JP3357405B2 - Method for producing flavin nucleotides - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to flavin nucleotides using microorganisms, especially flavin mononucleotides (F
MN) and / or a method for producing flavin adenine dinucleotide (FAD). FMN and FAD are
In particular, it is an important substance as a coenzyme form of vitamin B ₂ as a raw material for nutrients and various pharmaceuticals as well as a reagent for biochemical research, and is used in the industrial field of bioindustry.

[0002]

2. Description of the Related Art Various methods are known for producing flavin nucleotides. For example, it belongs to the genus Corynebacterium or the genus Brevibacterium, has the ability to produce ATP from adenosine-5'-3 phosphate (ATP) precursor, a phosphate donor and an energy donor, and A method using a microorganism capable of producing FAD from ATP (JP-A-59-132892), which is derived from a microorganism belonging to the genus Corynebacterium or Brevibacterium and belonging to the genus Corynebacterium or Brevibacterium. D containing a DNA fragment carrying the genetic information involved in the synthesis of FMN or FAD
A method using a microorganism having NA (JP-A-2-138988) can be used.

[0003] In microorganisms belonging to the genus Brevibacterium, FM is derived from riboflavin (FR) and ATP.
Flavokinase (FK) activity to produce N, FMN and A
It is known that flavin adenine dinucleotide synthetase (FADS) activity, which produces FAD from TP, exists on the same protein [Journal.
Of Biological Chemistry (J. Biol. Che
m.), 261 , 16169-16173 (1986)].

[0004] However, a method using a microorganism belonging to the genus Escherichia, Enterobacter or Pseudomonas and having a recombinant DNA containing a DNA fragment carrying the gene information involved in the synthesis of FMN and / or FAD is known. Absent.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to produce flavin nucleotides, which are important as raw materials for nutrients, various raw materials for pharmaceuticals, and reagents for biochemical research, in a higher yield and at lower cost.

[0006]

According to the present invention, there is provided a recombinant DNA containing a DNA fragment which belongs to the genus Escherichia, Enterobacter or Pseudomonas and carries the genetic information involved in the synthesis of FMN and / or FAD. Flavin nucleotide precursors and AT in an aqueous medium in the presence of cells, culture solutions or processed products thereof
A method for producing flavin nucleotides, comprising reacting P with flavin and collecting flavin nucleotides generated from the reaction solution.

As flavin nucleotide precursors,
Riboflavin, flavin mononucleotide,
Flavin nucleotides include flavin mononucleotides and flavin adenine dinucleotides.
As the DNA fragment, a DNA fragment containing all or a part of a gene encoding a protein having FK activity and FADS activity, or the FADS activity of the protein was reduced or eliminated while the FK activity remained. Examples include DNA fragments containing all or a part of the gene encoding the modified protein. As a DNA fragment containing all or a part of a gene encoding a protein having FK activity and FADS activity, protein X [Journal of Biological Chemistry (J. Biol. Chem.), 260 , 5616-5620] (1985)].

[0008] Further, for proteins having FK activity and FADS activity, FADS
Examples of the DNA fragment containing all or a part of the gene encoding the modified protein having reduced or eliminated activity include:
A DNA fragment containing all or part of a gene encoding an amino acid sequence obtained by substituting glycine, which is the 23rd amino acid for protein X, with another amino acid can be mentioned. Amino acids to be substituted include arginine, alanine, aspartic acid and the like.

[0009] Microorganisms serving as a source of DNA fragments include microorganisms having FK activity and FADS activity, microorganisms having FK activity but having reduced or eliminated FADS activity, or all or all of the genes encoding protein X. Any microorganism belonging to the genus Escherichia, Enterobacter or Pseudomonas can be used as long as it is a microorganism containing a part thereof.

