JPH089985A - Production of thiamine diphosphoric acid - Google Patents

Abstract

PURPOSE:To efficiently obtain the subject compound as a coenzyme-type vitamin B1 useful as nutrient agent, medicine, etc., by reacting thiamine with ATP in the presence of a microorganism of the genus Paracoccus capable of producing thiamine diphosphoric acid from thiamine and ATP. CONSTITUTION:Thiamine is made to react with ATP in an aqueous medium in the presence of a microbial cell, cultured liquid or their treated material of a microorganism belonging to the genus Paracoccus (e.g. Paracoccus dinitrificans ATCC 19367) or a microorganism derived from a strain belonging to the genus Paracoccus, having a transduced recombinant DNA containing a DNA fraction-carrying genes concerning thiamine diphosphoric acid synthesis, where the microorganism, etc., are effective as a catalyst for the reaction producing thiamine diphosphoric acid from thiamine and ATP. After the reaction, the product is separated from the reaction liquid to obtain the objective thiamine diphosphoric acid of the coenzyme-type thiamine (vitamin B1) useful as a nutrient preparation, a raw material for medicine, etc., a biochemical reagent, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing thiamine diphosphate (hereinafter abbreviated as TPP). TPP is a coenzyme type of thiamine (vitamin B ₁ ) and has uses as a raw material for nutrients and various raw materials for pharmaceuticals. It is also an important substance as a biochemical reagent.

[0002]

2. Description of the Related Art As a method for producing TPP, there are many known methods for chemically phosphorylating thiamine [British Patent No. 687673, Journal of the American Chemical Societies].
ty 60 , 2263 (1938), same magazine 63 , 1160 (1941), Japanese Examined Sho 49-
No. 14751, etc.]. Further, a method for phosphorylating thiamine by an enzymatic reaction using two kinds of enzymes, thiamin kinase and thiamine monophosphate kinase, has also been reported [JP-A-3-210190].

However, when the chemical synthesis method is used, a mixture of various phosphoric acid esters other than the desired product is produced, so that a complicated purification step is required, the yield is extremely low, and thiamine kinase, When two kinds of enzymes, thiamin monophosphate kinase, are used, thiamine monophosphate is by-produced due to the difference in the optimal reaction conditions of both enzymes, the yield is low, and both are difficult to industrialize. .

[0004]

An object of the present invention is to provide a method for industrially and efficiently producing TPP from thiamine by a one-step enzymatic reaction.

[0005]

According to the present invention, a microorganism belonging to the genus Paracoccus , or a DNA derived from a microorganism belonging to the genus Paracoccus, which carries the genetic information involved in the synthesis of thiamin diphosphate Microorganisms having a recombinant DNA containing a fragment, and having the activity of catalyzing the reaction of producing thiamine diphosphate from thiamine and adenosine-5'-triphosphate, the culture medium, or those In the presence of the treated product, thiamine and adenosine-5'-triphosphate (hereinafter abbreviated as ATP) are reacted in the culture or in an aqueous medium to produce TPP produced in the culture or in the aqueous medium. It is possible to provide a method for producing TPP, which comprises collecting

As a microorganism belonging to the genus Paracoccus, any microorganism can be used as long as it can catalyze the reaction of producing TPP from thiamine and ATP. As a specific example, Paracoccus denitrificans A
The TCC19367 strain can be mentioned. Recombinant D
As a microorganism having NA, Paracoc
A microorganism obtained by transforming a host microorganism with a recombinant DNA obtained by incorporating into a vector a DNA fragment that carries the genetic information involved in the synthesis of thiamine diphosphate derived from a microorganism belonging to the genus ( cus ).

Examples of the DNA fragment include a DNA encoding the amino acid represented by SEQ ID NO: 1, a DNA represented by SEQ ID NO: 2 or SEQ ID NO: 3 and a DNA hybridizing to these. The host microorganism used in the present invention may be any microorganism having recombinant DNA uptake ability and capable of expressing the tpk gene, such as Escherichia ( Escherichia ) genus, Enterobacter ( Enterobacter ) genus, Pseudomonas ( Pseudomonas )
Genus Corynebacterium (Corynebacterium) genus or Brevibacterium (Brevibacterium) a microorganism belonging to the genus belonging to and the like. Preferably Escherichia coli ( Esc
The LE392 strain of the herichia coli ) K-12 strain can be mentioned. The vector used in the present invention includes
The genus Escherichia , Enterobacter ( En
Terobacter) genus Pseudomonas (Pseudomonas) genus, so long as it is autonomously replicable in Corynebacterium (Corynebacterium) genus or Brevibacterium (Brevibacterium) genus microorganisms belonging to such use any phage vectors, plasmid vectors, etc. be able to. Preferably pBR322 [F. Bolivar et al; Gene, 2 , 95
(1977)], pUC19 [C. Yanisch-Perron et al; Gene, 3].
3 , 103 (1985)] and the like.

Recombinant DNA of tpk gene DNA fragment and vector DNA is cleaved in a test tube with a restriction enzyme which gives the same cut ends, and then ligated with DNA ligase to prepare various recombinant hybrids. Can be obtained with things. Using the obtained recombinant hybrid product, a host microorganism is transformed, a transformant strain carrying a recombinant DNA containing a tpk gene DNA fragment is selected, and a recombinant DNA is obtained from the strain. You can

As a transformation method, a known method [T. Mani
atis et al .; Molecular Cloning (Molecular
Cloning), Cold Spring Harbor Laboratory (1982), S. Cohen et al .; Proceeding of National Academic Science (Proc. Natl.
Acad. Sci.) USA, 69 , 2110 (1972)] and the like.

