[go: up one dir, main page]

WO2008038149A2 - Procédé de production de cytidine 5'-monophosphate - Google Patents

Procédé de production de cytidine 5'-monophosphate Download PDF

Info

Publication number
WO2008038149A2
WO2008038149A2 PCT/IB2007/003807 IB2007003807W WO2008038149A2 WO 2008038149 A2 WO2008038149 A2 WO 2008038149A2 IB 2007003807 W IB2007003807 W IB 2007003807W WO 2008038149 A2 WO2008038149 A2 WO 2008038149A2
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
cmp
protein
woiv
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2007/003807
Other languages
English (en)
Japanese (ja)
Other versions
WO2008038149A3 (fr
Inventor
Hisaya Tanaka
Akihiko Maruyama
Yoshiyuki Yonetani
Ritsuko Katahira
Shingo Kakita
Satoshi Mitsuhashi
Jun-Ichi Saito
Hideki Oda
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kyowa Hakko Bio Co Ltd
KH Neochem Co Ltd
Original Assignee
Kyowa Hakko Kogyo Co Ltd
Kyowa Hakko Bio Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyowa Hakko Kogyo Co Ltd, Kyowa Hakko Bio Co Ltd filed Critical Kyowa Hakko Kogyo Co Ltd
Priority to JP2008536202A priority Critical patent/JP5175734B2/ja
Publication of WO2008038149A2 publication Critical patent/WO2008038149A2/fr
Publication of WO2008038149A3 publication Critical patent/WO2008038149A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases

