JP2004344029A - Gene-destroyed microorganism and method for producing pyrimidine nucleoside compound therewith - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a nucleoside compound from the pyrimidine base derivative having an amino group at the 4-position without applying a complicated deaminase-deactivating treatment. <P>SOLUTION: A microorganism having originally had both a cytosine deaminase gene and a cytidine deaminase gene, but simultaneously lacking both the cytosine deaminase gene and the cytidine deaminase gene. The method for producing a nucleoside compound having an amino group at the 4-position is characterized by using the microorganism simultaneously lacking both the cytosine deaminase gene and the cytidine deaminase gene. A nucleoside compound having the amino group at the 4-position can efficiently be produced from the corresponding pyrimidine base derivative without applying a complicated deaminase-deactivating treatment. The strain in which both the genes are destroyed by a recombination exhibits the same proliferative property as that of a wild strain, and can easily be cultured in a profitable high concentration. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing a pyrimidine nucleoside compound useful as a raw material for synthesizing a drug or the like. A method for producing a pyrimidine nucleoside compound using a microorganism expressing an enzyme having an activity of synthesizing a corresponding nucleoside compound from a pyrimidine base derivative having an amino group at position 4 in a microorganism deficient in both cytosine deaminase and cytidine deaminase genes About.
[0002]
[Prior art]
As a method for producing a corresponding nucleoside compound from a pyrimidine base derivative having an amino group at the 4-position, a method using nucleoside deoxyribosyltransferase-II of a lactic acid bacterium is disclosed in JP-A-2002-17393. This publication does not disclose the decomposition of a product by a bacterial reaction since a purified enzyme is used.
[0003]
A method using E. coli purine nucleoside phosphorylase is disclosed in European Patent Publication No. 1254959A2. According to the publication, when a corresponding nucleoside compound is produced from a pyrimidine base derivative having an amino group at the 4-position, the pyrimidine base derivative having the amino group at the 4-position or the corresponding nucleoside compound is deaminated to obtain a reaction product. Although the rate may decrease, a method of performing a treatment for selectively inactivating deaminase is disclosed, but the treatment is complicated and is not necessarily an industrially advantageous method.
[0004]
In the above-described method, a condition must be found in which only the above deaminase is inactivated and an enzyme having an activity of synthesizing a corresponding nucleoside compound from a pyrimidine base derivative having an amino group at the 4-position does not inactivate. When an enzyme other than nucleoside phosphorylase is used, the method is not always available.
[0005]
Furthermore, when a nucleoside compound is produced as a pharmaceutical product, the incorporation of the analog into a product is a serious problem when producing a nucleotide or the like using the nucleoside compound as a raw material. In this publication, the deaminase activity cannot be completely eliminated, and there is also a problem that the purification step is complicated because a small amount of by-products is removed.
[0006]
The publication also discloses a method for preventing deamination by using a cytidine deaminase-deficient strain of the genus Bacillus. Since Bacillus has only cytidine deaminase, it can produce the corresponding nucleoside with a single defect. However, for industrial production, high-density cultivation is difficult, and the gene manipulation system is complicated.
[0007]
[Patent Document 1] JP-A-2002-17393
[Patent Document 2] European Patent Publication No. 1254959A2
[0008]
[Problems to be solved by the invention]
As described above, it has been suggested that deficiency of deaminase is effective in producing a corresponding nucleoside compound from a pyrimidine base derivative having an amino group at the 4-position, but both genes of cytosine deaminase and cytidine deaminase were simultaneously expressed. The effect of the deletion is not known, and cannot be simply predicted from conventionally known techniques.
Further, even if both the cytosine deaminase and the cytidine deaminase genes were simultaneously deleted, it was not always clear that only the above gene product had the enzyme activity.
[0009]
In view of the above situation, an object of the present invention is to provide a method for producing a corresponding nucleoside compound from a pyrimidine base derivative having an amino group at the 4-position without performing a complicated deaminase deactivation treatment.