Recombinant DN containing tpk gene DNA fragment
As a method for selecting a transformant carrying A, there is a method of directly measuring the enzyme activity of the transformant, but a more efficient method is to use a mutant strain having a mutation in a gene involved in TPP synthesis. It can be obtained by obtaining a transformant complementing the TPP auxotrophy. The thiamine pyrophosphokinase activity possessed by the microorganism including the obtained transformant is measured by the following method.

When a transformant is used as the microorganism to be measured, LB liquid medium [Bactotryptone (manufactured by Difco) 10 g, yeast extract (manufactured by Difco) 5 g,
To a medium (hereinafter, abbreviated as LBAp liquid medium) added with 75 μg / ml of ampicillin to 3 ml of a medium containing 5 g of NaCl in 1 liter of water and adjusted to pH 7.2, and 3 ml of LB liquid medium for other microorganisms, 1 platinum was added. Ear inoculate and shake culture at 30 ° C. overnight. 0.8 ml of this culture
40 ml of liquid medium of the same composition was inoculated with paracoccus ( P
A microorganism belonging to the genus aracoccus ) is cultured at 30 ° C. for 30 minutes at 37 ° C. with shaking, and then the microorganism is separated by centrifugation. 50mM to this cell
Phosphate buffer (pH 7.0) is added so that the bacterial cell concentration becomes 200 mg / ml, and after suspension, the bacterial cells are crushed by ultrasonic treatment. The supernatant fraction after removing the disrupted cells by centrifugation is used as a crude enzyme solution for activity measurement. Reaction buffer to the crude enzyme solution 5~600μl [100mM Tris (hydroxymethyl) aminomethane (hereinafter, abbreviated as _{Tris), 10mM MgSO 4 · 7H 2} 0,10mM AT
Buffer solution containing P, 10 mM thiamine and adjusted to pH 9.0 with hydrochloric acid] to make 1 ml, and allowed to stand at 40 ° C. to carry out TPP production reaction from thiamine. 10 from the reaction mixture over time (30, 60, 90 and 120 minutes)
After collecting 0 μl and adding 100 μl of 0.2N HCl,
After boiling at 100 ° C. for 2 minutes and centrifugation, the supernatant fraction is appropriately diluted, and the TPP produced is quantified by high performance liquid chromatography (hereinafter abbreviated as HPLC).

The analytical conditions by HPLC are as follows. HPLC analysis conditions: 1) Column: Inertosil ODS-2 (GL Science) 2) Mobile phase: 50 mM KH ₂ PO ₄ (pH with 10N KOH)
Adjusted to 6.0) 3) Column temperature: 35 ° C. 4) Flow rate: 1 ml / min 5) Sample amount: 20 μl 6) Detection: 254 nm Ultraviolet absorption In addition, the enzyme activity for purifying 1 μmol of TPP per minute is 1 unit. .

For the culture of the obtained transformant, an ordinary synthetic medium or natural medium containing a carbon source, a nitrogen source, an inorganic substance, an amino acid, a vitamin and the like is used, and the temperature, pH and the like are adjusted under aerobic conditions. It is done while doing. Examples of the carbon source used in the medium include glucose, fructose, sucrose, maltose, molasses, molasses, white rice bran, cassava, bagasse, carbohydrates such as starch hydrolysate, ethanol, glycerin, alcohols such as sorbitol, pyruvic acid, Any organic acid such as lactic acid, acetic acid and citric acid, and amino acids such as glutamic acid, methionine and lysine can be used as long as the microorganism can assimilate.
The use concentration of these is preferably 0.5 to 30%.

As the nitrogen source, various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate and ammonium acetate,
Amino acids such as glutamic acid, glutamine and methionine, or various substances such as peptone, NZ amine, corn steep liquor, meat extract, yeast extract, casein hydrolyzate, nitrogen-containing organic substances such as fish meal and its digested products are used. it can. The use concentration of these is preferably 0.5 to 10%.

As the inorganic substance, potassium dihydrogen phosphate,
Sodium monohydrogen phosphate, magnesium sulfate, sodium chloride, calcium chloride, iron chloride, copper sulfate, manganese chloride, ammonium molybdate, zinc sulfate and the like can be used. When the microorganisms used require specific nutrients such as vitamins, amino acids, minerals and nucleic acids for growth, appropriate amounts of these substances are added to the medium.

The culture is carried out under aerobic conditions such as shaking culture or aeration stirring culture. The culture temperature is preferably 20 to 50 ° C, more preferably 28 to 40 ° C. Cultivation time is usually 1
It ends in 48 hours. It is desirable that the pH of the medium during the culture is kept substantially neutral with ammonia, urea, sodium hydroxide solution or the like. The culture of the microorganism thus obtained can be used in the reaction as it is, or the treated product obtained by treating the culture can be used in the reaction. As the processed product, a concentrated and dried product of a culture solution, a surfactant-treated product or a lytic enzyme-treated product, and a bacterial cell, a frozen bacterial cell, a dried product of the bacterial cell, or a lyophilized product obtained by centrifuging the culture. , A surfactant-treated product, a solvent-treated product, an enzyme-treated product, an ultrasonically treated product, a mechanically ground product, a mechanical pressure-treated product, a microbial cell, and an immobilized product of the microbial cell-treated product.

The aqueous medium includes water, phosphates, carbonates, acetates, borates, citrates, buffers such as Tris, and alcohols such as methanol and ethanol.
Examples thereof include aqueous solutions containing organic solvents such as esters such as ethyl acetate, ketones such as acetone, and amides such as acetamide. The reaction is carried out in the culture or in an aqueous medium in the presence of the microbial cells, the culture solution or the processed product thereof.

