganism, and examples thereof include carbohydrates such as glucose, fructose, sucrose, molasses containing these, starch, and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.

[0147] Examples of nitrogen sources include 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 digested products thereof.

[0148] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0149] In the method for producing CDP-choline glycoside by fermentation, the microorganism used may be a microorganism having 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 having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound. The microorganism having 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.

[0150] Furthermore, in the method for producing CDP-choline glycoside by fermentation, when the microorganism used does not have the ability to produce sugar nucleotides such as UDP-glucose and / or CDP-choline, instead of adding sugar nucleotides such as UDP-glucose and / or CDP-choline to the medium, a microorganism capable of producing sugar nucleotides such as UDP-glucose and / or CDP-choline may be co-cultured with the microorganism (I) for producing CDP-choline glycoside, thereby supplying the microorganism with sugar nucleotides such as UDP-glucose and / or CDP-choline.

[0151] 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 (a) to (e), and these known methods can be used alone or in combination: (a) a method of alleviating or deactivating at least one of the mechanisms controlling the biosynthetic pathway of sugar nucleotides such as UDP-glucose and / or CDP-choline, (b) a method of enhancing the expression of at least one enzyme involved in the biosynthetic pathway of sugar nucleotides such as UDP-glucose and / or CDP-choline, (c) a method of increasing the copy number of at least one enzyme gene involved in the biosynthetic pathway of sugar nucleotides such as UDP-glucose and / or CDP-choline, (d) a method of weakening or blocking at least one metabolic pathway branching off from the biosynthetic pathway of sugar nucleotides such as UDP-glucose and / or CDP-choline to metabolites other than the target substance, and (e) a method of selecting a cell line that has a higher degree of tolerance to analogs of sugar nucleotides such as UDP-glucose and / or CDP-choline compared to a reference strain.

[0152] In the method for producing CDP-choline glycoside by fermentation, when the microorganism used does not have the ability to produce sugar nucleotides such as UDP-glucose and / or CDP-choline, 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., and CDP-choline is available, for example, as CDP-choline (product code: C3438) manufactured by Tokyo Chemical Industry Co., Ltd.

[0153] When two or more types of microorganisms are cultured in the same medium, these microorganisms may be cultured simultaneously, or one of the microorganisms may be cultured in the medium during or after the culture of the other microorganism has been completed.

[0154] Furthermore, when two or more types of microorganisms are combined to produce CDP-choline glycoside, a compound that serves as a substrate for CDP-choline may be added. Alternatively, when a microorganism has only 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 having CDP-choline-producing activity so that CDP-choline-producing activity can be obtained. Note that even when a microorganism has CDP-choline-producing activity, two or more types of microorganisms can be combined.

[0155] Furthermore, for example, when R of the CDP-choline glycoside represented by the general formula (1) is a hexose residue, after a sugar is transferred from a sugar nucleotide to CDP-choline, the R structure of the CDP-choline glycoside can be converted to a hexose residue by the action of an endogenous enzyme of the microorganism, and thus the CDP-choline glycoside represented by the general formula (1) can also be produced.

[0156] Specifically, when 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 enabling the production of the CDP-choline glycoside represented by the general formula (1).

[0157] 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 is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0158] If necessary, antibiotics such as ampicillin or tetracycline may be added to the medium during culture. When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as necessary.

[0159] For example, when a microorganism transformed with an expression vector using a lac promoter is cultured, isopropyl-β-D-thiogalactopyranoside or the like may be added to the medium, and when a microorganism transformed with an expression vector using a trp promoter is cultured, indoleacrylic acid or the like may be added to the medium.

[0160] By the above-mentioned culture, CDP-choline glycoside can be produced in the culture, and thus CDP-choline glycoside can be produced. The amount of CDP-choline glycoside produced can be quantified using HPLC (e.g., an analytical apparatus SPD-M20A manufactured by Shimadzu Corporation) according to the method described below in "Analysis Examples."

[0161] CDP-choline glycoside can be collected from the culture by a combination of conventional methods such as an ion exchange resin method, a precipitation method, etc. When CDP-choline glycoside accumulates in the cells, for example, the cells can be disrupted by ultrasonication or the like, and the cells can be removed by centrifugation, and the CDP-choline glycoside can be collected from the resulting supernatant by an ion exchange resin method or the like.

[0162] (II) Method for Producing CDP-choline glycoside by an Enzymatic Method 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.