Definitions

  • Patent application title Method for producing cytidine 5, monomonophosphate
  • the present invention relates to a method for producing a novel protein having an activity of producing cytidine 5, monomonophosphate (CMP) from cytosine, the protein, a DNA encoding the protein, a recombinant DNA containing the DNA, the assembly
  • CMP monomonophosphate
  • the present invention relates to a microorganism having a recombinant DNA, and a method for producing CMP using a culture of the microorganism.
  • CMP is used with other nucleotides, adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), and uridine 5'-monophosphate (UMP) as an additive to infant milk.
  • AMP adenosine 5'-monophosphate
  • GMP guanosine 5'-monophosphate
  • UMP uridine 5'-monophosphate
  • CMP has been produced by enzymatic degradation of yeast RNA (Non-patent Document 1).
  • this RNA degradation method has the disadvantage that four nucleotides are produced at the same time, resulting in a surplus in the product due to the difference between production and demand. For this reason, development of an inexpensive method for producing individual nucleotides is desired.
  • Patent Document 1 An industrial individual production method has been established that can replace the RNA degradation method for GMP (Patent Document 1).
  • inexpensive individual manufacturing methods for CMP, A MP, and UMP have not yet been established.
  • As an individual method for producing these nucleotides there is a method using a salvage synthesis route and using the corresponding base produced by the chemical synthesis method as a substrate.
  • An industrial production method of triphosphoric acid (ATP) is mentioned (Patent Document 2).
  • Enzymes involved in the salvage synthesis pathway include nucleotides corresponding to bases such as uracil phosphoribosyltransferase (UPRTase), which catalyzes the reaction of UMP and pyrophosphate from uracil and phosphoribosylpyrophosphate (PRPP). Enzymes that produce in one step are known. However, the enzymes of the salvage synthesis pathway that produce CMP are not known across all species.
  • UPRTase uracil phosphoribosyltransferase
  • PRPP phosphoribosylpyrophosphate
  • Non-Patent Document 1 J. Gen. Appl. Microbiol., 3, 55 (1957)
  • Patent Document 1 Japanese Patent Laid-Open No. 62-285797
  • An object of the present invention is to provide a method for producing a novel protein having an activity of generating CMP from cytosine, the protein, a DNA encoding the protein, a recombinant DNA containing the DNA, and the recombinant DNA. It is an object of the present invention to provide a method for producing CMP using a microorganism and a culture of the microorganism.
  • the present invention relates to the following (1) to (14).
  • CMP cytidine 5′-monophosphate
  • amino acid sequence represented by SEQ ID NO: 1, 2, or 30 one or more amino acid residues selected from the 36th, 94th, 102th, 141st, 143rd and 198th amino acid residues
  • amino acids other than the 36th, 94th, 102th, 141st, 143rd, 198th, 201st, 204th and 205th amino acids A protein having an amino acid sequence in which one or more amino acid residues selected from residues are substituted with other amino acid residues, and having an activity of generating CMP from cytosine.
  • a recombinant DNA comprising the DNA of (7) above.
  • microorganism (10) The microorganism according to (9) above, wherein the microorganism belongs to the genus Escherichia, Corynebacterium, or Brevibaterium.
  • the microorganism culture of (9) or (10) above or a treated product thereof, a culture of a microorganism belonging to the genus Corynepacteria or a treated product thereof, cytosine, phosphate group donation A method for producing CMP, comprising: a body and an energy donor in an aqueous medium, producing and accumulating CMP in the medium, and collecting CMP from the medium.
  • the present invention also relates to the following (15) to (24).
  • a method for producing a protein having an activity of producing cytidine 5, monomonophosphate (CMP) from cytosine comprising the following steps:
  • Uridine 5 'from uracil The UMP binding site in UPRTa se on a computer using part or all of the three-dimensional structural information of the protein (UPRTa se) that has the activity to generate monophosphate (UMP) Selecting one or more amino acid deletions, substitutions and / or additions in the amino acid sequence of UPRTase to stabilize the complex model of UPRTase and CMP produced by adapting CMP to [2] A step of producing a mutant protein of UP RTase containing the deletion, substitution and / or addition selected in step [1].
  • a method for producing a protein having an activity of producing cytidine 5′-monophosphate (CMP) from cytosine comprising the following steps:
  • Uridine 5 ' Urine binding site in UPRTa se on the computer using part or all of the 3D structure information of protein (UPRTa se) that has the activity to generate monophosphate (UMP) Selecting one or more amino acid deletions, substitutions and Z or additions in the amino acid sequence of UPRTase, which stabilizes a complex model of UPRTase and CMP produced by adapting CMP to
  • a step of producing a UPRTase mutant protein comprising the deletion, substitution and / or addition selected in the steps [11] and [12].
  • amino acid sequence described in SEQ ID NO: 15 the amino acid sequence has one to several amino acids deleted, substituted and / or added,
  • a part of the three-dimensional structural information of a protein comprising the amino acid sequence of SEQ ID NO: 15 represented by the three-dimensional coordinate atomic coordinates described in Table 1 and having an activity of generating UMP from uracil, or
  • substitution of amino acid in which the constituent atom exists in the range of 1 OA from the oxygen atom of the oxo group at the 4-position of pyrimidine ring of UMP There,
  • amino acid substitution is a substitution of tyrosin at position 201 and proline at position 204 of the amino acid sequence shown in SEQ ID NO: 15.
  • amino acid substitution is substitution of tyrosine at position 201 with lysine and substitution of proline at position 204 with leucine in the amino acid sequence shown in SEQ ID NO: 15.
  • a part of the three-dimensional structural information of a protein comprising the amino acid sequence of SEQ ID NO: 15 represented by the three-dimensional coordinate atomic coordinates described in Table 1 and having an activity of generating UMP from uracil, or
  • the protein according to any one of (18) to (22), which is an acid substitution, and comprises one or more amino acid substitutions that stabilize the complex model.
  • the present invention further includes a protein obtained by any one of the above-described methods for producing CP RTase; DNA encoding the protein; recombinant DNA containing the DNA; microorganism containing the recombinant DNA;
  • the present invention relates to CMP used and a method for producing useful substances produced using CMP as a substrate or via CMP.
  • FIG. 1 is a diagram showing the alignment results of amino acid sequences of various UPRTase.
  • FIG. 2 is a diagram showing a complex model of a mutant Escherichia coli iUPRTase having an amino acid substitution of G205D, M141 and A143V and CMP.
  • FIG. 3 is a diagram showing a tetramer model of a complex of Escherichia coli UPRTase and CMP.
  • the protein (CPRTa se) having the activity of producing cytidine 5′-monophosphate (CMP) from cytosine of the present invention is a protein (UPRTa se) having the activity of producing uridine 5′—monophosphate (UMP) from uracil. It can be obtained as a mutant protein.
  • the UPRTase mutated protein is a protein having an amino acid sequence in which one to several amino acids are deleted, substituted and / or added in the amino acid sequence of UPRTase. Note that the term “several” means that 30 may be sufficient, 20 is preferable, and 5 is more preferable.
  • the activity of generating cytidine 5′-monophosphate (CMP) from cytosine means the activity of synthesizing CMP and pyrophosphate from cytosine and PRPP.
  • CPRTase may have the activity to synthesize UMP from uracil and PRP P (UPRTase activity) at the same time, but when compared by a method that can be confirmed by the method performed in the examples described later, etc.
  • the CPRTase activity is higher than the UPRTase activity, more preferable that the CPRTase activity is 10 times higher than the UPRTase activity, and it is further preferable that the CPRTase activity does not substantially have the UPRTase activity.
  • amino acid mutations For the selection of amino acid mutations to be added to UPRTase for the production of CPRTase, it is possible to create mutant proteins at random and perform screening after confirming the activity of these mutant proteins by the method described below.
  • Amino acid mutation or The mutation means an amino acid deletion, substitution and / or addition added to the amino acid sequence of UP RTase.
  • the screening method is not particularly limited.