[0010]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, a microorganism in which both cytosine deaminase and cytidine deaminase genes have been simultaneously deleted by mutagenesis or homologous recombination has an amino group at position 4. By expressing an enzyme having an activity of synthesizing a corresponding nucleoside compound from a pyrimidine base derivative, it has been found that the corresponding nucleoside compound can be efficiently produced without performing a complicated deaminase deactivation treatment.
[0011]
In particular, if a microorganism that does not lack genes other than the cytosine deaminase and cytidine deaminase by using homologous recombination to destroy both genes, including trace amounts of the deamino form that was impossible with conventional techniques, is included. It has been found that a highly pure nucleoside compound can be obtained without employing a complicated purification step.
[0012]
That is, the present invention is as follows.
[1] A microorganism originally having both genes of cytosine deaminase and cytidine deaminase, and deficient in the cytosine deaminase and cytidine deaminase genes at the same time.
[2] The microorganism according to [1], wherein the microorganism is Escherichia coli.
[3] The microorganism according to [1] or [2], wherein both genes of cytosine deaminase and cytidine deaminase are disrupted by homologous recombination, and genes other than the gene are not deleted.
[4] The microorganism according to any one of [1] to [3], wherein an enzyme having an activity of synthesizing a corresponding nucleoside compound from a pyrimidine base derivative having an amino group at the 4-position is expressed.
[5] A method for producing a pyrimidine nucleoside compound having an amino group at the 4-position using the processed product of the microorganism.
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, cytosine deaminase is an enzyme having a function of removing an amino group at the 4-position of cytosine to generate uracil and ammonia. It is known that 5-methylcytosine other than cytosine also serves as a substrate for this enzyme. The enzyme is known to be present in yeast and bacteria.
[0014]
In the present invention, the cytosine deaminase gene is a gene that encodes a cytosine deaminase. For example, in the case of Escherichia coli, the gene is denoted by the code symbol codA and has a base sequence (GenBank accession No. S56903 (coding region is base numbers 130-1413)). Indicated by
[0015]
In the present invention, cytidine deaminase is an enzyme having a function of removing the amino group at position 4 of cytidine to generate uridine and ammonia. It is known that, besides cytidine, deoxycytidine, 5-halogenated cytidine, 5- or 6-azacytidine, and cytosine arabinoside also serve as substrates. The enzyme is widely distributed in the living kingdom, and is present in tissues such as mammals, birds and fish, yeast and bacteria.
[0016]
In the present invention, the cytidine deaminase gene is a gene that encodes cytidine deaminase. For example, in the case of Escherichia coli, it is represented by the gene symbol cdd and has a base sequence (GenBank accession No. X63144 (coding region is base number 67-1059)). Indicated by
[0017]
In the present invention, a microorganism originally having both the genes for cytosine deaminase and cytidine deaminase is defined as a species whose chromosomal DNA and the like are not originally introduced into the microorganism by an artificial method such as genetic manipulation but are introduced into the microorganism. Has both genes.
[0018]
In the present invention, the microorganism having both cytosine deaminase and cytidine deaminase simultaneously deficient refers to a microorganism originally having both of the above-mentioned cytosine deaminase and cytidine deaminase and further deficient both cytosine deaminase and cytidine deaminase by some means. Microorganisms The origin and type of the microorganism of the present invention are not particularly limited as long as the above-mentioned properties are maintained.
A typical example of the microorganism of the present invention is a microorganism having the above properties and classified taxonomically into Escherichia coli.
[0019]
The microorganism of the present invention can be obtained by, for example, isolating a microorganism lacking both genes originally present in nature for some reason, obtaining a mutant in which the gene is not expressed by mutagenesis, or using a transposon. Thus, a method of dropping the periphery of the gene and a method of deleting only the gene by homologous recombination can be used, but the method is not particularly limited as long as both genes can be deleted.
[0020]
Deletion of a gene other than the target gene by mutagenesis or transposon may deteriorate the growth of the microorganism. When a specific enzyme is expressed by genetic recombination, a wild-type strain often exhibits a higher expression level than a strain into which various mutations have been introduced. Furthermore, if the growth is good, such as a wild strain, high-density culturing becomes easy, which is industrially advantageous. Therefore, a method of deleting only a specific gene by homologous recombination can be said to be a preferable method.