When the treated cells of the microorganism and the culture of the microorganism having the recombinant DNA containing the tpk gene are used, the concentration is usually 5 to 200 g as wet cells.
/ L is desirable. The concentrations of thiamine and ATP used in the reaction are 0.1 to 30 g / l,
It is 0.1 to 100 g / l.

As the ATP source, in addition to highly purified products, an ATP-containing liquid obtained by contacting adenine with microbial cells in the presence of an energy donor (Japanese Patent Laid-Open No. 59-5179).
9), or a microbial cell removal solution thereof, or a concentrated solution thereof can also be used. Further, a microorganism having ATP regenerating activity (Japanese Patent Laid-Open No. 61-74595) can be added to the reaction system to synthesize and supply ATP from glucose and inorganic phosphoric acid. In this case, instead of ATP, the ATP precursor, A
TP regeneration energy donor, phosphate group donor and AT
A microorganism having P biosynthetic activity is present in the reaction solution.
Corynebacterium ammoniagenes (formerly Brevibacterium ammoniagenes: International Journal of
Systematic Bacteriology, 34 , 442 (1987)) ATC
C21170 stocks are included. Cultivation of a microorganism having an ATP regeneration activity can be carried out by the method described in JP-A-61-74595.

The reaction is carried out at a temperature of 20 to 50 ° C. and a pH of 6 to 10
For 1 to 72 hours. If necessary, a surfactant or an organic solvent is added during the cell treatment or reaction. As the surfactant, a cationic surfactant such as polyoxyethylene / stearylamine (for example, Nimine S-215, manufactured by NOF CORPORATION), cetyltrimethylammonium bromide, cetylpyridinium chloride, cation FB, cation F2-40E, and the like, Anionic surfactants such as sodium lauryl sulfate, sodium oleyl amide sulfate, Nurex TAB, and Lapizole 80, nonionic surfactants such as polyoxyethylene sorbitan monostearate (for example, Nonion ST221, manufactured by NOF CORPORATION), An amphoteric surfactant such as lauryl betaine (for example, Anon BF, manufactured by NOF CORPORATION) is used, and these are usually used at a concentration of 0.1 to 50 g / l, preferably 1 to 20 g / l.

As the organic solvent, toluene, xylene,
Acetone, aliphatic alcohol, benzene, ethyl acetate, etc. are used. Usually, it is used at a concentration of 0.1 to 50 μl / ml, preferably 1 to 20 μl / ml. As a method for recovering TPP from a culture or an aqueous medium,
Known ion exchange resin method, activated carbon adsorption method, solvent extraction precipitation method and the like can be used.

Examples of the present invention will be shown below.

[0023]

【Example】

Example 1 Cloning of tpk gene (1) Preparation of host microorganism for cloning tpk gene As a host microorganism, genes involved in TPP biosynthesis system phosphomethylpyrimidine kinase gene (thiD) and thiamine monophosphate kinase gene ( (thiL), a thiamine kinase gene (thiK) that is involved in assimilation of thiamine, and has mutations in three genes, Escherichia coli
A TPP auxotrophic mutant strain of E. coli was used.

Although the TPP auxotrophic mutant cannot grow without TPP even in the presence of thiamine in the medium, the microorganism which has thiamine pyrophosphokinase activity is only thiamin in the medium. If present, thiamine pyrophosphokinase will generate TPP from thiamine and thus will be able to grow. In this example, Escherichia coli NI512 strain (H. Nakayama et al .; J. Bacteriol., 151 , 708 (1982)) having a TPP requirement produced by Nakayama et al. (Genotype: F ⁻ , argG6, metB1, his-1, leu-6, trp-31, gal-
6, lacY1, rpsL104, thiD1, thiK1, thiL3), a transduction method using P1 phage (JHMiller, Experiments in
Molecular Genetics, Cold Spring Harbor Lab. (197
2)) was used to add the hsdR mutation to obtain a strain AI1102 in which the host restriction system was released, and this strain was used as a host microorganism.

The strain from which the host restriction system has been released is cultivated in LB liquid medium at 30 ° C. for 20 hours, and then the cultured bacterial cell fluid is subjected to Escherichia coli C600r ^- m ^- strain (AT
CC33525) known method (JH Miller, Experiments in
Molecular Genetics, Cold Spring Harbor Lab. (197
It can be detected by lysing by plating on the same LB plate medium (LB liquid medium with 1.5% agar manufactured by Difco) as the λ phage prepared in 2)). (2) Cloning of tpk gene by the shotgun method Paracoccus denitrificans
ificans ) ATCC19367 was inoculated into 1 ml of platinum loops and 10 ml of LB medium and cultured at 30 ° C. for 20 hours. A known method (H. Sato & KIMiura: Bioc
him. Biophys. Acta., 72 , 619 (1963))
NA was separated and purified.

5 μg of purified chromosomal DNA of Paracoccus denitrificans
Buffer for EcoRI (100 mM Tris, 50 mM Na
Cl, 6 mM MgCl ₂ , 1 mM dithiothreitol, 100 μg / ml bovine serum albumin, dissolved in 40 μl of a buffer adjusted to pH 7.5 with hydrochloric acid) and added with 20 units of restriction enzyme EcoRI (Takara Shuzo),
After performing a digestion reaction at 37 ° C. for 2 hours, the reaction was stopped by heating at 65 ° C. for 10 minutes. Distilled water 140 μl, 3M sodium acetate (pH
5.6) After adding 20 μl, 2 volumes of water-cooled ethanol was added, and the mixture was allowed to stand at −80 ° C. for 20 minutes. After standing, the precipitate formed in this ethanol mixture was obtained by centrifugation (hereinafter, this method is abbreviated as ethanol precipitation method). 20 μl of distilled water was added to this precipitate to dissolve the precipitate.

