JP2003230377A - New microorganism and method for producing pravastatin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new microorganism useful for effectively producing pravastatin from mevastatin in high production rate and to provide a method for producing the pravastatin by using the same. <P>SOLUTION: The sequence of 502 bases from the 5' end of a 16S rRNA gene of the microorganism is manifested in the sequence No 1 in a sequence table (refer to the specification), and the method for producing the pravastatins comprises acting the microorganism on the mevastatins. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a pravastatin useful as an antihyperlipidemic agent, a salt thereof or a lactone useful for obtaining a lactone ring closure thereof, and pravastatin using the microorganism, a salt thereof or a lactone thereof. The present invention relates to a method for producing a closed ring.

[0002]

2. Description of the Related Art As a method for producing pravastatin useful as an antihyperlipidemic agent, particularly as a bioconversion method, a method for converting mevastatin using dog and rabbit liver homogenate is disclosed in JP-B-61- It is disclosed in Japanese Patent No. 13699. As a method of converting from mevastatin using a microorganism, Absidi (Absidi)
a) Genus, Cunninghamella
a) Genus, Syncephalo
sporum and Streptomyces (Strep)
The production method using the genus
It is disclosed in Japanese Patent Publication No. 476 and Japanese Patent Publication No. 63-21672. In addition, Japanese Examined Patent Publication No. 63-28595 and Japanese Examined Patent Publication No. 03-54103 disclose that Mucor (Muc
or), Rhisopus, Zygorynchus, Circinera (Cire)
inella), actinomu call (Actinomu)
cor), Gongronella,
Phycomyces, Mortierella, Pitanoporus (Pyt)
hanoporus) and Rhizoctonia (Rhizoc)
A method of using a microorganism belonging to each genus of Tonia) is disclosed. Furthermore, Japanese Patent Publication No. 03-71116 discloses a manufacturing method using a genus Nocardia. In addition, Japanese Patent Publication No. 04-82135
Japanese Patent Publication No. 07-24579 and Japanese Patent Publication No. 07-24579 disclose a novel microorganism Streptomyces carbophilus (Strept).
omycescarbophilus) is 3 ", 6 '
Although disclosed as a dihydroxy-mevastatin derivative-producing bacterium, "Actinomycetes Encyclopedia" (ed.
997, Asakura Shoten, p129), and "The topic of medicine-Background of development and medicinal effects" (edited by Japan Society for Agricultural Chemistry, 19).
In 1994, the Society Publication Center, P76-81), etc., describes that the bacterium is a pravastatin-producing bacterium.
Japanese Patent Application Laid-Open No. 2001-286293 discloses a method for producing pravastatin using the genus Microtetraspora. However, the conventional manufacturing method is not satisfactory because the conversion rate is low and the production efficiency is poor.

[0003]

Accordingly, an object of the present invention is to provide a novel microorganism useful for efficiently producing pravastatin from mevastatin at a high production rate, and a method for producing pravastatin using the same. It is in.

[0004]

Means for Solving the Problems As a result of intensive studies for achieving the above-mentioned object, the present inventors have succeeded in converting the 6β-position hydrogen atom of mevastatin into a hydroxyl group, and are capable of producing pravastatin with high productivity. The present invention has been completed by finding the above microorganisms.

That is, the present invention provides a microorganism in which the sequence of 502 bases on the 5'end side of the 16S rRNA gene is the base sequence shown in SEQ ID NO: 1 in the sequence listing.
The present invention also allows the microorganism to act on mevastatin represented by the following formula (I), a lactone ring-opened body thereof or a salt thereof (hereinafter, these may be collectively referred to as mevastatins), A method for producing pravastatin, a salt thereof or a ring-closed lactone thereof, which is converted into pravastatin, a salt thereof or a ring-closed lactone thereof represented by (II) (hereinafter, these may be collectively referred to as pravastatins) Is. The present invention also provides
A mevastatin represented by the following formula (I) and its lactone were prepared from a microorganism in which the 5'terminal 502 base sequence of the 6S rRNA gene shows 99% or more homology with the base sequence represented by SEQ ID NO: 1 in the sequence listing. Pravastatin, which acts on a ring-opened compound or a salt thereof to convert pravastatin represented by the following formula (II), a salt thereof or a lactone ring-closed compound thereof, pravastatin:
The present invention provides a method for producing a salt thereof or a lactone ring-closed body thereof.