  • Py r G CTP synthetase
  • co dA cytosine deaminase
  • Cdd cytidine deaminase
  • Escherichia coli that has lost the three enzyme activities
  • the method of performing the transformation using the obtained transformant as an indicator whether or not it grows in a cytosine-containing minimal medium I can give you. It is also possible to prepare a protein having an arbitrary mutation, quantify the amount of CMP produced, and confirm the CPRTase activity.
  • Another method for selecting amino acid mutations to be added to UPRTase for the production of CPRTase is a method using a computer model created based on the three-dimensional structural information of UPRTase. .
  • the 3D structure information of UPRTase any information available to those skilled in the art may be used. Structure information obtained by crystal structure analysis or structure information that can be acquired from a database such as PDB (Protein Data Bank) Alternatively, the structure information created using a protein homology model creation program such as Insight 97 using the structure as a model may be used. Further, the three-dimensional structure information may be information on UP RT as monomers, or information on multimers such as dimers or tetramers. Further, the three-dimensional structure information may relate to a part of the UP RT as monomer or multimer, but preferably includes information on the UMP binding site described later. In addition, when the above three-dimensional information is used for creating a multimer model, it is preferable to include information on a boundary portion between monomers.
  • a composite model of UPRTase and CMP can be created using the above-described three-dimensional structural information of UPRTase.
  • the entire 3D structure information or a part of it may be used to create this model.
  • a site considered to be involved in stabilization of the complex of UPRTase and CMP may be selected in advance, and a complex model including the site may be created.
  • UPRT ase which uses three-dimensional structural information and is the basis for mutation, is a protein that has the activity of generating uridine 5'-monophosphate (UMP) from uracil.
  • UMP uridine 5'-monophosphate
  • UPRTa se derived from Toxoplasma gondii has already been reported (EMBO J. (1998) 17: 3219-3232) (In this UP RTase derived from Toxoplasma gondii, The carbonyl group oxygen atom at the 2-position of the pyrimidine ring in the amide hydrogen atom of phenylalanine, the hydrogen atom at the 3-position of the pyrimidine ring at the carbonyl group oxygen atom of the 234th glycine, and the amide hydrogen atom of the 229th isoleucine The pyrimidine ring stabilizes the UMP bond with the pyrimidine ring by forming a hydrogen bond with the 4-position carbonyl oxygen atom.
  • the UMP binding site can also be determined for other types of UPRTase. Specifically, the alignment of the amino acid sequence of UPRTase from Toxop lasma gondii and the amino acid sequence of UPRTase using three-dimensional structural information was performed (ClustalW [Nuceli c Acids Research 22, 4673, (1994)] European Bioinformatics Institute website http: ⁇ ⁇ .ebi.ac.uk (available from lustalw), gene analysis software included in Genetyx (Software Development Co., Ltd.) 3D structure information UMP The binding site can be determined. As one of the analysis software parameters for alignment, you can use the Deno Retoi Direct.
  • Figure 1 shows an example of alignment of amino acid sequences of various UPRTases including UPRTases derived from Toxoplasma gondii and Escherichia coli. From these results, it is considered that in the UPRTase derived from Escherichia coli, isoleucine at position 202, glycine at position 207, alanine at position 209, etc. are involved in the binding of the pyrimidine ring of UMP.
  • UMP is docked to the UPRT ase derived from Escherichia coli, and the structure that has been reported for the UPRT ase derived from Toxoplasma gondii has a conformation that maintains the hydrogen bond of the UPRT ase and the pyrimidine ring of UMP. After selection, use the UMP complex model created by minimizing the energy of the complex.
  • UMP is converted to CMP as CMP by substituting oxygen at the 4-position force of the pyrimidine ring of the UMP with an amino group and removing hydrogen at the 3-position of the pyrimidine ring. Furthermore, the energy minimization calculation is performed again for the model.
  • Selection of amino acid mutations in UPRT ase for production of CPRT ase using the complex model of UPRT ase and CMP created as described above is to select amino acid mutations in UPRT ase that stabilize the model. This can be done with
  • stabilizing the model means, for example, reducing ⁇ AG.
  • AG free energy change
  • UMP the energy of the complex of UPRTase and UMP minus the energy of the protein alone and the energy of UMP alone
  • ⁇ ⁇ is the value of UPRTase and CMP here. This means the AG force calculated from the complex structure minus the AG calculated from the complex structure of the mutant UPRTase and CMP.
  • the difference between the AG of the model before and after the mutation is calculated on the computer, and the energy level after the amino acid mutation is 5 kJ / mo 1, It can be carried out by selecting a mutation that preferably decreases 20 kJ / mo 1, more preferably 50 kJ / mo 1 or more. Such calculations can be performed with the docking program Glide.
  • selection of mutations such as amino acid substitutions that stabilize the model can be made between model components (between UPRT ase and CMP in a complex model of UPRT ase and CMP, or between monomers in a multimeric model). Etc.) can be used as indicators. In other words, a mutation that increases the affinity between components may be selected.
  • Selection of a mutation that increases affinity can be performed, for example, by visual observation of the model. Between components in the model (UPRT ase and CMP, between monomers Hydrophobic interaction, ⁇ electrostatic interaction ⁇ hydrogen bonding ⁇ ⁇ / ⁇ interaction (interaction between magnetic fields generated by ring currents of aromatic rings) ⁇ 01 / ⁇ interaction (ring current and ring of aromatic rings) Magnetic field interaction generated by electrons of the chill group) Select a mutation that increases the force S, increases, or enhances.
  • mutations can be selected by focusing on the structural differences between UMP and CMP.
  • UMP and CMP differ in the 4-position of the pyrimidine ring, except that UMP is an oxo group and CMP is an amino group. Therefore, for example, the center of the constituent atom exists in the vicinity of the 4-position amino group of the pyrimidine ring of CMP (about 1 OA, preferably about 8 A, more preferably about 5 A from the center of the nitrogen atom of the amino group).
  • Affinity between UPRT ase and CMP can be increased by changing the amino acid residue of ase to an amino acid residue that interacts with the CMP amino group.
  • UMP and CMP differ in whether the nitrogen atom at the 3-position of the pyrimidine ring has a hydrogen atom or not.
  • a UP RT ase in which the center of the constituent atom exists in the vicinity of the nitrogen atom at the 3-position of the CMP pyrimidine ring (about 1 OA, preferably about 8 A, more preferably about 5 A from the center of the nitrogen atom).
  • the affinity between UPRTase and CMP can be increased by changing the amino acid residue to an amino acid residue that interacts with the CMP amino group. Furthermore, it is also preferable to select a mutation by comprehensively judging the amino acid residues of UPRT as near the 4th and 3rd positions of the pyrimidine ring. This is because UMP and CMP can be thought that the relationship between the 2- and 3-position hydrogen bond donors and acceptors of the pyrimidine ring is reversed.
  • the 205 position Gly is changed to, for example, Asp or Glu.
  • Asp or Glu an electrostatic interaction occurs between this Asp or Glu residue and the amino group of cytosine, thereby stabilizing the complex model.
  • 201-position Tyr as Lys
  • the shape of the binding site becomes a flat shape suitable for the binding of the pyrimidine ring, and the complex model can be stabilized.
  • substitution of Gly at position 205 with Asp is G205D
  • a single letter abbreviation is used to indicate the amino acid before mutation, followed by the amino acid after mutation.
  • a complex model of UPRT a s e and UMP can also be used to select mutations that stabilize the complex model of UP R a s e and CMP. Since the binding position of UMP and CMP is considered to be almost the same, by using the complex model of UMP and UP RT ase created directly from the above three-dimensional structure information, CMP and CMP are substantially This is because the amino acid involved in the UPRTase interaction can be selected. In this model, for example, an amino acid residue in the vicinity of the 4-position oxo group of the pyrimidine ring of UMP (1 from the center of the oxygen atom of the oxo group)