In addition, 2 μg of the vector pUC19 was added to EcoR
Dissolve it in 40 μl of buffer solution for I, add 10 units of EcoRI, perform digestion reaction at 37 ° C. for 2 hours,
The reaction was stopped by heat treatment for minutes, and precipitation of the plasmid fragment was obtained by the ethanol precipitation method. 15 μl of distilled water was added to this precipitate to dissolve the precipitate. Paracoc denitrificans obtained in this way
cus denitrificans ) chromosomal DNA EcoRI digested fragment and pUC19 EcoRI digested fragment were mixed, and T4 ligase buffer [66 mM Tris / HCl buffer (pH 7.6),
6.6 mM MgCl ₂ , 10 mM dithiothreitol and 1 mM ATP in buffer] 50 μl and 2
Add a unit of T4 DNA ligase (Takara Shuzo)
Recombinant DNA is ligated at 18 ℃ for 18 hours.
Got Using this recombinant DNA, Escherichia
The Escherichia coli AI1102 strain was prepared according to the method of Cohen et al. [S. Cohen et al; Proc. Natl. Acad. Sci. USA, 69 , 2
110 (1972)] and transformed into ampicillin (75
A transformant strain that is resistant to μg / ml) and has lost the TPP requirement of the host microorganism was obtained.

From this transformant, a known method [Molecular Cloning, T. Maniatis e.
t al. Cold Spring Harbor Laboratory
(1982)], the plasmid was isolated and analyzed by restriction enzyme digestion and agarose gel electrophoresis. The size of the resulting plasmid is 4.4 kilobase pairs (kbp) and pU
1.7 kb from Paracoccus denitrificans at the EcoRI cleavage site of C19
had the EcoRI insert of p. P
It was named AI213. The restriction enzyme digestion map of this plasmid pAI213 is shown in FIG.

The transformant in which pAI213 was introduced was M9.
CAAp plate medium _{(NH 4 Cl 1g, Na 2} HPO 4 · 1
2H ₂ O 14.7 g, KH ₂ PO ₄ 3 g, NaCl 5
_{g, MgSO 4 · 7H 2 O} 0.25g, CaCl 2 · 2H
₂ O 15 mg, glucose 2 g, vitamin B _{1 1} m
g, casamino acid 2 g, thiamine 10 mg, and ampicillin 75 mg were dissolved in 1 liter of water and 1.5% of agar was added), and TPP requirement was not recognized. When the thiamine pyrophosphokinase activity was examined, thiamine pyrophosphokinase activity not found in the host microorganism AI1102 strain was found in the transformed strain. (3) Determination of nucleotide sequence of gene encoding thiamine pyrophosphokinase The DNA sequence of the tpk gene contained in pAI213 was determined by the dideoxy method (F. Sanger et al .; Proc. Natl. Acad. Sci. USA, 7
4 , 5463 (1977)). The amino acid sequence of thiamine pyrophosphokinase deduced from the DNA sequence is shown in SEQ ID NO: 1, and the structural gene region of the tpk gene is shown in SEQ ID NO: 2.
It was shown to. Example 2 Acquisition of a microorganism highly expressing thiamine pyrophosphokinase activity In order to enhance the expression of the thiamine pyrophosphokinase activity of the pAI213-carrying strain, a promoter derived from the tryptophan biosynthetic enzyme gene of Escherichia coli was added upstream of the tpk gene of pAI213. (Hereafter, tr
plasmid pTPK
1 was produced.

About 2 μg of pAI213 was added to 20 μm of a buffer containing 10 mM Tris / HCl (pH 7.5), 6 mM MgCl ₂ , 1 mM dithiothreitol, 100 μg / ml bovine serum albumin and 150 mM NaCl.
After dissolving in 1 and adding 5 units of BamHI and performing a digestion reaction at 30 ° C. for 1 hour, 5 units of MaeI (manufactured by Boehringer Mannheim) was further added and a digestion reaction was performed at 45 ° C. for 1 hour. This was separated by 0.7% agarose gel electrophoresis, the gel fragment at the position of 0.75 kbp containing the tpk gene was cut out, and 0.75 kbp DNA was recovered from this fragment using DNA Prep (Diatron). After purification, 20 μl of TE buffer (10 mM Tris / HCl and 1 mM EDTA / Na ₂ , pH 8.0)
Dissolved in. Since the MaeI cleavage site is present in the tpk gene, the tpk gene in the 0.75 kbp DNA fragment encodes thiamine pyrophosphokinase in which N-terminal 9 amino acid residues are deleted.

As a vector, pTrS31 having a trp promoter (JP-A-3-210190) was used. The plasmid pTrS31 has one HindIII and one BamHI cleavage site downstream of the trp promoter. pTrS3
2 μg of 1 plasmid DNA was added to 20 mM Tris / hydrochloric acid (pH 8.5), 10 mM MgCl ₂ , 100 mM K
Dissolve in 20 μl of buffer containing Cl, and then use the restriction enzyme HindII
5 units each of I and BamHI were added, and a digestion reaction was performed at 37 ° C. for 2 hours. This is separated by 0.7% agarose gel electrophoresis, the gel fragment at the position of 4.5 kbp is cut out, and it is 4.5 k using DNA Prep (Diatron).
The bp DNA was purified and dissolved in 20 μl TE buffer.