[0163] The microorganism used in producing the CDP-choline glycoside of the present disclosure by an enzymatic method (hereinafter, "CDP-choline glycoside-producing microorganism (II)") 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 a microorganism whose type strain is a cytidine residue-containing microorganism. In the case of a microorganism that does not originally have a protein having the activity of transferring a sugar from a sugar nucleotide to a 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 having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound 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.

[0164] More specifically, the microorganism (II) for producing CDP-choline glycoside is a microorganism in which, in the above-mentioned "genetically modified microorganism having enhanced activity of a target protein," the "target protein" is a protein having activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, and preferably the "target protein" is an enzyme classified into at least one of GT2 enzyme and GT28 enzyme in the above-mentioned CAZy classification, or at least one selected from the above-mentioned [1] to [3].

[0165] In the "genetically modified microorganism with enhanced activity of a target protein," when 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, 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 the 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, or the like.

[0166] In the "genetically modified microorganism with enhanced activity of a target protein," when the "target protein" is at least one selected from the above-mentioned [1] to [3], the microorganism can be produced by the above-mentioned method for producing the "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."

[0167] When a microorganism capable of producing a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is used as an enzyme source, the microorganism may be capable of producing not only the protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, but also an enzyme (e.g., CCT, pyrG, etc.) required for producing CDP-choline, which is a substrate of CDP-choline glycoside, and further, CDP-choline from its starting substrates, such as choline, phosphorylcholine, uridine-5'-triphosphate (also referred to as CTP), and UTP.

[0168] A specific example of a recombinant microorganism capable of producing a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is BL21(DE3)opgH::kan / pET21a-opgH, which will be described later in the Examples.

[0169] 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."

[0170] In the method for producing CDP-choline glycoside by an enzymatic method, the "enzyme source" refers to a culture obtained by culturing the genetically modified microorganism of the present embodiment described above, in which the activity of a protein having 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.

[0171] In this specification, 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.

[0172] Among these, preferred are 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. Most preferred are 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.

[0173] 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.

[0174] The concentration of the substrate is preferably 0.1 mM to 10 M, more preferably 1 mM to 1 M. Examples of the substrate include sugar nucleotides and CDP-choline. As the starting material for the substrate, the substances described in "(I) Method for producing CDP-choline glycoside by fermentation" may be used. Examples of sugar nucleotides include UDP-Glc, UDP-Gal, GDP-Man, UDP-GlcNAc, UDP-GalNAc, GDP-Fuc, UDP-GlcA, etc., with UDP-Glc being preferred.

[0175] Examples of aqueous media include water, buffer solutions such as phosphates, carbonates, acetates, borates, citrates, and Tris, alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, and amides such as acetamide. The culture medium of the microorganism used as the enzyme source can also be used as the aqueous medium.

[0176] In the reaction for producing CDP-choline glycoside, a chelating agent such as phytic acid, a surfactant, or an organic solvent may be added as needed. Examples of surfactants include nonionic surfactants such as polyoxyethylene octadecylamine (e.g., Nymeen S-215, manufactured by NOF Corporation), cationic surfactants such as cetyltrimethylammonium bromide and 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), as long as they promote the production of CDP-choline. These surfactants may be used alone or in combination. The surfactant is typically used at a concentration of 0.1 to 50 g / L.

[0177] Examples of 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. To promote the production reaction, adenine, adenosine-5'-monophosphate (AMP), adenosine-5'-triphosphate (ATP), magnesium sulfate, magnesium chloride, and the like may be added. Adenine, AMP, and ATP are typically used at concentrations of 0.01 to 100 mM.

[0178] 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."

[0179] [Genetically Modified Microorganism with Reduced or Absent Activity of Target Protein] The method for producing a cytidine residue-containing compound and the method for inhibiting production of glycosides of a cytidine residue-containing compound of the present disclosure may use a genetically modified microorganism. Examples of genetically modified microorganisms include genetically modified microorganisms in which the activity of a target protein is reduced or absent 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 absent. Here, the definition of such a microorganism and a method for producing the microorganism are described.

[0180] Examples of the target protein in the "genetically modified microorganism in which the activity of the target protein is reduced or deleted compared to that of the parent strain" include proteins described in "Proteins having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound and DNA encoding said protein".

[0181] As used herein, "reduced or absent protein activity" may mean that the activity of the protein is reduced or absent compared to that of the parent strain. "Reduced or absent protein activity" specifically means that the activity of the protein per cell is reduced or absent compared to that of 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 that of the parent strain.

[0182] In a genetically modified microorganism in which the activity of a protein of interest is reduced or deleted, 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 to which a modification other than the modification that reduces or deletes the activity of the protein of interest 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 deleted compared to the parent strain.

[0183] The term "activity" in the phrase "decreased or absent protein 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%.