[0021]
In the present invention, a pyrimidine base derivative having an amino group at the 4-position is a compound having a pyrimidine skeleton and having an amino group at the 4-position, such as cytosine, 5-azacytosine, 5-methylcytosine, 5-fluorocytosine, and the like. Can be exemplified.
[0022]
In the present invention, a nucleoside compound corresponding to a pyrimidine base derivative having an amino group at the 4-position is a compound in which a saccharide such as ribose or deoxyribose and a pyrimidine base derivative having an amino group at the 4-position are linked by an N-glucoside bond. And examples thereof include cytidine, deoxycytidine, 5-azacytidine, 5-fluorocytidine, and 5-methylsideoxytidine.
[0023]
In the present invention, the activity of synthesizing a corresponding nucleoside compound from a pyrimidine base derivative having an amino group at the 4-position refers to the above-mentioned corresponding activity from a phosphorylated sugar or a nucleoside and the above-described pyrimidine base derivative having an amino group at the 4-position. It refers to the function as a catalyst that promotes the reaction for synthesizing nucleoside compounds.
[0024]
Therefore, the enzyme having the activity of synthesizing the corresponding nucleoside compound from the pyrimidine base derivative having an amino group at the 4-position in the present invention refers to the enzyme having the above-mentioned activity. For example, a nucleoside phosphorylase capable of synthesizing a corresponding nucleoside compound from a phosphorylated sugar and a pyrimidine base derivative having an amino group at the 4-position, or a nucleoside ribosyl capable of synthesizing a corresponding nucleoside compound from a nucleoside and a pyrimidine base derivative having an amino group at the 4-position Transferase, nucleoside deoxyribosyltransferase and the like can be mentioned.
[0025]
A nucleoside phosphorylase capable of synthesizing a corresponding nucleoside compound from a phosphorylated sugar and a pyrimidine base derivative having an amino group at the 4-position is an enzyme that synthesizes the corresponding nucleoside compound from a phosphorylated sugar and a pyrimidine base derivative having an amino group at the 4-position. And any source of animals, plants and microorganisms may be used as long as this requirement is satisfied. Specific examples of such a nucleoside phosphorylase include enzymes known as conventional purine nucleoside phosphorylases of microorganisms belonging to the genus Escherichia such as Escherichia coli, which also have the above-mentioned activity. Examples can be given. As a specific example, the DNA base sequence of the purine nucleoside phosphorylase of Escherichia coli is shown in SEQ ID NO: 8, and the amino acid sequence translated from the base sequence is shown in SEQ ID NO: 9. In addition, recent advances in genetic engineering have made it easier to modify the amino acid sequence by inactivating, inserting, or substituting a part of the base sequence. In view of the state of the art, a modified amino acid sequence of a part of the base sequence within a range that does not affect the desired enzyme activity, for example, within a range that can maintain or improve the enzyme activity, is also described in the present invention. It is intended to be included in the cytosine nucleoside phosphorylase of the invention. For example, the amino acid sequence shown in SEQ ID NO: 9 has deletion, substitution or addition of 2 to 3 amino acids within a range that does not affect the desired enzyme activity, or SEQ ID NO: 8 A nucleotide sequence which does not affect the enzyme activity of interest in the nucleotide sequence of the nucleotide sequence, and which has a mutation such as deletion, substitution or addition within a range in which its complementary sequence can hybridize under stringent conditions. Can be used.
[0026]
The method for producing a corresponding nucleoside compound from a pyrimidine base derivative having an amino group at the 4-position using the microorganism of the present invention in the present invention is as follows.