Compensating for the deletion of thiamine pyrophosphokinase, which has a deletion of 7 N-terminal amino acid residues, the HindIII cleavage site of pTrS31 plasmid and Ma of 0.75 kbp DNA
The linker shown in FIG. 2 was used to connect to the eI cleavage site. The nucleotide sequence of the region that complements the deletion 9 amino acid residue portion of thiamin pyrophosphokinase in the linker is Paracoccus denitrif
icans ) derived from the chromosome was modified to a sequence corresponding to the optimal codon of E. coli (Ikemura; Cell Engineering, Vol. 5, No. 3, pages 212-221, (1986)). That is, the 2nd serine residue, the 3rd and 4th glutamine residues, the 5th glycine residue, the 6th threonine residue, and the 7th residue from the N-terminus of Paracoccus denitrificans thiamine pyrophosphokinase. Of the codon corresponding to the lysine residue of
A linker was used in which the first letter and the first letter of the codon corresponding to the 8th leucine residue were replaced with the base shown in SEQ ID NO: 2 by the base shown in FIG.

The triester method (R.
Crea et al .: Proc. Natl. Acad. Sci., 75 , 5765 (1978)), after synthesizing 33 bases of single-stranded DNA shown in SEQ ID NO: 4 and 31 bases of single-stranded DNA shown in SEQ ID NO: 5, Two
μg was mixed in 50 μl of water, heated at 65 ° C. for 10 minutes, and allowed to stand at room temperature for annealing to obtain a linker.

Paracoccus denitrificans
occus denitrificans ) -derived MaeI, Ba containing the tpk gene
HindIII, B of pTrS31 was added to 5 μl of DNA fragment digested with mHI.
3 μl of amHI digested DNA fragment, 2 μl of the above DNA linker
And 40 μl of T4 ligase buffer were added, and then 2 units of T4 DNA ligase (Takara Shuzo) was added to give 16
It was treated at ℃ for 18 hours. Using this treatment solution, Escherichia coli AI1102 strain was subjected to the method of Cohen et al. [S. Cohen et al; Proc. Natl. Acad. Sci. USA,
69 , 2110 (1972)] and transformed into M9CAAp
It was applied to a plate medium. After culturing at 37 ° C. for 24 hours, the grown TPP-non-requiring transformant was inoculated into a test tube containing 10 ml of LBAp liquid medium. The cells were cultured by shaking at 37 ° C. for 16 hours, and then collected by centrifugation. A known method [Molecular Cloning, T.
Plasmids were isolated by Maniatis et al. Cold Spring Harbor Laboratory (1982)]. Paracoccus denitrificans produced in this way
The plasmid containing the tpk gene from occus denitrificans ) was designated as pTPK1. This plasmid pTPK1
The restriction enzyme digestion map of the above is shown in FIG.

PTPK1 is a high expression type plasmid in which a thiamine pyrophosphokinase structural gene having a start codon is connected to the downstream region of the trp promoter via a linker. D of tpk gene contained in pTPK1
The NA sequence was determined by the dideoxy method (F. Sanger et al .; Proc. Natl. Ac
ad. Sci. USA, 74 , 5463 (1977)). The t
The modified structural gene region of the pk gene is shown in SEQ ID NO: 3.

Paracoccus denitrificans
occus denitrificans) ATCC19367 strain, AI1
102 / pAI213 strain and LE392 / pTPK1
After culturing the strain in LBAp liquid medium at 37 ° C. for 16 hours, the thiamine pyrophosphokinase activity was examined. LE392 / p
The TPK1 strain is about 14 of the AI1102 / pAI213 strains.
It had 0 times the activity (Table 1).

[0037]

[Table 1]

The strain was FERM BP- on June 8, 1994.
Deposited as 4692 at the Institute of Biotechnology, Institute of Biotechnology, AIST. Example 3 Paracoc denitrificans
cus denitrificans ) Production of TPP from thiamine and ATP using bacterial cells of ATCC 19367 strain Polypeptone (manufactured by Daigo Nutrition Chemical Co., Ltd.) 10 g / l, meat extract 7 g / l, yeast extract (manufactured by Daigo Nutrition Chemistry Co., Ltd.)
5 g / l and NaCl 3 g / l (pH with caustic soda
Liquid medium consisting of the composition of 7.2) was dispensed into large test tubes in 10 ml aliquots and steam sterilized at 120 ° C. for 20 minutes.
In the liquid medium, Paracoccus denitrificans ( Parac
occus denitrificans ) ATCC19367 strain was inoculated with one platinum loop and cultured with shaking at 30 ° C. and 300 rpm for 20 hours. After culturing, cells were obtained by centrifuging the culture solution, and 10 mM phosphate buffer solution (pH 7.2) was added and suspended so as to have a wet weight of 400 g / l.

1.25 ml of this suspension was placed in a large test tube and thiamin hydrochloride 25 mg, ATP 0.1 g, magnesium sulfate 25 mg, 0.3 M phosphate buffer (pH
7.2) 0.5 ml, 20 mg of Nymeen S-215 (manufactured by NOF CORPORATION) and 0.05 ml of xylene were added, and distilled water was added to bring the total volume to 5 ml. 1 of this mixture
The reaction was carried out at 30 ° C. with shaking and stirring at 00 rpm.
As a result, TPP was produced at 77 μM at 46 hours after the reaction. Example 4 Escherichia coli L
Thiamine and AT using bacterial cells of E392 / pTPK1 strain
Production of TPP from P. Escherichia col obtained in Example 2
i ) The LE392 / pTPK1 strain was inoculated into an LBAp liquid medium, shake-cultured at 37 ° C. for 20 hours, and then the culture solution was centrifuged to obtain bacterial cells.