A microorganism having FK activity and FADS activity
Specific examples of Escherichia coli K12 strain,
Escherichia coli C600, a mutant of K12 strain
Stock (Appleyard R.K., Genetics), 3
9, 440 (1954)]. These d
FK activity and FA in microorganisms belonging to the genus R. scherichia
Whether the DS activity is on the same protein or not
Although it was unknown whether it was protein,
Genes obtained from microorganisms belonging to the genus Escherichia and solved
As a result of the analysis,
Like, both activities are on the same protein (protein X)
And the domain responsible for FADS activity on the N-terminal side
However, a domain responsible for FK activity is located on the C-terminal side
It became clear. This is Brevibacterium
Microorganisms belonging to the genus Escherichia as well as microorganisms belonging to the genus
Generates FAD from FR and ATP via FMN
It shows that it has the activity of Such fine
If an organism is used, not only FR but also FMN is used as a substrate.
Production and production of FMN and FAD from FR.
And FAD production from FMN. I
However, Escherichia coli contains Brevibacterium
Hybridizes with genes derived from microorganisms belonging to the genus Aum
Gene belongs to the genus Brevibacterium
Genes derived from the microorganisms used
It is clear that

[0011] It should be noted that the FAD
The gene encoding the protein having reduced or eliminated S activity is obtained by using a Mutant-K kit (manufactured by Takara Shuzo) to substitute glycine, the 23rd amino acid of protein X, with another amino acid by site-specific mutation. And is effective for producing FMN alone from FR. As the host microorganism used in the present invention, any microorganism belonging to the genus Escherichia, Enterobacter, or Pseudomonas, and having a DNA uptake ability, such as a mutant strain having drug resistance, auxotrophy, etc. in addition to a wild strain can be used. May be used. Preferable examples include Escherichia coli DH5α strain [Bethesda Research Laboratories, Focus, 8 , 9 (1986)].

As the vector used in the present invention, any phage vector, plasmid vector, or the like can be used as long as it is capable of autonomous replication in Escherichia, Enterobacter or Pseudomonas species. Preferably, pBR322 [Gene (GE
NE), 2 , 95 (1977)], pUC19 [Gene (GE
NE), 33 , 103 (1985)]. In addition, host / vector systems of other strains such as genus Brevibacterium and Corynebacterium may be used.

[0013] FMN and / or FA as described above
Recombinant DNA of a DNA fragment carrying the genetic information involved in the synthesis of D and vector DNA can be prepared by cleaving both DNAs with a restriction enzyme that gives the same cleaved end in a test tube, and then performing a ligation reaction with DNA ligase. Together with a recombinant hybrid of Using the obtained recombinant hybrid, a host microorganism selected from microorganisms belonging to the genus Escherichia, Enterobacter or Pseudomonas is transformed, and FMN and / or FAD are transformed.
It is possible to obtain a recombinant plasmid containing the DNA by selecting a transformant having a recombinant plasmid containing a DNA fragment carrying the genetic information involved in the synthesis of the DNA, and isolating the plasmid from the strain. it can.

As a transformation method, the method of Cohen et al. [Cohen et al .; Proc. Of Natl. Acad. Sc.
i.) USA, 69 , 2110 (1972)]. As a method for selecting a transformant having a recombinant plasmid containing a DNA fragment carrying genetic information involved in the synthesis of FMN and / or FAD, a method for selecting a transformant in which all or a part of the amino acid sequence of the DNA is known, For example, a method of synthesizing a DNA probe having a base sequence corresponding to the amino acid sequence and selecting the same based on the property of hybridizing with the DNA is used.

Next, the DNA fragment is cut out from the obtained recombinant plasmid with an appropriate restriction enzyme, and this DNA fragment is inserted into a vector DNA capable of autonomously replicating in a host microorganism, whereby a recombinant containing the DNA is obtained. A plasmid is prepared, and a host microorganism is transformed by the method of Cohen et al. To obtain a transformed strain. Culture of the thus obtained transformant (a strain with high FK-FADS activity or a strain with high FK activity) contains a carbon source, a nitrogen source, an inorganic substance, an amino acid, a vitamin and the like according to a usual bacterial culture method. It is carried out in an ordinary synthetic or natural medium under aerobic conditions while adjusting the temperature, pH and the like. Preferably, a high-density culture method [Biotechnol.Bioeng., 17 , 227-239 (197
5)].

