JP2938497B2 - Method for producing cytidine by fermentation - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing cytidine by fermentation, which is useful as a raw material for synthesizing pharmaceuticals and the like. More specifically, the present invention relates to a method for mass-producing cytidine using a transformant of a microorganism having cytidine-producing ability.

(Prior art)

As a method for obtaining cytidine by culturing a microorganism, a method using a mutant strain of Bacillus subtilis or Proteus lettgelli (Japanese Patent Publication No. 36-21499); analog resistance to purine, pyrimidine and histidine belonging to the genus Brevibacterium Using a mutant strain of
No. 871); a method using a mutant strain that is analog-resistant to purines of the genus Microbacterium (JP-B-57-18872)
Japanese Patent Application Laid-Open No. 61-135597) and a method using a mutant strain having analog resistance to Bacillus pyrimidine.

In the metabolic pathway leading to the synthesis of cytidine in a microorganism, the uracil base portion of the uridine compound is aminated to produce a cytidine compound, and cytidine is derived from the cytidine compound.
The reaction at this time is performed by amination of uridine triphosphate (hereinafter, referred to as UTP) by cytidine triphosphate (hereinafter, CTP).
That. ) Is the only pathway. The enzyme that catalyzes this reaction is cytidine triphosphate synthase (hereinafter referred to as CT
It is called P synthetase. However, the activity of this enzyme is strictly regulated, such as being subject to feedback inhibition by the product CTP and its expression level being suppressed by the amount of cytidine in the medium. In addition, regarding the cloning of the gene for this enzyme, the CTP synthetase gene derived from Bacillus subtilis [Journal of Biologial Chemistry 261 , 5568 (198
6)], a CTP synthetase gene derived from Bacillus subtilis [Journal of Bacteriology 170 , 4194-4108 (198
8)] is reported.

However, none of the above cloning reports was CT
It is not intended for industrial use of the P synthetase gene, nor is the enzyme gene highly expressed using microorganisms, nor is it intended for industrial production of cytidine using it not.

[Object of the invention]

An object of the present invention is to provide a method capable of mass-producing cytidine by fermentation, which is useful as a raw material for synthesis of pharmaceuticals and the like.

Another object of the present invention is to use a cytidine fermentation method using a cytidine-producing bacterium having a high cytidine-producing ability obtained by a recombinant technique, instead of a conventional cytidine-producing bacterium that has relied only on random mutation. It is to provide a mass production method.

[Structure and effect of the invention]

The present inventors have focused on CTP synthetase among various enzyme groups in the synthesis system of cytidine-based compounds, and believed that enhancing this enzyme activity contributes to an increase in cytidine production, and cloned CTP synthetase belonging to the genus Bacillus. By incorporating the gene into a cytidine-producing bacterium belonging to the genus Bacillus and expressing it at a high level, it was found that the expression level of CTP synthetase was increased, and that the production amount of cytidine was dramatically increased.Based on the findings, the present invention was completed. I came to.

That is, the present invention transforms a microorganism having the ability to produce cytidine belonging to the genus Bacillus with a recombinant DNA containing the CTP synthetase gene belonging to the genus Bacillus, and cultivating the resulting transformant to produce cytidine in the culture. A method for producing cytidine, comprising accumulating and collecting cytidine from the culture.

The microorganism having cytidine-producing ability belonging to the genus Bacillus used in the present invention may be a microorganism originally having cytidine-producing ability, or a Bacillus to which cytidine-producing ability has been imparted by natural or artificial mutation. A microorganism belonging to the genus may be used. Examples of such microorganisms include Bacillus subtilis or a purine analog, a pyrimidine analog, or a histidine analog-resistant strain derived from Bacillus subtilis, preferably a Bacillus natto C-1 strain (Microtechnical Laboratories No. 11055). No.

The Bacillus natto C-1 is a bacterium having a cytidine-producing ability newly classified from soil, and its bacteriological properties are as follows.

(A) Morphological shape 1) Cell shape and size: short rod, 0.7-1.0 × 2
8μ 2) Polymorphs: Single rarely in duplicate 3) Motility: None 4) Spores: Endogenous spores of oval form are formed near the center of vegetative cells 5) Gram stain: positive 6) Acid-fast: negative ( b) Growth conditions 1) Gravy agar culture: Produces a flat colony with a grayish-white, dehiscence-like periphery. 2) Gravy liquid culture: Creates a film on the surface but is otherwise transparent. (c) Physiological properties 1) Nitrate reduction : Yes 2) Use of citric acid: Yes 3) Use of propionic acid: No 4) VP test: Positive 5) Starch hydrolysis: Yes 6) Hydrolysis of casein: Yes 7) Oxidase: Weak 8) Catalase: Yes 9) Production of indole: none 10) Attitude to enzyme: aerobic 11) Requirement of biotin: yes 12) Production of viscous substance in medium containing 1.5% glutamic acid: yes 13) Excretion / existence of cytidine to the medium Bacillus Natto C-1, the first year of Heisei 10
It was deposited with the Ministry of International Trade and Industry of the Ministry of International Trade and Industry on the date of March 19, 2009 under the deposit number of FERM P-11055.