[0184] Recombination that reduces the activity of a protein can be achieved by, for example, disrupting part or all of the region of the gene encoding the protein, introducing a stop codon, recombining expression regulatory sequences such as promoters and Shine-Dalgarno (SD) sequences, manipulating factors involved in expression control, introducing mutations into the coding region, mutagenesis, etc. These techniques are known.

[0185] Gene disruption can be achieved, for example, by deleting (deleting) a gene on a chromosome, such as by knocking out a specific gene using PCR [Baba T. et al., Mol Systems Biol (2006)] or by using the homologous recombination system of lambda phage [Proc. Natl. Acad. Sci. USA, 97, 6640-6645 (2000)].

[0186] 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. A decrease 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 assessing 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.

[0187] The reduction in the protein amount can be confirmed by Western blotting using an antibody (Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The protein amount (e.g., the number of molecules per cell) 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.

[0188] Gene disruption can be confirmed by determining the nucleotide sequence, restriction enzyme map, or full length of a part or all of the gene, depending on the means used for disruption.

[0189] The above-mentioned methods for reducing protein activity can be used to reduce the activity of any protein (e.g., by-product-producing enzyme) or the expression of any gene (e.g., a gene encoding such a protein).

[0190] [Method for producing a cytidine residue-containing compound with suppressed production of glycosides of the cytidine residue compound] 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.

[0191] The case where the cytidine residue-containing compound is CDP-choline will be described in detail below.

[0192] [Method for Producing Cytidine Diphosphate Choline] 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.

[0193] Specific examples of the method for producing CDP-choline according to the present disclosure include (III) a method for producing CDP-choline by a fermentation method, and (IV) a method for producing CDP-choline by an enzymatic method.

[0194] (III) Method for Producing CDP-choline by Fermentation 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.

[0195] A microorganism used in producing CDP-choline by fermentation (hereinafter also referred to as "CDP-choline-producing recombinant microorganism (III)") is 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 in a parent strain, and which has the ability to produce CDP-choline.

[0196] By using 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 deleted compared to the parent strain, the production of CDP-choline glycoside resulting from the transfer of glucose from UDP-glucose to CDP-choline can be suppressed, and CDP-choline can be produced efficiently.

[0197] An example of a recombinant microorganism (III) for producing CDP-choline is a microorganism in which, in the above-mentioned "genetically modified microorganism having reduced or deleted activity of a protein of interest," the "protein of interest" is a protein having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound, and preferably the "protein of interest" is an enzyme classified as at least one of GT2 enzyme and GT28 enzyme in the above-mentioned CAZy classification, or at least one selected from the above-mentioned [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 having reduced or deleted activity of a protein of interest."

[0198] Since the recombinant microorganism (III) for producing CDP-choline has the ability to produce 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. Furthermore, as described above in "(I) Method for producing CDP-choline glycoside by fermentation," CDP-choline may be produced by combining two or more types of microorganisms.

[0199] CDP-choline is synthesized from CTP and phosphorylcholine by choline phosphate cytidyltransferase [EC 2.7.7.15] (hereinafter abbreviated as CCT). Microorganisms capable of producing CDP-choline are required to have the activity to produce CTP and phosphorylcholine, as well as CCT activity.

[0200] Phosphorylcholine is produced from choline and ATP by choline kinase [EC 2.7.1.32] (hereinafter abbreviated as CKI), and therefore, microorganisms capable of producing phosphorylcholine are required to have CKI activity.

[0201] CTP is produced according to the biosynthetic pathway of pyrimidine nucleic acids, 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, followed by cytidine-5'-triphosphate synthetase [EC 6.3.4.2] (PyrG) which has the activity of producing CTP from UTP. For efficient synthesis of CTP, carbamoyl phosphate synthase [EC 6.3.5.5] for producing carbamoyl phosphate from glutamine and aspartate carbamoyltransferase [EC 6.3.5.5] for synthesizing carbamoyl phosphate from glutamine and N-carbamoyl aspartate from carbamoyl phosphate and aspartic acid are required. 2.1.3.2], 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 (hereinafter abbreviated as OMP) from orotate and PRPP, orotidine-5'-monophosphate decarboxylase [EC 4.1.1.23] that has the activity of producing uridine-5'-monophosphate (hereinafter abbreviated as UMP) from OMP, and uridine decarboxylase [EC 4.1.1.23] that has the activity of producing uridine from uracil. Phosphorylase [EC 2.4.2.3], uridine kinase [EC 2.7.1.48] having the activity of producing UMP from uridine, uridylate-cytidylate kinase [EC 2.7.1.48] having the activity of producing uridine-5'-diphosphate (hereinafter abbreviated as UDP) from UMP, nucleoside diphosphate kinase [EC 2.7.4.6] having the activity of producing uridine-5'-triphosphate (hereinafter abbreviated as UTP) from UDP, and cytidine-5'-triphosphate synthetase [EC 6.3.4.2] having the activity of producing CTP from UTP are required.