Examples of the carbon source used in the medium include glucose, fructose, sucrose, molasses, molasses,
Carbohydrates such as starch hydrolysates, alcohols such as ethanol, glycerin, and sorbitol; organic acids such as pyruvic acid, lactic acid, and acetic acid; and amino acids such as glycine, alanine, glutamic acid, and aspartic acid. Any can be used. 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 nitrate, ammonium carbonate, ammonium acetate and ammonium phosphate, urea, peptone, NZ amine, meat extract, yeast extract, corn steep Various substances such as nitrogen-containing organic substances such as liquor, casein hydrolyzate, fish meal or its digest, and various amino acids such as glycine and glutamic acid can be used. The use concentration is preferably 0.1 to 10%.

As inorganic substances, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, magnesium phosphate,
Sodium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, calcium carbonate and the like can be used. When the microorganism used requires specific nutrient substances such as amino acids, nucleic acids, and vitamins for growth, these substances are added to the medium in appropriate amounts.

The culture is carried out under aerobic conditions such as shaking culture or aeration and stirring culture. The optimal culture temperature is generally 28 to 42 ° C. Culture is usually completed in 1 to 24 hours. Medium
It is desirable to keep the pH almost neutral with ammonia, urea, sodium hydroxide solution, or the like. The culture of the microorganism obtained in this way can be used for the reaction as it is,
Further, a treated product obtained by variously treating the culture may be used for the reaction. As the processed product, a concentrated product of the culture, a dried product, a cell obtained by treating the culture with a centrifugal separator, a product treated with a surfactant or a bacteriolytic enzyme, a dried product of the cell, an acetone-treated product, Surfactant-treated products, lysed enzyme-treated products, immobilized cells, and the like.

The reaction can be carried out in any aqueous medium. Preferably, FR or FMN and ATP and, if necessary, a surfactant and / or an organic solvent are simultaneously present in the culture solution of the microorganism, or FR is added to the culture solution, the cells, or the processed product thereof after completion of the culture.
Alternatively, FMN and ATP, and if necessary, a surfactant and / or an organic solvent,
By reacting for ~ 72 hours, flavin nucleotides can be accumulated in the medium or the reaction solution. At this time, the pH was 6-9, and the oxidation-reduction potential was -250-
It is desirable to adjust to -400 mV. The concentration of each substrate in the medium and the reaction solution is usually FR 0.1 to 20 g / l, FM
N 0.1 to 40 g / l, ATP 0.1 to 100 g / l. A strain (transformant) having enhanced FK and / or FADS activity used as an enzyme source is 5 to 2 as wet cells.
It is desirable to use at a concentration of 00 g / l. Although FR is hardly soluble in water, the purpose can be achieved in a suspended state even if FR is not dissolved in water.

As the ATP source, in addition to a highly purified sample,
ATP-containing liquid obtained by contacting adenine with microbial cells in the presence of an energy donor (JP-A-59-517)
99) or a cell-removing solution thereof, and a concentrated solution thereof and the like can also be used. A microorganism having ATP regeneration activity (JP-A-61-74595) can be added to the reaction system to synthesize and supply ATP from glucose and inorganic phosphoric acid. In this case, an ATP precursor, an ATP regeneration energy donor, a phosphate group donor, and a microorganism having ATP biosynthesis activity are present in the reaction solution instead of ATP. The strain having ATP regeneration activity used herein includes Brevibacterium ammoniagenes [scientific name has been changed to Corynebacterium ammoniagenes, and International Journal of Systematic Bacteriology (I
nternational Journal of Systematic Bacteriology),
vol.34, No.4, pp442 (1987), hereinafter referred to as Corynebacterium ammoniagenes. ATCC21170 strain and Escherichia coli ATCC11303 strain. AT
Culture for the purpose of obtaining cells of a microorganism having P regeneration activity follows the method described in JP-A-61-74595.

Examples of the surfactant include cationic surfactants such as polyoxyethylene stearylamine (for example, Nymein S-215, manufactured by NOF Corporation), cetyltrimethylammonium bromide, cetylpyridium chloride, sodium lauryl sulfate, sodium oleyl Anionic surfactants such as amidosulfuric acid, polyoxyethylene sorbitan monostearate (for example, nonionic ST2
21, non-ionic surfactants such as Nippon Yushi Co., Ltd., and amphoteric surfactants such as lauryl betaine (eg, Anon BF, Nippon Yushi Co., Ltd.) are used, and these are usually 0.1
It is used at a concentration of 5050 g / l, preferably 1-20 g / l.