0.8 g of this microbial cell (weight of wet microbial cell) was added to 200
Put it in a beaker of ml volume, 272 mg of thiamine, ATP
936 mg (234 mg each added at 0, 1, 5 and 8 hours), 0.1 g of magnesium chloride, 2 ml of 1M Tris-hydrochloric acid (pH 9.0) and 0.2 ml of xylene were added, and distilled water was added to bring the total amount to 20 ml. did. The reaction was carried out at 40 ° C. while stirring this mixed solution at 300 rpm. As a result, 30 mM of TPP was produced 24 hours after the reaction. Example 5 Escherichia coli LE392 / p having thiamine pyrophosphokinase activity
TPP production from thiamine in a coupled system of TPK1 strain and Corynebacterium ammoniagenes ATCC21170 strain having ATP regeneration activity (1) Escherichia coli LE392 / pTP having thiamine pyrophosphokinase activity
Preparation of K1 strain Escherichia col obtained in Example 2
i ) The LE392 / pTPK1 strain was inoculated into an LBAp liquid medium, shake-cultured at 37 ° C. for 20 hours, and then the culture solution was centrifuged to prepare cells.

(2) Preparation of cells of Corynebacterium ammoniagenes ATCC 21170 strain having ATP regeneration activity Liquid medium for seed culture 1 [glucose 50 g / l, polypeptone (manufactured by Daigo Nutrition Chemical Co., Ltd.) 10 g / l, yeast Extract (manufactured by Daigo Nutrition Chemical Co., Ltd.) 10 g / l, urea 5 g /
1, (NH ₄ ) SO ₄ 5 g / l, KH ₂ PO ₄ 1 g / l,
K ₂ HPO ₄ 3 g / l, MgSO ₄ .7H ₂ O 1 g / l,
CaCl ₂ · 2H ₂ O 0.1g / l, FeSO ₄ · 7H ₂
O 10 mg / l, ZnSO ₄ .7H ₂ O 10 mg / l,
MnSO ₄ .4-6H ₂ O 20 mg / l, L-cysteine hydrochloride 20 mg / l, D-calcium pantothenate
10 mg / l, vitamin B ₁ 5 mg / l, nicotinic acid
Liquid medium consisting of 5 mg / l and biotin 30 μg / l (adjusted to pH 7.2 with caustic soda) was dispensed into large test tubes in 10 ml aliquots, steam sterilized at 120 ° C. for 20 minutes, and then corynebacteria was added to the liquid medium. Um. Ammoniagenes ATCC 21170 strain was inoculated and cultured at 28 ° C. for 24 hours with shaking.

20 ml of this culture broth was used as the seed culture medium 1
The seed culture solution 1 was obtained by inoculating a 2 liter baffled Erlenmeyer flask containing 230 ml of the above, and culturing with shaking at 28 ° C. for 24 hours. Liquid medium for seed culture 2 [glucose 100 g
/ L, meat extract 10g / l, polypeptone 10g /
1, KH ₂ PO ₄ 1 g / l, K ₂ HPO ₄ 3 g / l, Mg
_{SO 4 · 7H 2 O 1g /} l, CaCl 2 · 2H 2 O 0.1
_{g / l, FeSO 4 · 7H} 2 O20mg / l, ZnSO 4
_{· 7H 2 O 10mg / l,} MnSO 4 · 4~6H 2 O2
0 mg / l, β-alanine 15 mg / l, L-cysteine hydrochloride 20 mg / l, and biotin 100 μg
Urea which had been sterilized by steam sterilization of a medium having a composition of 1 / l at 120 ° C for 20 minutes and then steam sterilized at 120 ° C for 20 minutes / g
1 and vitamin B ₁ 5 mg / l were added to a 5 liter culture tank containing 2.5 liters of a liquid medium adjusted to pH 7.2 with caustic soda, and 250 ml of the above seed culture solution 1 was added.
Inoculate and culture at 32 ° C., 600 rpm, aeration rate of 2.5 liter / min, while adjusting the pH to 6.8 with concentrated ammonia water, and culturing until glucose in the culture solution is consumed, Culture liquid 2 was obtained.

Liquid medium for main culture [glucose 180 g
/ L, KH ₂ PO ₄ 1 g / l, K ₂ HPO ₄ 3 g / l, M
gSO ₄ .7H ₂ O 1 g / l, CaCl ₂ .2H ₂ O 0.
_{1g / l, FeSO 4 · 7H} 2 O 20mg / l, ZnS
_{O 4 · 7H 2 O 10mg /} l, MnSO 4 · 4~6H 2 O
20 mg / l, L-cysteine hydrochloride 20 mg / l,
Sodium glutamate 1g / l and biotin 10
Urea that had been sterilized by steaming a medium having a composition of 0 μg / l for 20 minutes at 120 ° C. and then for 20 minutes at 120 ° C.
2 g / l and vitamin B ₁ 5 mg / l were added, and the seed culture solution 2 was added to a 5 liter culture tank containing 2.5 liters of a liquid medium adjusted to pH 7.2 with caustic soda.
ml inoculated, and the pH was adjusted to 6.8 with concentrated ammonia water under the culture conditions of 32 ° C., 600 rpm, and aeration rate of 2.5 l / min.
While adjusting to, culture was performed until glucose in the culture solution was consumed to obtain a main culture solution. The cells were prepared by centrifuging this main culture solution.

(3) TPP production from thiamine LE392 / pTP prepared in the above (1) and (2)
200 ml of 0.4 g of K1 strain and 3 g of Corynebacterium ammoniagenes ATCC 21170 strain
Put in a beaker, glucose 0.6g, thiamine 2
_{02mg, MgSO 4 · 7H 2 O} 0.1g, KH 2 PO 4
272 mg and xylene 0.2 ml were added, and distilled water was added to make the total volume 20 ml. 400r of this mixture
The reaction was carried out at 37 ° C. while stirring at pm and adjusting the pH to 7.8 with 10 N potassium hydroxide. During the reaction, phosphoric acid
Glucose and thiamine were added as needed so that KH ₂ PO ₄ was maintained at 1 to ₅ g / l. As a result, 67 mM of TPP was produced 24 hours after the reaction.