[0202] Many microorganisms, including Escherichia coli, possess the CTP synthetic pathway, but it is preferable that the activity of this pathway is enhanced in the recombinant microorganism used in the method for producing CDP-choline.

[0203] That is, 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 necessary for the CTP synthetic pathway, including PyrG, and at least one of CCT and CKI, is enhanced. Such a microorganism is, for example, a microorganism in the above-mentioned "genetically modified microorganism in which the activity of a target protein is enhanced," in which the "target protein" is at least one of the enzymes necessary for the CTP synthetic pathway, including PyrG, and at least one of CCT and CKI.

[0204] Such a 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," a DNA encoding at least one of the above-mentioned enzymes necessary for the CTP synthetic pathway, including PyrG, and at least one of CCT and CKI.

[0205] Whether a microorganism is capable of producing CDP-choline can be confirmed by culturing the microorganism in a medium and detecting CDP-choline using HPLC (for example, an analytical device SPD-M20A manufactured by Shimadzu Corporation) as described below.

[0206] In "(III) Method for producing CDP-choline by fermentation," the culture conditions (aerobic conditions, temperature, time, pH), substances that may be added to the medium, and a method for collecting CDP-choline from the culture are the same as those described above in "(I) Method for producing CDP-choline by fermentation."

[0207] CDP-choline can be produced by culturing as described above in the culture medium. The amount of CDP-choline produced can be quantified using HPLC (e.g., an analytical apparatus SPD-M20A manufactured by Shimadzu Corporation) according to the method described in "Analysis Examples" below.

[0208] CDP-choline can be collected from the culture by a combination of conventional methods such as an ion exchange resin method, a precipitation method, and the like. When CDP-choline accumulates within the cells, for example, the cells can be disrupted by ultrasonication or the like, and then removed by centrifugation. CDP-choline can be collected from the resulting supernatant by an ion exchange resin method or the like.

[0209] (IV) Method for Producing CDP-choline by an Enzymatic Method Examples of methods for producing CDP-choline by an enzymatic method 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.

[0210] A microorganism used in enzymatic production of CDP-choline (hereinafter also referred to as "CDP-choline-producing recombinant microorganism (IV)") is a 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 a parent strain.

[0211] When a culture of a recombinant microorganism or a treated product of the culture is used as an enzyme source for a reaction producing CDP-choline, by using 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 in 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.

[0212] An example of a recombinant microorganism (IV) for producing CDP-choline is a microorganism in which the "target protein" in the above-mentioned "genetically modified microorganism having reduced or deleted 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 GT2 enzyme and GT28 enzyme in the above-mentioned CAZy classification, or at least one selected from the above-mentioned [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 deleted activity of the target protein."

[0213] In the method for producing CDP-choline by an enzymatic method, 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. Examples of enzymes involved in the CDP-choline biosynthetic pathway include pyrG, CCT, and CKI.

[0214] In 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 having 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 by 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 the presence of 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 having the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or eliminated compared to a parent strain and that serves as an enzyme source that can be used in a CDP-choline production reaction may be a recombinant microorganism that has the ability to produce a protein having the above-mentioned CCT activity, or, when another substrate is used, may be a recombinant microorganism that has the ability to produce a protein having enzymatic activity that can react with the substrate.

[0215] The method for culturing the microorganism and the medium are the same as those described above in "(I) Method for Producing CDP-choline Glycoside by Fermentation." The treatment product of the culture and the reaction conditions are the same as those described above in "(II) Method for Producing CDP-choline Glycoside by Enzymatic Method."

[0216] 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."

[0217] [Method for inhibiting the production of glycosides of cytidine residue-containing compounds] 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 the 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 the 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."

[0218] The reduced or absent activity of transferring a sugar from a sugar nucleotide in a genetically modified microorganism can be confirmed, for example, by comparing the activity of transferring a sugar from a sugar nucleotide between the genetically modified microorganism and a control microorganism that has not been modified to reduce the activity. Specifically, each microorganism is cultured and supplied with an appropriate substrate and sugar donor, e.g., CDP-choline and UDP-glucose to produce CDP-choline glucose glycoside when confirming the activity of a protein transferring glucose from UDP-glucose to CDP-choline. Finally, the amount of CDP-choline glucose glycoside produced in the reaction solution is analyzed using the analytical method described below in "Analysis Examples," and the amount of CDP-choline glucose glycoside produced between the genetically modified microorganism and a control microorganism that has not been modified to reduce the activity can be compared to confirm that the activity of transferring a sugar from a sugar nucleotide in the genetically modified microorganism is reduced or absent.