As the organic solvent, toluene, xylene,
Acetone, fatty acid alcohol, ethyl acetate and the like are used, and these are usually 0.1 to 50 ml / l, preferably 1 to 20 ml / l.
l is good. As a method for collecting the flavin nucleotides accumulated in the medium or the reaction solution, an ordinary method using activated carbon, an ion exchange resin, or the like can be used.

An embodiment of the present invention will be described below.

[0025]

【Example】

Example 1 Obtaining microorganisms with high FK-FADS activity and microorganisms with high FK activity (1) Purification of FK-FADS A culture of Escherichia coli C600 strain was centrifuged to obtain 30 g of wet cells. This was suspended in 100 ml of a purification buffer (I) [50 mM Tris-HCl buffer (pH 8), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol], and homogenized (Brown Biotech, glass bead diameter). (0.1 mm). The crushed liquid was centrifuged to obtain about 90 ml of the supernatant. In the supernatant, 0.4% final concentration
After adding protamine sulfate, the mixture was centrifuged to remove the high molecular nucleic acid component as a precipitate. The obtained supernatant was put into a cellulose membrane with the purification buffer (I) and dialyzed to obtain about 11 ml of a desalting active fraction. This fraction was added to a CM-Sepharose column (manufactured by Pharmacia) previously equilibrated with the purification buffer (I), and then 0M → 1M Na
The target enzyme was eluted with 300 ml of the purification buffer (I) having a linear gradient of Cl, and a 12 ml portion of the active elution fraction having the highest enzyme concentration was collected. This active fraction was added to a Sephacryl S-300 column (manufactured by Pharmacia), eluted with a purification buffer (I), subjected to gel filtration, and subjected to 5 ml of the active fraction.
I got This fraction was added to an ATP-agarose column previously equilibrated with the purification buffer (I), and then 0M → 1
Purification buffer (I) 1 having an M NaCl linear concentration gradient
The active fraction was collected and finally collected at 16.7 μg.
3.5 ml of protein / ml purified enzyme was obtained.

(2) Acquisition of FK-FADS gene The purified enzyme obtained above was electrophoresed by SDS-polyacrylamide gel electrophoresis, and was subjected to PVDF (polyvinylidene difluid).
oride) was blotted on a membrane and analyzed for N-terminal amino acids. As a result, the N-terminal 14 amino acids were consistent with protein X. Therefore, the protein X nucleotide sequence [Y. Kamio et al., Journal of Biological Chemistry (J. Bi.
ol. Chem.) , 260, 5616-5620 (1985)].
A 5 ′ primer for R (SEQ ID NO: 1) and a 3 ′ primer (SEQ ID NO: 2) were designed and synthesized by a solid phase synthesis method using the phosphoramidite method [SL Beaucage et al., Tetrahedron Lett., 22 , 1859 (1981)], an automated DNA synthesizer 380A manufactured by Applied Biosystems, Inc.
Was synthesized using PCR was performed using Gene AmpDNA Amplification Reagent Kit (Takara Shuzo), using chromosomal DNA of Escherichia coli C600 strain as a template, 1 ng / 100 μl, synthetic DNA primers each at 1.0 μM, dATP, dCTP,
dGTP and dTTP were each added to 200 μM and Taq DNA.
Polymerase (Takara Shuzo) was mixed at a ratio of 2.5 units / 100 μl and mixed at 92 ° C. for 1 minute, 37 ° C. for 2 minutes and 7 minutes.
The reaction at 0 ° C. for 3 minutes was repeated 25 times to obtain a target DNA fragment encoding protein X.

(3) Preparation of Recombinant DNA Hind III and Eco RI were added to 20 μl of a solution containing 1 μg of the DNA fragment obtained by the reaction as described above, and digestion was performed. Separately, HindIII and EcoRI were added to 20 μl of a solution containing 1 μg of the vector pUC19 for digestion. The digested DNA fragment and vector DNA were purified by phenol extraction and ethanol precipitation. 100 ng of purified DNA and 20 of purified vector DNA
ng was suspended in a solution containing 66 mM Tris-HCl buffer (pH 7.6), 66 mM magnesium chloride, 10 mM DTT and 0.1 mM ATP, and 10 units of T4 ligase was added thereto.
The reaction was performed at 4 ° C. for 16 hours to ligate both DNAs to obtain a recombinant DNA.

