JP2003093046A - Useful microorganism for transformation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain pravastatin in a short culturing time in the presence of ML-236B sodium salt in high concentration by biotransformation using a microorganism. SOLUTION: Pravastatin is produced in high yield by using a microorganism having ability to efficiently hydroxylate ML-236B sodium salt in the presence of ML-236B sodium salt in high concentration and belonging to the genus Streptomyces, such as Streptomyces rimosus subsp. rimosus GS00247 strain, Streptomyces lavendulae N-67 strain, Streptomyces sp. 34-3 strain, Streptomyces sp. 34-7 strain or Streptomyces sp. 974 strain.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently producing pravastatin (formula 2) by converting ML-236B sodium salt (formula 1) by the action of a microorganism or a microbial enzyme.

[0002]

2. Description of the Related Art ML-236B sodium salt is HMG
It is known that by inhibiting CoA reductase, cholesterol biosynthesis is reduced and a strong serum cholesterol lowering action is exhibited. On the other hand, ML-23
Some of the hydroxylated derivatives of 6B sodium salt are known to be more effective than ML-236B sodium salt as competitive inhibitors of HMG-CoA reductase, one representative of which is pravastatin. The hydroxylation of ML-236B sodium salt can be carried out by various fungi and actinomycetes such as Nocardia actinomycete (Japanese Patent Publication No. 3-71116) and Streptomyces carbophilus (Japanese Patent Publication No. 7-24579). Has been reported. Further, hydroxylation is carried out by culturing the microorganism in a medium containing ML-236B sodium salt, or by culturing and recovering the microorganism, and then using the bacterial cell or bacterial cell extract thereof.
The method of converting sodium salt is mainly used.

[0003]

However, in the conventionally known method, since the growth of the bacterium is inhibited by the action of ML-236B sodium salt, it is necessary to add ML-236B sodium salt from the beginning of the culture. However, since it was necessary to gradually add it at a concentration not exceeding 0.5 mg / ml, long-term culture was required, and efficient production of pravastatin was not possible.

[0004]

[Means for Solving the Problems] As a result of intensive studies on a method for efficiently producing pravastatin, the present inventors have found that Streptomyces rimosus subsp.
us) GS00247 strain, Streptomyces lavendulae N-67 strain, Streptomyces sp. 34-3
Strain, Streptomyces sp.
34-7 strain, or Streptomyces sp.
(Streptomyces sp.) No. Five 974 strains of actinomycetes were obtained. By culturing these microorganisms in a medium containing ML-236B sodium salt, ML-236B sodium salt can be converted from the initial medium even under high concentration conditions, and pravastatin can be obtained in a high yield in a short period of time. It was found that the present invention has been completed.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention, ML-236B sodium salt is used as a medium component.
Remosas Sub-SP Limosus (Streptomyces rimo
sus subsp. rimosus) GS00247 strain, Streptomyces lavendulae N-
67 strains, Streptomyces sp.
sp.) 34-3 strain, Streptomyces sp.
tomyces sp.) 34-7 strain, or Streptomyces sp. No. It is characterized in that pravastatin is accumulated in the culture medium by inoculating any of the 974 strains into a medium and culturing under a certain condition.