CT from microorganisms capable of producing cytidine of the present invention
The method for cloning the P synthetase gene can be obtained by the following method.

(1) The ability to express the gene of a microorganism capable of producing cytidine and introducing a whole DNA library of a microorganism capable of producing cytidine into a microorganism deficient in CTP synthetase, thereby eliminating the need for growth of cytidine. A method of selecting a colony using as a mark and recovering a DNA library from the colony.

(2) Purification of CTP synthetase of a microorganism having cytidine-producing ability, DNA that is complementary to an oligonucleotide having a DNA sequence of a gene deduced from a part of the amino acid sequence, and purified from the DNA of a microorganism having cytidine-producing ability. How to select from library.

(3) A method in which, when a CTP synthetase of a microorganism closely related to a cytidine-producing microorganism has been cloned, a clone exhibiting complementarity thereto is selected from a DNA library of the cytidine-producing microorganism.

The CTP synthetase gene thus obtained is expressed as it is or after being improved in a microorganism having cytidine-producing ability. The following method can be used as the method.

(1) For microorganisms with an established host-vector system, CTP
The synthetase gene is incorporated into this vector. At this time, if the vector has a high copy number, the same gene may be directly incorporated.

(2) In microorganisms that have established a method for integrating a target gene into a chromosome, CTP synthetase is directly integrated into the chromosome. At this time, if a plurality of target genes can be integrated per chromosome, the CTP synthetase gene may be integrated as it is.

As an improvement of the CTP synthetase gene, the enzyme is repressed by cytidine in the culture medium, so the gene promoter must be replaced with a constitutive (always expressed) one, or the enzyme can inhibit CTP feedback inhibition. As a result, a mutation, for example, a substitution, deletion or addition of a part of the open reading frame shown in FIG. 2, is made in the portion encoding the arostec site of the enzyme to construct a CTP-insensitive enzyme. And the like.

From the microorganisms containing the recombinant thus obtained, a strain having stable properties is selected and cultured in the same manner as in the usual culture of microorganisms. That is, as the medium, a carbon source, a nitrogen source, various inorganic salts, amino acids, vitamins, and the like necessary for the growth of the bacterium are appropriately selected and used alone or in combination. When a change in PH is observed in the medium, sulfuric acid, calcium carbonate, sodium hydroxide, aqueous ammonia, etc. are appropriately added. It is also effective to add a large amount of a compound such as uracil, uridine, UMP, or the like to a medium, and synthesize the compound with cytidine by salvage. The culture temperature is in the range of 20 ° C to 48 ° C, taking into account the growth of the microorganism used, the amount of cytidine accumulated, and the suppression of by-products.
It can be selected as appropriate. The cultivation time varies depending on the amount of cytidine accumulated and the amount of by-products produced, but is usually 1 to 7 days.

Methods for separating and collecting cytidine from culture include precipitation,
A known method such as treatment with an ion-exchange resin or extraction with a solvent can be used (see JP-A-61-135597). Examples The present invention will be specifically described with reference to examples.

Example 1 [Step 1] Bacillus natto C-1 strain [Microtechnical Laboratories No. 11055
Extraction of total DNA from

Except for using the 10% PEG precipitation method instead of the sucrose density gradient centrifugation method, the cells of Bacillus natto C-1 strain were obtained by the method of Kawamura et al.
3.13 mg of total DNA was extracted and purified from 7 g.

[Step 2] Preparation of total DNA library The total DNA obtained in step 1 was completely digested with the restriction enzyme Pst I, and then subjected to 1% agarose gel electrophoresis to obtain 6 to 9 kb.
From the gel part corresponding to the size of
The A fragment was recovered. 2 μg of the recovered DNA was mixed with 600 ng of vector PUC18 completely cleaved with PstI, and reacted with T4 DNA ligase at 15 ° C. for 12 hours to bind both DNAs.

Transformation of this recombinant DNA using CaCl ₂ [Molecular Cloning, page 249, Cold Spring
Harbor Laboratory], Escherichia coli
Introduced into MB65 [Microtechnical Laboratories No. 8900], ampicillin 50
L-broth agar medium containing 1 μg / ml [Bactotripton (manufactured by Difco) 1%, yeast extract 0.5%, NaCl 0.5%,
6800 colonies grown on 1.5% agar were divided into four equal parts, collected, and analyzed by the method of Bernboim et al. [Nucleicacid Research,
7 , 1513 (1979)] to prepare a chromosome library of Bacillus natto C-1.