(4) Preparation of Escherichia coli Transformed with Recombinant DNA Escherichia coli DH5α strain was added to an LB liquid medium [1%
Tryptone, 0.5% yeast extract, 1% sodium chloride (pH 7.5)] and cultured at 37 ° C for 4 hours. 3
Centrifuged at 000 rpm for 7 minutes and collected at 50
After suspending in 20 ml of a mM calcium chloride solution and allowing to stand at 0 ° C. for 20 minutes, the cells were collected by the same centrifugation operation as above, and the cells were collected at 0 ° C. and 50 ° C.
It was suspended in 40 ml of a mM calcium chloride solution. The suspension and the solution containing the recombinant DNA obtained in Example 1, paragraph (3) were mixed and allowed to stand at 0 ° C. for 10 minutes. Then at 42 ° C 9
After a 0 second heat treatment, the mixture was combined with 50 μg / ml ampicillin and 20 μg / ml 5-bromo-4-chloro-
It was spread on an LB agar plate medium (LB liquid medium + 1.5% agar) containing 3-indolyl-β-galactoside (Xgal). This plate medium was kept at 37 ° C. for 24-48 hours.

(5) DN encoding FK-FADS
Acquisition of A After incubation, colonies appeared on the plate medium. Of these, white colonies were separately cultured in an LB liquid medium to obtain wet cells of the transformed strain. The obtained cells were disrupted by the method described in Example 1, paragraph (1), and the supernatant was collected. The supernatant was used as a crude enzyme preparation, and the method of DJ Manstein et al. [Journal of
Biological chemistry (J. Biol. Chem.), 26
1 , 16169-16173 (1986)], the activity was measured both when FR was used as the substrate and when FMN was used.
As a result, as shown in Table 1, the FK activity was about 32
There was a strain with a 5-fold increase in FADS activity about 240-fold. The recombinant plasmid of this strain was called pFK5A
It was named. This strain is transformed into Escherichia coli DH5α
/ PFK5A strain.

The strain was deposited on November 7, 1991 under the Budapest Treaty with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as FERM BP-3643.

(6) Confirmation of the location of FK and FADS domains In the FK-FADS gene obtained in Example 1, section (5), FK-FADS was used to confirm the location of FK and FADS domains. A recombinant plasmid having an N-terminal and C-terminal deletion gene was constructed. That is, FK
The recombinant plasmid having the N-terminal deletion gene of -FADS is shown in SEQ ID NOS: 3, 4 and 5 by using pFK5A as a template and in the same manner as in Examples 1 (2) and (3). 5 ′ primer and 3 ′ shown in SEQ ID NO: 6
Hind III- Ba of the PCR product obtained using the
The mHI fragment was transferred to the ATG expression vector pTrS33 (Japanese Unexamined Patent Publication No.
No. 227075) to construct a recombinant plasmid, and the obtained recombinant plasmids are pND12 and pND12, respectively.
The mutant genes were designated as ND18 and pND30 (the mutant genes encode proteins in which 12, 18, and 30 amino acids at the N-terminal side of FK-FADS have been deleted, respectively). on the other hand,
The recombinant plasmid having the C-terminal deletion gene of FK-FADS was shown in SEQ ID NOS: 1 and 7 by using pFK5A as a template and in the same manner as in Sections (2) and (3) of Example 1. Hi of PCR product obtained using primers
The nd III- Bam HI fragment was inserted into pUC19 to construct a recombinant plasmid.
D20 (this mutant gene encodes a protein in which the 20 amino acids at the C-terminal side of FK-FADS have been deleted). These recombinant plasmids were introduced into Escherichia coli DH5α, and both enzyme activities were measured in the same manner as in Example 1 (5) using the crude enzyme solution of the obtained transformant. The results are shown in Table 1. As can be seen from these results, a FADS domain exists at the N-terminal side of protein X (FK-FADS), and at least N-terminal
Deletion of 18 amino acids from the terminal suggested that FADS activity was drastically reduced and that the FK domain was present on the C-terminal side. The activity of the mutant plasmid-introduced strain was pFK5
It is considered that the reason why the activity was lower than that of the A-introduced strain was that granules were formed during the culture, and the amount of the soluble enzyme was reduced.