[0219] An example of a "recombinant microorganism for inhibiting the production of glycosides of cytidine residue-containing compounds" is a microorganism in which the "target protein" in the aforementioned "genetically modified microorganism having reduced or deleted 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 GT2 enzyme and GT28 enzyme in the CAZy classification described above, or at least one selected from the above-mentioned [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 deleted activity of the target protein."

[0220] Furthermore, in the present disclosure, "inhibiting the production of glycosides of cytidine residue-containing compounds" may also mean that the ratio of CDP-choline glycoside to CDP-choline in a composition containing CDP-choline produced using a "genetically modified microorganism in which the activity of a target protein is reduced or deleted" is equal to or less than a certain value. In this specification, a composition produced using a recombinant microorganism for inhibiting the production of glycosides of cytidine residue-containing compounds is also referred to as CDP-choline-containing composition B. The "CDP-choline-containing composition B" of the present disclosure may be any composition containing CDP-choline and may further contain CDP-choline glycoside. CDP-choline-containing composition B 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; or it may be the aqueous medium in which CDP-choline is produced by the enzymatic method described in "(IV) Method for producing CDP-choline by enzymatic method" or a purified version of the aqueous medium. Here, "purification" refers to a procedure for 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.

[0221] Composition B produced by the method of this embodiment preferably has a ratio (e.g., which can be evaluated by HPLC peak intensity ratio) of the amount of CDP-choline glycoside produced to the amount of CDP-choline contained in Composition B of 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 under the same conditions, with the same type and amount of recombinant microorganism for inhibiting the production of glycoside of a cytidine residue-containing compound. When a sufficient amount of UDP-Glc is present, the amount of CDP-choline glycoside produced tends to be high, whereas when the amount of UDP-Glc is insufficient, the amount of CDP-choline glycoside produced also tends to be low.

[0222] 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. The 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. For example, the genetically modified microorganism described above in the "Method for producing cytidine diphosphate choline" can be used as a microorganism capable of producing cytidine residue-containing compounds, which are cytidine residue-containing compounds.

[0223] The inhibition of 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 above-mentioned HPLC (for example, an analytical apparatus SPD-M20A manufactured by Shimadzu Corporation), and comparing the detected and quantified glycosides with those of the parent strain.

[0224] In the method of the present disclosure for inhibiting the production of a glycoside of a cytidine residue-containing compound, the glycoside of the cytidine residue-containing compound is preferably CDP-choline glycoside, and more preferably CDP-choline glucose glycoside.

[0225] As explained above, the present specification discloses the following: <<1>> A cytidine diphosphate choline glycoside represented by the following general formula (1), a salt thereof, an N-oxide thereof, or a solvate thereof:

[0226]

[0227] (In formula (1), R is a sugar residue.)

[0228] <<2>> The cytidine diphosphate choline glycoside, a salt thereof, an N-oxide thereof, or a solvate thereof according to <<1>> above, wherein the cytidine diphosphate choline glycoside is a cytidine diphosphate choline glucose glycoside represented by the following general formula (2):

[0229]

[0230] <<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 the activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound. <<5>> A method for producing a glycoside of a cytidine residue-containing compound according to <<4>> above, wherein the glycoside of the cytidine residue-containing compound is the cytidine diphosphate choline glycoside according to <<1>> or <<2>> above. <<6>> A 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 a cytidine residue-containing compound is an enzyme classified as at least one of GT2 enzyme and GT28 enzyme in the CAZy classification. <<7>> A 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 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 the 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 cytidine residue-containing compound. [3] A homologous protein comprising an amino acid sequence having 60% or more identity to 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. <<8>> A genetically modified microorganism of the following (A) or (B): (A) A genetically modified microorganism in which the activity of a protein having an activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or absent compared to that of a parent strain, and which has the ability to produce a cytidine residue-containing compound. (B) A genetically modified microorganism in which the activity of a protein having an activity of transferring a sugar from a sugar nucleotide to a cytidine residue-containing compound is reduced or absent compared to that of a parent strain. <<9>> The genetically modified microorganism according to <<8>> above, wherein the cytidine residue-containing compound is cytidine diphosphate choline.<<10>> The genetically modified microorganism according to <<8>> or <<9>> above, 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]: [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 cytidine residue-containing compound. [3] A homologous protein comprising an amino acid sequence having 60% or more identity to 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. <<11>> The genetically modified microorganism according to <<8>> or <<9>> 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 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 <<8>> to <<13>> above, and producing a cytidine residue-containing compound in at least one of a culture supernatant and intracellular space using the genetically modified microorganism. <<15>> A method for producing a cytidine residue-containing compound according to <<14>> above, 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):

[0231]

[0232] (In formula (1), R is a sugar residue.)