  • An amino acid residue in which the center of the constituent atom is present at about 0 A, preferably about 8 A, more preferably about 5 A can be selected.
  • the amino acid after mutation may be selected assuming that the 4-position oxo group of the pyrimidine ring is replaced with an amino group.
  • an amino acid residue in the vicinity of the nitrogen atom at the 3-position of the pyrimidine ring of UMP an amino acid residue in which the center of the constituent atom exists about 10 A, preferably about 8 A, more preferably about 5 A from the center of the nitrogen atom) ) Can be selected.
  • the amino acid after mutation may be selected on the assumption that the hydrogen atom bonded to the nitrogen atom at the 3-position of the pyrimidine ring disappears.
  • a complex model of UPRTase and cytosine or uracil can also be used to select a mutation that stabilizes the complex model of UPRTase and CMP.
  • the uracil bond in U P R T a s e is reported.
  • a mutation that stabilizes can be selected.
  • a complex model of the mutation UPRTase and CMP having the mutation selected as described above is prepared in the same manner as described above, and additional amino acid mutations that stabilize the model are prepared. You may choose. Alternatively, based on the amino acid sequence of the mutation UPRTase having a mutation selected using any one of the above complex models, random screening can be performed to obtain CPRTase with higher activity.
  • DNA having a mutation selected as described above is used as a saddle type DNA encoding the mutation UPRTase, and DNA having random mutations is prepared by the error-prone PC method.
  • Recombination including Screening can be carried out using transformants prepared using body DNA.
  • mutations selected by focusing on the structural differences between UMP and CMP using the complex model of UP RT ase and CMP or the complex model of UPRT ase and UMP other models Mutation that stabilizes can be selected. It is preferable that other mutations coexist with mutations selected by focusing on the structural differences between UMP and CMP.
  • mutations selected by focusing on the structural differences between UMP and CMP for example, in the complex model of UP RTase and CMP, within the range of about 1 OA, preferably about 8 A, more preferably about 5 A from the center of any atom constituting CMP
  • the amino acid residue of UP RTase in which the center exists can be changed to an amino acid residue that interacts with the CMP amino group.
  • the center of the constituent atom exists in the range of about 10 A, preferably about 8 A, more preferably about 5 A from the center of any atom constituting UMP.
  • An amino acid residue of UP RTase can be selected as an amino acid residue to be mutated.
  • UPRTase usually has a dimer-forming force S and GTP, which binds to form a tetramer, increasing the number of amino acids that interact with PRP P and increasing UPRTase activity (Proc Natl. Acad. Sci. USA (2002) 99: 78-83.).
  • a similar trend can be predicted for CPRT a s e, so it can be predicted that CPRT a s e activity is also increased by mutations that stabilize multimers. Therefore, amino acid mutations added to UPRTase for the production of CPRTase can be performed by selecting mutations that stabilize UPRTase or any of the aforementioned multimeric models of the complex. it can.
  • Mutations that stabilize the multimeric model include examining the amino acid at the boundary between each monomer on computer graphics and energeticly unstable elements between them (for example, electrostatic repulsion, three-dimensionality). Mutations that remove unstable elements or enhance interactions.
  • an amino acid having a center of a constituent atom in the range of about 1 OA, preferably about 8 A, more preferably about 5 A from any atom of the adjacent UP RTase monomer is added to the adjacent atom. It can be changed to an amino acid residue that interacts with the UP RTase monomer.
  • the i3 arm consisting of the second and third strands ( ⁇ 2, ⁇ 3) and the second of the other monomer
  • the dimer is formed by the interaction between the third and fourth ⁇ -helices ( ⁇ 2, A3, ⁇ 4). Therefore, these areas including the J3 arm
  • the multimer model may be created based on the three-dimensional structure information of UPRTase or preferably one of the complex models described above.
  • the multimeric structure is thought to contribute to increasing the binding site with the substrate, it is also preferable to create the multimeric model using all or part of the complex model of UPRTase and PRPP.
  • Random screening can be performed based on the UPRTase mutant protein having the mutation selected as described above to obtain CPRT ase having higher activity.
  • mutant protein of UPRTase obtained as CPRTa se is introduced as a secondary mutation, as well as substitution with mutually convertible amino acids as described below.
  • Other mutations may be included.
  • the production method of the variant protein is not particularly limited, but it can be produced by a method using genetic recombination described in the production of a specific protein described later.
  • Examples of CP RT as of the present invention include the proteins described in the following (a) to (d).
  • the 36th amino acid residue is cysteine, the 94th amino acid residue is tyrosin, the 102nd amino acid residue is cysteine, and the 141st amino acid residue is Leucine, a protein of (b) above having an amino acid sequence in which the 1st to 3rd amino acid residues are substituted with valine, or the 1st to 8th amino acid residues of glycine, and (d) the above (a) to (c) 3 6th, 9 4th, 1 0 2nd, 1 4 1st, 1 4 3rd, 1 9 8th, 2 0 1st, 2 0 4th and 2 0
  • the “other amino acid residue” in (b) and (d) above may be either a natural amino acid or a non-natural amino acid.
  • Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L monoglutamic 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, L-cysteine, etc.
  • the 36th amino acid residue is cysteine
  • the 94th amino acid residue is tyrosine or phenylalanine
  • the 102nd amino acid residue is cysteine.
  • the first amino acid residue is derived from oral isine, isoleucine, norleucine, valine, norpaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine and cyclohexanalanalanin Selected amino acids
  • 1 4 3rd amino acid residue is valine, leucine, isoleucine, norleucine, norpaline, alanine, 2_aminobutanoic acid, methionine, 0-methylserine, t-butylglycine, t-butylalanine and cyclohe Amino acid selected from xylalanin, 1 9 8
  • amino acids that can be substituted for each other in the protein (d) are shown below. Amino acids included in the same group can be substituted for each other.
  • Group A Leucine, Isoleucine, Norleucine, Norrin, Norpaline, Alanine, 2-Aminobutanoic acid, Methionine, O-Methylserine, t-Butylglycine, t-Butylalanin, Cyclohexylalanin
  • Group B aspartic acid, gnoretamic acid, isoaspartic acid, isodartamic acid, 2-aminoadipic acid, 2-aminosuberic acid
  • Group C Asparagine, glutamine Group D: Lysine, Arginine, Ornitine, 2,4-Diaminobutanoic acid, 2,3-Diaminopropionic acid
  • Group E Proline, 3-hydroxyproline, 4-hydroxyproline
  • Group F serine, threonine, homoserine
  • the DNA of the present invention includes the DNAs described in the following (e) and (f)
  • the DNA of the present invention can be obtained by introducing a mutation into DNA encoding UPR T ase derived from Escherichia coli.
  • chromosomal DNA is prepared from Escherichia coli W3110 strain, and DNA having the nucleotide sequences represented by SEQ ID NOs: 6 and 7, for example, is chemically synthesized using the DNA as a cage, and the DNA is used as a primer set. It can be obtained by PCR. Specific DNA that can be obtained by the above method includes DNA having the base sequence represented by SEQ ID NO: 5.
  • DNA encoding UP RT ase derived from Escherichia coli is also obtained by a hybridization method using a part or all of the DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 5 as a probe. Can get it can.
  • the hybridization method can be performed in accordance with the method described in Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press x 2001 (hereinafter abbreviated as molecular cloning). .
  • DNA encoding UP RT ase derived from Escherichia coli can also be obtained by chemically synthesizing DNA having the base sequence by a known method based on the base sequence represented by SEQ ID NO: 5.
  • the DNA of the present invention can be obtained by adding molecular cloning and Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter referred to as “DNA” encoding UP RTase derived from Escherichia coli obtained in (1) above). It can be obtained by introducing a site-specific mutation according to the method described in Current Protocols' In Molecular Biology and Abbreviations, etc.
  • the error-prone PCR method [Proc. Sci. USA, 90, 5618 (1 982)] can also be obtained by introducing a random mutation into DNA having the base sequence represented by SEQ ID NO: 5.
