Recombinant microorganism used for producing CDP-choline, and method for producing CDP-choline using said recombinant microorganism

Abstract

A recombinant microorganism having a capability of producing xenogeneic CTP:phosphocholine cytidylyltransferase, wherein the CTP:phosphocholine cytidylyltransferase is a polypeptide formed of a specific amino acid sequence and having a CTP:phosphocholine cytidylyltransferase activity. A method for producing CDP-choline, the method comprising producing CDP-choline by using said recombinant microorganism.

Description

Recombinant microorganism for use in producing CDP-choline and method for producing CDP-choline using said recombinant microorganism

[0001] The present invention relates to a transformant capable of producing a protein having excellent substrate specificity and activity to synthesize cytidine diphosphate choline, and an efficient method for producing cytidine diphosphate choline using the transformant.

[0002] Cytidine 5'-diphosphate choline (hereinafter referred to as 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).

[0003] Despite its usefulness, it is difficult to obtain large amounts of CDP-choline by extraction from natural sources or chemical synthesis, and it is mainly synthesized by enzymatic reactions in living organisms using cytidine monophosphate (hereinafter referred to as CMP) or orotic acid as starting materials (Patent Documents 1 and 2 and Non-Patent Documents 2 and 3).

[0004] When producing CDP-choline from orotic acid, first, cytidine triphosphate (hereinafter referred to as CTP) is synthesized from orotic acid through multiple enzymatic reactions. Then, the obtained CTP is condensed by the action of CTP:phosphocholine cytidyltransferase in the presence of phosphocholine (hereinafter referred to as P-choline) synthesized by an enzymatic reaction from choline, thereby synthesizing CDP-choline (Non-Patent Document 3).

[0005] Similarly, when CMP is used as a starting material, CTP is produced from CMP and then condensed with CTP:phosphocholine cytidylyltransferase in the presence of P-choline to obtain CDP-choline (Non-Patent Document 3).

[0006] From the above, it is clear that CTP:phosphocholine cytidylyltransferase plays an important role in the production of CDP-choline using an enzymatic reaction.

[0007] CTP:phosphocholine cytidylyltransferases are widely distributed in mammals including humans, eukaryotes such as yeast, and bacteria (Non-Patent Documents 4, 5, and 6). These CTP:phosphocholine cytidylyltransferases basically have the activity of synthesizing CDP-choline from CTP and P-choline.

[0008] However, when deoxycytidine triphosphate (hereinafter referred to as dCTP) is present, it is known that dCTP is accepted as a substrate instead of CTP, and deoxycytidine diphosphate choline (hereinafter referred to as dCDP-choline) is produced from dCTP and P-choline (Non-Patent Document 7).

[0009] International Publication No. WO 2003 / 095660 International Publication No. WO 2007 / 023830

[0010] Nutrients (2020) Vol. 12 (3), 793, p. 1-17Bull. Inst. Chem. Res. Kyoto U. (1976) Vol. 53, p. 546-562Appl. Microbiol. Biotechnol. (2017) Vol. 101, p. 1409-1417J. Bio. Chem. (1998) Vol. 273, p. 14022-14029Eur. J. Biochem. (1987) Vol. 169, p. 477-486J. Bio. Chem. (2002) Vol. 277, p. 4343-4350Eur. J. Biochem. (1983) Vol. 131, p. 223-229

[0011] Since dCTP is a compound essential for life activities as a raw material for DNA synthesis, it is not possible to completely suppress the by-production of dCDP-choline in the production of CDP-choline using microorganisms, which hinders efficient CDP-choline production. In addition, dCDP-choline has physical properties very similar to those of CDP-choline, making it difficult to separate the two.

[0012] Therefore, a CTP:phosphocholine cytidyltransferase that exhibits higher substrate specificity for CTP than for dCTP is required, but no such enzyme has been known to date. Furthermore, although the crystal structure of CTP:phosphocholine cytidyltransferase from Streptococcus pneumoniae has been elucidated (Non-Patent Document 6), its reactivity for dCTP has not been predicted.

[0013] An object of the present invention is to provide a recombinant microorganism for use in producing CDP-choline and a method for producing CDP-choline using the recombinant microorganism.

[0014] As a result of investigating the above-mentioned problems, the present inventors have discovered a CTP:phosphocholine cytidylyltransferase that exhibits excellent substrate specificity, being more reactive with CTP than with dCTP. Furthermore, they have found that CDP-choline can be efficiently produced by utilizing a recombinant microorganism capable of producing a heterologous CTP:phosphocholine cytidylyltransferase. Based on these findings, the present invention has been completed.