[0006]

[Chemical 7]

[0007]

[Chemical 8]

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism of the present invention is 16S rR.
As long as the 502 base sequence on the 5'end side of the NA gene is the base sequence shown in SEQ ID NO: 1 in the sequence listing, it is derived by a wild strain, a mutant strain, or a genetic method such as cell fusion or gene manipulation method. It may be any strain such as a recombinant strain. These microorganisms of the present invention have the ability to convert mevastatins to pravastatins.

The microorganism having the above base sequence can be obtained from soil. In particular, as a strain having a high ability to convert mevastatins to pravastatins, Amycolatopsis sp.
G6506 strain and G6508 strain can be illustrated. These microorganisms are labeled as “Amycolate” on the Patent Biological Depositary Center of the National Institute of Advanced Industrial Science and Technology as an indication of the microorganisms (indication for identification given by the depositor).
opsis sp. G6506 ”Contract number“ FERM ”
P-18682 "and" Amycolatopsis s
p. G6508 Accession number "FERM P-1868"
3 ”has been deposited.

The G6506 strain is a strain isolated from soil in Ehime prefecture. As a method of separating this strain,
A method of applying the soil suspension to a selective medium having the following composition and aerobically culturing was used. [Composition of selective medium: glycerin
1%, vitamin-free casein 0.03%, KNO ₃
0.2%, 0.2% NaCl, MgSO 4 · 7H 2 O
0.005%, CaCO ₃ 0.002%, FeSO ₄
・ 7H ₂ O 0.001%, agar 1.8%, (pH
7.1)]

The G6508 strain is a strain isolated from soil in Tokyo. This strain was isolated in the same manner as the G6506 strain using the selective medium having the above composition.

The mycological properties of both strains of the present invention are as follows. 1. Morphological cells of both strains are divided into filamentous cells. It forms aerial and basal hyphae and does not form sporangia or motile spores.

2. Physiological property test (1) Degradation of citric acid: negative (2) Growth at 45 ° C: negative (3) Acid production from inositol: negative

3. Chemotaxonomic properties Both strains are Gram-positive strains, and the cell wall component diaminopimericac
id) isomer type is meso type and cell wall chemical type is II
It was type I. Arabinose and galactose were detected as the characteristic sugar composition of all the cells, and the cells were of type A. Mycolic acid was not detected as a bacterial cell component. The phospholipid was PII type and the menaquinone composition was MK-9.

4.16S rRNA (ribosome RN
Genetic analysis of A) For the G6506 strain and G6508 strain described above, 1
The nucleotide sequence of about 500 bp at the 5'end of the 6S rRNA gene was determined. Specifically, the base sequence was determined by extracting DNA from both strains, amplifying the base of 16S rDNA corresponding to 16S rRNA by a conventional polymerase chain reaction (PCR) method, and determining by a sequencer. The sequences of 502 bases on the 5'end side of the 16S rRNA gene of both strains were completely the same. The determined nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing. Furthermore, based on the obtained nucleotide sequence, a database (MicroSeq ^™ Bacterial 500 Library v.0.0023 and GenB
ank / EMBL / DDBJ international DNA database)
The homology between both strains and the related strains was examined. The results are shown in Table 1. In addition, a reference strain (Type S
Fig. 1 shows the phylogenetic tree by the neighborhood joining method with the train).

[0016]

[Table 1]

Based on the determined nucleotide sequence (SEQ ID NO: 1), homology search results (Table 1), and a phylogenetic tree of the closely related strain with the reference strain by the neighborhood conjugation method (FIG. 1), the above-mentioned G6506 strain and G
The 6508 strain is Amycolatopsis.
Amycolatopsis, which has a high degree of affinity with A.
homology with rubidus) is 98.04%,
No related species forming a cluster were found in the phylogenetic tree.