Streptomyces rimosu (Streptomyces rimosu) used in the present invention
s subsp. rimosus) GS00247 strain, Streptomyces lavendulae N-
67 strains, Streptomyces sp.
sp.) 34-3 strain, Streptomyces sp.
tomyces sp.) strain 34-7, and Streptomyces sp.
Streptomyces sp. No. The identification results of the 974 strain are as follows. 1. GS00247 strain (1) Growth on agar medium: On an agar medium (yeast-starch medium), colonies show a gray color, and both growth and formation of aerial hyphae are vigorous. (2) Cell wall composition: Main component LL-diaminopimelic acid,
Glycine (3) Main fatty acid composition: 16: 0 ISO 50%, 16: 1 CIS 9
13%, 14: 0 Not applicable stock in ISO 5% MIDI database. (4) 16S rRNA nucleotide sequence: Microseq
As a result of collation by the database, Streptomyces rimosus
95.6% homology with subsp. rimosus. 2. N-67 strain (1) Growth on agar medium: On an agar medium (yeast-starch medium), colonies show cream to beige color, and both growth and formation of aerial hyphae are vigorous. (2) Cell wall composition: Main component LL-diaminopimelic acid,
Glycine (3) Main fatty acid composition: 16: 0 ISO 28%, 15: 0 ANTEIS
O 24% Streptomyces exfol in MIDI database
Valid for iatus 0.546. (4) 16S rRNA nucleotide sequence: Microseq
As a result of collation by database, lavendulae and 98.6
% Homology. 3.34-3 strain (1) Growth on agar medium: On an agar medium (yeast-starch medium), colonies show a gray color, and both growth and formation of aerial hyphae are vigorous. (2) Cell wall composition: Main component LL-diaminopimelic acid,
Glycine (3) Main fatty acid composition: 15: 0 ANTEISO 28%, 16: 0 IS
O 19% Identified as Streptomyces in the MIDI database. (4) 16S rRNA nucleotide sequence: Microseq
As a result of collation by the database, it is found that the homology is 96.6%.
Presented by Streptomyces anulatus. Meanwhile, BLAST SE
In the ARCH database, Streptomyces sp. OB-23 (A
ccession No. AF112177.1) and 98.7% homology. 4.34-7 strain (1) Growth on agar medium: Colonies appear gray on an agar medium (yeast starch medium), and both growth and formation of aerial hyphae are vigorous. (2) Cell wall composition: Main component LL-diaminopimelic acid,
Glycine (3) Main fatty acid composition: 15: 0 ANTEISO 39%, 16: 0 IS
O 18%, 17: 0 ANTEISO12% S at MIDI database
Identified as genus treptomyces. (4) 16S rRNA nucleotide sequence: Microseq
As a result of collation with the database, the homology of 99.6%
Presented by Streptomyces anulatus. Meanwhile, BLAST SE
In the ARCH database, Streptomyces sp. OB-23 (A
ccession No. AF112177.1) and 99.6% homology. 5. No. Strain 974 (1) Growth on agar medium: Colonies are gray on an agar medium (yeast-starch medium), and both growth and formation of aerial hyphae are vigorous. (2) Cell wall composition: Main component LL-diaminopimelic acid,
Glycine (3) Main fatty acid composition: 15: 0 ANTEISO 38%, 16: 0 IS
O 18%, 17: 0 ANTEISO10% S at MIDI database
Identified as genus treptomyces. (4) 16S rRNA nucleotide sequence: Microseq
As a result of collation with the database, the homology of 99.6%
Presented by Streptomyces anulatus. Meanwhile, BLAST SE
In the ARCH database, Streptomyces sp. OB-23 (A
ccession No. AF112177.1) and 99.6% homology.

From the above results and colony findings, G
The S00247 strain is Streptomyces rimosus subsp. Rim.
osus) and N-67 strain were identified as Streptomyces lavendulae.

[0008] 34-3 strain, 34-7 strain, No. 3 strains of 974 strains were micros based on 16S rRNA nucleotide sequence.
Streptomyces by seq database search
Although annulus (Streptomyces anulatus) was presented, a large difference was observed in the mycological findings. On the other hand, BL
In the AST SEARCH database, all 3 strains are Streptomyces sp. OB-23 (Streptomyces sp. OB-
It was confirmed that the homology with 23) was extremely high. From the above results, we determined that these 3 strains were closely related to Streptomyces sp. OB-23 (Streptomyces sp. OB-23), but the OB-23 strain is a new strain that is being applied for registration. , 34-3 strain, 34-7 because the name is not registered
Stock, No. Three of the 974 strains were Streptomyces sp.

The transformant strain used in the present invention is the above-mentioned GS00.
247 strains, N-67 strains, 34-3 strains, 34-7 strains, N
o. Not only the 974 strain, but also the artificial mutant or natural mutant can be used as long as it has the conversion ability. GS00247 strain, N-67 strain, 34-3 strain,
34-7 strain, No. The artificial mutant strain of strain 974 can be easily obtained by, for example, ultraviolet irradiation, cobalt 60 irradiation, or a chemical mutagenesis agent. It is also possible to introduce this conversion ability into other microorganisms by a genetic engineering technique.

In the culture for conversion of ML-236B sodium salt into pravastatin, any condition under which the conversion reaction to pravastatin proceeds can be selected. As a culturing method, an ordinary culturing method of filamentous fungi can be used, but a method of shaking culture or stirring culture using a liquid medium is preferable. Further, in addition to ML-236B sodium salt, a carbon source, a nitrogen source, vitamins and minerals necessary for the growth of microorganisms, an additive that promotes the conversion reaction to pravastatin may be used during the culture. The preferred culture temperature is 18 ° C to 50 ° C, more preferably 25 ° C to 33 ° C.

The present invention is directed to Streptomyces rimosus subs.
p. rimosus) GS00247 strain, Streptomyces
Lavendulae (Streptomyces lavendulae) N-67
Strain, Streptomyces sp.
34-3 strain, Streptomyces sp.
ces sp.) 34-7 strain, and Streptomyces sp. It includes any conversion rate of pravastatin by strain 974, preferably 30% or more, more preferably 60% or more,
Most preferably, it is at least%.