[0045]

According to the present invention, TPP can be industrially efficiently produced from thiamine and ATP.

[0046]

[Sequence list]

SEQ ID NO: 1 Sequence length: 218 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Ser Gln Gln Gly Thr Lys Leu Ala Ser Ala Arg Pro Val Thr 1 5 10 15 Leu Ile Gly Gly Ala Pro Val Gly Arg Ala Asp Leu Glu Gln Ala 20 25 30 Leu Ala Leu Ala Pro Val Val Ala Ala Ala Asp Ser Gly Ala Asp 35 40 45 Thr Ala Leu Ser His Gly Leu Met Pro Ala Ala Val Trp Gly Asp 50 55 60 Phe Asp Ser Ile Ser Ala Arg Ala Arg Ala Glu Ile Pro Leu Ala 65 70 75 Asn Gln His Pro Ile Ala Glu Gln Asn Ser Thr Asp Phe Glu Lys 80 85 90 Cys Leu Ser Asn Leu Asp Ala Pro Phe Val Val Ala Leu Gly Phe 95 100 105 Ser Gly Ala Arg His Asp His Phe Leu Ser Val Leu Asn Val Leu 110 115 120 Ala Arg Arg Ile Gly Pro Pro Cys Ile Leu Ile Ala Gly Glu Asp 125 130 135 Val Ile Thr Leu Ala Pro Pro Arg Leu Ala Leu Asp Leu Ala Pro 140 145 150 Gly Thr Arg Val Ser Leu Phe Pro Met Gly Pro Ala Thr Gly Arg 155 160 165 Ser Gln Gly Leu Lys Trp Pro Ile Asp Gly Leu Ala Phe Ala Pro 170 175 180 Gly Gly Ar g Ser Gly Thr Ser Asn Gln Ala Thr Gly Pro Val Thr 185 190 195 Leu Glu Cys Asp Gly Pro Met Leu Leu Ile Leu Pro Arg Ser Glu 200 205 210 Leu Ala Thr Leu Ala Arg Ala Leu 215 SEQ ID NO: 2 Sequence Length S: 654 Sequence type: Nucleic acid Topology: Linear Sequence type: Genomic DNA Origin organism name: Paracoc denitrificans
cus denitrificans ) Strain name: ATCC19367 Sequence features Symbols representing features: mat peptide Location: 1.654 Method of determining features: E sequence ATG AGC CAG CAA GGG ACC AAG TTG GCT AGT GCC CGC CCG GTC ACG CTG 48 ATC GGC GGT GCG CCG GTC GGC CGC GCC GAT CTG GAG CAG GCG CTG GCC 96 CTG GCG CCG GTG GTG GCC GCC GCC GAT AGC GGC GCC GAT ACC GCG CTG 144 AGC CAC GGG TTG ATG CCG GCG GCG GTC TGG GGC GAT TTC GAC AGC ATT 192 TCC GCC CGC GCC CGG GCC GAG ATC CCG CTG GCG AAC CAG CAC CCC ATC 240 GCC GAG CAG AAC AGC ACC GAT TTC GAG AAA TGC TTG TCC AAT CTC GAT 288 GCT CCC TTC GTC GTC GCC CTG GGC TTT TCG GGC GCG CGC CAT GAC CAT 336 TTC CTG TCG GTG CTC AAT GTG CTC GCC CGC CGT ATC GGC CCG CCC TGC 384 ATC CTG ATC GCG GGC GAG GAC GTG ATC ACG CTT GCC CCG CCG CGC CTC 432 GCG CTG GAT CTC GCG CCC GGC ACG CGG GTC TCG CTG TTC CTC GGC 480 CCG GCC ACC GGC CGC TCG CAG GGC CTG AAA TGG CCC ATC GAC GGC CTG 528 GCC TTC GCG CCG GGC GGC CGC AGC GGC ACC TCG AAC CAG GCC ACG GGT 576 CCG GT C ACG CTG GAA TGC GAC GGC CCG ATG TTG CTG ATC CTG CCG CGC 624 TCG GAA CTG GCG ACG CTC GCC CGC GCC CTC 654 SEQ ID NO: 3 Sequence length: 654 Sequence type: Nucleic acid Topology: Linear sequence type : Genomic DNA Origin organism name: Paracoccu
s denitrificans ) Strain name: ATCC19367 Sequence features Symbols representing features: mat peptide Location: 1.654 Method of determining features: E sequence ATG AGT CAA CAG GGC ACT AAA CTG GCT AGT GCC CGC CCG GTC ACG CTG 48 ATC GGC GGT GCG CCG GTC GGC CGC GCC GAT CTG GAG CAG GCG CTG GCC 96 CTG GCG CCG GTG GTG GCC GCC GCC GAT AGC GGC GCC GAT ACC GCG CTG 144 AGC CAC GGG TTG ATG CCG GCG GCG GTC TGG GGC GAT TTC GAC AGC ATT 192 TCC GCC CGC GCC CGG GCC GAG ATC CCG CTG GCG AAC CAG CAC CCC ATC 240 GCC GAG CAG AAC AGC ACC GAT TTC GAG AAA TGC TTG TCC AAT CTC GAT 288 GCT CCC TTC GTC GTC GCC CTG GGC TTT TCG GGC GCG CGC CAT GAC CAT 336 TTC CTG TCG GTG CTC AAT GTG CTC GCC CGC CGT ATC GGC CCG CCC TGC 384 ATC CTG ATC GCG GGC GAG GAC GTG ATC ACG CTT GCC CCG CCG CGC CTC 432 GCG CTG GAT CTC GCG CCC GGC ACG CGG GTC TCG CTG TTC CTC GGC 480 CCG GCC ACC GGC CGC TCG CAG GGC CTG AAA TGG CCC ATC GAC GGC CTG 528 GCC TTC GCG CCG GGC GGC CGC AGC GGC ACC TCG AAC CAG GCC ACG GGT 576 CCG GTC ACG CTG GAA TGC GAC GGC CCG ATG TTG CTG ATC CTG CCG CGC 624 TCG GAA CTG GCG ACG CTC GCC CGC GCC CTC 654 SEQ ID NO: 4 Sequence length: 33 Sequence type: Nucleic acid Topology: Linear Sequence type: Other Nucleic Acids Synthetic DNA Hypothetical Sequence: Yes Sequence features Characteristic symbols: insertion seq Location: 1.33 Method of determining features: E sequence AGCTTAAATG AGTCAACAGG GCACTAAACT GGC 33 SEQ ID NO: 5 Sequence length: 31 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Hypothetical sequence: Yes Sequence features Characteristic symbol: insertion seq Location: 1..31 Method for determining features: E Array TAGCCAGTTT AGTGCCCTGT TGACTCATTT A 31