[0015] That is, the present invention relates to the following: 1. A recombinant microorganism capable of producing a heterologous CTP:phosphocholine cytidylyltransferase, wherein the CTP:phosphocholine cytidylyltransferase is any one of polypeptides selected from the following (A1) to (A3) and (B1) to (B3): (A1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10. (A2) A polypeptide consisting of an amino acid sequence having at least 60% identity to the amino acid sequence shown in SEQ ID NO: 10 and having CTP:phosphocholine cytidylyltransferase activity. (A3) A polypeptide consisting of an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 10 and having CTP:phosphocholine cytidylyltransferase activity. (B1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14. (B2) A polypeptide consisting of an amino acid sequence having at least 60% identity to the amino acid sequence shown in SEQ ID NO: 14 and having CTP:phosphocholine cytidylyltransferase activity. (B3) A polypeptide consisting of an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 14 and having CTP:phosphocholine cytidylyltransferase activity. 2. 2. The recombinant microorganism according to 1 above, into which any one DNA selected from the following (a1) to (a3) ​​and (b1) to (b3) has been introduced in an expressible manner:(a1) DNA encoding the amino acid sequence shown in SEQ ID NO: 10; (a2) DNA consisting of an amino acid sequence having at least 60% identity to the amino acid sequence shown in SEQ ID NO: 10 and encoding a polypeptide having CTP:phosphocholine cytidylyltransferase activity; (a3) ​​DNA consisting of an amino acid sequence obtained by deleting, substituting, inserting or adding 1 to 20 amino acids in the amino acid sequence shown in SEQ ID NO: 10 and encoding a polypeptide having CTP:phosphocholine cytidylyltransferase activity; (b1) DNA encoding the amino acid sequence shown in SEQ ID NO: 14; (b2) DNA consisting of an amino acid sequence obtained by deleting, substituting, inserting or adding 1 to 20 amino acids in the amino acid sequence shown in SEQ ID NO: 14 and encoding a polypeptide having CTP:phosphocholine cytidylyltransferase activity; (b3) DNA consisting of DNA having at least 60% identity to the DNA encoding the amino acid sequence shown in SEQ ID NO: 14 and encoding a polypeptide having CTP:phosphocholine cytidylyltransferase activity. 3. The recombinant microorganism according to 1 or 2 above, which is Escherichia coli. 4. 4. The recombinant microorganism according to any one of 1 to 3 above, wherein the reactivity of the CTP:phosphocholine cytidylyltransferase to dCTP is 0.75 or less when the reactivity to CTP is taken as 1. 5. A method for producing CDP-choline, comprising producing CDP-choline using the recombinant microorganism according to any one of 1 to 4 above.

[0016] The recombinant microorganism of the present invention is capable of producing a heterologous CTP:phosphocholine cytidylyltransferase that exhibits excellent substrate specificity, i.e., higher reactivity with CTP than with dCTP. By utilizing the recombinant microorganism of the present invention, CDP-choline can be efficiently produced while suppressing the by-production of dCDP-choline.

[0017] 1 shows the reactivity of SplicC, LslicC, and AhlicC to CTP or dCTP. The vertical axis represents the ratio of the amount of dCDP-choline to the amount of CDP-choline generated using CTP or dCTP and P-choline as substrates. SplicC, LslicC, and AhlicC represent CTP:phosphocholine cytidylyltransferase derived from Streptococcus pneumoniae, Lactobacillus salivarius, and Anaerococcus hydrogenalis, respectively.

[0018] 1. Recombinant Microorganism and Method for Producing the Same The recombinant microorganism of the present invention is a recombinant microorganism capable of producing a heterologous CTP:phosphocholine cytidylyltransferase. In the present invention, the CTP:phosphocholine cytidylyltransferase is any one of polypeptides selected from the following (A1) to (A3) and (B1) to (B3): (A1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10. (A2) A polypeptide consisting of an amino acid sequence having at least 60% identity to the amino acid sequence shown in SEQ ID NO: 10, and having CTP:phosphocholine cytidylyltransferase activity. (A3) A polypeptide consisting of an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 10, and having CTP:phosphocholine cytidylyltransferase activity. (B1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14. (B2) A polypeptide consisting of an amino acid sequence having at least 60% identity to the amino acid sequence shown in SEQ ID NO: 14, and having CTP:phosphocholine cytidylyltransferase activity. (B3) A polypeptide consisting of an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 14, and having CTP:phosphocholine cytidylyltransferase activity.

[0019] The polypeptides described in (A2) or (B2) above include polypeptides consisting of an amino acid sequence having at least 60% 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 shown in SEQ ID NO: 10 or 14, in the following order, and having CTP:phosphocholine cytidylyltransferase activity.

[0020] The polypeptide described in (A3) or (B3) above includes a polypeptide consisting of an amino acid sequence in which 1 to 20, preferably 1 to 10, more preferably 1 to 8, and most preferably 1 to 5 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 10 or 14, and which has CTP:phosphocholine cytidylyltransferase activity.

[0021] The identity of amino acid sequences and nucleotide sequences can be determined using the Lipman-Pearson method [Science, 227(4693), 1435-41(1985)], the BLAST algorithm by Karlin and Altschul [Pro. Natl. Acad. Sci. USA, 90, 5873(1993)], or FASTA [Methods Enzymol., 183, 63(1990)]. Based on the BLAST algorithm, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403(1990)]. When analyzing a base sequence using BLASTN based on BLAST, the parameters are, for example, score = 100 and wordlength = 12. When analyzing an amino acid sequence using BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known.

[0022] An amino acid sequence in which amino acids have been deleted, substituted, inserted or added refers to an amino acid sequence obtained by artificially deleting or substituting amino acid residues in the original amino acid sequence, or by artificially inserting or adding amino acid residues into the amino acid sequence.