  • UPRT ase and UMP such as Toxoplasma gondii and Bacillus caldolyticus derived from various organisms is used as a model, and a homology model of Escherichia coli is created based on this model. Extract amino acid residues in the vicinity of the UMP binding site, select amino acid residues presumed to play an important role in converting substrate specificity from the original substrate uracil to cytosine, and site-specific It is also possible to select a protein having an activity of converting cytosine to CMP by introducing a genetic mutation. For details, refer to the description of “CP RT as design” above.
  • the protein of the present invention of 1 above is obtained by using the method described in Molecular Cloning Current Protocols 'In' Molecular Biology etc., for example, by It can be made to express in.
  • a DNA fragment of an appropriate length containing a portion encoding the protein of the present invention is prepared as necessary, and the DNA fragment is used as an appropriate expression vector.
  • a transformant can be prepared by introducing the silkworm recombinant DNA into a host cell suitable for the expression vector.
  • the protein of the present invention can also be efficiently produced by preparing DNA in which the base sequence of the DNA fragment is substituted so that it is an optimal codon for host cell expression.
  • Any host cell can be used as long as it can express the target gene.
  • the expression vector a vector that can replicate autonomously in the above-described host cell and contains a promoter at a position where the DNA encoding the polypeptide of the present invention can be transcribed is used.
  • the recombinant vector containing the DNA encoding the protein of the present invention is capable of autonomous replication in a prokaryotic organism, and at the same time a promoter, a ribosome binding sequence, A vector composed of the DNA of the present invention and a transcription termination sequence is preferred. It may contain a gene that controls the promoter.
  • pBTrp2 when a microorganism belonging to the genus Escherichia is used as a host cell, pBTrp2, pBTacl, pBTac2 (all available from Boehringer Mannheim), pTrc99A (Pharmacia), pSE280 (Invitrogen), pGEM EX-1 (Promega), QE82L (QIAGEN), pKYPIO (JP-A 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSAl [Agric. Bi ol. Chem., 53, 277 (1989)], pGELl (Proc. Natl. Acad. Sci.
  • pGKA2 prepared from Escherichia coli IGKA2 (FERM BP-6798), JP 60-221091)
  • pTerm2 US 4686191, US4939094, US5160735)
  • pSupex pUB110
  • pTP5 pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)]
  • pGEX Pharmacia
  • pET system Novagen
  • the host cell belongs to the genus Corynebacterium
  • pCGl Japanese Unexamined Patent Publication No. 57-134500
  • pCG2 Japanese Unexamined Patent Publication No.
  • any promoter can be used as long as it functions in the host cell.
  • trp promoter P TRD
  • lac promoter P L promoter
  • P R promoter can be mentioned promoter one terpolymer derived from such T7 promoter, E. coli or phage, or the like.
  • artificially designed and modified promoters such as two promoters in series (PX 2), tac promoter, lacT7 promoter, let I promoter, and the like can also be used.
  • a transcription termination sequence is not necessarily required for the expression of the DNA of the present invention, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.
  • Host cells include microorganisms belonging to Escherichia, Corynebacterium, or Brevibacterium, such as Escnerichia coli, Gorynebacterium ammoniagenes ⁇ Corvneo acterium glutamicum ⁇ Brevibacterium iramariophilum, Brevibacterium iramariophilum sacch arolyticum ⁇ Brevibacterium f lavum and Brevibacterium lactofermentum and the like can be mentioned, more preferably Escherichia coli XL1- Blue, Escheri chia coli X and 2 - Blue, Escherichia coli DH1, Escherichia coli DH5 CK, Esche richia coli MCI 000 ⁇ Escherichia coli MM294, Escherichia coli W1485, Esc herichia coli JM109, Escherichia coli HB101, Escherichia coli No.
  • Escnerichia coli such
  • any method can be used as long as it is a method for introducing DNA into the host cell.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 ( 1972)], protoplast method (JP-A 57-186492, JP-A 57-18649), electroporation [for example, Journa 1 of Bacteriology, 175, 4096 (1993), Appl. Microbiol. Biotechnol. , 52. 541 (1999)], Gene, 17, 107 (1982) and Molecular & General Genetics, 168, 111 (1979).
  • the protein of the present invention in addition to producing it as it is, it can be produced as a secreted production or fusion protein according to the method described in Molecular Cloning.
  • the protein of the present invention can be obtained by culturing the transformant obtained by the above method in a medium, producing and accumulating the protein of the present invention in the culture, and collecting the protein of the present invention from the culture. Can do.
  • the transformant can be cultured by a normal culture method.
  • a natural medium or a synthetic medium may be used as long as it contains a carbon source, a nitrogen source, and an inorganic salt that can be assimilated by the transformant, and can efficiently culture the transformant. Any of these may be used.
  • the carbon source for example, sugars such as glucose, fructose, sucrose, maltose and starch hydrolysate, alcohols such as ethanol, and organic acids such as acetic acid, lactic acid and succinic acid can be used.
  • concentration is preferably 5 to 400 g / l.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, various inorganic and organic ammonium salts, urea, other nitrogen-containing compounds, meat extract, yeast extract, corn steep liquor, Nitrogen-containing organic substances such as soybean hydrolyzate can be used.
  • the concentration is preferably 1-100 g / l.
  • inorganic salts include primary hydrogen phosphate lithium, secondary hydrogen phosphate lithium, ammonium sulfate, sodium chloride, magnesium sulfate, calcium carbonate, and the like.
  • trace nutrient sources such as piotin and thiamine can be added as needed.
  • These micronutrient sources can be substituted with natural products such as meat extract, yeast extract, corn 'steep' liquor and casamino acids.
  • Cultivation is performed under aerobic conditions such as shaking culture and deep aeration stirring culture.
  • Culture temperature is 20 ° (:. ⁇ 50 ° C, preferably preferably 30 ° C ⁇ 42 ° C, pH of the medium is 4 to 10, good Mashiku is maintained at 5-8 Adjustment of P H is , Using inorganic or organic acid, alkaline solution, urea, calcium carbonate, ammonia, etc.
  • Culturing time is 12 to 6 days, preferably 1 to 3 days. Antibiotics such as may be added to the medium. [0 0 5 9]
  • an inducer may be added to the medium if necessary.
  • an inducer may be added to the medium if necessary.
  • an inducer when cultivating a microorganism transformed with a recombinant vector using a promoter, when culturing a microorganism transformed with isopropyl- ⁇ -D-thiogalatatoviranoside, etc. Indoleacrylic acid or the like may be added to the medium.
  • a method for producing the protein of the present invention there are a method of producing in the host cell, a method of secreting it outside the host cell, and a method of producing it on the host cell outer membrane.
  • the protein of the present invention is produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Lou et al. [Proc. Natl Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (199 0)], or by applying the method described in JP-A-5-336963, W094 / 23021, etc.
  • the protein can be actively secreted outside the host cell.
  • the protein of the present invention can be actively expressed outside the host cell by expressing in a form in which a signal peptide is added in front of the polypeptide containing the active site of the protein of the present invention. Can be secreted.
  • the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.
  • the cells are collected by centrifugation after culturing, suspended in an aqueous buffer solution, an ultrasonic crusher, a French press, Manton Gaurin. Crush the cells with a homogenizer, dynomill, etc. to obtain a cell-free extract.
  • an ordinary enzyme isolation and purification method that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Jetylaminoethyl (DEAE) — Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei Co., Ltd.) and other anion-exchange chromatography using a resin, S- Sepharose FF (Pharmacia) etc.
  • a solvent extraction method a salting-out method using ammonium sulfate
  • a desalting method a precipitation method using an organic solvent
  • Jetylaminoethyl (DEAE) — Sepharose Jetylaminoethyl (DEAE) — Sepharose
  • DIAION HPA-75 manufactured by Mitsubishi Kasei Co., Ltd.
  • other anion-exchange chromatography using a resin S- Sepharose FF (Pharmacia) etc.
  • Cation exchange chromatography hydrophobic chromatography using resins such as butyl sepharose and phenycephalose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric
  • a purified product can be obtained by using a method such as electrophoresis such as point electrophoresis alone or in combination.