Next, in addition to the gene analysis of rRNA, morphological analysis, physiological property tests, and mycological properties such as chemotaxonomic properties are taken into consideration, and the belonging species of both strains are identified by the Berjay bacterial classification ( Bergey's manual of determinative bacteriol
ogy, ninth edition, 1994), and as a result, both strains (G6506 strain and G6508 strain) were examined.
Is Amycolatopsis
A new species belonging to the genus Amycolatopsis sp.
colitopsis sp. ) Was determined.

The genus Nocardia described in Japanese Patent Publication No. 03-71116, which has been known as a pravastatin-producing bacterium, is known to have a cell wall of the IV type (Japanese Patent Publication No. 03-71116). 71116 and The Journal of Antibiotics, 36 (9), 1176-11.
83 (1983)). Moreover, the novel microorganism Streptomyces carbophilus (Streptom) described in Japanese Patent Publication No. 04-82135 and Japanese Patent Publication No. 07-24579.
yes carbophilus) is known to have a cell wall chemical type of type I. However, the chemical type of the cell wall of both strains belonging to the genus Amycolatopsis of the present invention is type III, which is clearly different from these strains.

The microorganism of the present invention is useful for converting the 6β-position hydrogen atom of the mevastatin represented by the formula (I) into a hydroxyl group and producing the pravastatin represented by the formula (II). Mevastatin may be mevastatin, a lactone ring-opened body thereof or a salt thereof, and pravastatin, a salt thereof or a lactone ring-closed body thereof is produced as pravastatin.

A preferred embodiment of the method for producing pravastatin of the present invention will be described below. In the method for producing pravastatin of the present invention, the microorganism of the present invention, or 502 at the 5'end of the 16S rRNA gene
A microorganism having a base sequence having 99% or more homology with the base sequence represented by SEQ ID NO: 1 in the sequence listing is used. Any of these microorganisms may be allowed to act on mevastatin, and the mevastatin that is a starting material (substrate) may be subjected to an incubation treatment. Microorganisms to be acted on include strains, cultured bacterial cells, cultured bacterial cell preparations (homogenized bacterial cells, etc.), and treated cultured bacterial cells (immobilized bacterial cells, etc.).
Any of the above can be used, and when the microorganism is allowed to act, an enzyme derived from the microorganism (crude enzyme, immobilized enzyme, etc.) may be present.

The method of incubating the substrate by allowing the microorganisms to act on the substrate includes a method of adding the substrate to a culture solution of the strain when the strain is cultivated, the cultured bacterial cell, and the cultured bacterial cell preparation. Alternatively, a method may be mentioned in which a substrate is added to the suspension of the treated product of cultured cells and the mixture is incubated while a gas containing oxygen, for example, air is aerated.

In the above-mentioned method, the substrate may be added to the culture broth at any time when the culture is started or when a certain period has elapsed after the start of the culture. The culture of the strain and the culture of the strain performed in a state where the substrate is added can be carried out in principle according to a general microorganism culture method, but usually, shaking culture by liquid culture, aeration stirring culture. It is preferable to carry out under aerobic conditions such as.

The medium used for the culture may be any medium containing a nutrient source that can be utilized by microorganisms belonging to the genus Amycolatopsis, and various synthetic media, semi-synthetic media, natural media and the like can be used. It is available. Glucose, maltose, xylose, fructose, sucrose and the like can be used as the carbon source in the medium alone or in combination. These carbon sources may be added at any time during the culturing. For example, when glucose is fed so as to have a concentration range of 3 to 7 g / l, the conversion rate of mevastatins to pravastatins relatively increases. You can Also, as a nitrogen source, peptone, meat extract, soybean powder, casein,
Amino acids, malt extracts, yeast extracts, organic nitrogen sources such as urea, and inorganic nitrogen sources such as sodium nitrate and ammonium sulfate can be used alone or in combination. Others, such as sodium chloride, potassium chloride, calcium carbonate,
If necessary, salts of magnesium sulfate, sodium phosphate, potassium phosphate, cobalt chloride, etc., heavy metal salts, and vitamins can be added and used. When foaming is remarkable during culture, various known defoaming agents can be appropriately added to the medium.