[0023] 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.

[0024] 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: 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

[0025] The CTP:phosphocholine cytidylyltransferase activity refers to the activity of synthesizing CDP-choline from CTP and P-choline.

[0026] The presence of CTP:phosphocholine cytidylyltransferase activity in the above polypeptide can be confirmed, for example, by the following method. First, a recombinant DNA containing DNA encoding the polypeptide is prepared by the method described below. Next, a microorganism in which CTP:phosphocholine cytidylyltransferase activity cannot be confirmed, such as Escherichia coli BL21 strain, is transformed with the recombinant DNA to produce a microorganism, and a cell extract containing the polypeptide is prepared from the resulting culture. Next, the cell extract is contacted with the substrates CTP and P-choline to produce CDP-choline. Finally, CDP-choline in the reaction solution is detected by a common analytical method using high-performance chromatography or gas chromatography, thereby confirming that the target polypeptide has CTP:phosphocholine cytidylyltransferase activity.

[0027] A "recombinant microorganism capable of producing CTP:phosphocholine cytidylyltransferase" can be obtained by introducing recombinant DNA having DNA encoding CTP:phosphocholine cytidylyltransferase into a parent strain in an expressible manner.

[0028] A parent strain refers to an original strain that is the target of genetic modification, transformation, etc. An original strain that is the target of transformation by introducing DNA is called a host strain.

[0029] 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.

[0030] As parent strains, specifically, for example, Escherichia coli BL21 codon plus, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (both manufactured by Agilent Technologies), Escherichia coli BL21(DE3)pLysS (manufactured by Merck Millipore), 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, 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 acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, or Pseudomonas sp. D-0110, or yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, or Candida utilis.

[0031] Furthermore, a bred strain to which the ability to produce CTP and / or P-choline, which are substrates of CTP:phosphocholine cytidylyltransferase, has been artificially imparted or enhanced can also be suitably used as a parent strain.

[0032] Methods for artificially imparting or enhancing the ability to produce CTP and / or P-choline to a microorganism used as a parent strain include the following (1) to (5), and the above known methods can be used alone or in combination: (1) a method for alleviating or eliminating at least one of the mechanisms controlling the CTP and / or P-choline biosynthetic pathway; (2) a method for enhancing the expression of at least one enzyme involved in the CTP and / or P-choline biosynthetic pathway; (3) a method for increasing the copy number of at least one enzyme gene involved in the CTP and / or P-choline biosynthetic pathway; (4) a method for weakening or blocking at least one metabolic pathway branching off from the CTP and / or P-choline biosynthetic pathway to a metabolite other than the target substance; and (5) a method for selecting a cell line having a higher degree of resistance to CTP and / or P-choline analogues compared to a wild-type strain.

[0033] Specific examples of the method for artificially imparting or enhancing the ability to produce CTP and P-choline include known methods such as various gene manipulation methods (Patent Document 1, Patent Document 2, Non-Patent Document 3).

[0034] Whether a microorganism is capable of producing CTP and P-choline can be confirmed by culturing a breeding strain and a parent strain in a medium and comparing the amounts of CTP and P-choline produced. Alternatively, it can be confirmed by transforming the microorganism with a recombinant DNA containing a DNA encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 10 or 14, culturing the transformed microorganism in a medium, and detecting CDP-choline accumulated in the culture using HPLC as described below.

[0035] When the microorganism used as a parent strain has the ability to produce a polypeptide having CTP:phosphocholine cytidylyltransferase activity other than those described above in (A1) to (A3) and (B1) to (B3), CDP-choline can be efficiently produced while suppressing the by-production of dCDP-choline by using a strain in which the function of the polypeptide has been reduced or deleted as a parent strain. The method for reducing or deleting the function of the polypeptide is not particularly limited, as long as it reduces or completely stops the function of the polypeptide. For example, any known method, such as removing all or part of the DNA encoding the polypeptide, can be used as appropriate.

[0036] The DNA encoding CTP:phosphocholine cytidylyltransferase may be any one selected from the following (a1) to (a3), (b1) to (b3), and (c1) to (c3): (a1) DNA encoding the amino acid sequence shown in SEQ ID NO: 10. (a2) DNA consisting of an amino acid sequence having at least 60% identity to the amino acid sequence shown in SEQ ID NO: 10 and encoding a polypeptide having CTP:phosphocholine cytidylyltransferase activity. (a3) ​​DNA consisting of an amino acid sequence obtained by deleting, substituting, inserting or adding 1 to 20 amino acids in the amino acid sequence shown in SEQ ID NO: 10 and encoding a polypeptide having CTP:phosphocholine cytidylyltransferase activity. (b1) DNA encoding the amino acid sequence shown in SEQ ID NO: 14. (b2) DNA consisting of an amino acid sequence obtained by deleting, substituting, inserting or adding 1 to 20 amino acids in the amino acid sequence shown in SEQ ID NO: 14 and encoding a polypeptide having CTP:phosphocholine cytidylyltransferase activity. (b3) DNA consisting of a DNA having at least 60% identity to the DNA encoding the amino acid sequence shown in SEQ ID NO: 14 and encoding a polypeptide having CTP:phosphocholine cytidylyltransferase activity. (c1) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 7 or 11. (c2) DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 7 or 11 and encodes a polypeptide having CTP:phosphocholine cytidylyltransferase activity. (c3) DNA that consists of a nucleotide sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the nucleotide sequence shown in SEQ ID NO: 7 or 11 and encodes DNA having CTP:phosphocholine cytidylyltransferase activity.