That is, a microorganism expressing an enzyme capable of synthesizing a corresponding nucleoside compound using a pyrimidine base derivative having an amino group at position 4 as a substrate, the microorganism originally having both genes of cytosine deaminase and cytidine deaminase, and The reaction is carried out by selecting the appropriate reaction conditions, such as pH and temperature, in an aqueous medium using the cells of the microorganism, the culture solution, and the processed product of the microorganisms simultaneously deficient in the cytosine deaminase and cytidine deaminase genes. In this case, the aqueous medium is water or a solvent containing water as a main component and having a pH buffering ability.
[0027]
The reaction can be carried out usually at a pH of 4 to 11, preferably 7 to 10 and a temperature of 10 to 80C, preferably 30 to 50C. Although the reaction time is optional, it can be carried out for about 1 to 20 hours.
In other reactions, it is sufficient that stirring can be performed to such an extent that cells used as a substrate or a catalyst do not precipitate.
[0028]
In the present invention, microbial cells, culture solution, and their processed products are obtained by destroying microbial cells or microbial cells in the culture solution by ultrasonic waves, osmotic shock, etc. And those obtained by immobilizing the disrupted substance with an immobilization carrier or the like, or enzymes purified from the disrupted substance or the like.
[0029]
The amount of the pyrimidine base derivative having an amino group at the 4-position, which is used as a substrate during the reaction, can be generally used in the range of 0.1 to 50 (wt)%, preferably 5 to 20 (wt)%. .
[0030]
The amount of microbial cells, culture solution, or their processed products used for the substrate may be any amount as long as the reaction proceeds.However, since they do not have substrate / product decomposition activity, they can be added in an excessive amount. It does not reduce the reaction yield. The catalyst concentration can be generally set at about 0.1 (weight)% to 10 (weight)%.
[0031]
The method for collecting a nucleoside compound corresponding to a pyrimidine base derivative having an amino group at the 4-position from the reaction solution obtained as described above can be performed by utilizing the difference in solubility between the derivative and the product in an aqueous medium or by ion exchange. Or a method using an adsorption resin. For example, high-purity nucleosides can be obtained by adding activated carbon to the reaction solution, removing bacterial cells and the like in the reaction solution by filtration, and concentrating and crystallizing the filtrate. Since the content of the deamino compound is significantly suppressed by the reaction as compared with the conventional technique, complicated purification for removing the deamino compound is unnecessary.
[0032]
【Example】
Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
[0033]
[Analysis method]
All the generated nucleoside compounds were quantified by high performance liquid chromatography. The analysis conditions are as follows.
Column: Develosil ODS-MG-5 4.6 × 250 mm (Nomura Chemical)
Column temperature; 40 ° C
Pump flow rate: 1.0 ml / min.
Detection; UV 254 nm,
Eluent: 50 mM monopotassium phosphate: methanol = 8: 1 (V / V)
[0034]
Example 1 (Production of cytosine deaminase-deficient strain)
Escherichia coli K-12 W3110 strain (ATCC 27325) (ATCC stands for American Type Culture Collection) was inoculated into 50 ml of LB medium, cultured at 37 ° C. overnight, collected, and collected with a lysate containing 1 mg / ml of lysozyme. Lysed. After the lysate was treated with phenol, DNA was precipitated by ethanol precipitation according to a conventional method. The resulting DNA precipitate was collected by winding it around a glass rod, washed, and used for PCR.
[0035]
Primers for PCR include a base sequence encoding a known cytosine deaminase (hereinafter referred to as codA) from a base sequence encoding a known cytosine permease (hereinafter referred to as codB) of Escherichia coli (GenBank accession No. 1). X63656) and oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 1 and 2 (synthesized by consignment to Hokkaido System Science Co., Ltd.) were used. These primers have EcoRI and HindIII restriction enzyme recognition sequences near the 5 ′ end and near the 3 ′ end, respectively.
[0036]
Using a 0.1 ml PCR reaction solution containing 6 ng / μl of the E. coli chromosomal DNA and 3 μM of each primer completely digested with the restriction enzyme HindIII, denaturation: 96 ° C. for 1 minute, annealing: 55 ° C. for 1 minute, extension reaction : PCR was performed under the condition of a reaction cycle consisting of 74 ° C. for 1 minute and 30 cycles.