[0033]

[Table 1]

(7) Acquisition of strains with reduced FADS activity The Kunkel method (Methods in Euzymology, 154 , 367 (1987)) using the primers shown in SEQ ID NOs: 8, 9 and 10, respectively. To obtain the mutant plasmid (SEQ ID NOs: 8, 9 and 1).
The primer shown at 0 is designed to replace glycine at position 23 of protein X with arginine, alanine and aspartic acid, respectively). Natural type F as a template
The plasmid having the K-FADS gene was prepared by the same method as in Example 1, (2) and (3), using the primers shown in SEQ ID NOs: 2 and 11 for PC.
The Pst I- Eco RI fragment of the R product was ligated with pTZ19R [Protein Engineering,
1 , 67 (1986)]. On this occasion,
The introduction of the mutation was confirmed by extracting a plasmid from the obtained recombinant and using an automatic DNA sequencer.
Plasmids in which a mutation in which the glycine at position 23 in the amino acid to be encoded is substituted with arginine, alanine or aspartic acid were introduced into pKK11 and pKK11, respectively.
12, and named pKK13. Escherichia coli DH5α was transformed using each of the mutation-introduced plasmids. The resulting transformant (DH5α / pKK11
Strain, DH5α / pKK12 strain, DH5α / pKK13
The activity of both enzymes was measured in the same manner as in Example 1, Section (5) using the crude enzyme solution of Strain Co., Ltd. As a result, as shown in Table 2, when glycine at position 23 was replaced with arginine, alanine or aspartic acid, FK activity remained, but FADS
Activity had dropped dramatically.

Escherichia coli DH5α / pKK1
1 strain, DH5α / pKK12 strain, DH5α / pKK13
The strains were submitted to the Institute of Microbial Industry and Technology by the Ministry of Industrial Science and Technology on October 13, 1992, based on the Budapest Treaty.
No. (FERM BP-4032), Microfabrication Kenjo No. 4033 (FERMBP
-4033).

[0036]

[Table 2]

Example 2 100 g / l FR of Escherichia coli DH5α / pFK5A cultured by high-density culture, FR
10 g / l, ATP 15 g / l, xylene 10 ml / l
, Nimein S-215 4g / l in a large test tube (21mm inner diameter) at 46 ° C for 30 hours at 0.2N
The pH was adjusted to 7.0 to 7.2 with a potassium hydroxide solution, and the reaction was carried out with occasional shaking to such an extent that the cells did not settle. Table 3 shows the amounts of the generated FMN · Na ₂ and FAD · Na ₂ .
Instead of using Escherichia coli DH5α / pFK5A strain, Escherichia coli DH5α strain, DH5α /
pKK11 strain, DH5α / pKK12 strain and DH5α
Table 3 also shows the results obtained by using the / pKK13 strain.

[0038]

[Table 3]

Example 3 Escherichia coli cultured by the high-density culture method
Cultured cells of DH5α / pFK5A strain 100 g / l, FM
N20g / l, ATP 25g / l, xylene 10ml /
l, reaction solution 10 containing 4 g / l of Nimeen S-215
ml into a large test tube, 0.2N hydroxylation at 46 ° C for 24 hours
Adjust the pH to 7.0 to 7.2 with a potassium solution to prevent cell sedimentation.
The reaction was made with occasional shaking. Generated FA
D ・ Na _TwoThe amounts are given in Table 4. Escherichia coli D
Instead of using the H5α / pFK5A strain, Escherich
A. coli DH5α strain and JP-A-2-138988
F from Corynebacterium ammonigenes listed
Plasmid containing K-FADS gene was
Recombinant ATCC21 introduced into Umm Ammonigenes
170 / pKH43 strain (however, the cell mass is 200
g / l, FMN is 12 g / l, ATP is 24 g / l,
(The reaction temperature is 37 ° C and the reaction time is 20 hours.)
Table 4 also shows the results.