[0037] "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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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 / L denatured salmon sperm DNA, followed by washing the filter in a 0.2x SSC solution at about 65°C.

[0042] 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.

[0043] 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 consisting of 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.

[0044] Among the DNAs encoding CTP:phosphocholine cytidyltransferase, the DNA described in (a1) or (c1) above can be obtained by Southern hybridization to the chromosomal DNA library of microorganisms, for example, using the probe DNA that can be designed based on the amino acid sequence represented by SEQ ID NO: 10 or the base sequence represented by SEQ ID NO: 7, or by PCR [PCR Protocols, Academic Press (1990)] using the chromosomal DNA of microorganisms as a template, using the primer DNA that can be designed based on this base sequence.The origin of the chromosomal DNA of the microorganisms used in the above-mentioned operation is not particularly limited, but can be, for example, Lactobacillus genus lactic acid bacteria, Anaerococcus genus, Streptococcus genus, Fusobacterium genus, Bacillus genus, Paenibacillus genus, Granulicatella genus, Clostridium genus bacteria.Among these, Lactobacillus genus lactic acid bacteria and Anaerococcus genus bacteria are preferred, and Lactobacillus salivarius is more preferred. Lactobacillus salivarius strain ATCC 11741 is available from the American Type Culture Collection (ATCC).

[0045] Among the DNAs encoding CTP:phosphocholine cytidyltransferase, the DNA described in (b1) or (c1) above can be obtained by Southern hybridization of a microbial chromosomal DNA library using a probe DNA that can be designed based on the amino acid sequence represented by SEQ ID NO: 14 or the nucleotide sequence represented by SEQ ID NO: 11, or by PCR using a primer DNA that can be designed based on the nucleotide sequence and the chromosomal DNA of the microorganism as a template. The origin of the chromosomal DNA of the microorganism used in the above operation is not particularly limited, but examples include lactic acid bacteria of the genus Lactobacillus, Anaerococcus, Streptococcus, Fusobacterium, Bacillus, Paenibacillus, Granulicatella, and Clostridium. Among these, lactic acid bacteria of the genus Lactobacillus and bacteria of the genus Anaerococcus are preferred, and Anaerococcus hydrogenalis is more preferred. Anaerococcus hydrogenalis ATCC49630 strain can be obtained from the American Type Culture Collection (ATCC).

[0046] Among the DNAs encoding CTP:phosphocholine cytidylyltransferase, the DNAs described in (a2), (b2), or (c2) above can be obtained by, for example, searching various protein sequence databases for amino acid sequences that have 60% or more identity to the amino acid sequence represented by SEQ ID NO: 10 or 14, 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, in that order; or by searching various gene sequence databases for nucleotide sequences that have 95% or more identity to the nucleotide sequence represented by SEQ ID NO: 7 or 11, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity; and using probe DNA or primer DNA that can be designed based on the amino acid sequence or nucleotide sequence obtained by the search, and a microorganism containing the DNA, by a method using Southern hybridization or PCR, similar to the method for obtaining the above-mentioned DNA.

[0047] Among DNAs encoding CTP:phosphocholine cytidylyltransferase, the DNA described in (a3), (b3), or (c3) above can be obtained, for example, by subjecting a DNA consisting of a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 10 or 14, or a nucleotide sequence represented by SEQ ID NO: 7 or 11, to error-prone PCR or the like as a template.

[0048] Alternatively, the DNA described in (a3), (b3), or (c3) 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). That is, first, PCR is performed using the DNA as a template with a sense primer corresponding to the 5' end of DNA consisting of a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 10 or 14 or a nucleotide sequence shown in SEQ ID NO: 7 or 11, and an antisense primer corresponding to the sequence immediately preceding (on the 5' side) the mutation introduction site and having a sequence complementary to the mutation sequence at its 5' end, thereby amplifying fragment A (in which a mutation has been introduced on the 3' side) extending from the 5' end of the DNA to the mutation introduction site. Next, PCR is performed using the DNA as a template with a sense primer corresponding to the sequence immediately (3' side) of the mutation site having 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 PCR is performed without adding a template or primer. Since the sense strand of amplified fragment A and the antisense strand of amplified fragment B share the mutation site, they hybridize and the PCR reaction proceeds with them acting as both primer and template, amplifying the mutated DNA.

[0049] The obtained DNAs described in (a1) to (a3), (b1) to (b3), and (c1) to (c3) above can be used as they are or cleaved with an appropriate restriction enzyme or the like, and then inserted into a vector by a conventional method. The resulting recombinant DNA is then introduced into a host cell, and the DNA can be analyzed using a commonly used base sequence analysis method, for example, 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.

[0050] 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.

[0051] 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)].