[0233] <<18>> A composition comprising cytidine diphosphate choline produced using the genetically modified microorganism according to any one of <<8>> to <<13>> above, wherein the abundance ratio of the amount of cytidine diphosphate choline glycoside produced relative to the amount of cytidine diphosphate choline produced is 35% or less. <<19>> A method for producing a composition, comprising: preparing the genetically modified microorganism according to any one of <<8>> to <<13>> above, and producing a composition using the microorganism, wherein the abundance ratio of cytidine diphosphate choline glycoside relative to the amount of cytidine diphosphate choline in the composition is 35% or less. <<20>> The composition according to <18>, wherein the abundance ratio of the amount of cytidine diphosphate choline glycoside produced relative to the amount of cytidine diphosphate choline produced is 0.0005% or more and 35% or less. <<21>> A method for producing the composition according to <19>, wherein the abundance ratio of cytidine diphosphate choline glycoside relative to the amount of cytidine diphosphate choline in the composition is 0.0005% or more and 35% or less.

[0234] The present invention will be specifically explained below by way of examples, but the present invention is not limited to the following examples as long as it does not depart from the gist of the invention.

[0235] [Analysis Examples] In the examples, CDP-choline and CDP-choline glycoside were analyzed using an analytical instrument SPD-M20A manufactured by Shimadzu Corporation under the following conditions: Analysis conditions Column: Shodex Asahipak Analysis temperature: 50°C Flow rate: 0.5 ml / min Eluent composition: 30 mM potassium dihydrogen phosphate / 10% acetonitrile (pH 3.5) Detector: SPD-M20A

[0236] 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.

[0237] This fragment was used to disrupt the opgH gene of the BL21(DE3) strain using the Lambda-Red recombination system (Datsenko K. A. and Wanner B. L. (2000) Proc. Natl. Acad. Sci. USA 97:6640-6645). Specifically, the pKD46 plasmid was introduced into the BL21(DE3) strain, and the strain was cultured in the presence of arabinose to express the λ 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 eliminate pKD46, thereby deleting the opgH gene and obtaining an opgH-non-expressing strain (BL21 (DE3) opgH::kan) in which opgH is not expressed.

[0238] Example 2: Construction of opgH Expression Plasmid A plasmid expressing the E. coli opgH gene (SEQ ID NO: 3) was constructed by the following procedure. An opgH gene fragment was obtained by amplification using E. coli genomic DNA as a template and primers of SEQ ID NO: 4 and SEQ ID NO: 5. Next, PCR was performed using plasmid pET21a (Novagen) as a template and primers of SEQ ID NO: 6 and SEQ ID NO: 7. The obtained 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.

[0239] 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).

[0240] Example 4 Evaluation of the Effect of OpgH Expression on the Amounts of CDP-Choline and CDP-Choline Glycoside The above-mentioned BL21(DE3)opgH::kan / pET21a and BL21(DE3)opgH::kan / pET21a-opgH were inoculated into wide test tubes containing 5 ml of LB medium [10 g / L Bactotryptone (manufactured by Difco), 5 g / L yeast extract (manufactured by Difco), 10 g / L sodium chloride] containing 100 mg / mL ampicillin, and cultured at 30°C for 16 hours. 500 μL 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. 50 ml of culture for each strain was suspended in 5 ml of suspension buffer (50 mM phosphate buffer (pH 7.0)) and disrupted by sonication. After disruption, the cells were centrifuged at 5,800 × g for 10 minutes to precipitate undisrupted cells, and the supernatant was collected. The supernatant was subjected to quantification of protein amount by the Bradford method and used for the reaction.

[0241] The 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 allowing the mixture to stand at 30°C. 16 hours after the start of the reaction, the reaction was stopped by heating at 99°C for 10 minutes, and the amounts of CDP-choline and CDP-choline glycoside were analyzed using a Shimadzu SPD-M20A analyzer according to the method described in [Analysis Example].

[0242] The results are shown in Table 1. As shown in Table 1, it was demonstrated that CDP-choline glycoside was produced from CDP-choline by the opgH-expressing strain. On the other hand, no CDP-choline glycoside was produced in the opgH-disrupted strain.