[0040]

[Table 4]

Example 4 Cultured Escherichia coli DH5α / pFK5A 100 g / l, FR, cultured by high-density culture
1 g / l, xylene 10 ml / l, Nymeen S-21
5 Place 10 ml of the reaction solution containing 4 g / l in a large test tube.
48 hours at 6 ° C, pH 7.0 with 0.2N potassium hydroxide solution.
The mixture was adjusted to 7.2, and the reaction was carried out with occasional shaking so that the cells did not settle. Generated FMN ・ Na ₂ , FAD ・
Table 5 shows the amounts of Na ₂ . Escherichia coli DH5
Instead of using α / pFK5A strain, Escherichia coli DH5α strain, DH5α / pKK11 strain, DH5α / p
Table 5 also shows the results obtained by using KK12 strain and DH5α / pKK13 strain.

[0042]

[Table 5]

Example 5 Escherichia coli cultured by the high-density culture method
Cultured cells of DH5α / pFK5A strain 20 g / l,
Corynebacterium cultured by the method described in 61-74595
Cultured cells of Terium ammoniagenes ATCC21170 strain 1
60 g / l, glucose 100 g / l, FR 16 g / l
l, KH_TwoPO_Four20 g / l, MgCl _Two・ 7H_TwoO
3 g / l, xylene 10 ml / l, Nymeen S-215
 500 liters of reaction solution containing 4 g / l in 2 liter mini-volume
Put in a fermentation tank at 46 ° C for 48 hours, 5N potassium hydroxide solution
Adjust the pH to 7.0 with redox while stirring at 600 rpm.
The reaction was carried out while maintaining the original potential at -350 to -400 mV.
FMN ・ Na generated_Two, FAD ・ Na_TwoTable 6 shows the amount
Show. Escherichia coli DH5α / pFK5A strain
Instead of using Escherichia coli DH5α strain and
Similar results using DH5α / pKK12 strain
Are also shown in Table 6.

A recombinant AT obtained by introducing a plasmid having the FK-FADS gene derived from Corynebacterium ammonigenes into Corynebacterium ammonigenes
Table 6 also shows the results of similar experiments performed using the CC21170 / pKH43 strain. The reaction was performed as follows. 160 g of cultured cells of ATCC 21170 / pKH43 strain
/ L, glucose 100 g / l, FR 8 g / l, KH ₂
PO ₄ 20 g / l, MgSO ₄ .7H ₂ O 5 g / l,
ZnSO ₄ 10 g / l, sodium pyrophosphate 40 g /
l, xylene 10 ml / l and Nymeen S-215
500 ml of the reaction solution containing 4 g / l was placed in a 2 liter mini-fermenter, adjusted to pH 7.8 with 5N potassium hydroxide solution at 38 ° C. for 55 hours, and stirred at 600 rpm to adjust the oxidation-reduction potential to −300 to −400 mV. While maintaining the soluble KH ₂
KH ₂ PO _{4 is} appropriately adjusted to keep the PO ₄ concentration at about 30 g / l.
Was added and reacted.

[0045]

[Table 6]

[0046]

[Sequence list]

SEQ ID NO: 1 Sequence length: 26 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCGGTTAAGC TTGACCGCTG TACAAG 26

SEQ ID NO: 2 Sequence length: 25 Sequence type: Number of nucleic acid chains: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCCTGTTTCC GGCGAATTCA GGGTT 25

Sequence number: 3 Sequence length: 50 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATCGTATCGA TAAGCTTATG GCCCCGCAAG AAGGGTGTGT GCTGACTATT 50

SEQ ID NO: 4 Sequence length: 50 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATCGTATCGA TAAGCTTATG GTGCTGACTA TTGGTAATTT CGACGGCGTG 50

SEQ ID NO: 5 Sequence length: 50 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence ATCGTATCGA TAAGCTTATG GGTCATCGCG CGCTGTTACA GGGCTTGCAG 50

SEQ ID NO: 6 Sequence length: 41 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GATTTATAGG CATGCATCAG ATTCTCGGAT CCGTATTTCG G 41

SEQ ID NO: 7 Sequence length: 53 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CGATGAATTC GGATCCGCTC TTATTTCAGT TCGTCCAGCG ACGCAAATCG CTG 53

SEQ ID NO: 8 Sequence length: 33 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GTGCTGACTA TTCGTAATTT CGACGGCGTC CAT 33

SEQ ID NO: 9 Sequence length: 33 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GTGCTGACTA TTGCTAATTT CGACGGCGTC CAT 33