  • the insoluble form of the protein when expressed in an insoluble form in the cell, the insoluble form of the protein can be recovered as a precipitate fraction by similarly collecting the cell, crushing, and centrifuging. . Solubilize the recovered protein insolubles with a protein denaturant.
  • the protein can be returned to a normal three-dimensional structure by diluting or dialyzing the solubilized solution and reducing the concentration of the protein denaturant in the solubilized solution. After the operation, a purified sample of the protein can be obtained by the same isolation and purification method as described above.
  • the protein of the present invention or a derivative such as a protein having a sugar chain added to the protein is secreted extracellularly, the protein or the protein derivative can be recovered in the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a method such as centrifugation as described above, and a purified sample is obtained from the culture supernatant by using an isolation purification method similar to the above. Can be obtained.
  • a culture of the transformant obtained by the above method 4 or a treated product of the culture, cytosine, a phosphate group donor and an energy donor are present in an aqueous medium, and CMP is produced in the medium;
  • the CMP can be acquired by accumulating and collecting the CMP from the medium.
  • a culture of a transformant obtained by the above method 4 a treated product of the culture, a culture of a microorganism belonging to the genus Corynepacteria, or a treated product of the culture, cytosine, a phosphate group donor CMP can also be obtained by allowing the energy donor to be present in an aqueous medium, generating and storing CMP in the medium, and collecting CMP from the medium.
  • the transformant is used as wet cells at a concentration of 0.5 to 500 g / l, preferably 5 to 200 g / l.
  • the cytosine used as a substrate may be synthetic or natural. In addition, if it contains cytosine, refined products, cytosine-containing crude products obtained from natural products, etc. Either is acceptable. Cytosine is used at a concentration of 1 to 100 g / l, preferably about 1 to 50 g / l.
  • Energy donors include glucose, fructose, sucrose, molasses, starch hydrolyzed carbohydrates, pyruvic acid, lactic acid, acetic acid, organic acids such as ketoglutaric acid, amino acids such as glycine, alanine, aspartic acid, and glutamic acid. Any substance can be used as long as it can metabolize the above-described transformant and produce ATP in the cell. These are used at a concentration of 5 to 200 g / l.
  • Phosphoric acid group donors include orthophosphoric acid, pyrophosphoric acid, tripolyphosphoric acid, tetrapolyphosphoric acid, tetrapolymetaphosphoric acid, etc., polymetaphosphoric acid, monopotassium phosphate, dipotassium phosphate, monosodium phosphate, phosphoric acid Inorganic phosphates such as disodium can be listed. These are used at a concentration of 1-50 g / l.
  • microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes and Corynebacterium glutamicum, and more preferable examples include Corynebacterium ammonia3 ⁇ 4enes ATCC6872, Corynebacterium aramoniagenes ATCC21170, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamic icum ATCC21171 and the like.
  • Microorganisms belonging to the genus Corynebacterium can be cultured in the same manner as in 4 above.
  • Microorganisms belonging to the genus Corynebatarum are used as wet cells at a concentration of 0.5 to 500 g / l, preferably 5 to 200 g / l.
  • aqueous medium examples include aqueous solutions containing salts and salts that do not inhibit the CMP production reaction, such as water, phosphate buffer, tris buffer, and borate buffer.
  • aqueous solutions containing salts and salts that do not inhibit the CMP production reaction such as water, phosphate buffer, tris buffer, and borate buffer.
  • the supernatant of a culture obtained by culturing a transformant or a microorganism belonging to the genus Corynebataterium can be used as an aqueous medium as it is.
  • a surfactant or an organic solvent may be added to the aqueous medium as necessary.
  • a surfactant or an organic solvent By adding a surfactant or an organic solvent, the production efficiency of CMP can be increased.
  • surfactants include cationic surfactants such as polyethylene glycol stearylamine (eg, Nimine S-215, manufactured by Nippon Oil & Fats Co., Ltd.) and alkyldimethylbenzyl ammonium mucolide (eg, Sanizol B-50, manufactured by Kao Corporation).
  • Anionic surfactants such as sodium lauryl sulfate (for example, Persoft SL, manufactured by NOF Corporation), polyethylene glycol sorbitan monostearate (for example, Nonionic surfactants such as nonionic ST221 (manufactured by NOF Corporation) and amphoteric surfactants such as dimethyllauryl betaine (eg NISSAN ANON BL, manufactured by NOF Corporation), etc. It is used at a concentration of ⁇ 20 g, preferably 1 to 10 g / l.
  • organic solvent examples include toluene, xylene, aliphatic alcohol, acetone, ethyl acetate and the like, and these are usually used at a concentration of 0.1 to 50 ml / l, preferably 1 to 20 ml / l.
  • the production reaction of CMP is carried out under aerobic conditions such as by aeration stirring.
  • the reaction temperature is maintained at 20 ° C to 50 ° C, preferably 30 ° C to 42 ° C, and the pH in the medium is maintained at 4 to 10, preferably 5 to 8.
  • the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.
  • the reaction time is usually 1 to 48 hours.
  • CMP can be isolated and purified from the reaction solution obtained by removing precipitates such as bacterial cells by using a known method such as activated carbon treatment or ion exchange resin treatment.
  • Examples of useful substances produced as described above include cytidine mono 5′-triphosphoric acid, CMP-sialic acid, C D P-choline and the like.
  • Examples of a method for producing cytidine 15,5 tritriphosphate using CMP as a substrate include the methods described in JP-A-2002-085087 and JP-A-2000-217593, and CMP-sialic acid.
  • a method for producing CDP-choline for example, methods described in JP-A-5-276973, W02004009830 and JP-A-2002-085087 can be mentioned.
  • -313 The method described in 594 gazette and Japanese Patent Publication No. 48-2358 gazette can be mentioned. ⁇ Example ⁇
  • the host strain MC1000 A codA Acdd A pyrG:: Km was constructed as follows to detect the enzymatic activity to produce CMP from cytosine and phosphoribosyl pyrophosphate (PRPP). (1) Preparation of a strain deficient in the gene encoding cytosine deaminase (codA gene) and the gene encoding cytidine deaminase
  • a strain lacking the codA gene and cdd gene on the chromosomal DNA of Escherichia coli MC1000 can be obtained by a method using the homologous recombination system of lambda phage (Proc. Natl. Acad. Sci. USA, 97, 6640-6645 (2000)) It produced according to.
  • Plasmid P KD46, pKD3, 'pKD4 and pC P20 were obtained from the Escherichia coli strain carrying the plasmid from the Escherichia coli Collagenetic Stock Center (Yale University, USA) and extracted from the Escherichia coli. Was used.
  • nucleotide sequence containing the Escherichia coli codA gene represented by SEQ ID NO: 6 the nucleotide sequences represented by nucleotide numbers 1-1031 and 2094-3105 were amplified by PCR reaction. These amplified fragments have pKD4 as a saddle and have FRT (FLP recognition target) sites at both ends obtained by amplifying DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 7 and 8 as a primer set. (ami nog lycoside phosphotransferase) Ligated with iiis fragment.
  • the ligated fragment was introduced by electroporation into Escherichia coli MC1000 strain into which pKD46 had been introduced in advance and ⁇ Red recombinase was induced and expressed.
  • a transformant was selected using kanamycin resistance as an index, and a strain in which the L gene was inserted into the codA gene on the chromosome to destroy the gene was obtained.
  • the base sequence portions represented by the base numbers 1-1011 and 1656-2572 were amplified by PCR reaction. These amplified fragments were obtained by amplifying pKD3 as a saddle, using the DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 10 and 11 as a primer set, and having FRT sites at both ends.
  • the (chloramphenicol acetyltrans ferase) gene fragment was ligated.
  • the ligated fragment was introduced by the electroporation method into the kanamycin resistant strain obtained as described above in which ⁇ Red recombinase was inducibly expressed.
  • Transformants were selected using kanamycin and kulam lamfenicol resistance as indicators, and a strain in which the cat gene was disrupted by insertion of the cat gene into the gene on the chromosome was obtained.
  • pCP20 was introduced, FLP recombinase was induced and cultured, and then the strain from which the gene was removed from the chromosomal DNA After selecting Ramfenicol sensitivity as an index, pCP20 was removed.