[0243]

[0244] Therefore, it was revealed that CDP-choline glycoside is produced by glycosyltransferase of UDP-glucose to CDP-choline glycoside when opgH, an E. coli enzyme, is expressed in microorganisms, and that the production of CDP-choline glycoside is suppressed when opgH is not expressed in microorganisms. This finding is thought to contribute to the production and suppression of CDP-choline glycoside.

[0245] 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. When an opgH-expressing strain is used, the abundance ratio of CDP-choline glycoside relative to the amount of CDP-choline produced is 36%, whereas when an opgH non-expressing strain is used, the abundance ratio of CDP-choline glycoside relative to the amount of CDP-choline is 0%. In another embodiment, when an opgH-expressing strain is used, the abundance ratio of CDP-choline glycoside relative to the amount of CDP-choline produced is 4.3%, whereas when an opgH non-expressing strain is used, the abundance ratio of CDP-choline glycoside relative to the amount of CDP-choline is 0%.

[0246] That is, in the production of a cytidine residue-containing compound, 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 parent strain can suppress the production of glycosides of the cytidine residue-containing compound. This demonstrates that CDP-choline can be efficiently produced by using a strain 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 reduced or deleted compared to the parent strain.

[0247] [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.

[0248] The conditions for the direct injection-mass spectrometry (DI-MS) and nuclear magnetic resonance spectrometry (NMR) described below are as follows: Direct injection-mass spectrometry (DI-MS) Liquid chromatograph section Apparatus: Waters ACQUITY UPLC type Mobile phase: water:acetonitrile=1:1 Flow rate: 0.2 ml / min

[0249] 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

[0250] Nuclear magnetic resonance analysis (NMR) Apparatus: 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

[0251] 6-1. Estimation of the molecular formula To investigate the molecular formula of RT22, direct injection mass spectrometry (DI-MS) was performed. As a result, the mass difference between the ions (m / z 651 and m / z 649) detected in ESI positive mode and ESI negative mode was 2, which indicated that the former was a protonated molecule ([M+H] ＋ ), the latter is the deprotonated molecule ([M−H] － Considering the difference in H from the protonated and deprotonated molecules, the molecular formula is C ２０ H ３６ N ４ O １６ P ２ It was estimated that:

[0252] 6-2. Structural Estimation by MS / MS Measurements To investigate the structure of RT22, MS / MS analysis was performed on RT22 (molecular weight: 650) and citicoline (molecular weight: 488). As a result, the product ion patterns below m / z 489 were similar, suggesting that RT22 has a citicoline skeleton. Furthermore, as a result of estimating the composition from the mass difference of 162 between m / z 489 and m / z 651, C ６H １０ O ５ was proposed as a candidate, and it was therefore predicted that RT22 has a structure in which a monosaccharide is added to the citicoline skeleton.

[0253] 6-3. Nuclear Magnetic Resonance Analysis The 1H-NMR spectrum and 13C-NMR spectrum of RT22 were evaluated, and the following structure was predicted.

[0254]

[0255] From the results of 1D-TOSCY spectrum, selective excitation of a signal corresponding to the 1st position of a monosaccharide resulted in the detection of signals presumed to correspond to the 2nd to 6th positions, supporting the presence of a monosaccharide. Furthermore, analysis of signals other than monosaccharides revealed no inconsistency with the citicoline skeleton. Furthermore, analysis of the remote correlation in the HMBC spectrum revealed a remote correlation between signal A of the monosaccharide and signal B of the citicoline skeleton, suggesting that they are bound within a few bonds. The bond was presumed to be as follows:

[0256]

[0257] 1H-NMR(D2O)δppm:δ = 8.10 (1H, d, J = 8.0 Hz), 6.26 (1H, d, J = 8.0 Hz), 6.22 (1H, d, J = 4.5 Hz), 4.66 (1H, d, J = 8.0 Hz), 4.54 (1H, t, J = 4.8 Hz), 4.49 (1H, t, J = 5.0 Hz), 4.45-4.15 (5H, m), 3.80-3.60 (4H, m), 3.55-3.30 (4H, m), 3.23 (9H, s)

[0258] 13C-NMR(D2O) δppm:164.7, 154.8, 146.5, 106.0, 98.8, 91.0, 85.9, 84.8, 78.9, 78.4, 76.1, 72.2, 71.8, 68.9, 67.5, 63.4, 62.9, 56.9 ESI+MS m / z 651([M+H] + ) ESI-MS m / z 649([M-H] - )

[0259] It is clear that a person skilled in the art can conceive of various modifications and alterations within the scope of the claims, and it is understood that these also naturally fall within the technical scope of the present invention. Furthermore, the components in the above-described embodiments may be combined in any manner without departing from the spirit of the invention.