SEQ ID NO: 10 Sequence length: 33 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GTGCTGACTA TTGATAATTT CGACGGCGTC CAT 33

SEQ ID NO: 11 Sequence length: 26 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCGTTAATCT GCAGGCACTC GTTACG 26

[0058]

According to the present invention, flavin nucleotides, which are important as raw materials for nutrients, various pharmaceutical materials, and reagents for biochemical research, can be produced at a higher yield and at lower cost.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI (C12P 19/32 C12R 1:19) (56) References JP-A-2-138988 (JP, A) JP-A-3-139283 (JP, A) J, Biol. Chem. Vol. 9, (1985), p. 5616− 5620 (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12P 19/32 CA (STN) REGISTRY (STN) BIOSIS / WPI (DIALOG)

Claims (13)

(57) [Claims] 1. A microorganism derived from a microorganism belonging to the genus Escherichia .
In the presence of microorganisms belonging to the genus Escherichia having a recombinant DNA containing a DNA fragment containing a gene encoding protein X, a microorganism, a culture solution or a processed product thereof,
Riboflavin and / or flavin mono in aqueous medium
The nucleotide is reacted with adenosine-5'-3 phosphate, and flavin mononucleotide and flavin mononucleotide generated from the reaction solution are reacted .
Flavin mononucleotide and collecting the pre / or flavin adenine dinucleotide and /
Or a method for producing flavin adenine dinucleotide . 2. The flavin mononucleotide and / or plasmid according to claim 1, wherein the recombinant DNA is pFK5A.
A method for producing rabin adenine dinucleotide . 3. The DNA fragment belongs to the genus Escherichia.
Per protein X derived from that microorganism, while keeping remain flavokinase activity, flavin mononucleotide according to claim 1, wherein a DNA fragment containing the gene encoding the modified protein having reduced or lost flavin adenine dinucleotide synthetase activity and / Or flavin
A method for producing adenine dinucleotide . 4. The flavin mononucleotide and the flavin mononucleotide according to claim 3 , wherein the gene encodes an amino acid sequence in which protein X derived from a microorganism belonging to the genus Escherichia is deleted with an amino acid within 30 amino acids from the N-terminal .
And / or a method for producing flavin adenine dinucleotide . 5. The recombinant DNA is pND18 or pN18.
The flavin mononucleotide according to claim 4, which is D30.
And / or a method for producing flavin adenine dinucleotide . 6. The gene, wherein glycine, which is the 23rd amino acid of protein X derived from a microorganism belonging to the genus Escherichia, is replaced with arginine, alanine or asparagine.
A gene encoding the amino acid sequence is substituted with an acid according to claim 3, wherein the flavin mononucleotide and / or
A method for producing flavin adenine dinucleotide . 7. The recombinant DNA is pKK11, pKK1
7. Flavin mono according to claim 6, which is 2 or pKK13.
The nucleotide and / or Furabin'adeni Njinukure
Otide manufacturing method. 8. A microorganism derived from a microorganism belonging to the genus Escherichia.
While keeping remain flavokinase activity per protein X, reduced flavin adenine dinucleotide synthetase activity or abolished, and protein X N-from end 3
A recombinant DNA of a DNA fragment containing a gene encoding an amino acid sequence in which no more than 0 amino acids have been deleted and a vector DNA fragment. 9. The recombinant DNA is pND18 or pN18.
The recombinant DNA according to claim 8, which is D30. 10. A microorganism derived from the genus Escherichia.
A recombinant DNA of a DNA fragment containing a gene encoding an amino acid sequence obtained by substituting arginine, alanine or aspartic acid, which is the 23rd amino acid for protein X, with a vector DNA fragment. 11. The recombinant DNA is pKK11, pKK
The recombinant DNA according to claim 10, which is 12 or pKK13. A microorganism belonging to the genus Escherichia, comprising the recombinant DNA according to any one of claims 8 to 11 . 13. The microorganism belonging to the genus Escherichia is the Escherichia coli DH5α / pKK11 strain (FERM BP-
4031), Escherichia coli DH5α / pKK12 strain (FERM BP-4032) or Escherichia coli DH5α
The microorganism according to claim 12, which is a / pKK13 strain (FERM BP-4033).