[0037]
The above-mentioned PCR reaction product and temperature-sensitive plasmid pTH18cs1 (A set of temperature sensitive-replication / -segregation and temperature-resistant plasmid-vector-one-course-first-course-difference-economy-for-the-difference-for-the-difference-for-the-first-order) After digestion with HindIII and ligation using Ligation High (Toyobo Co., Ltd.), Escherichia coli DH5α was transformed at 30 ° C. using the obtained recombinant plasmid. The transformant was cultured on an LB agar medium containing 10 μg / ml of chloramphenicol (Cm) to obtain a Cm-resistant transformant. The obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 10 μg / ml of Cm, and a plasmid was recovered from the obtained cells.
This plasmid was digested with PstI, a large fragment was recovered and ligated using Ligation High (Toyobo Co., Ltd.), and the resulting recombinant plasmid was used to transform Escherichia coli DH5α. The transformant was cultured on an LB agar medium containing 10 μg / ml of chloramphenicol (Cm) to obtain a Cm-resistant transformant. The obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 10 μg / ml of Cm, and a plasmid was recovered from the obtained cells. The plasmid having the designed size was transformed into the W3110 strain at 30 ° C. to obtain a transformant growing on an LB agar plate containing 10 μg / ml of Cm. The obtained transformant was spread on an agar plate and cultured at 30 ° C. overnight. Next, these cultured cells were applied to an LB agar plate containing 10 μg / ml of Cm, and colonies growing at 42 ° C. were obtained.
The operation of obtaining a single colony growing at 42 ° C. was repeated once again, and a clone in which the entire plasmid was integrated into the chromosome by homologous recombination was selected. It was confirmed that no plasmid was detected from this clone even when ordinary plasmid extraction was performed.
[0038]
Next, the above clone was spread on an LB agar plate, cultured at 30 ° C. overnight, inoculated into an LB liquid medium (3 ml / test tube), and cultured with shaking at 42 ° C. for 3 to 4 hours. This was appropriately diluted so as to obtain a single colony, applied to an LB agar plate, and cultured at 42 ° C. overnight to obtain a colony. From the emerged colonies, 100 colonies were picked at random and grown on LB agar plates and LB agar plates containing 10 μg / ml of Cm, respectively, and Cm-sensitive clones that grow only on the LB agar plates were selected.
[0039]
These target clones were cultured in 5 mL of LB liquid medium containing 10 μg / ml of Cm at 37 ° C. for 20 hours, and the cells were separated by centrifugation at 10,000 rpm for 5 minutes. The collected cells were suspended in 1 mL of 10 mM Tris-HCl buffer (pH 8.0).
The activity was measured by adding 0.1 mL of the cell suspension to 1 mL of 10 mM cytosine and 100 mM Tris-HCl buffer, keeping the mixture at 50 ° C. for 1 hr, diluting it 10-fold, and analyzing it by liquid chromatography.
Of the 10 clones obtained, 2 clones completely lacked codA activity and uracil could not be detected. This strain was designated as a W3110 / codA-deficient strain.
[0040]
Example 2 (Production of cytosine deaminase / cytidine deaminase deficient strain)
The primers for PCR include the nucleotide sequence of the cdd gene encoding a known cytidine deaminase of Escherichia coli (hereinafter referred to as cdd) (the nucleotide sequences shown in SEQ ID NOs: 4 and 5 designed based on GenBank accession No. X63144). These primers were synthesized (consigned to Hokkaido System Science Co., Ltd.). These primers have EcoRI and HindIII restriction enzyme recognition sequences near the 5 ′ end and near the 3 ′ end, respectively.
[0041]
Using a 0.1 ml PCR reaction solution containing 6 ng / μl of the E. coli chromosomal DNA and 3 μM of each primer completely digested with the restriction enzyme HindIII, denaturation: 96 ° C. for 1 minute, annealing: 55 ° C. for 1 minute, extension reaction : PCR was performed under the condition of a reaction cycle consisting of 74 ° C. for 1 minute and 30 cycles.