[0052] Any method for introducing a recombinant DNA obtained by incorporating the DNA of the present invention into a host cell can be used, as long as it is a method for introducing DNA into a host cell, such as a method using calcium ions (Proc. Natl. Acad. Sci., USA, 69, 2110, 1972), a protoplast method (Japanese Patent Laid-Open Publication No. 63-248394), or an electroporation method (Nucleic Acids Res., 16, 6127, 1988).

[0053] 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.

[0054] 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: 7 or 11.

[0055] The recombinant DNA having the DNA described in (a1) to (a3), (b1) to (b3), and (c1) to (c3) above refers to, for example, a DNA that is autonomously replicable in a parent strain, and in which the DNA described in any one or more of (a1) to (a3), (b1) to (b3), and (c1) to (c3) above has been incorporated into an expression vector containing a promoter at a position where the DNA described in any one or more of (a1) to (a3), (b1) to (b3), and (c1) to (c3) above can be transcribed.

[0056] DNA that can be integrated into the chromosome of a parent strain and is described in any one or more of (a1) to (a3), (b1) to (b3), and (c1) to (c3) above is also a recombinant DNA having the DNA described in (a1) to (a3), (b1) to (b3), and (c1) to (c3) above. When the recombinant DNA is DNA that can be integrated into the chromosomal DNA of a parent strain, it does not need to contain a promoter.

[0057] When a prokaryote such as a bacterium is used as a parent strain, the recombinant DNA capable of autonomous replication in the parent strain is preferably recombinant DNA comprising a promoter, a ribosome binding sequence, one or more of the DNAs described in (a1) to (a3), (b1) to (b3), and (c1) to (c3) above, 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 the ribosome binding sequence, and the start codon is adjusted to an appropriate distance (e.g., 6 to 18 bases).

[0058] In the case of recombinant DNA capable of autonomous replication in a parent strain, 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.

[0059] The expression vector is not particularly limited as long as it is a suitable nucleic acid molecule for introducing, amplifying, and expressing the target DNA into a host, and not only plasmids but also, for example, artificial chromosomes, vectors using transposons, and cosmids may be used.

[0060] 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.

[0061] 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 modified 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.

[0062] 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)].

[0063] 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)].

[0064] 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.

[0065] 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.

[0066] The recombinant DNA of the present invention can be prepared, for example, by treating the DNA fragment prepared by the above-mentioned method with a restriction enzyme and inserting it downstream of the promoter of the above-mentioned appropriate expression vector.

[0067] 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. Information on codon usage frequencies in parent strains used in the production method of the present invention is available through public databases.

[0068] Methods for introducing a recombinant DNA having a DNA encoding CTP:phosphocholine cytidylyltransferase into a host cell as an autonomously replicable plasmid include, for example, the above-mentioned calcium ion method, the protoplast method, the electroporation method, the spheroplast method [Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)], the lithium acetate method [J. Bacteriol., 153, 163 (1983)], and the like.

[0069] Furthermore, when inserting recombinant DNA containing DNA encoding CTP:phosphocholine cytidylyltransferase into the genome of a parent strain, a method such as homologous recombination may be used. That is, DNA containing a portion of the chromosomal region that will induce introduction of the target DNA can be incorporated into the genome by being incorporated into a microbial cell and then inducing homologous recombination in a portion of the chromosomal region. 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)]. Here, the chromosomal region that will induce introduction is not particularly limited, but is preferably a non-essential gene region or a non-gene 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 the microbial cell, and examples thereof include the above-mentioned method using calcium ions, the protoplast method, and the electroporation method.

[0070] Whether a microorganism has been obtained by introducing recombinant DNA having a DNA encoding CTP:phosphocholine cytidylyltransferase into a parent strain in an expressible manner can be confirmed, for example, by comparing the transcription level of the DNA in the microorganism with that of the parent strain by Northern blotting or the production level of the protein in the microorganism with that of the parent strain by Western blotting.

[0071] Whether the microorganism constructed by the above method is a recombinant microorganism capable of producing CTP:phosphocholine cytidylyltransferase can be confirmed, for example, by the following method. First, the parent strain and the constructed microorganism are cultured in media, and a cell extract containing CTP:phosphocholine cytidylyltransferase is prepared from the resulting culture. Next, the cell extract is contacted with the substrates CTP and P-choline to produce CDP-choline. Finally, CDP-choline in the reaction solution is detected by a general analytical method using high-performance chromatography or gas chromatography, thereby confirming that the constructed microorganism is a recombinant microorganism capable of producing CTP:phosphocholine cytidylyltransferase.

[0072] Specific examples of recombinant microorganisms capable of producing heterologous CTP:phosphocholine cytidylyltransferase include BL21 / pET21a-LslicC and BL21 / pET21a-AhlicC, which will be described later in the Examples section.

[0073] The CTP:phosphocholine cytidylyltransferase of the present invention preferably has high substrate specificity and a higher reactivity to CTP than to dCTP. Specifically, for example, when the reactivity to CTP is taken as 1, the reactivity to dCTP is preferably 0.75 or less, more preferably 0.60 or less, 0.50 or less, 0.40 or less, even more preferably 0.30 or less, particularly preferably 0.20 or less, and most preferably 0.15 or less. The reactivity to dCTP when the reactivity to CTP is taken as 1 can be calculated, for example, from [the amount (mM) of dCDP-choline produced when CTP:phosphocholine cytidylyltransferase is allowed to coexist with P-choline and dCTP] / [the amount (mM) of CDP-choline produced when CTP:phosphocholine cytidylyltransferase is allowed to coexist with P-choline and CTP].