[Step 3] Selection and separation of CTP synthetase clone from DNA library The CTP synthetase-deficient Escherichia coli JF618 [CGSC5566 strain: USA] was transformed from the chromosome library DNA obtained in step 2 by a transformation method using CaCl _2. Yale University,
E.Coli genetic stock center), containing 80 μg / ml of ampicillin and cytidine-free M9 medium (Na ₂ HPO
₄ 6g /, KH ₂ PO ₄ 3g /, NaCl 0.5g /, NH ₄ Cl 1g /, 1mM
MgSO ₄ , 0.1 mM CaCl ₂ 200 μg / ml threonine, 30 μg / ml
Leucine, 50 μg / ml proline, 20 μg / ml histidine, 20
A strain growing on [μg / ml arginine, 2 μg / ml thiamine, 20 μg / ml uracil] was selected, and a 7.6 kb plasmid DNA containing a Bacillus natto-derived DNA fragment was recovered by the method of Barnboim et al. This plasmid is called BamHI
And a 3.0 kb fragment derived from Bacillus natto C-1 obtained by digestion with Pvu II was recovered by agarose gel electrophoresis, and 1 μg was recovered. 4.9 kb DNA fragment 200 cut with I and Sma I
ng was mixed, 15 ° C. at T ₄ DNA ligase, reacted for 12 hours,
Both DNAs were ligated. The recombinant DNA was introduced into Escherichia coli JF618 by transformation method using CaCl _2,
The target plasmid having 7.9 kb was obtained by selecting in M9 medium containing 80 μg / ml of ampicin and no cytidine.

This plasmid, pFS037, stably complements the CTP synthetase deficiency of JF618, resulting in a Bacillus natto C.
The CTP synthetase gene was found to be contained in this 3.0 kb fragment derived from -1.

[Step 4] Determination of base sequence of CTP synthetase gene derived from Bacillus natto C-1 The sequence of the 3.0 kb DNA fragment was determined by the method of Sanger et al.
atl. Acad. Sci. USA 74,5463 (1977)] (FIG. 2). As a result, there was a portion encoding the same amino acid as Bacillus subtilis CTP synthetase. However, its base sequence was different from that of Bacillus subtilis, and the frequency of use of its genetic code was unique to Bacillus natto C-1 as shown in Table 1. The promoter region and the terminator region were also unique to this strain.

[Step 5] Southern hybridization with chromosomal DNA using CTP synthetase gene derived from Bacillus natto C-1 as a probe 1.8 kb including the entire region encoding Bacillus natto CTP synthetase cut out with restriction enzymes Dra I and Acc I DN
Using the A fragment (see FIG. 2) as a probe (probe I), the chromosome of Bacillus natto C-1 was Pst I, Hind
Southern hybridization was performed with the DNA cut in III. As a result, 7.6 kb of Pst I digest and Hind III digest
A band was generated at the expected position of 0.5 kb. Also, a 840 bp DN downstream of the AccI recognition site 40 bp downstream of the terminal codon
When the A fragment (see FIG. 2) was used as the probe (probe II), a band was generated at the expected position of 7.6 kb in the Pst I fragment. This confirmed that the 3.0 kb fragment thus cloned was derived from Bacillus natto C-1. Meanwhile, Bacillus subtilis 168 strains (Bacillus Genetic stock center, Ohio University, USA)
IAI strain). Using Probe I, a band was generated at the expected position of 0.5 kb after Hind III cleavage, but with Probe II, Pst I, Hind III, Eco
No band was detected in DNA cut with any of the RIs.

[Step 6] Introduction of plasmid pFS037 into Bacillus natto C-1 15 μg of pFS037 extracted from Escherichia coli pFS037 / JF618 by the method of Burnboim et al. Using a protoplast of Yamane et al. [Gene Engineering, P173, Kyoritsu Shuppan,
1987] and introduced into Bacillus natto C-1 strain,
43 g / ml tetracycline resistant colonies were obtained. Plasmids of 5 colonies were extracted by the method of Barnboim et al., Digested with BamHI and EcoRI, and the electrophoresis pattern was observed on an agarose gel. All the patterns were the same as those of pFS037.

One of these five colonies was designated as Bacillus natto C-1 (pFS037) on January 29, 1990 by the Ministry of International Trade and Industry at the National Institute of Microbial Industry and Technology. No. (FERM P-11211).