The culture conditions of the strain can be appropriately selected within a range where the strain can grow well. Usually p
It is advisable to culture at H6 to 9.5, 25 to 35 ° C for about 2 to 8 days. The various culture conditions described above can be appropriately changed according to the type and characteristics of the microorganism used, external conditions, etc., and the optimum conditions can be selected. The amount of the strain to be inoculated into the medium is preferably 1 platinum loop in the case of flask culture, and 1 to 5% (v / v) of the main culture solution in the case of scale-up, but substantially This does not apply if it can be cultured.

The substrate, mevastatin, can be added to the culture solution as a powder or dissolved in a water-soluble organic solvent such as ethanol, and the addition amount thereof is 0.5 to 1 liter of the culture solution. 4.0 g is preferred. When the amount added is more than 4.0 g / l, the conversion rate becomes remarkably slow, which is not preferable. After adding the substrate, preferably 25 to 35
By converting the substrate mevastatin to the desired pravastatin by performing an operation such as shaking or aeration and stirring at 1 ° C for 1 to 7 days, particularly about 2 to 5 days, and proceeding the reaction under aerobic conditions. can do.

The cultured cells in the above method are
It can be obtained by culturing the above-mentioned strain as described above and then separating from the culture solution by centrifugation or filtration. The cultured bacterial cell preparation is, for example, a homogenized bacterial cell or the like, and the treated cultured bacterial cell is obtained by immobilizing the cultured bacterial cell on, for example, an ion exchange resin, a ceramic, or chitosan. Immobilized cells and the like. These cultured cells,
As a solution that can be used for preparing a suspension of a cultured cell preparation or a treated product of a cultured cell, the above-mentioned medium, or Tris-acetic acid, Tris-hydrochloric acid, sodium succinate, sodium citrate, sodium phosphate, phosphorus is used. Examples include buffer solutions such as potassium acid alone or in a mixture. The pH of the buffer solution is preferably 6.0 to 9.0, more preferably 7.0 to 8.5. The amount of cultured cells in the suspension is preferably about 0.1 to 5% in terms of wet volume ratio. Further, the addition of the substrate to the suspension may be carried out in the same manner as the addition of the substrate to the culture solution in the above method, and the addition amount of the substrate per one time can maintain the activity of the bacterial cells. The range is preferable, but 0.1 to 5 g per liter of the suspension may be added once or several times, or may be continuously added at about 0.5 to 5 g / day. The incubation in the above method may be performed in the same manner as the culture of the strain in the above method.

In order to isolate the desired pravastatins thus produced from the reaction system, it is preferable to select and combine various known purification means. For example, adsorption / elution on a hydrophobic adsorption resin, solvent extraction using ethyl acetate, n-butanol, etc., column method using silica gel or thin layer chromatography, preparative high performance liquid chromatography using a reversed phase column, etc. Can be separated and purified by using the above, alone or in combination as appropriate, and if necessary repeatedly used.

[0029]

EXAMPLES The present invention will be described in more detail below with reference to specific examples, but the present invention is not intended to be limited to these examples. The percentages (%) in the following examples are W / V percentages unless otherwise specified.

Example 1 Glucose 2.0%, Polype
Puton 1.5%, yeast extract 0.05%, NaH ₂PO_Four
・ 2H₂O 0.05%, MgSO_Four・ 7H₂O 0.05
Add 10 ml of medium containing 10% to a 24φ test tube,
It was sterilized by heating at ℃ for 20 minutes. Amycolatopsis
Inoculated with the strain G6506 (1 platinum loop)
Grow on shaker (310rpm) for 4 days at 27 ℃.
I nourished. The final concentration of mevastatin is 500mg /
1), and cultivated under the same conditions for 3 days
It was The obtained culture solution is centrifuged at 14000 rpm for 10 minutes.
The supernatant obtained by separation was analyzed by HPLC under the following conditions.
Analysis revealed that the concentration of pravastatin was 430 mg
/ L, the conversion of mevastatin to pravastatin
The rate was 86%.