[0042]
The above reaction product and the temperature-sensitive plasmid pTH18cs1 prepared in Example 1 were digested with EcoRI and HindIII, ligated using Ligation High (Toyobo Co., Ltd.), and then used for the obtained recombinant plasmid. E. coli DH5α was transformed. The transformant was cultured on an LB agar medium containing 10 μg / ml of chloramphenicol (Cm) to obtain a Cm-resistant transformant. The obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 10 μg / ml of Cm, and a plasmid was recovered from the obtained cells.
This plasmid was digested with BglII, the ends were filled in with a BKL kit (Takara Shuzo), ligated using Ligation High (Toyobo Co., Ltd.), and the resulting recombinant plasmid was used to transform Escherichia coli DH5α was transformed. The transformant was cultured on an LB agar medium containing 10 μg / ml of chloramphenicol (Cm) to obtain a Cm-resistant transformant. The obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 10 μg / ml of Cm, and a plasmid was recovered from the obtained cells.
After confirming that this plasmid was not digested with BglII, this plasmid was transformed into a W3110 / docA-deficient strain at 30 ° C. to obtain a transformant growing on an LB agar plate containing 10 μg / ml of Cm. The obtained transformant was spread on an agar plate and cultured at 30 ° C. overnight. Next, these cultured cells were applied to an LB agar plate containing 10 μg / ml of Cm, and colonies growing at 42 ° C. were obtained.
The operation of obtaining a single colony growing at 42 ° C. was repeated once again, and a clone in which the entire plasmid was integrated into the chromosome by homologous recombination was selected. This clone was confirmed to have no plasmid in the cytoplasm.
[0043]
Next, the above clone was spread on an LB agar plate, cultured at 30 ° C. overnight, inoculated into an LB liquid medium (3 ml / test tube), and cultured with shaking at 42 ° C. for 3 to 4 hours. This was appropriately diluted so as to obtain a single colony, applied to an LB agar plate, and cultured at 42 ° C. overnight to obtain a colony. From the emerged colonies, 100 colonies were picked at random and grown on an LB agar plate and an LB agar plate containing 10 μg / ml of Cm, respectively, and a Cm-sensitive clone growing only on the LB agar plate was selected.
[0044]
These target clones were cultured in 5 mL of an LB liquid medium containing 10 μg / ml of Cm at 37 ° C. for 20 hours, and the cells were separated by centrifugation at 10,000 rpm for 5 minutes. The collected cells were suspended in 1 mL of 10 mM Tris-HCl buffer (pH 8.0), and the activity of cdd was measured.
The activity was measured by adding 0.1 mL of the cell suspension to 1 mL of 10 mM cytidine and 100 mM Tris-HCl buffer, keeping the mixture at 50 ° C. for 1 hr, diluting it 10-fold, and analyzing it by liquid chromatography.
Of the 10 clones obtained, 8 clones completely lacked cdd activity and uridine could not be detected. This strain was designated as W3110 / codA.cdd-deficient strain.
[0045]
Example 3 (Preparation of cytosine phosphorylase-producing bacteria)
Primers for PCR are based on the nucleotide sequence of the deoD gene encoding a known purine nucleoside phosphorylase (hereinafter referred to as PNP) from Escherichia coli (GenBank accession No. AE000508 (coding region is base numbers 11531-12250)). Oligonucleotides having the designed base sequences shown in SEQ ID NOs: 6 and 7 (synthesized by consignment to Hokkaido System Science Co., Ltd.) were used. They have EcoRI and HindIII restriction enzyme recognition sequences, respectively.
[0046]
Using a 0.1 ml PCR reaction solution containing 6 ng / μl of the E. coli chromosomal DNA and 3 μM of each primer completely digested with the restriction enzyme HindIII, denaturation: 96 ° C. for 1 minute, annealing: 55 ° C. for 1 minute, extension reaction : PCR was performed under the condition of a reaction cycle consisting of 74 ° C. for 1 minute and 30 cycles.