[Step 7] CTP synthetase activity of Bacillus natto C-1 having pFS037 Bacillus natto C-1 having pFS037 was SEED liquid medium containing 20 μg / ml tetracycline [glucose 2%,
NaCl 0.25%, Polypeptone (Wako Pure Chemical) 1%, Yeast Extract D3 (Wako Pure Chemical) 1%, Biotin 100μg / ml,
Inoculate one platinum loop into 50 ml of uracil 100 µg / ml, PH7.3]
Incubate at 18 ° C for 18 hours. Bacteria were collected from 5 ml of this culture, and 2 ml of a homogenate buffer [Tris-HCl 20 mM (PH8.0) EDT
A1mM, ATP1mM, mercaptoethanol 20mM, glutamine
2mM], and subjected to sonication in ice to obtain a cell homogenate. This was added to an equal volume of 2 × reaction buffer [Tris-HCl 50 mM (PH8.0), MgCl ₂ 25 mM, UTP 10 mM, ATP 8 mM
M, GTP1mM, glutamine 20mM, EDTA1mM), 37 ° C
After heating for 5 minutes, the reaction is stopped by boiling for 3 minutes. The reaction solution was analyzed by HPLC (Whatman SAX-10-25 column, 0.4
M phosphoric acid-2.5% acetonitrile buffer (PH3.3),
Flow rate of 1.5 ml per minute at a wavelength of 921 mm], the amount of CTP produced is quantified, and the activity to produce 1 μmol CTP per minute is 1 unit.
Is defined.

As a result, Bacillus natto C-1 having pFS037 was 118.6 × 10 ⁻³ units per 1 mg of the homogenate protein, whereas Bacillus natto C-1 having only the vector pHY300PLK was 10.5 × 10 ⁻³ units. Met.

Thus, about 30 copies of plasmid pHY300PLK
By incorporating the CTP synthetase gene above and introducing it into Bacillus natto C-1, an increase in CTP synthetase activity of 10-fold or more was confirmed.

[Step 8] Production of cytidine using Bacillus natto C-1 having pFS037 Bacillus natto C-1 having pFS037 was cultured at 37 ° C. for 18 hours in a SEED liquid medium containing 20 μg / ml of tetracycline, and then 20 μg of tetracycline / ml of cytidine production medium [glucose 10%, CaCO ₃ 1%, yeast extract D3 (Wako Pure Chemical) 0.5%, KH ₂ PO ₄ 0.8%, (NH ₄ ) ₂ SO ₄ 0.4%, (NH
₂ ) 0.4% ₂ CO, biotin 100 ng / ml, (pH 7.2)] 2% inoculation in 50 ml and cultivation in a 500 ml flask at 37 ° C. for 3 days; 0.24 mg / ml cytidine accumulated Was confirmed.

As a control, when Bacillus natto C-1 described in Step I was cultured under the same conditions except that tetracycline was not contained, the accumulated amount of cytidine was 0.10 mg / ml.

Example 2 Bacillus natto C-1 having pFS037 obtained in Example 1 was cultured under the conditions of Example 1 except that uracil 2 mg / ml was contained in the culture solution.
It was 0.4 mg / ml.

As a control, Bacillus natto C-1 was cultured under the same conditions as above except that tetracycline was not contained, and the amount of cytidine accumulated in the medium was 0.12 mg / ml.

Reference Example 1 CTP synthetase activity of Escherichia coli JF618 having pFS037 Escherichia coli JF61 having pFS037 obtained in Example 1
8 in L-broth liquid medium 100 containing 50 μg / ml of ampicillin
Inoculate one platinum loop per ml and incubate at 37 ° C for 18 hours. Bacteria are collected from 100 ml of this culture, and Bacillus natto C-1
The CTP synthetase activity is measured in the same manner as in Example. As a result, pFS037 / JF618 contained 103 mg / mg of the homogenate protein.
× to 10 ^-3 of the units, Escherichia coli JF618 with pHY300pLK alone was 9.7 × 10 ^-3 units. Thus, the Bacillus natto C-1 CTP synthetase gene can be highly expressed in heterologous organisms.

[Summary of Effects of the Invention] According to the present invention, the productivity of cytidine is significantly increased by using a microorganism transformed with a recombinant DNA containing a CTP synthetase gene. When a uracil-based compound is added to a culture solution, the amount of accumulated cytidine-based compound is further increased by a pyrimidine salvage route.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing construction of a plasmid pFS037 containing a CTP synthetase gene derived from Bacillus natto C-1. FIG. 2 shows the nucleotide sequence of the CTP synthetase gene derived from Bacillus natto C-1.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁶ , DB name) C12N 15/00-15/90 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A microorganism having the ability to produce cytidine belonging to the genus Bacillus is transformed with a recombinant DNA containing the CTP synthetase gene belonging to the genus Bacillus, and the resulting transformant is cultured to produce and accumulate cytidine in the culture. And collecting cytidine from the culture. 2. The CTP synthetase gene according to claim 1, wherein the CTP synthetase gene belonging to the genus Bacillus is a CTP synthetase gene in which the open reading frame of the base sequence shown in FIG. Cytidine production method.