<HPLC analysis conditions> Column: symmetry (registered trademark, manufactured by Waters) C18 5 μm (3.9φ × 150 mm) Mobile phase: acetonitrile / PIC-A reagent (5 mM)
(360: 640) Flow rate of mixed solution: 1.0 ml / min Detection: 237 nm

[Examples 2 to 8] 10 ml of a medium containing the same components as in Example 1 was placed in a 24φ test tube and kept at 121 ° C and 20 ° C.
Heat sterilized for 1 minute. This was inoculated with the strains shown in Table 2 (inoculation amount: 1 platinum loop), and cultured at 28 ° C. for 4 days on a shaking culture machine (310 rpm). Mevastatin was added to this so that the concentration was as shown in Table 2, and the cells were further cultured for 3 days under the same conditions. The results of HPLC analysis of the obtained culture solution in the same manner as in Example 1 are shown in Table 2. The amount of pravastatin produced increased up to the mevastatin addition amount of 2000 mg / l, but decreased conversely at 4000 mg / l.

[0033]

[Table 2]

Example 9 Glucose 2.0%, Polype
Puton 1.5%, yeast extract 0.05%, NaH ₂PO_Four
・ 2H₂O 0.05%, MgSO_Four・ 7H₂O 0.05
%, LG-294 (Asahi Denka Kogyo KK) 0.05%
50 ml of preculture medium consisting of
It was put in a box and sterilized by heating at 121 ° C. for 20 minutes. A
Inoculated with mycotopsis SP G6506 strain (contact
Seed amount 1 platinum loop), 27 ° C for 2 days, rotary shaker
(220 rpm) to prepare a preculture liquid.
Next, a 2.6-liter jar fermenter (MD-2
50: manufactured by Marubishi Bioengineering Co., Ltd.) with a pre-culture medium
Prepare 1.5 liters of medium for conversion culture of the same composition, 121 ° C,
Heat sterilized for 30 minutes. This is contacted with 150 ml of preculture liquid
Seed, temperature 27 ℃, stirring number 300 rpm, aeration 1.0
It was cultured under the condition of vvm. After 2 days, the cells grew well
Confirm that the concentration of mevastatin is 2000 mg / l
Was added and the culture was continued. Mevastatin
Three days after the addition, 1770 mg / l pravastati
Were produced. The conversion rate at this time was 88.5%.
It was

[Example 10] The same procedure as in Example 9 was carried out except that the Amycolatopsis SP G6506 strain of Example 9 was changed to the Amycolatopsis SP G6508 strain. Three days after the addition of mevastatin, 1790
mg / l pravastatin was produced. The conversion rate at this time was 89.5%.

[0036]

According to the present invention, it is possible to provide a novel microorganism useful for efficiently producing pravastatin from mevastatin at a high production rate, and a method for producing pravastatin using the same.

[0037]

[Sequence list] <110> ASAHI DENKA KOGYO KABUSHIKI KAISHA <120> Novel microorganism and method for producing pravastatin <130> A0206 <160> 1 <210> 1 <211> 502 <212> RNA <213> Amycolatopsis sp. <400> 1 tggagagttt gatcctggct caggacgaac gctggcggcg tgcttaacac atgcaagtcg 60 aacgctgaac cggtttcggc cggggatgag tggcgaacgg gtgagtaaca cgtgggtaat 120 ctgccttgta ctctgggata agcctgggaa actgggtcta ataccggata tgactcttca 180 ccgcatggtg gggggtggaa agttccggcg gtacaggatg aacccgcggc ctatcagctt 240 gttggtgggg taatggccca ccaaggcgac gacgggtagc cggcctgaga gggtgaccgg 300 ccacactggg actgagacac ggcccagact cctacgggag gcagcagtgg ggaatattgc 360 acaatgggcg caagcctgat gcagcgacgc cgcgtgaggg atgacggcct tcgggttgta 420 aacctctttc gccagggacg aagcgcaagt gacggtacct ggataagaag caccggctaa 480 ctacgtgcca gcagccgcgg ta 502