  • the strain obtained by the above operation is the 1032-2093 in the nucleotide sequence represented by SEQ ID NO: 6 on the chromosomal DNA of Escherichia coli MC1000 strain, and 1012-1655 in the nucleotide sequence represented by SEQ ID NO: 9.
  • the resulting recombinant plasmid DNA was digested with and then smoothed, and PUC4K (purchased from Amersham Biosciences) digested with ⁇ ⁇ and smoothed, containing about 1.3 kb containing the gene derived from PUC4K. And ligated with the DNA fragment.
  • the obtained plasmid DNA was digested with ⁇ and ⁇ nl and purified to obtain a defective eiil gene fragment in which the aph gene was inserted into the gene and the gene was destroyed.
  • the DNA fragment was introduced into Escherichia coli JC7623 by electroporation, a transformant was selected using kanamycin resistance as an indicator, and a strain exhibiting cytidine requirement was isolated.
  • PI lysate is prepared from the isolated strain, infected with 294 Escherichia coli strains, and a strain exhibiting resistance to kanamycin is selected.
  • a strain in which the above gene was disrupted was obtained, and the strain was named Escherichia coli MM294ApyrG :: Km.
  • PI lysate was prepared from the Escherichia coli MM294 pyrG :: Km strain obtained in (2) above, infected with the MClOOOAcodAAcdd strain obtained in (1) above, and the transduced strain was selected using kanamycin resistance as an indicator. Selected.
  • the obtained transduced strain was a strain in which the codA gene and the cdd gene were deleted and the pyrG gene was disrupted, and the strain was named Escherichia coli MClOOOAcodAAcddApyrG :: Km.
  • Escherichia coli MC1000 AcodA Acdd ApyrG Km strain is Cytidine-required, M 9 minimal agar medium containing 50 mg / l cytosine [glucose 1.8 g, sodium monohydrogen phosphate 6 g, dihydrogen phosphate 3 g, salt Contains 11 g of sodium chloride 0.5 g, ammonium chloride lg, magnesium sulfate heptahydrate 0.25 g, calcium chloride dihydrate 15 mg, thiamine hydrochloride 10 mg, Agar-Noble (Difco) 20 g] It was confirmed that it could not grow.
  • a transformant obtained by introducing a plasmid expressing a mutant UPRTase into Escherichia coli MCI 000 ⁇ codA Acdd ApyrG:: Km strain has the activity that the mutant enzyme generates CMP from cytosine. Can grow on M9 minimal medium containing cytosine only if
  • a chromosomal DNA of Escherichia coli W3110 strain is prepared by a known method, and then the chromosomal DNA is used as a saddle and DNA comprising the nucleotide sequence represented by SEQ ID NOs: 13 and 14 is used as a primer set. PCR was performed using Pyrobest DNA polymerase (Takara Shuzo).
  • the approximately 0.7 kb amplification product obtained by PCR was purified using Qiagen PCR Purificatin Kit (manufactured by Qiagen) and cleaved with 1 and ⁇ .
  • the restriction enzyme-treated product was subjected to agarose gel electrophoresis, and then extracted and purified using Qiagen Gel Extraction Kit (manufactured by Qiagen).
  • the DNA fragment was ligated with the expression vector pTrc99A (manufactured by Pharmacia), which was also cleaved with EcoRI and BamHI, using DNA Ligatin Kit ver.2.0 (manufactured by Takara Shuzo).
  • DH5a Toyobo Co., Ltd. was transformed.
  • a transformant was selected using ampicillin resistance as an indicator, and the obtained transformant was cultured overnight in an LB medium containing 10 ⁇ / g / ml ampicillin.
  • plasmid DNA is prepared using Qiagen Plasmid Miniprep Kit (Qiagen), and the nucleotide sequence is analyzed, so that the plasmid contains the target Escherichia coli UPRTase gene. It was confirmed that the plasmid had a structure in which a DNA fragment of about 0.7 kb was inserted, and the plasmid was named pEUPl.
  • Recombinant wild-type UPRTase used for crystal preparation for X-ray crystal structure analysis was prepared as follows.
  • the reading frame of the upp gene that encodes UPRTase (open re- reading frame) is assumed to start with Met-10, and Cys-160 is expected to be located on the surface of the molecule, causing aggregation when the purified enzyme is concentrated. This was replaced with Ser.
  • fusion PCR was performed using the following four types of primers and the wild-type upp gene cloned in plasmid pQE82L (Qiagen) as a mirror type.
  • PQE_C160S_Nl and pQE—C160S—N2 having the nucleotide sequences represented by SEQ ID NOs: 3 and 4 and pQE_C160S_Cl and pQE—C160S—C2 having the nucleotide sequences represented by SEQ ID NOs: 3 and 3 and 7 were used as primers, respectively.
  • PCR was performed separately and each product was purified by agarose electrophoresis. Subsequently, PCR was performed by mixing them into a cage and using pQE_C160S_Nl and pQE_C160S_C2 as primers.
  • the amplified fragment was digested with the restriction enzyme BamHI, purified by agarose electrophoresis, then previously quenched with the restriction enzyme BamHI, and then ligated with plasmid pQE82L treated with alkaline phosphatase.
  • Escherichia E. coli DH5 ⁇ strain was transformed.
  • the UPRTase having the amino acid sequence represented by SEQ ID NO: 38 was expressed as a fusion protein having a histidine tag at the amino terminal. did.
  • Escherichia coli BL21 strain was transformed, and the obtained transformant was used as the following UPRTase expression strain. Substitution of C160S had little effect on the specific activity of the enzyme (data not shown).
  • the cells were disrupted by ultrasonication while being cooled in an ice bath. This was centrifuged at lOOOOrpm for 1 hour to obtain a cell extract in the supernatant.
  • TAL0N resin (Clonetech) was used for purification of the histidine tag fusion protein. 200 / L TAL0N resin was used per 50 mL of culture medium. After washing with washing buffer (50 raM Na-phospha'te pH 7.0, 15 raM imidazole, 10 mM 2-mercaptoethanol), elution buffer (50 mM Na-phosphate pH 7.0, 150 mM imidazole, Elution with 10 mM 2-mercaptoethanol). The above operation was performed at room temperature.
  • the following operations were performed at 4 ° C.
  • the collected fraction was dialyzed against anion chromatography equilibration buffer (20 mM Tris-HCl, pH 8.5) for 1 hour.
  • the dialyzed solution was centrifuged at lOOOOrpm for 10 minutes, and only the supernatant fraction was collected.
  • An anion exchange column (monoQ ram, GE Healthcare Bioscience).
  • elution was performed with a linear concentration gradient of 0 ⁇ 0.5M NaCl using UV absorption at a wavelength of 280 nm as an index.
  • the active fraction eluted in the range of 250-350 mM NaCl.
  • a stock solution was added to a concentration of 0.5 mM DTT and 0.5 mM GTP, followed by concentration using ami con ultra-15, and further concentration buffer (20 raM Tris- HCl pH 8.5, 20 mM NaCl, 0.5 mM DTT, 0.5 mM GTP) was added, and the solution was concentrated to a concentration of 18 mg / mL while exchanging the original buffer.
  • Crystallization was performed by a sitting drop vapor diffusion method.
  • the recombinant UPRTase concentrated solution prepared above and a solution containing 0.2 raM di-ammonium tartrate and 15-20% (w / v) PEG3350 are mixed in equal amounts and left to stand for about 1 week.
  • 05 X 0. 05 X 0. 15 About 3 columnar crystals were precipitated.
  • X-ray diffraction data was collected using the Quantum 315 detector manufactured by ADSC at the Beamline BL5A of the Synchrotron Radiation Research Laboratory, Institute for Materials Structure Science, High Energy Accelerator Research Organization.
  • the wavelength was set to 1.0000 A and the distance from the crystal to the detector was set to 352.4 mm.
  • the measurement temperature was 100K.
  • the exposure was performed for 20 seconds at a vibration angle of 1 ° per frame.
  • the Emerge value was 10.8% at 100-2. 3A resolution, and 2.4.2-2. 3 ⁇ resolution at the outermost shell was 61.1%. became.
  • the asymmetric unit of the crystal contained 4 UPRTase molecules, and the solvent content of the crystal was about 45%.
  • the structure was determined by a molecular replacement method using the monomer structure of Thermotoga maritima UPRTase (PDB code 1050) as a search model.
  • MolRep CCP4 package
  • CNX Acelrys
  • Coot CCP4 package
  • a refined structure with 2.3 A resolution including water and GTP molecules was determined, and the Rf actor was 21.0% (Rfree 28.3%).
  • PR0CHECK 90.6% of the residues other than glycine were located in the most preferred region, and 99.9% of the total residues were acceptable. It has been shown to have a bond dihedral angle.
  • Table 1 shows the obtained three-dimensional structure information (structure coordinates).
  • ATOM 272 ALA A 414.22, 476311602282.481 00.6--
  • OZOZOZ 2E ZOOOZOOOS OOOS: O
  • ATOM >>>>>>>>>>>>>>>>>>>> ooooooooooooooooooooo ooooooooooo co ccccccc rND rsj rNo r rsj hh rsj ho r ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ - ⁇ — »-oooooo
  • IIII f IIIIIIIIIIIIIIIIII IIIIIIII 1 I cccor ro ho ccccccrcr ro rhrr ho ho rrrhhrrao ro or one--j oo oo co o one or one oo one o co o 3 ⁇ 4 " ⁇ 3 ⁇ 4;- ⁇ ⁇ 3 ⁇ 4 ⁇ >