[0074] Thus, by utilizing a recombinant microorganism capable of producing CTP:phosphocholine cytidylyltransferase that exhibits substrate specificity with higher reactivity to CTP than to dCTP, CDP-choline can be efficiently produced while suppressing the by-production of dCDP-choline.

[0075] 2. Method for Producing CDP-choline The method for producing CDP-choline of the present invention comprises producing CDP-choline using a recombinant microorganism capable of producing the heterologous CTP:phosphocholine cytidylyltransferase described above.

[0076] Conventional methods for producing CDP-choline include fermentation and biocatalysis (see, for example, Japanese Patent No. 3369236; Y. Liu et al., Appl. Microbiol. Biotechnol., 101, 1409, 2017). The method for producing CDP-choline of the present invention utilizes a recombinant microorganism capable of producing a heterologous CTP:phosphocholine cytidylyltransferase that is more reactive with CTP than with dCTP, thereby effectively suppressing the by-production of dCDP-choline and enabling CDP-choline to be produced more efficiently than conventional methods. Specific examples of such methods are described in Japanese Patent No. 3369236 and Y. Liu et al., Appl. Microbiol. Biotechnol., 101, 1409, 2017. The effects of the present invention can be obtained by using the CTP:phosphocholine cytidylyltransferase of the present invention in place of CCT (choline phosphate cytidyltransferase) in the method for producing CDP-choline described in 1.

[0077] Methods for producing CDP-choline of the present invention include (I) a method for producing CDP-choline by fermentation, and (II) a method for producing CDP-choline using a culture of a recombinant microorganism and a treated product of the culture. Each production method will be described below.

[0078] (I) Method for Producing CDP-choline by Fermentation The method for producing CDP-choline by fermentation includes a method for producing CDP-choline, which comprises culturing in a medium a recombinant microorganism capable of producing a heterologous CTP:phosphocholine cytidylyltransferase 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.

[0079] As a recombinant microorganism used in a method for producing CDP-choline by fermentation, it is preferable to use the recombinant microorganism of the present invention described above in 1. Furthermore, it is preferable that the recombinant microorganism is a microorganism capable of producing CTP and P-choline, which are substrates for CTP:phosphocholine cytidylyltransferase, and / or a microorganism in which the decomposition activity of CTP, P-choline, or CDP-choline has been artificially weakened.

[0080] 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 CTP:phosphocholine cytidylyltransferase or a starting material for the substrate, a carbon source, a nitrogen source, inorganic salts, etc. that can be utilized by the microorganism, and allows efficient cultivation of the microorganism.

[0081] Substrates for CTP:phosphocholine cytidylyltransferase include, for example, CTP and P-choline. Starting materials for the substrate include, for example, orotic acid, CMP, choline chloride, glucose, etc. 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.

[0082] 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.

[0083] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0084] In a method for producing CDP-choline by fermentation, when the microorganism used does not have the ability to produce CTP and P-choline, which are substrates for CTP:phosphocholine cytidylyltransferase, CTP and P-choline are added to the medium.

[0085] Furthermore, in the method for producing CDP-choline by fermentation, when the microorganism used does not have the ability to produce CTP and P-choline, which are substrates for CTP:phosphocholine cytidylyltransferase, instead of adding CTP and P-choline to the medium, CTP and P-choline may be supplied to the recombinant microorganism of the present invention by co-culturing a microorganism capable of producing CTP and / or P-choline with the recombinant microorganism of the present invention.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] CDP-choline can be produced by culturing as described above and thereby producing CDP-choline in the culture. The amount of CDP-choline can be determined using HPLC (for example, an analytical device SPD-M20A manufactured by Shimadzu Corporation).

[0090] 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.

[0091] (II) Method for Producing CDP-choline Using a Culture of a Recombinant Microorganism and a Treated Product of the Culture The method for producing CDP-choline using a culture of a recombinant microorganism and a treated product of the culture is characterized by causing a culture of a recombinant microorganism capable of producing a heterologous CTP:phosphocholine cytidylyltransferase or a treated product of the culture, as an enzyme source, and a substrate to be present in an aqueous medium, and producing CDP-choline in the aqueous medium. The production method may include, for example, producing CDP-choline in the aqueous medium, allowing it to accumulate, and collecting CDP-choline from the aqueous medium.

[0092] The method and medium for culturing the microorganism are the same as those described above in (I).

[0093] 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.

[0094] 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.

[0095] The amount of CTP:phosphocholine cytidyltransferase as an enzyme source is preferably 1 to 500 g / L, more preferably 1 to 300 g / L.

[0096] 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 CTP and P-choline.

[0097] 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.

[0098] In the CDP-choline production reaction, 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.

[0099] 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.

[0100] The CDP-choline produced in the aqueous medium can be quantified and collected by the method described above in (I).