[Brief description of drawings]

FIG. 1 is a schematic diagram of Amycolatopsis (Amyc) of the present invention.
It is a figure which shows the phylogenetic tree based on the homology of the nucleotide sequence of 16S rRNA gene of a new-type microorganism which belongs to the genus Olatopsis), and a closely related strain.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] February 19, 2002 (2002.2.1
9)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0037

[Correction method] Change

[Correction content]

[0037]

[Sequence list] <110> ASAHI DENKA KOGYO KABUSHIKI KAISHA <120> Novel microorganism and method for producing pravastatin <130> A0206 <160> 1 <210> 1 <211> 502 <212> D NA <213> Amycolatopsis sp. <400> 1 tggagagttt gatcctggct caggacgaac gctggcggcg tgcttaacac atgcaagtcg 60 aacgctgaac cggtttcggc cggggatgag tggcgaacgg gtgagtaaca cgtgggtaat 120 ctgccttgta ctctgggata agcctgggaa actgggtcta ataccggata tgactcttca 180 ccgcatggtg gggggtggaa agttccggcg gtacaggatg aacccgcggc ctatcagctt 240 gttggtgggg taatggccca ccaaggcgac gacgggtagc cggcctgaga gggtgaccgg 300 ccacactggg actgagacac ggcccagact cctacgggag gcagcagtgg ggaatattgc 360 acaatgggcg caagcctgat gcagcgacgc cgcgtgaggg atgacggcct tcgggttgta 420 aacctctttc gccagggacg aagcgcaagt gacggtacct ggataagaag caccggctaa 480 ctacgtgcca gcagccgcgg ta 502

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 4B024 AA01 AA11 CA11 HA11                 4B064 AD65 BH20 CA02 CB12 CD04                       DA01                 4B065 AA01X BA22 BB09 CA12                       CA44

Claims (7)

[Claims] 1. A microorganism in which the sequence of 502 bases on the 5'end side of the 16S rRNA gene is the base sequence shown in SEQ ID NO: 1 in the sequence listing. 2. A compound having the ability to convert a mevastatin represented by the following formula (I), an opened lactone thereof or a salt thereof into a pravastatin represented by the following formula (II), a salt thereof or a closed lactone thereof. 1. The microorganism according to 1. [Chemical 1] [Chemical 2] 3. Amycolatosis
The microorganism according to claim 1, which belongs to the genus (psis). 4. Amycolatopsis SP
olatopsis sp. ) G6506 (FERM P
-18682) The microorganism according to claim 1, which is a strain. 5. Amycolatopsis SP
olatopsis sp. ) G6508 (FERM P
-18683) The microorganism according to claim 1, which is a strain. 6. A compound represented by the following formula (II) by reacting the microorganism according to any one of claims 1 to 5 with mevastatin represented by the following formula (I), a lactone ring-opened product thereof or a salt thereof. A method for producing pravastatin, a salt thereof, or a lactone ring-closed product thereof, which comprises converting pravastatin, a salt thereof or a ring-closed lactone thereof. [Chemical 3] [Chemical 4] 7. A microorganism having a 502 base sequence on the 5'end side of the 16S rRNA gene showing 99% or more homology with the base sequence shown in SEQ ID NO: 1 in the sequence listing is represented by the following formula (I). A method for producing pravastatin, a salt thereof or a closed lactone thereof, which comprises converting pravastatin represented by the following formula (II), a salt thereof or a closed lactone thereof to a pravastatin, a salt thereof or a closed lactone thereof, by acting on mevastatin, a lactone opened ring thereof or a salt thereof. [Chemical 5] [Chemical 6]