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne une protéine ayant pour activité la production de cytidine 5'-monophosphate (CMP) à partir de cytosine. Cette invention concerne spécifiquement une protéine comprenant une séquence d'acides aminés représentée par SEQ ID NO:1, 2 ou 30, ou une protéine comprenant une séquence d'acides aminés dans laquelle au moins un résidu d'acide aminé sélectionné parmi des résidus d'acides aminés situés aux positions 36, 46, 94, 102, 121, 141, 143, 167 et 198 dans une séquence d'acides aminés représentée par SEQ ID NO:1, 2 ou 30 est substitué par un résidu d'acide aminé différent.
PCT/IB2007/003807 2006-09-27 2007-09-27 Procédé de production de cytidine 5'-monophosphate Ceased WO2008038149A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2008536202A JP5175734B2 (ja) 2006-09-27 2007-09-27 シチジン５’−モノリン酸の製造法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2006262666 2006-09-27
JP2006-262666 2006-09-27

Publications (2)

Publication Number Publication Date
WO2008038149A2 true WO2008038149A2 (fr) 2008-04-03
WO2008038149A3 WO2008038149A3 (fr) 2008-11-13

Family

ID=39230606

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2007/003807 Ceased WO2008038149A2 (fr) 2006-09-27 2007-09-27 Procédé de production de cytidine 5'-monophosphate

Country Status (2)

Country Link
JP (1) JP5175734B2 (fr)
WO (1) WO2008038149A2 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7693230B2 (en) 1999-04-16 2010-04-06 Parkervision, Inc. Apparatus and method of differential IQ frequency up-conversion
US7072427B2 (en) 2001-11-09 2006-07-04 Parkervision, Inc. Method and apparatus for reducing DC offsets in a communication system

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1747270A2 (fr) * 2004-05-10 2007-01-31 Fondazione Centro San Raffaele Del Monte Tabor Enzyme synthetique activant la fluoro-uridine dans les therapies anticancereuses

Also Published As

Publication number Publication date
WO2008038149A3 (fr) 2008-11-13
JP5175734B2 (ja) 2013-04-03
JPWO2008038149A1 (ja) 2010-01-28

Similar Documents

Publication Publication Date Title
JP6789436B2 (ja) Ａｔｐホスホリボシル転移酵素変異体及びそれを用いたｌ−ヒスチジンの生産方法
EP3315610B1 (fr) Procédé de production d'oligosaccharides fucosylés
KR102074957B1 (ko) 타가토스 생산용 조성물 및 이를 이용한 타가토스 제조방법
US11180785B2 (en) Composition for producing tagatose and method of producing tagatose using the same
Choi et al. Biosynthesis of the human milk oligosaccharide 3‐fucosyllactose in metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis and β‐galactosidase modification
BR112019014131A2 (pt) Variante de sintetase de adenil succinato, polinucleotídeo, vetor, microorganismo do gênero corynebacterium e método para preparar nucleotídeos de purina
EP3697805A1 (fr) Production fermentative d'acide n-acétylneuraminique
JP2021506337A (ja) シアリルトランスフェラーゼおよびその使用
ES2955417T3 (es) Variante fosforribosil pirofosfato amidotransferasa y método para producir nucleótido de purina usando la misma
KR102076288B1 (ko) 타가토스 생산용 조성물 및 이를 이용한 타가토스 제조방법
Li et al. Semi-rationally designed site-saturation mutation of Helicobacter pylori α-1, 2-fucosyltransferase for improved catalytic activity and thermostability
ES2656892T3 (es) Microorganismo recombinante que produce ácido quinolínico y procedimiento de producción de ácido quinolínico mediante su uso
KR102308173B1 (ko) 신규 과당-4-에피머화 효소 및 이를 이용한 타가토스의 제조 방법
US20180195051A1 (en) Methods and organism with increased ethanol production
WO2008038149A2 (fr) Procédé de production de cytidine 5'-monophosphate
Goyal et al. Crystallization and preliminary X-ray crystallographic analysis of the curli transporter CsgG
Cui et al. Avermectin biosynthesis: stable functional expression of branched chain α-keto acid dehydrogenase complex from Streptomyces avermitilis in Escherichia coli by selectively regulating individual subunit gene expression
CN119614406B (zh) 一种克鲁维酵母基因修饰细胞
KR101998477B1 (ko) 클로스트리디움 스터코라리움 유래 엘-람노스 이성화효소 변이체 및 이를 이용한 알룰로스로부터 알로스의 생산 방법
KR102232837B1 (ko) 글루코실글리세롤 생산 활성을 가지는 신규한 폴리펩티드 및 이를 이용한 글루코실글리세롤 제조방법
KR101617526B1 (ko) 활성이 개선된 대장균 유래의 돌연변이 당 이성질화 효소 및 그를 이용한 l-굴로스의 생산
KR102219195B1 (ko) 신규 과당-4-에피머화 효소 및 이를 이용한 타가토스의 제조 방법
KR20230143916A (ko) 신규한 시스 아코니트산 합성 효소 및 이의 용도
WO2009057230A1 (fr) Procédé de fabrication de cytidine 5'-monophosphate
CN119162144A (zh) 一种高产3′-唾液酸乳糖的唾液酸转移酶突变体及其构建方法和应用

Legal Events

Date Code Title Description
DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07825771

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 2008536202

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07825771

Country of ref document: EP

Kind code of ref document: A2

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)