[0047]
The PCR reaction product and plasmid pUC18 (Takara Shuzo Co., Ltd.) were digested with EcoRI and HindIII and ligated using Ligation High (Toyobo Co., Ltd.). E. coli DH5α was transformed. The transformant was cultured on an LB agar medium containing 50 μg / ml of ampicillin (Am) and X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside), and was resistant to Am and a white colony. Was obtained.
[0048]
A plasmid was extracted from the thus obtained transformant, and the plasmid into which the target DNA fragment was inserted was named pUC-PNP73. pUC-PNP73 was transformed into a W3110 / codA.cdd deficient strain.
The obtained transformant was cultured with shaking in 100 mL of an LB medium containing 50 μg / ml of Am at 37 ° C. overnight. The culture solution was centrifuged at 13000 rpm for 10 minutes, and the obtained cells were suspended in 20 mL of 100 mM Tris-HCl buffer (pH 8.0). The suspension was centrifuged again at 13000 rpm for 10 minutes, and the obtained cells were treated with 2 mL of 100 mM Tris-HCl buffer (pH 8.0) and 10 mM of 2′-deoxyribose 1-phosphate di (monocyclohexylammonium) salt ( SIGMA) and stored frozen at -20 ° C.
[0049]
Example 4 (Synthesis of deoxycytidine)
20 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 20 mM cytosine (manufactured by Wako Pure Chemical Industries, special grade), 100 mM Tris-HCl buffer (pH 8.0), Example 3 Was reacted at 50 ° C for 20 hours. When the reaction solution was diluted and analyzed, 10.7 mM deoxycytidine was produced, but no deoxyuridine or uracil could be detected.
[0050]
Example 5 (Synthesis of 5-fluorodeoxycytidine)
20 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 20 mM 2-fluorocytosine (SIGMA), 100 mM Tris-HCl buffer (pH 8.0), obtained in Example 3. A reaction solution (1.0 ml) containing 0.1 ml of the obtained bacterial cell solution was reacted at 50 ° C. for 20 hours. When the reaction solution was diluted and analyzed, 10.7 mM of 2-fluorodeoxycytidine was produced, but 2-fluorodeoxyuridine and 2-fluorouracil could not be detected at all.
[0051]
【The invention's effect】
According to the present invention, a microorganism that originally has both genes of cytosine deaminase and cytidine deaminase, and that is deficient in the cytosine deaminase and cytidine deaminase genes at the same time, without performing a complicated deaminase inactivation treatment as in the prior art. The corresponding nucleoside compound can be efficiently produced from a pyrimidine base derivative having an amino group at the 4-position.
[0052]
Furthermore, the strain in which the gene has been disrupted by homologous recombination has completely lost both enzyme activities of cytosine deaminase and cytidine deaminase. A trace amount of a deamino compound is not detected in the nucleoside compound obtained by carrying out the reaction using such a microorganism, and the nucleoside compound produced by the production method of the present invention is a nucleoside compound obtained by a conventional production method. Since the complicated purification step can be greatly simplified, it is particularly excellent as a pharmaceutical or an intermediate for producing a pharmaceutical.
[0053]
Furthermore, a strain in which the gene has been disrupted by homologous recombination exhibits the same growth property as a wild strain, and industrially advantageous high-concentration culture can be easily carried out.
[0054]
[Sequence list]

Claims (5)

A microorganism originally having both the cytosine deaminase and cytidine deaminase genes, and having the cytosine deaminase and cytidine deaminase genes simultaneously deficient. The microorganism according to claim 1, wherein the microorganism is Escherichia coli. The microorganism according to claim 1 or 2, wherein both genes of cytosine deaminase and cytidine deaminase are disrupted by homologous recombination, and genes other than said gene are not deleted. The microorganism according to any one of claims 1 to 3, wherein an enzyme having an activity of synthesizing a corresponding nucleoside compound from a pyrimidine base derivative having an amino group at the 4-position is expressed. A method for producing a pyrimidine nucleoside compound having an amino group at the 4-position using the processed product of the microorganism.