Pravastatin produced by conversion culture is often purified. Purification literally also means complete homogenization, but concentration, precipitation, and
It also includes those that have undergone steps such as extraction with an organic solvent, chromatography (thin layer chromatography, column chromatography, high performance liquid chromatography), and drying.

[0013]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0014]

Example 1 Streptomyces sp. 34-3 strain was inoculated into 20 500 mL Erlenmeyer flasks containing 100 mL of a medium having the composition shown in Table 1 below, and the strains were incubated at 30 ° C. for 21 hours.
The cells were cultivated with rotary shaking at 0 rpm and ML-23 at the same time as the inoculation.
6B sodium salt was added to a final concentration of 2.0 mg / mL, and the mixture was further cultivated for 5 days at 30 ° C. and 210 rpm with rotary shaking.

[0015]

[Table 1] Medium composition

The concentration of pravastatin in the culture medium at the end of the culture was determined by HPLC to be about 1.10 mg / mL, and the conversion rate from ML-236B sodium salt was about 55.
%Met.

After the completion of the culture, the converted culture solution was filtered, and the obtained filtrate was adjusted to pH 3 with trifluoroacetic acid. About 2 L of the pH-adjusted filtrate was extracted three times with about 1 L of ethyl acetate, and the ethyl acetate layer was dehydrated with anhydrous sodium sulfate, filtered and concentrated to 1 L, and a catalytic amount of trifluoroacetic acid was added for lactonization. Next, the lactonized ethyl acetate layer was washed with 1 L of a 5% sodium hydrogen carbonate solution, the ethyl acetate layer was dehydrated with anhydrous sodium sulfate, and then dried under reduced pressure. The dried product was subjected to silica gel column chromatography to collect a fraction containing a lactone form of pravastatin. The obtained fraction was further subjected to a reversed phase HPLC column (Shiseido Capcellpa
(K C18 UG120) was separated and collected to obtain 1.22 g of pravastatin lactone.

[0018]

Example 2 Streptomyces remosus subsp. Remosus GS00247 strain was used in the same manner as in Example 1 to obtain 1.25 pravastatin lactone form.
g was obtained.

[0019]

[Example 3] Streptomyces lavendulae N-
Using 67 strains, the same procedure as in Example 1 was carried out to obtain 1.30 g of pravastatin lactone.

[0020]

Example 4 Using Streptomyces sp. Strain 34-7 and in the same manner as in Example 1, 1.13 g of pravastatin lactone was obtained.

[0021]

[Embodiment 5] Streptomyces sp. 97
Using the 4 strains, the same procedure as in Example 1 was carried out to obtain 1.21 g of pravastatin lactone.

[0022]

Example 6 Streptomyces sp. 34-3 strain was inoculated into three 500 mL Erlenmeyer flasks containing 100 mL of the medium having the medium composition shown in Example 1, 30 ° C, 210 r.
The cells were cultivated with rotary shaking at pm and inoculated into a 30 L jar fermenter containing 15 L of a medium having the composition shown in Table 2 below (inoculation amount 2%), temperature 30 ° C., stirring number 300 rpm, aeration 0 The culture was performed with stirring at 8 v / v / m for 4 days. ML-236B sodium salt at a final concentration of 2.0 mg / mL at the same time as inoculation
Was added. The concentration of pravastatin in the culture broth obtained after the completion of the culturing was determined by HPLC analysis to be about 1.05 mg / mL, and the conversion rate from ML-236B sodium salt was about 53%.

[0023]

[Table 2] Medium composition

After the culture was completed, the converted culture solution was filtered, and the obtained filtrate was adjusted to pH 3 with trifluoroacetic acid. About 15 L of the pH-adjusted filtrate was extracted twice with about 15 L of ethyl acetate,
Then, it was washed with 15 L of saturated saline. The ethyl acetate layer was dehydrated over anhydrous sodium sulfate, filtered, and concentrated to 10 L, to which a catalytic amount of trifluoroacetic acid was added for lactonization. Next, the lactonized ethyl acetate layer was washed twice with 10 L of a 5% sodium hydrogen carbonate solution, the ethyl acetate layer was dehydrated with anhydrous sodium sulfate, and then dried under reduced pressure. The dried product was subjected to silica gel column chromatography to collect a fraction containing a lactone form of pravastatin. The obtained fraction was further subjected to a reversed phase HPLC column (Shiseido CapcellpakC
18 UG120) and fractionated to obtain 9.61 g of pravastatin lactone.

[0025]

Example 7 Using Streptomyces remosus subsp. Remosus GS00247 strain, the procedure similar to that of Example 6 was repeated to obtain the lactone form of pravastatin of 9.52.
g was obtained.