[Brief description of drawings]

Figure 1: Paracoccus denitrificans ATCC
3 shows a restriction map of plasmid pAI213 containing a gene encoding thiamine pyrophosphokinase derived from 19367.

FIG. 2: 0.75k containing the gene encoding thiamine pyrophosphokinase from Paracoccus denitrificans ATCC19367 in plasmid pTrS31
The linker used when inserting the bp DNA fragment is shown.

Figure 3: Paracoccus denitrificans ATCC
3 shows a restriction map of plasmid pAI213 containing a gene obtained by modifying a part of the gene encoding the thiamine pyrophosphokinase derived from 19367.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location // C12N 9/12 (C12P 17/16 C12R 1:19) (C12N 1/21 C12R 1:19) ) (C12N 15/09 ZNA C12R 1:01) C12R 1:01)

Claims (16)

[Claims] Bearing 1. A Paracoccus (Paracoccus) a microorganism belonging to the genus or Paracoccus (Paracoccus) derived from a microorganism belonging to the genus recombinant DNA comprising a DNA fragment carrying genetic information responsible for the synthesis of thiamine diphosphate, In the presence of a microorganism, which has an activity of catalyzing the reaction of producing thiamine diphosphate from thiamine and adenosine-5'-triphosphate, in the presence of an microbial cell, a culture solution or a treated product thereof in an aqueous medium. A method for producing thiamine diphosphate, which comprises reacting thiamine with adenosine-5′-triphosphate and collecting the thiamine diphosphate produced from the reaction solution. 2. A tp that encodes the amino acid represented by SEQ ID NO: 1 by a microorganism belonging to the genus Paracoccus.
The production method according to claim 1, which is a microorganism having a k gene. 3. A microorganism carrying the recombinant DNA is, Escherichia (Escherichia) genus Enterobacter (Ente
Robacter) genus Pseudomonas (Pseudomonas) genus Corynebacterium (Corynebacterium) genus or process of claim 1 which is Brevibacterium (Brevibacterium) microorganisms belonging to the genus. 4. A DNA fragment containing a gene encoding a protein having thiamine pyrophosphokinase activity.
The method according to claim 1 or 3, which is an A fragment. 5. Adenosine-5′-triphosphate has an activity of producing adenosine-5′-triphosphate from a precursor of adenosine-5′-triphosphate, a phosphate donor and an energy donor. The adenosine-5'-triphosphate supplied by the microorganism that has the above-mentioned, claim 2, claim 3 or claim 3.
Alternatively, the production method according to claim 4. 6. A recombinant DNA comprising a DNA fragment derived from a microorganism belonging to the genus Paracoccus and containing a gene encoding a protein having thiamine pyrophosphokinase activity, and a vector DNA. 7. The recombinant DNA according to claim 6, wherein the gene is a gene encoding the amino acid sequence represented by SEQ ID NO: 1 or a gene hybridizing therewith. 8. The recombinant DNA according to claim 6, wherein the gene is a gene represented by the nucleotide sequence of SEQ ID NO: 2 or a gene hybridizing therewith. 9. The recombinant DNA according to claim 6, wherein the gene is the gene represented by the nucleotide sequence of SEQ ID NO: 3 or a gene that hybridizes with it. 10. A gene derived from a microorganism belonging to the genus Paracoccus and encoding a protein having a thiamine pyrophosphokinase activity, or a gene hybridizing therewith. 11. A gene encoding the amino acid sequence represented by SEQ ID NO: 1 or a gene hybridizing therewith. 12. A gene represented by the nucleotide sequence of SEQ ID NO: 2 or a gene which hybridizes with this. 13. A gene represented by the nucleotide sequence of SEQ ID NO: 3 or a gene that hybridizes with the gene. 14. A recombinant DNA, which is derived from a microorganism belonging to the genus Paracoccus and which encodes a gene encoding a protein having thiamine pyrophosphokinase activity or a DNA fragment containing a gene hybridizing thereto and a vector DNA. Microorganisms possessed. 15. The microorganism according to claim 14, wherein the gene is a gene encoding the amino acid sequence represented by SEQ ID NO: 1 or a gene hybridizing therewith. 16. The microorganism according to claim 14, wherein the gene is a gene represented by the nucleotide sequence of SEQ ID NO: 3 or a gene that hybridizes therewith.