[0101] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0102] [Analysis Example] Analysis of CDP-choline and dCDP-choline by HPLC was carried out using an analyzer SPD-M20A manufactured by Shimadzu Corporation under the following conditions.

[0103] 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

[0104] Solutions containing CDP-choline (Kyowa Hakko Bio Co., Ltd.) at concentrations of 0, 0.005, 0.01, and 0.02 g / L were analyzed as standard samples, and a calibration curve was created from the area value of the peak at a retention time of 10.56 min. The samples were diluted to concentrations of 0.005 to 0.02 g / L and analyzed, and the CDP-choline contained in the samples was quantified using the calibration curve based on the area value of the peak at a retention time of 10.56 min. Because standard samples of dCDP-choline are difficult to obtain, dCDP-choline was quantified using the CDP-choline calibration curve based on the area value of the peak at a retention time of 9.37 min.

[0105] Example 1: Construction of a microorganism obtained by transforming a parent strain with DNA encoding Streptococcus pneumoniae-derived CTP:phosphocholine cytidylyltransferase (SplicC) PCR was performed using chromosomal DNA (GenBank: CP053210.1) having the nucleotide sequence represented by SEQ ID NO: 1 as a template and DNAs consisting of the nucleotide sequences represented by SEQ ID NOs: 2 and 3 as a primer set. A DNA fragment of approximately 700 bp containing DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 was purified.

[0106] PCR was performed using the expression vector pET21a (Novagen) as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 4 and 5 as a primer set, and a DNA fragment of approximately 5.2 kbp (hereinafter referred to as the pET21a inverse fragment) was purified.

[0107] The above two types of DNA fragments were ligated using In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.), and the ligation reaction solution was used to transform Escherichia coli BL21 strain to construct BL21 / pET21a-SplicC (the amino acid sequence of the insert is set forth in SEQ ID NO: 6).

[0108] Example 2: Construction of a microorganism obtained by transforming a parent strain with DNA encoding CTP:phosphocholine cytidylyltransferase (LslicC) derived from Lactobacillus salivarius PCR was performed using chromosomal DNA (GenBank: CP011403.1) having the base sequence represented by SEQ ID NO: 7 as a template and DNAs consisting of the base sequences represented by SEQ ID NOs: 8 and 9 as a primer set. A DNA fragment of approximately 700 bp containing DNA consisting of the base sequence represented by SEQ ID NO: 7 was purified.

[0109] The DNA fragment and the pET21a Inverse fragment were ligated using an In-Fusion HD Cloning Kit, and the ligation reaction solution was used to transform Escherichia coli BL21 strain to construct BL21 / pET21a-LslicC (the amino acid sequence of the insert is shown in SEQ ID NO: 10).

[0110] Example 3: Construction of a microorganism obtained by transforming a parent strain with DNA encoding CTP:phosphocholine cytidylyltransferase (AhlicC) derived from Anaerococcus hydrogenalis PCR was performed using chromosomal DNA (GenBank: NZ_ABXA01000041.1) having the nucleotide sequence represented by SEQ ID NO: 11 as a template and DNAs consisting of the nucleotide sequences represented by SEQ ID NOs: 12 and 13 as a primer set. A DNA fragment of approximately 700 bp containing DNA consisting of the nucleotide sequence represented by SEQ ID NO: 11 was purified.

[0111] The DNA fragment and the pET21a Inverse fragment were ligated using an In-Fusion HD Cloning Kit, and the ligation reaction solution was used to transform Escherichia coli BL21 strain to construct BL21 / pET21a-AhlicC (the amino acid sequence of the insert is shown in SEQ ID NO: 14).

[0112] The identity of the amino acid sequences encoding SplicC, LslicC, and AhlicC was analyzed by the Lipman-Pearson method, and the results are shown in Table 1.

[0113]

[0114] As shown in Table 1, LslicC and AhlicC showed 60% or more identity. On the other hand, SplicC showed low identity of about 30% with LslicC and AhlicC, less than 60%.

[0115] Example 4 Production of CDP-choline or dCDP-choline Using CTP or dCTP and P-choline as Substrates BL21 / pET21a-SplicC, BL21 / pET21a-LslicC, and BL21 / pET21a-AhlicC were inoculated into a wide test tube containing 5 ml of LB medium [10 g / L Bactotryptone (Difco), 5 g / L Yeast Extract (Difco), 10 g / L Sodium Chloride] containing 100 mg / mL ampicillin, and cultured at 30°C for 16 hours. The culture was inoculated at 1% into a baffled Erlenmeyer flask containing 50 ml of LB medium containing 100 mg / mL ampicillin, and cultured at 30°C and 220 rpm for 24 hours. Two hours after the start of culture, 0.1 mM IPTG was added. 50 ml of the culture medium was centrifuged to obtain wet cells.

[0116] 50 ml of each culture medium was suspended in 5 ml of suspension buffer [50 mM phosphate buffer (pH 7.0)] and subjected to ultrasonic disruption. After disruption, the suspension was centrifuged at 5,800 × g for 10 minutes to precipitate undisrupted cells, and the supernatant was collected. The protein content of the supernatant was quantified using the Bradford method and used in the reaction.