[0026]

[Embodiment 8] Streptomyces lavendulae N-
Using strain 67, the same procedure as in Example 6 was carried out to obtain 9.35 g of a lactone form of pravastatin.

[0027]

[Example 9] Streptomyces sp. 34-3 strain was inoculated into three 500 mL Erlenmeyer flasks containing 100 mL of the medium having the medium composition shown in Example 1 at 30 ° C and 210 r.
Culture was performed by rotary shaking at pm. After 48 hours, 30 L jars containing 15 L of the medium having the composition shown in Table 2 similar to Example 6 were inoculated (inoculation amount 2%), temperature 30 ° C., stirring number 300 r.p.
The culture was carried out with stirring for 5 days at a flow rate of 0.8 v / v / m. Simultaneously with the inoculation, ML-236B sodium salt was added to a final concentration of 0.8 mg / mL. During this period, feed culture was performed so that the glucose concentration was 1% and the ML-236B concentration was 0.8 mg / mL. As a result, the concentration of pravastatin in the culture broth obtained after the completion of the culture was determined by HPLC analysis to be about 1.76 mg / mL, and M
The conversion rate from the L-236B sodium salt was about 55%.

After the culture was completed, the converted culture solution was filtered, and the obtained filtrate was adjusted to pH 4 with trifluoroacetic acid. About 15 L of pH-adjusted filtrate was adsorbed on DIAION HP-20 (manufactured by Mitsubishi Kasei), washed with water, and then acetone / 0.2N NaOH.
= 50/50, the acetone was distilled off with an evaporator, and the mixture was concentrated. The pH was adjusted to 4 again with trifluoroacetic acid, extracted twice with 10 L of ethyl acetate, and washed with 10 L of saturated saline. The ethyl acetate layer was dehydrated with anhydrous sodium sulfate, filtered, and lactonized by adding a catalytic amount of trifluoroacetic acid. Next, the lactonized ethyl acetate layer was washed twice with 10 L of a 5% sodium hydrogen carbonate solution, the ethyl acetate layer was dehydrated with anhydrous sodium sulfate, and then dried under reduced pressure. This dried product was subjected to silica gel column chromatography,
Fractions containing the lactone form of pravastatin were collected. The obtained fraction was further subjected to a reversed phase HPLC column (Shiseido Cap
Cellpak C18 UG120) was separated and collected to obtain 15.2 g of pravastatin lactone.

[0029]

[Example 10] 100 mL of medium having the medium composition shown in Example 1
Streptomyces sp. 34-3 strain was inoculated into 20 500 mL Erlenmeyer flasks containing
The cells were cultivated with rotary shaking at 0 rpm and ML-23 at the same time as the inoculation.
6B sodium salt was added so that the final concentration was 2.0 mg / mL, and the mixture was further cultured for 5 days at 30 ° C. and 210 rpm with rotary shaking.

After the completion of the culture, the culture solution was filtered, the filtrate was adsorbed on Diaion HP-20 (manufactured by Mitsubishi Kasei), washed with water, and the fraction containing pravastatin sodium salt was eluted with 50% acetone. As a lyophilized product, 2.6 g was obtained. HPLC (column: Shiseido Capcellpak C18 U)
The fractionation was repeated with G120, 40% methanol) to obtain 314 mg of pravastatin sodium salt.

[0031]

INDUSTRIAL APPLICABILITY The present invention uses the novel microorganism ML-23.
By culturing in a medium containing 6B sodium salt,
It is useful in that ML-236B sodium salt can be converted to pravastatin at a high yield, and pravastatin can be obtained in a state where there are few related compounds. According to the present invention, it becomes possible to efficiently produce pravastatin, its salts, and lactones, which are useful as therapeutic agents for hyperlipidemia.

Claims (3)

[Claims] 1. The following structural formula (1): ML-236B sodium salt represented by the following general formula (2) , A microorganism belonging to the genus Streptomyces having the activity of converting to pravastatin, Streptomyces risemus subsp.
mosus subsp. rimosus) GS00247 strain (FERM
P-18537), Streptomyces lavendulae N-67 strain (FERM)
P-18532), Streptomyces sp. (Str
eptomycess p.) 34-3 strain (FERM P-1853
6), Streptomyces sp
p.) 34-7 strain (FERM P-18535), or Streptomyces sp. N
o. 974 strain (FERM P-18533). 2. A method for producing pravastatin represented by formula (2) by culturing the microorganism according to claim 1 in a medium containing ML-236B sodium salt represented by formula (1). 3. Derived from the microorganism according to claim 1,
Pravastatin is produced by adding ML-236B sodium salt to a reaction solution containing an enzyme source having an activity of converting ML-236B sodium salt represented by formula (1) into pravastatin represented by formula (2). Method.