[0260] This application is based on a Japanese patent application (Patent Application No. 2024-062224) filed on April 8, 2024, the contents of which are incorporated herein by reference.

[0261] 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 a fragment for opgH SEQ ID NO: 5: Nucleotide sequence of primer Rv for amplifying a fragment for opgH SEQ ID NO: 6: Nucleotide sequence of primer Fw for amplifying a fragment for pET21a SEQ ID NO: 7: Nucleotide sequence of primer Rv for amplifying a fragment for pET21a SEQ ID NO: 8: Amino acid sequence of opgH derived from Escherichia coli (Accession No.: WP_001295445.1) SEQ ID NO: 9: Amino acid sequence of opgH derived from Bacillus SEQ ID NO: 10: Nucleotide sequence of UgtP derived from Bacillus subtilis (Accession No.: NC_000964.3 2306514-2307662) SEQ ID NO: 11: Amino acid sequence of UgtP derived from Bacillus subtilis (Accession No.: NP_390075.1)

Claims

1. Cytidine diphosphate choline glycoside represented by the following general formula (1), a salt thereof, an N-oxide thereof or a solvate thereof: (In formula (1), R is a sugar residue.) 2. The cytidine diphosphate choline glycoside, its salt, its N-oxide or its solvate according to claim 1, wherein the cytidine diphosphate choline glycoside is a cytidine diphosphate choline glucose glycoside represented by the following general formula (2):

3. A composition containing at least one of the cytidine diphosphate choline glycoside according to claim 1 or 2, 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 the activity of transferring a sugar from a sugar nucleotide to the cytidine residue-containing compound.

5. A method for producing a glycoside of a cytidine residue-containing compound according to claim 4, wherein the glycoside of the cytidine residue-containing compound is the cytidine diphosphate choline glycoside according to claim 1 or 2.

6. A method for producing a glycoside of a cytidine residue-containing compound according to claim 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 GT2 enzyme and GT28 enzyme in the CAZy classification.

7. The method for producing a glycoside of a cytidine residue-containing compound according to claim 4, 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]: [1] A protein comprising the amino acid sequence represented by SEQ ID NO: 8 or 10. [2] A mutant protein comprising the 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 cytidine residue-containing compound. [3] A homologous protein comprising an amino acid sequence having 60% or more identity to 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.

8. Genetically modified microorganisms that fall under either (A) or (B) below. (A) 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, and which has the ability to produce 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.

9. The genetically modified microorganism of claim 8, wherein the cytidine residue-containing compound is cytidine diphosphate choline.

10. The genetically modified microorganism according to claim 8, 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]: [1] A protein comprising the amino acid sequence represented by SEQ ID NO: 8 or 10. [2] A mutant protein comprising the 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 cytidine residue-containing compound. [3] A homologous protein comprising an amino acid sequence having 60% or more identity to 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.

11. The genetically modified microorganism according to claim 8, 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 GT2 enzyme and GT28 enzyme in the CAZy classification.

12. The genetically modified microorganism according to claim 8, 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 of claim 8, 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 claims 8 to 13, and using the genetically modified microorganism to produce the cytidine residue-containing compound in at least one of a culture supernatant and intracellular space.

15. The method for producing a cytidine residue-containing compound according to claim 14, wherein the cytidine residue-containing compound is cytidine diphosphate choline.

16. A method for inhibiting the production of glycosides of compounds containing cytidine residues, comprising preparing the genetically modified microorganism of any one of claims 8 to 13.

17. A method for inhibiting the production of a glycoside of a cytidine residue-containing compound according to claim 16, wherein the glycoside of the cytidine residue-containing compound is a cytidine diphosphate choline glycoside represented by the following general formula (1): (In formula (1), R is a sugar residue.) 18. A composition containing cytidine diphosphate choline produced using a genetically modified microorganism according to any one of claims 8 to 13, wherein the ratio of the amount of cytidine diphosphate choline glycoside produced to the amount of cytidine diphosphate choline produced is 35% or less.

19. A method for producing a composition, comprising: preparing a genetically modified microorganism according to any one of claims 8 to 13; and producing a composition using said microorganism, wherein the ratio of cytidine diphosphate choline glycoside to cytidine diphosphate choline in said composition is 35% or less.

20. The composition described in claim 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. A method for producing a composition according to claim 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.

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