[0117] The reaction was carried out by adding the supernatant protein solution to a reaction solution containing 150 mM dipotassium hydrogen phosphate-HCl (pH 7.0) (Fujifilm Wako Pure Chemical Industries, Ltd.), 25 mM magnesium chloride hexahydrate (Kanto Chemical Co., Ltd.), 5 mM CTP or dCTP (both manufactured by Sigma-Aldrich), and 5 mM p-choline (manufactured by Tokyo Chemical Industry Co., Ltd.) to a final concentration of 0.1 mg / ml, followed by shaking at 30°C and 1,000 rpm. Ten minutes after the start of the reaction, the reaction was terminated by adding 1 / 5 the amount of 0.6 N acetic acid (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0118] The test was carried out three times independently, and CDP-choline and dCDP-choline were quantified by HPLC to calculate the mean ± standard deviation. The results are shown in Table 2 and Figure 1.

[0119]

[0120] As shown in Table 2 and FIG. 1 , LslicC and AhlicC had lower dCDP-choline / CDP-choline values ​​than SplicC, whose crystal structure has been elucidated, and when the reactivity to CTP is defined as 1, the reactivity to dCTP was 0.15 or less. These results demonstrated that LslicC and AhlicC exhibit excellent substrate specificity, that is, higher reactivity to CTP than to dCTP. Furthermore, considering the identity of the amino acid sequences shown in Table 1, it was demonstrated that polypeptides having 60% or more identity to the amino acid sequence of LslicC (SEQ ID NO: 10) or the amino acid sequence of AhlicC (SEQ ID NO: 14) exhibit excellent substrate specificity, that is, higher reactivity to CTP than to dCTP.

[0121] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese patent application (Patent Application No. 2021-147985) filed on September 10, 2021, the entirety of which is incorporated by reference. All references cited herein are incorporated in their entirety.

[0122] SEQ ID NO: 1: Nucleotide sequence of SplicC SEQ ID NO: 2: Nucleotide sequence of primer Fw for SplicC cloning SEQ ID NO: 3: Nucleotide sequence of primer Rv for SplicC cloning SEQ ID NO: 4: Nucleotide sequence of primer Fw for pET21a Inverse SEQ ID NO: 5: Nucleotide sequence of primer Rv for pET21a Inverse SEQ ID NO: 6: Amino acid sequence of insert in pET21a-SplicC SEQ ID NO: 7: Nucleotide sequence of LslicC SEQ ID NO: 8: Nucleotide sequence of primer Fw for LslicC cloning SEQ ID NO: 9: Nucleotide sequence of primer Rv for LslicC cloning SEQ ID NO: 10: Amino acid sequence of insert in pET21a-LslicC SEQ ID NO: 11: Nucleotide sequence of AhlicC SEQ ID NO: 12: Nucleotide sequence of primer Fw for AhlicC cloning SEQ ID NO: 13: Nucleotide sequence of primer Rv for AhlicC cloning SEQ ID NO: 14: Amino acid sequence of the insert of pET21a-AhlicC

Claims

1. A recombinant microorganism capable of producing a heterologous CTP:phosphocholine cytidylyltransferase, wherein the CTP:phosphocholine cytidylyltransferase is any one of polypeptides selected from the following (A1) to (A3) and (B1) to (B3): (A1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 (A2) A polypeptide consisting of an amino acid sequence having at least 60% identity with the amino acid sequence shown in SEQ ID NO: 10 and having CTP:phosphocholine cytidylyltransferase activity. (A3) A polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 10 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having CTP:phosphocholine cytidylyltransferase activity. (B1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14 (B2) A polypeptide consisting of an amino acid sequence having at least 60% identity to the amino acid sequence shown in SEQ ID NO: 14 and having CTP:phosphocholine cytidylyltransferase activity. (B3) A polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 14 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having CTP:phosphocholine cytidylyltransferase activity.

2. 2. The recombinant microorganism according to claim 1, into which any one DNA selected from the following (a1) to (a3) and (b1) to (b3) has been introduced in an expressible manner: (a1) DNA encoding the amino acid sequence shown in SEQ ID NO: 10 (a2) DNA encoding a polypeptide consisting of an amino acid sequence having at least 60% identity with the amino acid sequence shown in SEQ ID NO: 10 and having CTP:phosphocholine cytidylyltransferase activity. (a3) DNA encoding a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 10 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having CTP:phosphocholine cytidylyltransferase activity. (b1) DNA encoding the amino acid sequence shown in SEQ ID NO: 14 (b2) DNA encoding a polypeptide consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 14 and having CTP:phosphocholine cytidylyltransferase activity. (b3) DNA having at least 60% identity to the DNA encoding the amino acid sequence shown in SEQ ID NO: 14 and encoding a polypeptide having CTP:phosphocholine cytidylyltransferase activity.

3. The recombinant microorganism of claim 1, which is Escherichia coli.

4. 2. The recombinant microorganism according to claim 1, wherein the reactivity of the CTP:phosphocholine cytidylyltransferase to dCTP is 0.75 or less when the reactivity to CTP is taken as 1.

5. A method for producing CDP-choline, comprising producing CDP-choline using the recombinant microorganism according to claim 1.